WO2000033067A1

WO2000033067A1 - Method for diagnosing neoplasia

Info

Publication number: WO2000033067A1
Application number: PCT/US1999/028099
Authority: WO
Inventors: Li Liu; Bing Zhu; Han Li; W. Joseph Thompson; Gary A. Piazza; Rifat Pamukcu
Original assignee: Cell Pathways, Inc.
Priority date: 1998-11-25
Filing date: 1999-11-24
Publication date: 2000-06-08
Also published as: EP1133689A1; AU1833000A; JP2002531826A; EP1133689A4

Abstract

This invention provides a method for diagnosing a patient with neoplasia.

Description

METHOD FOR DIAGNOSING NEOPLASIA

BACKGROUND OF THE INVENTION

In recent years, new types of neoplasia inhibitors have been emerging. Such compounds selectively induce apoptosis (a form of cell death) in neoplastic, but not in normal cells. Neoplasia - which includes both precancerous and cancerous conditions ~ was historically treated chemotherapeutically only at the cancerous stage. Treatment with chemotherapeutics induced cell death (whether by apoptosis or necrosis) in rapidly proliferating cells indiscriminately (i.e., whether those cells were neoplastic or normal). As a result, most conventional chemotherapeutics caused significant cell death in normal tissues such as hair follicles, intestinal lining, skin and the like, that regenerate rapidly in the body. The side effects (e.g., hair loss, and skin and digestive disorders) of such conventional chemotherapeutics reflect non-specific cell death. As a result, conventional chemotherapeutics are used only on an acute (i.e., short-term) basis.

Because conventional chemotherapeutics non-specifically induce cell death, in both neoplastic and normal cells, such compounds are not recommended for use against precancerous conditions even in patients with the most severe forms of precancerous conditions. For example, in familial polyposis patients — who can each form thousands of colonic polyps — surgical removal of the colon is standard practice

(because of the extremely high cancer risk) whereas conventional chemotherapy is virtually unheard of.

As reported in pending U.S. Patent Application Serial No. 09/216,070, filed December 12, 1998, (Method For Identifying Compounds For Inhibition Of Cancerous Lesions, Pamukcu, et al. (Case No. P-l 19 OP)), which is incorporated herein by reference, the selective neoplasia inhibitors described therein induce apoptosis in neoplastic cells, but not in normal proliferating cells. Thus, as reported in Serial No. 09/216,070, even patients with precancerous lesions can take such inhibitors without the side effects of conventional chemotherapeutics. Given the other attributes of such compounds, they can be taken by patients even on a chronic (i.e., long-term) basis. As reported in that application, a common attribute of such selective neoplasia inhibitors is that they inhibit cyclic GMP (cGMP)-specific phosphodiesterases (PDEs).

Cyclic GMP-specific PDEs include the GMP-binding, cyclic GMP-specific phosphodiesterase (designated cGB-PDE) which is a phosphodiesterase gene family 5 isoenzyme (hereinafter "PDE5"). PDE5 is described more fully, wter alia, by Beavo, et al., in U.S. Patent Nos. 5,652,131 and 5,702, 936, that are incorporated herein by reference. Phosphodiesterase gene families 6 and 9 are also cGMP-specific isoforms. Another cGMP-specific PDE includes one of the types of PDE2 described below. The novel form of PDE2 disclosed herein is fully described by Liu, et al., in pending

U.S. Patent Application Serial No. 09/173,375, A Novel Cyclic GMP-Specific Phosphodiesterase And Methods For Using Same In Pharmaceutical Screening For Identifying Compounds For Inhibition Of Neoplastic Lesions (Case No. P-143), and pending U.S. Patent Application Serial No. 09/414,628, Methods for Identifying Compounds For Inhibition of Neoplastic Lesions, And Pharmaceutical Compositions

Containing Such Compounds (Case No. P-179A), both of which are herein incorporated by reference.

For general background on phosphodiesterases, see, Beavo, J. A. (1995) Cyclic Nucleotide Phosphodiesterases: Functional Implications of Multiple Isoforms, Physiological Reviews 75:725-747; and the web site

<http://weber.u. washington.edu/~pde/pde.html> (Nov. 1998).

BRIEF SUMMARY OF THE INVENTION

This invention involves methods of determining whether a patient with neoplasia has a type of neoplasia that is likely to respond to treatment with a cyclic

GMP-specific PDE inhibitor.

In one aspect, this invention involves exposing a neoplastic tissue sample from a patient to a cyclic GMP-specific PDE inhibitor and monitoring whether the neoplastic tissue sample exhibits a sensitivity to treatment with that inhibitor. Preferably, the cGMP-specific PDE inhibitor used herein has an inhibitory effect on at least the novel PDE2-like enzyme described hereinafter and in U.S. Patent Application Serial No. 09/173,375. In another aspect of the invention, the cGMP- specific PDE inhibitor used herein has an inhibitory effect on at least PDE5 and the PDE2-like enzymes described hereinafter and in U.S. Patent Application Serial Nos. 09/173,375 and 09/414,628.

In another aspect, this invention involves exposing a neoplastic tissue sample from a patient to a SAAND, such as exisulind, and monitoring whether the neoplastic tissue sample exhibits a sensitivity to treatment with that inhibitor by evaluating whether PKG activity increases. The evaluation of PKG activity can include the detection of PKG activation, or the amount of PKG enzyme, or a combination of the two.

In another aspect, this invention involves exposing a neoplastic tissue sample from a patient to an antineoplastic drug, and monitoring whether the neoplastic tissue sample exhibits a sensitivity to treatment with that inhibitor by detection of the levels of beta-catenin.

In another aspect, this invention includes the use of one or more antibodies that are immunoreactive with cGMP-specific PDEs to detect the presence of elevated cGMP-specific PDEs in a neoplastic tissue sample. Preferably, the antibodies are immunoreactive with the PDE2-like enzymes described hereinafter and in U.S. Patent Application Serial Nos. 09/173,375 and 09/414,628. Alternatively, the antibodies preferably are immunoreactive with at least the PDE2-like enzymes described herein and PDE5. Antibodies specific for cGMP-specific PDEs, including PDE5 and the PDE2-like enzymes described herein, can be used in a variety of immunoassay methods, such as EIAs, ELISAs, or RIAs, to detect both the presence and the quantity of cGMP-specific PDEs in a tissue sample. The presence of elevated cGMP-specific

PDE protein in the neoplastic tissue is indicative that the neoplasia is likely to respond to treatment with a cGMP-specific PDE inhibitor.

In another aspect, this invention provides for diagnostic kits for ascertaining whether a particular neoplasia is a type of neoplasia that would respond to treatment with a cGMP-specific PDE inhibitor. Diagnostic kits may be used, for example, to detect the level of cGMP-specific PDE protein, to detect the activity and/or level of PKG protein, or to detect the level of β-catenin protein, in a neoplastic tissue sample.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from SW480 neoplastic cells, as assayed from the eluent from a DEAE- Trisacryl M column.

Figure 2 is a graph of cGMP activities of the reloaded cGMP phosphodiesterases obtained from SW480 neoplastic cells, as assayed from the eluent from a DEAE-Trisacryl M column.

Figure 3 is a graph of the kinetic behavior of the novel PDE of this invention.

Figures 4A and 4B illustrate the effects of sulindac sulfide and exisulind on apoptosis and necrosis of HT-29 cells.

Figure 5A and 5B illustrate the effects of sulindac sulfide and exisulind on HT-29 cell growth inhibition and apoptosis induction as determined by DNA fragmentation.

Figure 6A is a SDS protein gel of SW480 cell lysates from drug-treated cell lysates in the absence of added cGMP, where cells were treated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2), exisulind (200, 400 and 600μM; lanes 3, 4, 5) and E4021 (0.1 , 1 and 1 OμM, lanes 6, 7, 8).

Figure 6B is a SDS (X-ray film exposure) gel PKG assay of SW480 cell lysates from drug-treated cell lysates in the presence of added cGMP, where cells were treated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2), exisulind (200, 400 and 600μM: lanes 3, 4, 5) and E4021 (0.1 , 1 and l OμM, lanes 6, 7, 8). Figure 7 is a bar graph of the results of Western blot experiments of the effects of exisulind on β-catenin and PKG levels in neoplastic cells relative to control.

Figure 8 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from HTB-26 neoplastic cells, as assayed from the eluent from a DEAE- Trisacryl M column. Figure 9 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from HTB-26 neoplastic cells, as assayed from the eluent from a DEAE- Trisacryl M column with low and high substrate concentration.

Figure 10 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from LnCAP neoplastic cells, as assayed from the eluent from a DEAE-

Trisacryl M column.

Figure 1 1 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from LnCAP neoplastic cells, as assayed from the eluent from a DEAE- Trisacryl M column with low and high substrate concentration. Figure 12 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from SW480 neoplastic cells, as assayed from the eluent from a DEAE- Trisacryl M column using ethylene glycol in the buffer.

Figure 13 is a graph of the cGMP activities of the cGMP phosphodiesterases obtained from SW480 neoplastic cells grown in roller bottles, as assayed from the eluent from a DEAE-Trisacryl M column.

Figures 14A and 14B are photographs illustrating the elevated amount of PDE present in prostate cancer tissue sample (Figure 14B) compared to "normal" benign prostatic hypertrophy sample (Figure 14A) from humans, utilizing an antibody test according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves diagnostic methods to determine whether a patient with neoplasia has a type of neoplasia that is likely to respond to treatment with a cGMP-specific PDE inhibitor. As mentioned above, there are a new class of inhibitors that induce apoptosis in neoplastic tissues, but not in normal tissues. The inhibition of cyclic GMP-specific PDEs, including PDE5 and the novel PDE described below, with such inhibitors is a powerful new tool in the treatment neoplasia. I. INHIBITION OF CELL GROWTH

To determine whether a patient has a type of neoplasia that is likely to respond to treatment with a cGMP-specific PDE inhibitor, a neoplastic tissue sample from the patient is exposed to such an inhibitor and is tested to determine whether the neoplastic tissue sample exhibits sensitivity to treatment with the cGMP-specific PDE inhibitor.

For example, in a patient with familial polyposis, a suspected neoplastic tissue sample is obtained, processed, and cultured in appropriate tissue culture medium and conditions in the presence and absence of a cGMP-specific PDE inhibitor to determine whether the neoplastic tissue sample is sensitive to treatment with such an inhibitor. Sensitivity to a cGMP-specific PDE inhibitor can be characterized by growth inhibition or by an increase in apoptosis in the neoplastic cells treated with the inhibitor, relative to the untreated tissue sample.

In one embodiment, the diagnostic method of this invention involves determining whether a neoplastic tissue sample is responsive to treatment with a cGMP-specific PDE inhibitor by exposing the neoplastic tissue sample to a cGMP- specific PDE inhibitor and determining whether such treatment reduces the growth of tumor cells in vitro.

Briefly, suspected neoplastic tissue samples are removed from a patient and grown as explants in vitro. The tissue samples are grown in the presence and absence of a cGMP-specific PDE inhibitor. After being grown in culture, cells are fixed by the addition of cold trichloroacetic acid. Protein levels are measured using the sulforhodamine B (SRB) colorimetric protein stain assay as previously described by Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S., and Boyd, M.R., "New Colorimetric Assay For

Anti cancer-Drug Screening," J. Natl. Cancer lnst. 82: 1107-1 1 12. 1990, which is incorporated herein by reference.

In addition to the SRB assay, a number of other methods are available to measure growth inhibition and can be used instead of the SRB assay. These methods include counting viable cells following trypan blue staining, labeling cells capable of DNA synthesis with BrdU or radiolabeled thymidine, neutral red staining of viable cells, or MTT staining of viable cells.

Inhibition of cell growth indicates that the neoplasia in question is sensitive to anti-neoplastic cGMP-specific PDE inhibitors. Inhibition of cell growth is indicative that the patient would be an appropriate candidate for treatment with an anti- neoplastic cGMP-specific PDE inhibitor.

As described by Pamukcu, et al., in the pending U.S. Patent Application Serial No. 09/216,070, filed December 12, 1998 (Method For Identifying Compounds For Inhibition Of Cancerous Lesions, (Case No. P-l 19 OP)), a number of compounds potentially useful as PDE inhibitors in the diagnostic method of this invention were tested on a number of neoplastic cell lines representing various cell types. For example, these cell lines include: SW-480 - colonic adenocarcinoma; HT-29 - colonic adenocarcinoma; A-427 - lung adenocarcinoma; MCF-7 - breast adenocarcinoma; UACC-375 - melanoma line; and DU145 - prostrate carcinoma. Growth inhibition data obtained using these cell lines indicate an inhibitory effect by cGMP-specific PDE inhibitors on neoplastic lesions. These cell lines are well characterized, and are used by the United States National Cancer Institute in their screening program for new anti-cancer drugs.

To show the effectiveness of cGMP-specific PDE inhibition on various forms of neoplasia, (and, therefore, the usefulness of the diagnostic methods of this invention) cGMP-specific PDE inhibitors were tested on a number of neoplastic cell lines. The effects of sulindac sulfide and exisulind, two cGMP-specific PDE inhibitors, were determined. Exisulind is defined as (Z)-5-fluoro-2-methyl-l-[[4- (methylsulfonyl)phenyl] methylene]indene-3-yl acetic acid or a salt thereof. (See, Pamukcu and Brendel, U.S. Patent No. 5,401,774.) The data are shown in Table 1 below. The IC₅₀ values were determined by the SRB assay. These data indicate that such cGMP-specific PDE inhibitors are effective in the treatment of neoplastic conditions. Table 1 : Growth Inhibitory Data of Various Cell Lines

Cell Tvpe/ IC™ , 'uMV

Tissue specificity Sulindac sulfide Exisulind

HT-29, Colon 60 120

HCT1 16, Colon 45 90

MCF7/S, Breast 30 90

UACC375, Melanoma 50 100

A-427, Lung 90 130

Bronchial Epithelial Cells 30 90

NRK, Kidney (non ras-transformed) 50 180

KNRK, Kidney (ras transformed) 60 240

Human Prostate Carcinoma PC3 82

*Determined by neutral red assay as described by Schmid et al., in Proc. AACR Vol 39, p. 195 (1998).

II. APOPTOSIS

In another aspect of the diagnostic method of this invention, sensitivity of a neoplastic tissue to treatment with a cGMP-specific PDE inhibitor is tested with an apoptosis assay. For example, a suspected neoplastic tissue sample is processed and exposed to a cGMP-specific PDE inhibitor. Sensitivity to a cGMP-specific PDE inhibitor is characterized by an increase in apoptosis in the neoplastic tissue sample treated with the inhibitor relative to the untreated tissue sample.

Two distinct forms of cell death may be described by morphological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased permeability of the plasma membrane; the cells swell and the plasma membrane ruptures within minutes. Apoptosis is characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases. Apoptosis occurs naturally during normal tissue turnover and during embryonic development of organs and limbs. Apoptosis also is induced by cytotoxic T-lymphocytes and natural killer cells, by ionizing radiation, and by certain chemotherapeutic drugs. Inappropriate regulation of apoptosis is thought to play an important role in many pathological conditions including cancer, AIDS, Alzheimer's disease, etc. Patients with neoplasias that exhibit an increase in cell death through apoptosis after treatment with a cGMP-specific PDE inhibitor are candidates for treatment with a cGMP-specific PDE inhibitor.

In one type of apoptosis assay, suspected neoplastic cells are removed from a patient. The cells are then grown in culture in the presence or absence of a cGMP- specific PDE inhibitor. Apoptotic cells are measured by combining both the attached and "floating" compartments of the cultures. The protocol for treating tumor cell cultures with PDE inhibitors and related compounds to obtain a significant amount of apoptosis has been described in the literature. (See, Piazza, G.A., et al., Cancer Research, 55:31 10-16, 1995, which is incorporated herein by reference). The novel features of this assay include collecting both floating and attached cells, identification of the optimal treatment times and dose range for observing apoptosis, and identification of optimal cell culture conditions.

A. ANALYSIS OF APOPTOSIS BY MORPHOLOGICAL

OBSERVATION Following treatment of neoplastic and normal cells with a test compound, cultures can be assayed for apoptosis and necrosis by fluorescent microscopy following labeling with acridine orange and ethidium bromide. The method for measuring apoptotic cell number has previously been described by Duke & Cohen,

"Morphological And Biochemical Assays Of Apoptosis," Current Protocols In Immunology, Coligan et al., eds., 3.17.1 -3.17.16 (1992, which is incorporated herein by reference).

For example, floating and attached cells are collected, and aliquots of cells are centrifuged. The cell pellet is then resuspended in media and a dye mixture containing acridine orange and ethidium bromide. The mixture is then examined microscopically for morphological features of apoptosis.

B. ANALYSIS OF APOPTOSIS BY DNA FRAGMENTATION Apoptosis can also be quantified by measuring an increase in DNA fragmentation in cells which have been treated with cGMP-specific PDE inhibitors. Commercial photometric EIAs for the quantitative in vitro determination of cytoplasmic histone-associated-DNA-fragments (mono- and oligonucleosomes) are available (Cell Death Detection ELISA^{o ys}, Cat. No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay is based on a sandwich-enzyme- immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates.

According to the vendor, apoptosis is measured in the following fashion. The sample (cell-lysate) is placed into a streptavidin-coated microtiter plate (MTP).

Subsequently, a mixture of anti-histone-biotin and anti-DNA peroxidase conjugate are added and incubated for two hours. During the incubation period, the anti-histone antibody binds to the histone-component of the nucleosomes and simultaneously fixes the immunocomplex to the streptavidin-coated MTP via its biotinylation. Additionally, the anti-DNA peroxidase antibody reacts with the DNA component of the nucleosomes. After removal of unbound antibodies by washing, the amount of nucleosomes is quantified by the peroxidase retained in the immunocomplex. Peroxidase is determined photometrically with ABTS7 (2,2'-Azido-[3- ethylbenzthiazolin-sulfonate]) as substrate.

C. APOPTOSIS ASSAY

Increases in apoptosis are indicative that the neoplasia in question is sensitive to treatment with a cGMP-specific PDE inhibitor.

A colon carcinoma cell line, HT-29, was treated with the cGMP-specific PDE inhibitors, sulindac sulfide and exisulind in accordance with the protocols for the assay mentioned above. (See, Piazza, G.A., et al., Cancer Research, 55:3110-16, 1995.) In accordance with those protocols, Figures 4 A and 4B show the effects of sulindac sulfide and exisulind on apoptotic and necrotic cell death. HT-29 cells were treated for six days with the indicated dose of either sulindac sulfide or exisulind. Apoptotic and necrotic cell death was determined as previously described (Duke and

Cohen, In: Current Protocols in Immunology, 3.17.1 - 3.17.16, New York, John Wiley and Sons, 1992). The data show that both sulindac sulfide and exisulind are capable of causing apoptotic cell death without inducing neoplastic cell necrosis. All data were collected from the same experiment. Figures 5 A and 5B show the effect of sulindac sulfide and exisulind on tumor growth inhibition and apoptosis induction as determined by DNA fragmentation. The top figure (5A) shows growth inhibition (open symbols, left axis) and DNA fragmentation (closed symbols, right axis) by exisulind. The bottom figure (5B) shows growth inhibition (open symbols) and DNA fragmentation (closed symbols) by sulindac sulfide. Growth inhibition was determined by the SRB assay after six days of treatment. DNA fragmentation was determined after 48 hours of treatment. All data was collected from the same experiment.

The diagnostic method of this invention is used to determine whether a particular neoplasia is sensitive to treatment with a cGMP-specific PDE inhibitor. The apoptosis inducing activity for a series of phosphodiesterase inhibitors, specific for different PDEs, was determined. The data are shown in Table 2 below. HT-29 cells were treated for 6 days with various inhibitors of phosphodiesterase. Apoptosis and necrosis were determined morphologically after acridine orange and ethidium bromide labeling in accordance with the assay described, supra. The data show cGMP-specific PDE inhibition represents a unique and valuable pathway to induce apoptosis in neoplastic cells. Table 2: Apoptosis Induction Data for PDE Inhibitors

Inhibitor Reported Selectivity % Apoptosis % Necrosis

Vehicle 8 6

8-methoxy-IBMX PDE1 2 1

Milrinone PDE3 18 0

RO-20-1724 PDE4 1 1 2

MY5445 PDE5 80 5

IBMX Non-selective 4 13

D. APOPTOSIS CLINICAL STUDY

Increases in apoptosis are indicative that the neoplasia in question is sensitive to treatment with a cGMP-specific PDE inhibitor, such as sulindac sulfone (exisulind). A human in vivo exisulind-induced selective induction of apoptosis in colonic polyps is described below. Six familial polyposis patients per group were administered one of three doses of exisulind (200 mg, 400 mg which was later lowered to 200 mg for most patients in the group, or 300 mg) twice daily (BID). 1. METHODS

Biopsies are taken from patients and used to investigate possible cellular mechanisms of apoptosis. Biopsy samples are placed in transfer media (500 ml RPMI 1640 containing 50 ml fetal calf serum, 5x10 units penicillin G, and 5x10 μg streptomycin) and kept on ice for less than 1 hour until transfer to the pathology department. Upon receipt in the pathology department, samples are removed from the transfer media and oriented mucosa up, serosa down on filter paper, placed between biopsy sponges in a tissue cassette, and fixed in 10% neutral buffered formalin for 24 hours. Samples are then transferred to 70% ethanol and embedded in paraffin. Samples are oriented perpendicularly to the tissue cassette during final orientation in paraffin for longitudinal crypt exposure and easy visualization of mucosa and the relation to the basement membrane.

Four micron sections of tissue were cut, mounted, deparaffmized, rehydrated in graded alcohol, and treated with pepsin (5mg/ml) to digest protein in the tissue. Sections were washed and treated with 2% hydrogen peroxide (H₂0₂) in PBS to quench endogenous peroxidase and washed again. Tissue samples were then circled with a PAP pen (Research Products Int., 800-323-9814) to produce a hydrophobic barrier to concentrate reagents on the sample. If a DNase positive control is desired, the sample is treated with DNase for 10 minutes, equilibrated in transferase buffer, and treated using 100 enzyme units/ml terminal transferase enzyme (TdT) at 37°C for

60 minutes. Samples are washed and anti-digoxigenin-peroxidase is applied. Each sample is then covered with a coverslip and left in a humid box at room temperature for 30 minutes. After washing three times peroxidase is developed using DAB for nine minutes. After sufficient color development, the slides are washed and counterstained with hemotoxylin and eosin.

Apoptotic and nonapoptotic cells are counted on the basis of staining and morphology. An apoptotic labeling index (ALI) is calculated by dividing the total number of apoptotic cells counted by the total number of epithelial cells counted and expressing the quotient as a percentage. 2. RESULTS

Baseline ALI were measured in both normal samples and paired polyp samples. Baseline ALI in normal tissue was determined to be 0.61% ± 0.05 (mean ± SEM), a nine-fold lower level of apoptosis than in polyp samples which had a mean apoptotic level of 5.60% ± 0.74. (Table 3).

TABLE 3

There was no significant change in normal mucosa ALI versus baseline ALI during treatment over time for any of the treatment groups. However, dysplastic tissue taken from patients in the 400/200 mg BID group demonstrated a two-fold elevation in ALI following drug treatment when the group was uniformly dosed at 400 mg BID. A two fold increase in ALI was also noted in polyps following six months of treatment on the 300 mg BID dose. The 200 mg BID group did not demonstrate any elevation in ALI following treatment with exisulind. (Table 4).

TABLE 4

This study shows that over six months of treatment, apoptosis levels are doubled in regressing polyps, and indicates that exisulind, a cGMP-specific PDE inhibitor can effectively induce the regression of neoplasia, such as adenomatous lesions, by apoptosis. This selective induction of apoptosis in polyps by exisulind and the accompanying diminution of polyp size and decrease in polyp number is an important discovery for the treatment of neoplasias. III. PHOSPHODIESTERASE ACTIVITY

A. PHOSPHODIESTERASE ENZYME ASSAY

In one embodiment of this invention, the presence of cGMP-specific PDEs in a neoplastic tissue sample is determined by performing a phosphodiesterase enzyme assay. If cGMP-specific PDE activity is elevated in a neoplastic tissue sample, compared to cGMP-specific PDE activity in normal tissue, it is indicative that the neoplasia in question can be treated with an anti-neoplastic cGMP-specific PDE inhibitor. The normal tissue used in this assay, and in the other assays described herein which employ normal tissue, is optionally from the same patient as the neoplastic tissue sample or from a reference standard which may be based on a population of patients, and optionally is the same type of tissue as the neoplastic tissue. Additionally, if the neoplastic cells in a sample are exposed to an antineoplastic cGMP-specific PDE inhibitor and the cGMP-specific hydrolytic activity of the sample decreases, it is further indicative that the neoplasia in question is a candidate for treatment with a cGMP-specific PDE inhibitor.

Phosphodiesterase activity (whether in a mixture or separately) can be determined using methods known in the art, such as a method using a radioactively labeled form of cGMP as a substrate for the hydrolysis reaction. Cyclic GMP labeled with tritium ( H-cGMP) is used as the substrate for the PDE enzymes. (Thompson, W.J., Teraski, W.L., Epstein, P.M., Strada, S.J., Advances in Cyclic Nucleotide

Research, K): 69-92, 1979, which is incorporated herein by reference). In this assay, cGMP-PDE activity is determined by quantifying the amount of cGMP substrate that is hydrolyzed either in the presence or absence of a cGMP-specific PDE inhibitor. In brief, a solution of defined substrate H-cGMP specific activity is mixed with a cGMP-specific PDE inhibitor. The control sample contains no inhibitor. The mixture is incubated with cell lysates from neoplastic tissue samples. The degree of phosphodiesterase inhibition is determined by calculating the amount of radioactivity released in samples that include a cGMP-specific PDE inhibitor and comparing those against a control sample which contains no inhibitor. B. CYCLIC NUCLEOTIDE MEASUREMENTS Alternatively, the sensitivity of a neoplastic tissue sample to treatment with a cGMP-specific PDE inhibitor is reflected by an increase in the levels of cGMP in neoplastic cells exposed to the cGMP-specific PDE inhibitor. The amount of PDE activity can be determined by assaying for the amount of cyclic GMP in the extract of neoplastic cells treated with a cGMP-specific PDE inhibitor using a radioimmunoassay (RIA). In this procedure, cells from a neoplastic tissue are incubated with a cGMP-specific PDE inhibitor. After about 24 to 48 hours, the cells are solubilized, and cyclic GMP is purified from the cell extracts. The cGMP is acetylated according to published procedures, such as using acetic anhydride in triethylamine (Steiner, A.L., Parker, C.W., Kipnis, D.M., J. Biol Chem., 247(4): 1 106- 13, 1971, which is incorporated herein by reference). The acetylated cGMP is quantitated using radioimmunoassay procedures (Haφer, J., Brooker, G., Advances in Nucleotide Research, l_0:l-33, 1979, which is incorporated herein by reference). In addition to observing increases in the content of cGMP in neoplastic cells as a result of treatment with a cGMP-specific PDE inhibitor, decreases in the content of cAMP have also been observed. It has been observed that treatment of a neoplastic tissue sample with a cGMP-specific PDE inhibitor initially result in an increased cGMP content within minutes, and secondarily, there is a decreased cAMP content within 24 hours. To determine the cyclic AMP content in cell extracts, radioimmunoassay techniques similar to those described above for cGMP are used.

IV. ANTIBODY TECHNIQUES

In another aspect, the present invention includes the use of one or more antibodies that are immunoreactive with cGMP-specific PDEs. Antibodies that are immunoreactive with cGMP-specific PDEs specifically recognize and bind to cGMP- specific PDEs. Antibodies reactive to cGMP-specific PDEs are used to detect and quantify the various cGMP-specific PDEs present in a suspected neoplastic tissue sample. The presence of cGMP-specific PDEs in a neoplastic tissue sample is - 18 -

indicative that the particular neoplasia is a candidate for treatment with a cGMP- specific PDE inhibitor.

Antibodies can be generated individually against PDE5, individually against classic PDE2 or the novel PDE2-like enzyme described below and in pending application Serial No. 09/173,375 (Case No. P-143), or they can be generated against a mixture of cGMP phosphodiesterases, including PDE5 and PDE2. Antibodies can also be generated against other proteins of interest, such as PKG and β-catenin, using these methods. Means for preparing and characterizing antibodies are well known in the art. (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incoφorated herein by reference.)

Several antibodies that are useful in the diagnostic methods of the present invention are available commercially. Anti-PKG l β and anti-β-catenin antibodies can be purchased from StressGen Biotechnologies Coφ, BC, Canada and Upstate Biotechnology, NY, respectively.

A. ANTIBODY GENERATION

1. POLYCLONAL ANTIBODIES Antibodies can be either polyclonal or monoclonal. Briefly, a polyclonal antibody is prepared by immunizing an animal with immunogenic protein or polypeptide and collecting antisera from that immunized animal. A wide range of animal species are used for the production of antisera, and the choice is based on the phylogenetic relationship to the antigen. Typically the animal used for production of anti-antisera is a rabbit, a guinea pig, a chicken, a goat, or a sheep. Because of the relatively large blood volume of sheep and goats, these animals are preferred choices for production of polyclonal antibodies.

As is well known in the art, a given antigenic composition may vary in its ability to generate an immune response. It is often necessary, therefore, to boost the host immune system by coupling a peptide or polypeptide immunogen to a carrier. Examples of common carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Means for conjugating a polypeptide to a carrier protein are well known in the art and include MBS [m-Malecimidobenzoyl-N-hydroxysuccimide ester], EDAC [l-ethyl-3-(3-Dimethylaminopropyl) carbodiimide hydrochloride], and bisdiazotized benzidine.

As is also well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Cytokines, toxins or synthetic compositions may also be used as adjuvants. The most commonly used adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis) and incomplete Freund's adjuvant. Milligram quantities of antigen are preferred although the amount of antigen administered to produce polyclonal antibodies varies upon the nature and composition of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies

(MAbs).

For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. Sterility is maintained throughout this preparation. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography. 2. MONOCLONAL ANTIBODIES

MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent No. 4,196,265, incoφorated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide, peptide or domain. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rates are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed., 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions. The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen is typically mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen are made at approximately two week intervals. Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. Antibody-producing B cells are usually obtained by disbursement of the spleen, but tonsil, lymph nodes, or peripheral blood may also be used. Spleen cells are preferred because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma- producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as is known to those of skill in the art (Goding, pp. 65-66, 1986).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in about a 2:1 proportion in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. The original fusion method using Sendai virus has largely been replaced by those using polyethylene glycol (PEG), such as 37%o (v/v) PEG, as has been described in the art. The use of electrically-induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

A preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which particular clones are selected. The selection of hybridomas is performed by culturing the cells in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for antibody producers using ELISA IgG assays. Antibody positive hybridomas are screened further for MAbs with the desired reactivity using antigen based assays. Such assays are normally sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, clones of which are then propagated indefinitely to provide MAbs. The cell lines can be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histo-compatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the antibody producing hybridoma. The ascites fluid of the animal, and in some cases blood, can then be tapped to provide MAbs in high concentration.

The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

3. ANTIBODY CONJUGATES The present invention further provides antibodies against GMP PDE proteins that are linked to one or more other agents to form an antibody conjugate. Any antibody of sufficient selectivity, specificity, and affinity may be employed as the basis for an antibody conjugate.

Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins." In the context of the present invention, immunotoxins are generally less preferred.

Antibody conjugates are thus preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in in vivo diagnostic protocols, generally known as "antibody-directed imaging."

Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Patents Nos. 5,021,236 and 4,472,509, both incoφorated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. Fluorescent labels include rhodamine, fluorescein isothiocyanate and renographin.

The preferred antibody conjugates for diagnostic use in the present invention are those intended for use in vitro, where the antibody is linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or streptavidin compounds.

B. IMMUNOASSAYS

In another aspect, the present invention concerns immunoassays for binding, purifying, quantifying and otherwise generally detecting PDE protein components. As detailed below, immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunoadsorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot and slot blotting, FACS analyses, and the like may also be used. The steps of various useful immunoassays have been described in the scientific literature, such as, e.g., Nakamura et al, In; Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Chapter 27 (1987), incoφorated herein by reference.

In general, the immunobinding methods include obtaining a sample suspected of containing a protein or peptide, in this case, cGMP-specific PDEs, and contacting the sample with a first antibody immunoreactive with cGMP-specific PDEs under conditions effective to allow the formation of immunocomplexes.

Immunobinding methods include methods for purifying PDE proteins, as may be employed in purifying protein from patients' samples or for purifying recombinantly expressed protein. They also include methods for detecting or quantifying the amount of a cGMP-specific PDE in a tissue sample, which requires the detection or quantification of any immune complexes formed during the binding process.

The biological sample analyzed may be any sample that is suspected of containing a cGMP-specific PDE such as a homogenized neoplastic tissue sample.

Contacting the chosen biological sample with the antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e. , to bind to, any cGMP-specific PDEs present. The sample-antibody composition is washed extensively to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are based upon the detection of radioactive, fluorescent, biological or enzymatic tags. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

The cGMP-specific PDE antibody used in the detection may itself be conjugated to a detectable label, wherein one would then simply detect this label. The amount of the primary immune complexes in the composition would, thereby, be determined.

Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are washed extensively to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is detected.

1. ELISAs

An enzyme linked immunoadsorbent assays (ELISAs) is a type of binding assay. In one type of ELISA, the cGMP-specific PDE antibodies used in the diagnostic method of this invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a suspected neoplastic tissue sample is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound cGMP-specific PDE may be detected. Detection is generally achieved by the addition of another anti-PDE antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by the addition of a second anti- PDE antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another type of ELISA, the neoplastic tissue samples are immobilized onto the well surface and then contacted with the anti-PDE antibodies used in this invention. After binding and washing to remove non-specifically bound immune complexes, the bound cGMP-specific PDE antibodies are detected. Where the initial anti-PDE antibodies are linked to a detectable label, the immune complexes may be detected directly. Alternatively, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-PDE antibody, with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. 2. RIAs The radioimmunoassay (RIA) is an analytical technique which depends on the competition (affinity) of an antigen for antigen-binding sites on antibody molecules. Standard curves are constructed from data gathered from a series of samples each containing the same known concentration of labeled antigen, and various, but known, concentrations of unlabeled antigen. Antigens are labeled with a radioactive isotope tracer. The mixture is incubated in contact with an antibody. Then the free antigen is separated from the antibody and the antigen bound thereto. Then, by use of a suitable detector, such as a gamma or beta radiation detector, the percent of either the bound or free labeled antigen or both is determined. This procedure is repeated for a number of samples containing various known concentrations of unlabeled antigens and the results are plotted as a standard graph. The percent of bound tracer antigens is plotted as a function of the antigen concentration. Typically, as the total antigen concentration increases the relative amount of the tracer antigen bound to the antibody decreases. After the standard graph is prepared, it is thereafter used to determine the concentration of antigen in samples undergoing analysis. In an analysis, the sample in which the concentration of antigen is to be determined is mixed with a known amount of tracer antigen. Tracer antigen is the same antigen known to be in the sample but which has been labeled with a suitable radioactive isotope. The sample with tracer is then incubated in contact with the antibody. Then it can be counted in a suitable detector which counts the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent may also be similarly counted. Then, from the standard curve, the concentration of antigen in the original sample is determined.

C. EXPERIMENTAL PROCEDURES

Cyclic GMP-binding cGMP-specific phosphodiesterase (cGB-PDE or PDE5) specifically hydrolyzes cGMP into 5'-GMP. It has two allosteric (non-catalytic) cGMP-binding sites located in the N-terminal region of the protein (K_d = 1.3 mM), and a C-terminal catalytic domain which shows a strong preference for cGMP as a substrate (K_m = 5.6 mM). Cyclic GMP-dependent protein kinase (PKG) specifically phosphorylates PDE5 at Serine-92 in the bovine sequence (Thomas, M.K. et al., J. Biol. Chem. 265: 14971-14978 (1990)). Generally, PDEs are difficult to express in their entirety in bacterial expression systems. There has been, however, greater success in the expression of recombinant proteins containing different functional domains of PDEs.

1. ANTIGEN PRODUCTION

Two polyclonal antibodies reactive with PDE5/PDE2 were produced. The glutathione-S-transferase (GST) fusion gene system (Pharmacia) was used to express a portion of the cGMP-binding domain of PDE5. Advantages of the GST expression system include its high yield and ease of purification of the GST fusion protein from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B.

The first antibody, designated PDE5(1), was made using a short peptide of 17 amino acids as a hapten. The peptide sequence, CAQLYETSLLENKRNQV, corresponds to amino acids 307 to 322 of the cGMP high affinity binding domain of the bovine PDES. (See. Beavo, et al., U.S. Patent Nos. 5,652,131 and 5,702,936.) The peptide was synthesized using a Rainen Symphony Multiple Peptide Synthesizer, analyzed by mass spectrometry, and purified to greater than 90% purity using HPLC.

The peptide was synthesized to contain an N-terminal cysteine in order to produce a conjugated peptide. The purified peptide was linked via the sulfahydro of the N-terminal cysteine to maleimide-activated keyhole limpet hemocyanin (KLH,

Pierce), yielding a KLH-PDE peptide conjugate.

A second polyclonal antibody, PDE5(2), was also prepared as a GST fusion protein. The antigen for PDE5(2) is designated PDE5cg. RT-PCR methods, discussed in greater detail below, were used to obtain the putative cGMP-binding domain of PDE5. Forward and reverse primers were designed to specifically amplify a region of the PDE5 cDNA sequence (McAllister-Lucas L.M., et al., J Biol. Chem. 268, 22863-22873, 1993) and were not directed at conserved sequences among the PDE1 - PDE7 families.

RNA from HT-29 cells was isolated using 5'-3', Inc. kits for total RNA preparation followed by oligo (dT) column purification of mRNA. The forward primer (GAA-TTC-CGT-CAC-AGC-CTT-ATG-TCA-C, corresponding to the bovine PDES A cDNA sequence, nucleotides 561-579) and the reverse primer (CTC-GAG- TGC-ATC-ATG-TTC-CCT-TG, corresponding to the bovine PDE5A cDNA sequence, nucleotides 1264-1280) were used to obtain a 720 base pair fragment coding for the high affinity cGMP-binding domain of PDE5. The 720 base pair amplification product has 94% sequence homology with bovine PDES (nucleotides 561-1280) and codes for 240 amino acids with 98%> similarity to the bovine amino acid sequence.

The 720 base pair fragment was cloned into the pGEX-5X-3 glutathione-S- transferase (GST) fusion vector (Pharmacia Biotech) using the EcoRI and Xhol restriction sites. The GST- fusion protein was expressed in E. coli BL21 cells under IPTG (lOOμM) induction for 24 hrs. Then the fusion proteins were purified from the supernatant of the bacterial cell extract using a Glutathione Sepharose 4B affinity column and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) according to the manufacturers instructions (GST Gene Fusion System, Pharmacia Biotech). Two milligrams of purified GST-cGMP binding domain fusion protein were obtained from one liter of bacterial culture. The GST-cGMP binding domain fusion protein yields a 56 KDa product on an SDS-PAGE gel.

The purified GST-PDE5 binding domain fusion protein is characterized by its cGMP specificity and its high affinity binding of cGMP. A cyclic GMP binding assay (Francis S.H., et al., J. Biol. Chem. 255, 620-626, 1980) was used to determine the K_m of the fusion protein for cGMP. The assay was performed in a total volume of 100 μL containing 5 mM sodium phosphate buffer (pH 6.8), 1 mM EDTA and 0.25 mg/ml BSA and H³-cGMP (5.8 Ci/mmol, NEN). The purified soluble GST-PDE5 binding domain fusion protein (5 to 50 μg/assay) was incubated at 22°C for one hour and then transferred to a Brandel MB-24 Cell Harvester with GF/B as the filter membrane. Next the fusion protein was washed twice with 10 mL of cold 5 mM potassium buffer, pH 6.8. The membranes were cut out and transferred to scintillation vials, then 1 ml of H₂O and 6 ml of Ready Safe liquid scintillation cocktail was added and the samples were counted on a Beckman LS 6500 scintillation counter. A ³H- cGMP saturation binding curve at 25 °C was generated. The GST-cGMP binding domain fusion protein displays one high affinity binding site for cGMP. The K_m for cGMP is 0.41 ± 0.08 μM, which is similar to the high affinity binding site of the bovine PDE5 (K_d = 0.5 μM). As a control, a blank sample was prepared by boiling the fusion protein for five minutes. The radioactivity detected for the boiled sample was less than one percent of that detected for the unboiled protein. The scintillation counting results were calibrated for quenching by filter membrane or other debris.

The fusion protein showed binding activity similar to that of the native enzyme. This includes specificity for cGMP over cAMP and 2'-substituted cyclic nucleotide analogs. These data suggest that the recombinant GST-cGMP binding domain fusion protein has high affinity cGMP binding characteristics similar to those of the cGMP binding site of PDE5. 2. ANTIBODY PRODUCTION For the production of PDE5(1), sheep were injected with lOOμg of the KLH- conjugated peptide mixed with complete Freund's Adjuvant (Difco) for the initial injection. For subsequent injections, sheep were injected with the KLH-conjugated peptide mixed with incomplete Freund's Adjuvant every two weeks. Bleedings for antiserum were taken seven days after each injection, starting with the third injection. Pre-immunization serum was collected two weeks before antigen injection as a control for the antibody specificity assay. The test bleed was monitored by ELISA to determine the antibody titer. The immunization procedure for preparation of the PDE5(2) antibody was the same as that described above for the PDE5(1) antibody except 100 μg of affinity column purified GST-PDE5cg fusion protein (MW = 56 KDa) was used as an antigen in each injection.

Immunoblots for human PDE5 were carried out by using PDE5(1) and PDE5(2) antisera from sheep. Pre-injection antiserum was used as a pre-immune control. Both PDE5(1) and PDE5(2) showed specific binding for the GST-cGMP binding fusion protein (56 KDa) and for the native PDE5 protein (-93 KDa) isolated from HT-29 cell extracts. As negative controls, pre-immune serum did not bind to these proteins and pre-incubation of the immune serum with an excess of the GST- cGMP binding domain fusion protein also blocked binding of the antibody to the

PDE5 proteins. These results indicate that PDE5(1) and PDE5(2) antisera contain antibodies for human PDE5.

3. cGMP PDE LOCALIZATION IN NORMAL AND NEOPLASTIC TISSUE Figures 14A and 14B are photographs illustrating the elevated amount of PDE present in prostate cancer tissue sample (Figure 14B) compared to "normal" benign prostatic hypertrophy sample (Figure 14A) from humans, utilizing an antibody test according to the present invention. This experiment was performed on those tissue samples by exposing the samples to the PDE5(1) sheep antibody described above, and removing excess, unbound PDE5(l)antibody. Then a second biotinylated anti-sheep antibody is added. Any unbound second antibody is then removed. Next, avidin-DH, which binds to the biotinylated anti-sheep antibody is added. Biotinylated horseradish peroxidase, which binds to the avidin is added next. Finally, the colorimetric substrate diaminobenzimidine tetrahydrochloride (DAB) is added. From the photos taken (Figures 14A and 14B), it can clearly be seen that the prostate cancer tissue (Figure 1 B) has considerably more cGMP PDE present than the corresponding non- neoplastic tissue (Figure 14B). Of course, the PDE5(1) is cross-reactive with the PDE2-type enzymes disclosed herein, so this assay establishes that cGMP PDE is present in elevated levels in the prostate cancer tissue.

V. NUCLEIC ACID DETECTION

In another aspect, this invention includes the use of nucleic acid detection techniques to detect the level of cGMP-specific PDEs in a suspected neoplastic tissue sample. The nucleic acid sequences disclosed herein can be used in hybridization techniques such as slot and northern blots or in amplification techniques such as reverse transcriptase polymerase chain reaction (RT-PCR).

A. PCR AMPLIFICATION

The level of cGMP-specific PDE mRNA in a neoplastic tissue sample can correspond to the level of expression of the protein. The presence of high levels of cGMP-specific PDE mRNA in a neoplastic tissue relative to normal tissue can indicate that the neoplasia will respond to treatment with a cGMP-specific PDE inhibitor.

Nucleic acid used as a template for amplification is isolated from suspected neoplastic tissue samples. The nucleic acid may be genomic DNA or whole cell or fractionated RNA. Methods of nucleic acid isolation are well know in the art. (See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 1989.)

In the diagnostic method if this invention, it is preferred that RNA is isolated from a tissue sample. The RNA can then further fractionated to isolate messenger RNA by selecting for polyadenylated RNA (poly-A RNA). Then the mRNA can be converted into complementary DNA (cDNA). Briefly, in PCR, two oligonucleotide primers are synthesized whose sequences are complementary to sequences that are on opposite strands of the template DNA and flank the segment of DNA that is to be amplified. The template DNA is denatured by heating in the presence of an excess of the two primers, the four deoxynucleotide triphosphates, and magnesium. As the reaction is cooled, the primers anneal to their target sequences. Then the annealed primers are extended with DNA polymerase. The initial round can potentially double the product and each successive round of amplification can potentially lead to a logarithmic increase in amount of the amplification product because the product of one round can serve as template in the next round. Multiple rounds of amplification (denaturation, annealing, and DNA synthesis) are conducted until a sufficient amount of amplification product is produced. Finally, the amplification product is detected, usually by visual means or indirectly through chemiluminescence, or detection of a radioactive label or fluorescent label, or the like. There are a number of template dependent amplification processes. One of the best known and most widely used is the polymerase chain reaction which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159, which are incoφorated herein by reference. The thermostable Tag DNA polymerase is most commonly used in the PCR process because it remains active at the high temperatures used in the amplification process.

Reverse transcriptase PCR (RT-PCR) can be used to estimate semiquantitative levels of mRNA of cGMP-specific PDEs in neoplastic tissue samples. Methods of reverse transcribing RNA into cDNA are well known and are described in Sambrook, et al., 1989.

B. EXPERIMENTAL PROCEDURES

RNA was prepared from cells in culture or human and mouse tissue obtained from autopsy by using the QIAGEN (Valencia, CA) RNeasy Mini Kit. RNA then was treated with RNase-free DNase to eliminate genomic DNA contamination. cDNA was synthesized in a 30 μl reaction using 2 μg of total RNA. The RNA was heated for 5 minutes at 70°C with random hexamers (Life Technologies, Inc.) and cooled on ice. Reverse transcription was performed at 42°C for 1 hour with 0.5 mM dNTPs, 10 mM DTT, IX reverse transcription buffer (Stratagene, La Jolla, CA), and 200 units of Superscript II (Stratagene, La Jolla, CA) in the presence of RNase Inhibitors (Stratagene, La Jolla, CA). Seven percent of the cDNA was used for PCR amplification. PCR was performed for 30 cycles as follows: initial denaturation at 94°C for 5 minutes, 94°C for 1 minute, 55°C for 2 minutes, 72°C for 1 minute and extension at 72°C for 7 minutes. PCR products were separated on a 1 % agarose gel and electrophoresed in IX TBE buffer. PCR products were purified using Geneclean (Bio 101 , Inc.) and then sequenced.

Primers were synthesized to amplify a region of the human PDES mRNA which corresponds to the coding region for the N-terminal portion of the protein. The first set of primers, hV sense 1 and hV antisense 1 (s 1/as 1) generate a 385 base pair RT-PCR product which aligns with the human PDE5 sequence (Genbank accession # D 89094) from base pairs 432 to 816. Primers hV sense 2 and hV antisense 2 (s 2/as

2) generate a 174 base pair RT-PCR product which aligns with a human PDES splice variant, 5A2, (Genbank accession # Af043732) from base pairs 41 to 214.

Primer hV s 1 : GGG ACT TTA CCT TCT CTT AC Primer hV as 1 : GTG ACA TCC AAG AAG TGA CTA GA

Primer hV s 2: CCC GAA GCC TGA GGA ATT GAT GC Primer hV as 2: CTC CTC GAC CAT CAC TGC CG

VI. DIAGNOSTIC KITS

In another aspect, this invention provides for diagnostic kits for ascertaining whether a patient has a neoplasia. Diagnostic kits may be used to detect the level of cGMP-specific PDE protein in a patient suspected of having a neoplasia.

In another aspect, this invention provides for diagnostic kits for ascertaining whether a particular neoplasia is a type of neoplasia that would respond to treatment with a cGMP-specific PDE inhibitor. Diagnostic kits may be used to detect the level of mRNA encoding for cGMP-specific PDEs or the level of cGMP-specific PDE protein in a suspected neoplastic tissue sample.

The immunodetection kit includes an antibody or antibodies specifically reactive with cGMP-specific PDEs and an immunodetection reagent, and a means for containing each. The immunodetection reagent most commonly has a label associated with the antibody, or associated with a second binding ligand. An immunodetection kit can also utilize a antibodies to other species, such as the anti-PKG 1 β and anti-β- catenin antibodies mentioned above. The nucleic acid detection kit includes an isolated cGMP-specific PDE nucleic acid segment or nucleic acid primers that hybridize to distant sequences of a cGMP- specific PDE, capable of amplifying a nucleic acid segment of a cGMP-specific PDE.

Such kits are used to detect the amount of cGMP-specific PDE protein or mRNA, respectively, in a neoplastic tissue sample. The detection of elevated amounts of cGMP-specific PDE protein or mRNA in a neoplastic tissue relative to normal tissue is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

VII. THE NOVEL cGMP-SPEOFIC PHOSPHODIESTERASE A. IN GENERAL

As mentioned above, a new PDE2-like enzyme has been discovered in neoplastic cells. As discussed below, this new cGMP-PDE is appears to be a novel conformation of PDE2. Treatment of cells with a compound that inhibits both PDE5 and this novel PDE2-like enzyme leads to apoptosis of the neoplastic cells, as described below, whereas PDE5 inhibitors that do not induce apoptosis have not been found to inhibit this PDE2-like enzyme..

B. ISOLATION OF THE NOVEL PDE CONFORMATION The novel cGMP-specific phosphodiesterase (which appears to be a novel conformation of PDE2) was first prepared from the human carcinoma cell line commonly referred to as SW480 available from the American Tissue Type Collection in Rockville, Maryland, U.S.A. SW480 is a human colon cancer cell line that originated from moderately differentiated epithelial adenocarcinoma. As discussed below, a similar conformation has also been isolated from neoplasias of the breast (i.e., HTB-26 cell line) and prostate (i.e., LNCAP cell line).

By "isolated" we mean (as is understood in the art) not only isolated from neoplastic cells, but also made by recombinant methods (e.g., expressed in a bacterial or other non-human host vector cell lines). However, we presently believe isolation from the human neoplastic cell line is preferable since we believe that the target protein so isolated has a structure (i.e., a conformation or topography) that is closer to, if not identical with, one of the native conformations in the neoplastic cell as possible. The novel PDE activity was first found in SW480 colon cancer cell lines. To isolate the novel phosphodiesterase from SW480, approximately four hundred million SW480 cells were grown to confluence in and were scraped from 150 cm² tissue culture dishes after two washes with 10 mL cold PBS and pelleted by centrifugation.

The cells were re-suspended in homogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc₂, 0.1 mM EDTA, 0.8% Triton-100, lOμM benzamidine, 1 OμM TLCK, 2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A) and homogenized on an ice bath using a polytron tissumizer (three times, 20 seconds/pulse). The homogenized material was centrifuged at 105,000 g for 60 minutes at 4°C in a Beckman L8 ultracentrifuge, and the supernatant was diluted with TMPI-EDTA (60 mL) and applied to a 10-milliliter DEAE-Trisacryl M column pre- equilibrated with TMPI-EDTA buffer. The loaded column was washed with 60 mL of TM-EDTA, and PDE activities were eluted with a 120 mL linear gradient of NaOAC (0-0.5 M) in TM-EDTA, at a flow rate of 0.95 mL/minute, 1.4 mL/fraction.

Eighty fractions were collected and assayed for cGMP hydrolysis immediately (i.e. within minutes). Figure 1 shows the column's elution profile, revealing two initial peaks of cGMP PDE activity, peaks A and B, which were eluted by 40-50 mM and 70-80 mM NaOAC, respectively. As explained below, peak A is PDE5, whereas peak B is a novel cGMP-specific phosphodiesterase activity. Cyclic nucleotide PDE activity of each fraction was determined using the modified two-step radio-isotopic method of Thompson et al. (Thompson W.J., et al., Adv. Cyclic Nucleotide Res. 10: 69-92, 1979), as further described below. The reaction was in 400 μl containing Tris-HCl (40mM; pH 8.0), MgCl₂ (5mM), 2- mercaptoethanol (4 mM), bovine serum albumin (30 μg), cGMP (0.25μM-5μM) with constant tritiated substrate (200,000 cpm). The incubation time was adjusted to give less than 15% hydrolysis. The mixture was incubated at 30°C followed by boiling for 45 seconds to stop the reaction. Then, the mixture was cooled, snake venom (50μg) added, and the mixture was incubated at 30°C for 10 minutes. MeOH (1 mL) was added to stop the reaction, and the mixture was transferred to an anion-exchange column (Dowex 1-X8, 0.25 mL resin). The eluent was combined with a second mL of MeOH, applied to the resin, and after adding 6 mL scintillation fluid, tritium activity was measured using a Beckman LS 6500 for one minute.

To fractionate the cGMP hydrolytic activities of peaks A and B further, fractions 15 to 30 of the original 80 were reloaded onto the DEAE-Trisacryl M column and eluted with a linear gradient of NaOAC (0-0.5 M) in TM-EDTA. Fractions were again immediately assayed for cGMP hydrolysis (using the procedure described above with 0.2, 2, 5μM substrate), the results of which are graphically presented in Figure 2. One observation about peak B illustrated in Figure 2 is that increasing substrate concentration of cGMP dramatically enhanced activity when contrasted to peak A. While this observation is consistent with its being a PDE2, the fact that the enzyme characterized in Figure 2 is cGMP-specific (see below) suggests that it has a novel conformation compared to the classic PDE2 reported in the literature. Peak A activity shows apparent substrate saturation of high affinity catalytic sites.

C. THE ISOLATION OF CLASSIC PDE2 FROM SW480 Two methods were found that allowed "peak B" to be isolated from SW480 so that the enzyme had the classical PDE2 activity (i.e. was not cGMP-specific, but was cGMP stimulated). The first method involved growing the SW480 in 850 cm² Corning roller bottles instead of 150 cm² tissue culture flasks. SW480 were grown in roller bottles at 0.5 φm with each bottle containing 200 mL of RPMI 1640, 2 mM glutamine, and 25 mM HEPES. Cells were harvested by the following procedure. PBS media was warmed to 37°C for at least 15 minutes. 200 mL of 5% FBS/RPMI 1640 complete media is prepared and 5 mL of glutamine were added. 5 mL of antibiotic/antimycotic were also added.

70 mL of the PBS solution was added to 10 mL of 4X Pancreatin. The mixture was maintained at room temperature. The media was removed and the flask was rinsed with 4 mL of PBS being sure the bottom of the flask was covered. All solution was removed with a pipet. 4 mL of diluted Pancreatin was added to the flask, and the flask was swished to cover its bottom. The flask was incubated at 37°C for 8- 10 minutes. After the incubation, the flask was quickly checked under an inverted microscope to make sure all cells were rounded. The flask was hit carefully on its side several times to help detach cells. 10 mL of cold complete media were added to the flask to stop the Pancreatin proteolysis. The solution was swirled over the bottom to collect the cells. The media was removed using a 25 mL pipet, and the cells placed in 50 mL centrifuge tubes on ice. The tubes were spun at 1000 m at 4°C for 5 minutes in a clinical centrifuge to pellet cells. The supernatant was poured off and each pellet frozen on liquid nitrogen for 15 seconds. The harvested cells can be stored in a -70°C freezer.

The PDEs from the harvested SW480 cells were isolated using a FPLC procedure. A Pharmacia AKTA FPLC was used to control sample loading and elution on an 18 mL DEAE TrisAcryl M column. About 600 million cells of SW480 were used for the profiles. After re-suspending cells in homogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc₂, 0.1 mM EDTA,

0.8% Triton-100, lOμM benzamidine, l OμM TLCK, 2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A), samples were manually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loaded onto the column at 1 mL per minute, followed by a wash with 60 mL buffer A at 1 mL per minute. A gradient was run from 0-15% buffer B in 60 mL, 15-50% buffer B in 60 mL, and 50-100% buffer B in 16 mL. During the gradient, 1.5 mL fractions were collected.

The profile obtained was similar (Figure 13) to the profile for the novel PDE activity (see, e.g., Figure 1) obtained above, except that peak B isolated in this manner showed cAMP hydrolytic activity at 0.25 μM substrate that could be activated 2-3 fold by 5 μM cGMP.

A second method used to isolate classic PDE2 from SW480 was done using a non-FPLC DEAE column procedure described above (see Section VIIB) with the modification that the buffers contained 30% ethylene glycol, 10 mM TLCK and 3.6 mM β-mercaptoethanol. The addition of these reagents to the buffers causes a shift in the elution profile (see Figure 12) from low to high sodium acetate so that peak A moves from 40 to 150 mM, peak B from 75 to 280 mM and peak C from 200 to 500 mM Na acetate (see Figure 12). Peak B in Figure 12 was assayed with 2 μM cAMP substrate and showed a two-fold activation by 5 μM cGMP (see Figure 13). The selective PDE2 inhibitor EHNA inhibited 2 μM cGMP PDE activity in this peak B with an IC₅₀ of 1.6 μM and inhibited 2.0 μM cAMP PDE activity in peak B with an IC₅₀ of 3.8 μM (and IC₅₀ of 2.5 μM with addition of 10 μM rolipram).

D. cGMP-SPECIFICITY OF PDE PEAK A

AND THE NOVEL PEAK B ACTIVITY Each fraction from the DEAE column from Section VIIB was also assayed for cGMP-hydrolysis activity (0.25μM cGMP) in the presence or absence of Ca⁺⁺, or

Ca⁺⁺-CaM and/or EGTA and for cAMP (0.25μM cAMP) hydrolysis activity in the presence or absence of 5μM cGMP. Neither PDE peak A and peak B (fractions 5-22; see Figure 1) hydrolyzed cAMP significantly, establishing that neither had the activity of a classic cAMP-hydrolyzing family of PDE (i.e. a PDE 1, 2, 3).

Ca⁺⁺ (with or without calmodulin) failed to activate either cAMP or cGMP hydrolysis activity of either peak A or B, and cGMP failed to activate or inhibit cAMP hydrolysis. Such results establish that peaks A and B constitute cGMP- specific PDE activities but not classic or previously known PDE1, PDE2, PDE3 or PDE4 activities.

For the novel PDE peak B, as discussed below, cyclic GMP activated the cGMP hydrolytic activity of the enzyme, but did not activate any cAMP hydrolytic activity (in contrast with the peak B from Section VIIC above). This reveals that the novel PDE peak B ~ the novel phosphodiesterase of this invention — is not a cGMP- stimulated cAMP hydrolysis ("cGS") or among the classic or previously known PDE2 family activities because the known isoforms of PDE2 hydrolyze both cGMP and cAMP.

E. PEAK A IS A CLASSIC PDE5, BUT THE NOVEL PEAK B-

A NEW cGMP-SPECIFIC PDE-IS NOT To characterize any PDE isoform, kinetic behavior and substrate preference should be assessed. Peak A showed typical "PDE5" characteristics. For example, the K_m of the enzyme for cGMP was 1.07 μM, and Vmax was 0.16 nmol/min/mg. In addition, as discussed below, zaprinast (IC₅o=1.37μM) and E4021 (IC₅o=3 nM) and sildenafil inhibited activity of peak A. Further, zaprinast showed inhibition for cGMP hydrolysis activity of peak A, consistent with results reported in the literature. PDE peak B from Section VIIB showed considerably different kinetic properties as compared to PDE peak A. For example, in Eadie-Hofstee plots of peak A, cyclic GMP hydrolysis shows single line with negative slope with increasing substrate concentrations, indicative of Michaelis-Menten kinetic behavior. Peak B, however, shows the novel property for cGMP hydrolysis in the absence of cAMP of a decreasing (apparent K_m = 8.4), then increasing slope (K_m < 1) of Eadie-Hotfstee plots with increasing cGMP substrate (see, Figure 3). Thus, this establishes peak B's submicromolar affinity for cGMP (i.e., where K_m < 1).

Consistent with the kinetic studies (i.e., Figure 3) and positive-cooperative kinetic behavior in the presence of cGMP substrate, was the increased cGMP hydrolytic activity in the presence of increasing concentrations of cGMP substrate.

This was discovered by comparing 0.25 μM, 2 μM and 5 μM concentrations of cGMP in the presence of PDE peak B after a second DEAE separation to rule out cAMP hydrolysis and to rule out this new enzyme being a previously identified PDE5. Higher cGMP concentrations evoked disproportionately greater cGMP hydrolysis with PDE peak B, as shown in Figure 2. These observations suggest that cGMP binding to the peak B enzyme causes a conformational change in the enzyme. This confirms the advantage of using the native enzyme from neoplastic cells, but this invention is not limited to the native form of the enzyme having the characteristics set forth above.

F. ZAPRINAST- AND SILDENAFIL-INSENSITIVITY OF PDE PEAK

B RELATIVE TO PEAK A, AND THEIR EFFECTS ON OTHER PDE INHIBITORS Different PDE inhibitors were studied using twelve concentrations of drug from 0.01 to 100 μM and substrate concentration of 0.25 μM ³H-cGMP. IC₅₀ values were calculated with variable slope, sigmoidal curve fits using Prism 2.01

(GraphPad). The results are shown in Table 5. While compounds E4021 and zaprinast inhibited peak A, (with high affinities) IC5₀ values calculated against the novel PDE activity in peak B (Section VIIB) are significantly increased (>50 fold).

This confirms that peak A is a PDE5. These data further illustrate that the novel PDE activity of this invention is, for all practical puφoses, zaprinast-insensitive and

E4021 -insensitive.

Table 5 Comparison of PDE Inhibitors Against Peak A and Section VIIB Peak B (cGMP Hydrolysis)

PDE Family IC₅₀ IC₅o Ratio (IC₅₀

Compound Inhibitor Peak A (μM) Peak B (μM) PeakA/Peak B)

E4021 5 0.003 8.4 0.0004

Zaprinast 5 1.4 >30 <0.05

Compound E 5 and others 0.38 0.37 1.0

Sulindac sulfide 5 and others 50 50 1.0

Vinpocetine 1 >100 >100

EHNA 2 >100 3.7

Indolidan 3 31 >100 <0.31

Rolipram 4 >100 >100

Sildenafil 5 .0003 >10 <.00003

By contrast, sulindac sulfide and Compound E and competitively inhibited both peaks A and B phosphodiesterases at the same potency (IC₅₀=0.38 μM for PDE peak A; 0.37 μM for PDE peak B). Compound E is defined as (Z)-5-Fluoro-2- methyl-l-(3,4,5-trimethoxybenzylidene)-3-indenylacetamide, N-benzyl.

There is significance for the treatment of neoplasia and the selection of useful compounds for such treatment in the fact that peak B (either form of it) is zaprinast- insensitive whereas peaks A and B are both sensitive to sulindac sulfide and Compound E. We have tested zaprinast, E4021 and sildenafil to ascertain whether they induce apoptosis or inhibit the growth of neoplastic cells, and have done the same for Compound E. As explained below, zaprinast by itself does not have significant apoptosis-inducing or growth-inhibiting properties, whereas sulindac sulfide and Compound E are precisely the opposite. In other words, the ability of a compound to inhibit both PDE peaks A and B correlates with its ability to induce apoptosis in neoplastic cells, whereas if a compound (e.g., zaprinast) has specificity for PDE peak A only, that compound will not by itself induce apoptosis. G. INSENSITIVITY OF THE NOVEL PDE PEAK B TO INCUBATION

WITH cGMP-DEPENDENT PROTEIN KINASE G Further differences between PDE peak A and the novel peak B (Section VIIB) were observed in their respective cGMP-hydrolytic activities in the presence of varying concentrations of cGMP-dependent protein kinase G (which phosphorylates typical PDE5). Specifically, peak A and peak B fractions from Section VIIB were incubated with different concentrations of protein kinase G at 30°C for 30 minutes.

Cyclic GMP hydrolysis of both peaks has assayed after phosphorylation was attempted. Consistent with previously published information about PDE5, peak A showed increasing cGMP hydrolysis activity in response to protein kinase G incubation, indicating that peak A was phosphorylated. Peak B was unchanged, however (i.e., was not phosphorylated and insensitive to incubation with cGMP- dependent protein kinase G). These data are consistent with peak A being an isoform consistent with the known PDE5 family and peak B from Section VIIB being a novel cGMP-specific PDE activity.

H. NOVEL PEAK B IN PROSTATE AND BREAST CANCER CELL LINES The novel peak B was also isolated from two other neoplastic cell lines, a breast cancer cell line, HTB-26 and a prostate cancer cell line, LnCAP by a procedure similar to the one above used to isolate it from SW480. The protocol was modified in several respects. To provide even greater reproducibility to allow comparison of different cell lines, a Pharmacia AKTA FPLC was used to control sample loading and elution on an 18 mL DEAE TrisAcryl M column. SW840 was run by this same procedure multiple times to provide a reference of peak B. 200-400 million cells of SW480 were used for the profiles. 70 million cells of LnCAP were used for a profile (see Figures 10 and 11), and in a separate experiment 32 million cells of HTB-26 were used for a profile (see Figures 8 and 9). After re-suspending cells in homogenization buffer, samples were manually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loaded onto the column at 1 mL per minute, followed by a wash with 60 mL buffer A at 1 mL per minute. A gradient was run from 0-15%) buffer B in 60 mL, 15-50% buffer B in 60 mL, and 50-100% buffer B in 16 mL. During the gradient 1.5 mL fractions were collected. Peaks of cGMP PDE activity eluted around fraction 65 that was at 400 mM Na acetate (see Figures 8-11). This activity was measured at 0.25 μM cGMP (indicating submicromolar affinity for cGMP). Rolipram, a PDE4-specific drug, inhibited most of the cAMP PDE activity (i.e. the cAMP activity was due to PDE4), indicating that the peak B's cGMP activity were specific for cGMP over cAMP. All three peak B's (from SW480, HTB-26, and LnCAP) did not show stimulation with calcium/calmodulin and were resistant to 100 nM E4021, a specific PDE5-specific inhibitor like zaprinast (see Figures 8 and 10). The peak B's also showed a dramatic increase in activity when substrate was increased from 0.25 μM to 5 μM cGMP (suggesting positively cooperative kinetics) (see Figures 9 and 11). Also, the three peaks show similar inhibition by exisulind and Compound I. Compound I is defined as (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N- benzyl)indenylacetamide hydrochloride.

VIII. PROTEIN KINASE G AND β-CATENIN INVOLVEMENT A. IN GENERAL A series of experiments were performed to ascertain what effect, if any, an anti-neoplastic cGMP-specific PDE inhibitor such as exisulind had on cGMP- dependent protein kinase G ("PKG") in neoplastic cells containing either the adenomatous polyposis coli gene ("APC gene") defect or a defect in the gene coding for β-catenin. As explained below, such an inhibitor causes an elevation in PKG activity in such neoplastic cells. That increase in activity was not only due to increased activation of PKG in cells containing either defect, but also to increased expression of PKG in cells containing the APC defect. In addition, when PKG from neoplastic cells with either defect is immunoprecipitated, it precipitates with β- catenin. β-catenin has been implicated in a variety of different cancers because researchers have found high levels of it in patients with neoplasias containing mutations in the APC tumor-suppressing gene. People with mutations in this gene at birth often develop thousands of small tumors in the lining of their colon. When it functions properly, the APC gene codes for a normal APC protein that is believed to bind to and regulate β-catenin. Thus, the discovery that PKG in neoplastic cells containing either the APC gene defect or the β-catenin defect is bound to β-catenin indeed strongly implicates PKG in one of the major cellular pathways that leads to cancer. In addition, because of the relationship between cGMP-specific inhibition and PKG activity elevation upon treatment with SAANDs links cGMP to the PKG/ β- catenin/APC defect in such cells.

This latter link is further buttressed by the observation that β-catenin itself is reduced when neoplastic cells containing the APC defect or the β-catenin defect are exposed to a SAAND. This reduction in β-catenin is initiated by PKG itself. PKG phosphorylates β-catenin ~ which is another novel observation associated with this invention. The phosphorylation of β-catenin allows β-catenin to be degraded by ubiquitin-proteasomal system.

This phosphorylation of β-catenin by PKG is important in neoplastic cells because it circumvents the effect of the APC and β-catenin mutations. The mutated APC protein affects the binding of the β-catenin bound to the mutant APC protein, which change in binding has heretofore been thought to prevent the phosphorylation of β-catenin by GSK-3b kinase. In the case of mutant β-catenin, an elevation of PKG activity also allows the mutant β-catenin to be phosphorylated. By elevating PKG activity in neoplasia with cGMP-PDE inhibition allows for β-catenin phosphorylation (leading to its degradation) in neoplastic cells containing either type of mutation.

These findings buttress the role of cGMP-specific PDE inhibition in therapeutic approaches to neoplasia, and lead to new pharmaceutical diagnostic methods to identify further neoplasias responsive to treatment with cGMP-specific PDE inhibitors. These observations may also explain the unexpectedly broad range of neoplasias SAANDs can inhibit since both neoplasia with and without the APC defect can be treated, as explained above.

B. THE NOVEL PKG ASSAY The novel PKG assay of this invention involves binding to a solid phase plural amino acid sequences, each of which contain at least the cGMP binding domain and the phosphorylation site of phosphodiesterase type 5 ("PDE5"). That sequence is known and described in the literature below. Preferably, the bound PDE5 sequence does not include the catalytic domain of PDE5 as described below. One way to bind the PDE5 sequences to a solid phase is to express those sequences as a fusion protein of the PDE5 sequence and one member of an amino acid binding pair, and chemically link the other member of that amino acid binding pair to a solid phase (e.g., beads). One binding pair that can be used is glutathione S-transferase ("GST") and glutathione ("GSH"), with the GST being expressed as a fusion protein with the PDE5 sequence described above, and the GSH bound covalently to the solid phase. In this fashion, the PDE5 sequence/GST fusion protein can be bound to a solid phase simply by passing a solution containing the fusion protein over the solid phase, as described below.

RT-PCR method is used to obtain the cGB domain of PDE5 with forward and reverse primers designed from bovine PDE5A cDNA sequence (McAllister-Lucas L.

M. et al, J. Biol. Chem. 268, 22863-22873, 1993) and the selection among PDE 1-10 families. 5'-3', Inc. kits for total RNA followed by oligo (dT) column purification of mRNA are used with HT-29 cells. Forward primer (GAA-TTC-TGT-TAG-AAA- AGC-CAC-CAG-AGA-AAT-G, 203-227) and reverse primer (CTC-GAG-CTC- TCT-TGT-TTC-TTC-CTC-TGC-TG, 1664-1686) are used to synthesize the 1484 bp fragment coding for the phosphorylation site and both low and high affinity cGMP binding sites of human PDE5A (203-1686 bp, cGB-PDE5). The synthesized cGB- PDE5 nucleotide fragment codes for 494 amino acids with 97% similarity to bovine PDE5A. It is then cloned into pGEX-5X-3 glutathione-S-transferase (GST) fusion vector (Pharmacia Biotech )with tac promoter, and EcoRI and Xhol cut sites. The fusion vector is then transfected into E. Coli BL21 (DE3) bacteria (Invitrogen). The transfected BL21 bacteria is grown to log phase and then IPTG is added as ah inducer. The induction is carried at 20°C for 24 hrs. The bacteria are harvested and lysated. The soluble cell lysate is incubated with GSH conjugated Sepharose 4B (GSH- Sepharose 4B). The GST-cGB-PDE5 fusion protein can bind to the GSH-Sepharose beads and the other proteins are washed off from beads with excessive cold PBS. The expressed GST-cGB-PDE5 fusion protein is displayed on 7.5% SDS- PAGE gel as a 85 Kd protein. It is characterized by its cGMP binding and phosphorylation by protein kinases G and A. It displays two cGMP binding sites and the K_d is 1.6±0.2 μM, which is close to K_d=1.3 μM of the native bovine PDE5. The

GST-cGB-PDE5 on GSH conjugated sepharose beads can be phosphorylated in vitro by cGMP-dependent protein kinase and cAMP-dependent protein kinase A. The K_m of GST-cGB-PDE5 phosphorylation by PKG is 2.7μM and Vmax is 2.8 μM, while the K_m of BPDEtide phosphorylation is 68μM. The phosphorylation by PKG shows one molecular phosphate incoφorated into one GST-cGB-PDE5 protein ratio.

To assay a liquid sample believed to contain PKG using the PDE5-bound solid phase described above, the sample and the solid phase are mixed with phosphorylation buffer containing ³²P-γ-ATP. The solution is incubated for 30 minutes at 30°C to allow for phosphorylation of the PDE5 sequence by PKG to occur, if PKG is present. The solid phase is then separated from solution (e.g., by centrifugation or filtration) and washed with phosphate-buffered saline ("PBS") to remove any remaining solution and to remove any unreacted P-γ-ATP.

The solid phase can then be tested directly (e.g., by liquid scintillation counter) to ascertain whether P is incoφorated. If it does, that indicates that the sample contained PKG since PKG phosphorylates PDE5. If the PDE5 is bound via fusion protein, as described above, the PDE5-containing fusion protein can be eluted from the solid phase with SDS buffer, and the eluent can be assayed for³²P incoφoration. This is particularly advantageous if there is the possibility that other proteins are present, since the eluent can be processed (e.g., by gel separation) to separate various proteins from each other so that the fusion protein fraction can be assayed for³²P incoφoration. The phosphorylated fusion protein can be eluted from the solid phase with SDS buffer and further resolved by electrophoresis. If gel separation is performed, the proteins can be stained to see the position(s) of the protein, and ³²P phosphorylation of the PDE5 portion of the fusion protein by PKG can be measured by X-ray film exposure to the gel. If ³²P is made visible on X-ray film, that indicates that PKG was present in the original sample contained PKG, which phosphorylated the PDE5 portion of the fusion protein eluted from the solid phase.

Preferably in the assay, one should add to the assay buffer an excess (e.g., 100 fold) of protein kinase inhibitor ("PKI") which specifically and potently inhibits protein kinase A ("PKA") without inhibiting PKG. Inhibiting PKA is desirable since it may contribute to the phosphorylation of the PKG substrate (e.g., PDE5). By adding PKI, any contribution to phosphorylation by PKA will be eliminated, and any phosphorylation detected is highly likely to be due to PKG alone. A kit can be made for the assay of this invention, which kit contains the following pre-packaged reagents in separate containers:

1. Cell lysis buffer: 50 mM Tris-HCl, 1 % NP-40, 150 mM NaCl, 1 mM EDTA, ImM Na₃VO₄, 1 mM NaF, 500μM IBMX, proteinase inhibitors. 2. Protein kinase G solid phase substrate: recombinant GST-cGB-PDE5 bound Sepharose 4B (50%> slurry). 3. 2x Phosphorylation buffer: ³²P-γ-ATP (3000 mCi/mmol, 5-10 μCi/assay), 10 mM KH₂PO₄, 10 mM K₂HPO₄, 200 μM ATP, 5 mM MgCl_2. 4. PKA Protein Kinase I Inhibitor

Disposable containers and the like in which to perform the above reactions can also be provided in the kit. From the above, one skilled in the analytical arts will readily envision various ways to adapt the assay formats described to still other formats. In short, using at least a portion of PDE5 (or any other protein that can be selectively phosphorylated by PKG), the presence and relative amount (as compared to a control) of PKG can be ascertained by evaluating phosphorylation of the phosphorylatable protein, using a labeled phosphorylation agent.

C. SAANDS INCREASE PKG ACTIVITY IN NEOPLASTIC CELLS

Using the PKG assay described above, the following experiments were performed to establish that SAANDs increase PKG activity due either to increase in PKG expression or an increase in cGMP levels (or both) in neoplastic cells treated with a SAAND. TEST PROCEDURES

Two different types of PDE inhibitors were evaluated for their effects on PKG in neoplastic cells. A SAAND, exisulind, was evaluated since it is anti-neoplastic. Also, a non-SAAND classic PDE5 inhibitor, E4021 , was evaluated to ascertain whether PKG elevation was simply due to classic PDE5 inhibition, or whether PKG elevation was involved in the pro-apoptotic effect of SAANDs inhibition of PDE5 and the novel PDE disclosed in United States Patent Application No. 09/173,375 to Liu et al, filed October 15, 1998.

To test the effect of cGMP-specific PDE inhibition on neoplasia containing the APC mutation, SW480 colon cancer cells were employed. SW 480 is known to contain the APC mutation. About 5 million SW480 cells in RPMI 5% serum are added to each of 8 dishes:

2 - 10cm dishes — 30 μL DMSO vehicle control (without drug),

3 - 10cm dishes —200 μM, 400 μM. 600 μM exisulind, and 3 - 10cm dishes — E4021 ; 0.1 μM, 1 μM and 10 μM. The dishes are incubated for 48 hrs at 37°C in 5% CO₂ incubator.

The liquid media are aspirated from the dishes (the cells will attach themselves to the dishes). The attached cells are washed in each dish with cold PBS, and 200 μL cell lysis buffer (i.e., 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, ImM Na₃VO , 1 mM NaF, 500μM IBMX with proteinase inhibitors) is added to each dish. Immediately after the cell lysis buffer is added, the lysed cells are collected by scraping the cells off each dish. The cell lysate from each dish is transferred to a microfuge tube, and the microfuge tubes are incubated at 4°C for 15 minutes while gently agitating the microfuge tubes to allow the cells to lyse completely. After lysis is complete, the microfuge tubes are centrifuged full speed (14,000 r.p.m.) for 15 minutes. The supernatant from each microfuge tube is transferred to a fresh microfuge tube.

A protein assay is then performed on the contents of each microfuge tube because the amount of total protein will be greater in the control than in the drug- treated samples, if the drug inhibits cell growth. Obviously, if the drug does not work, the total protein in the drug-treated samples should be virtually the same as control. In the above situation, the control and the E-4021 microfuge tubes needed dilution to normalize them to the high-dose exisulind-treated samples (the lower dose groups of exisulind had to be normalized to the highest dose exisulind sample). Thus, after the protein assays are performed, the total protein concentration of the various samples must be normalized (e.g., by dilution).

For each drug concentration and control, two PKG assays are performed, one with added cGMP, and one without added cGMP, as described in detail below. The reason for performing these two different PKG assays is that cGMP specifically activates PKG. When PKG activity is assayed using the novel PKG assay of this invention, one cannot ascertain whether any increase the PKG activity is due to increased cGMP in the cells (that may be caused by cGMP-specific PDE inhibition) or whether the PKG activity level is due to an increased expression of PKG protein. By determining PKG activity in the same sample both with and without added cGMP, one can ascertain whether the PKG activity increase, if any, is due to increased PKG expression. Thus, if an anti-neoplastic drug elevates PKG activity relative to control, one can establish if the drug-induced increase is due to increased PKG protein expression (as opposed to activation) in the drug-treated sample if (1) the drug-treated sample with extra cGMP exhibits greater PKG activity compared to the control sample with extra cGMP, and (2) the drug-treated sample without extra cGMP exhibits greater PKG activity relative to control. After, parallel samples with and without added cGMP are prepared, 50 μL of each cell lysate is added to 20 μL of the PDE5/GST solid phase substrate slurry described above. For each control or drug cell lysate sample to be evaluated, the reaction is started by adding phosphorylation buffer containing 10 μCi ³²P-γ-ATP solution (200 μM ATP, 4.5 mM MgCl; 5 mM KH₂PO₄; 5 mM K₂HPO₄;) to each mixture. The resultant mixtures are incubated at 30°C for 30 minutes. The mixtures are then centrifuged to separate the solid phase, and the supernatant is discarded. The solid phase in each tube is washed with 700 μL cold PBS. To the solid phase, Laemmli sample buffer (Bio-Rad) (30 μL) is added. The mixtures are boiled for 5 minutes, and loaded onto 7.5%) SDS-PAGE. The gel is run at 150 V for one hour.

The bands obtained are stained with commassie blue to visualize the 85 Kd GST- PDE5 fusion protein bands, if present. The gel is dried, and the gel is laid on x-ray film which, if the PDE5 is phosphorylated, the film will show a corresponding darkened band. The darkness of each band relates to the degree of phosphorylation. As shown in Figures 6A and 6B, the SAAND exisulind causes PKG activity to increase in a dose-dependent manner in both the samples with added cGMP and without added cGMP relative to the control samples with and without extra cGMP. This is evidenced by the darker appearances of the 85 Kd bands in each of the drug- treated samples. In addition, the SW480 samples treated with exisulind show a greater PKG phosphorylation activity with added cGMP in the assay relative to the samples treated with vehicle with added cGMP. Thus, the increase in PKG activity in the drug-treated samples is not due only to the activation of PKG by the increase in cellular cGMP when the SAAND inhibits cGMP-specific PDE, the increase in PKG activity in neoplasia harboring the APC mutation is due to increased PKG expression as well.

Also the fact that the E4021 -treated SW480 samples do not exhibit PKG activation relative to control (see Figures 6A and 6B) shows that the increased PKG activation caused by SAANDs in neoplasia containing the APC mutation is not simply due to inhibition of classic PDE5. As an analytic technique for evaluating PKG activation, instead of x-ray film exposure as described above, the 85 Kd band from the SDS page can be evaluated for the degree of phosphorylation by cutting the band from the gel, and any ³²P incoφorated in the removed band can be counted by scintillation (beta) counter in the ³²P window. To test the effect of cGMP-specific PDE inhibition on neoplasia containing the β-catenin mutation, HCT1 16 colon cancer cells were employed. HCT1 16 is known to contain the β-catenin mutation, but is known not to contain the APC mutation.

The same procedure is used to grow the HCT1 16 cells as is used in the SW480 procedure described above. In this experiment, only exisulind and controls were used. The exisulind-treated cells yielded PKG that was phosphorylated to a greater extent than the corresponding controls, indicating that PKG activation occurred in the drug-treated cells that is independent of the APC mutation.

Thus, for the puφoses of the present invention, we refer to "reducing β- catenin" in the claims to refer to wild type and/or mutant forms of that protein.

D. CONFIRMATION OF INCREASED PKG EXPRESSION AND DECREASED β-CATENIN IN SW 480 BY WESTERN BLOT As demonstrated above, SAANDs cause an increase in PKG expression and an increase in cGMP level, both of which cause an increase in PKG activity in SAANDs- treated neoplastic cells. This increase in PKG protein expression was further verified by relatively quantitative western blot, as described below.

SW480 cells treated with exisulind as described previously are harvested from the microfuge tubes by rinsing once with ice-cold PBS. The cells are lysed by modified RIPA buffer for 15 minutes with agitation. The cell lysate is spun down in a cold room. The supernatants are transferred to fresh microcentrifuge tubes immediately after spinning. BioRad DC Protein Assay (Temecula, CA) is performed to determine the protein concentrations in samples. The samples are normalized for protein concentration, as described above. 50 μg of each sample is loaded to 10% SDS gel. SDS-PAGE is performed, and the proteins then are transferred to a nitrocellulose membrane. The blotted nitrocellulose membrane are blocked in freshly prepared TBST containing 5% nonfat dry milk for one hour at room temperature with constant agitation.

A goat-anti-PKG primary antibody is diluted to the recommended concentration/dilution in fresh TBST/5% nonfat dry milk. The nitrocellulose membrane is placed in the primary antibody solution and incubated one hour at room temperature with agitation. The nitrocellulose membrane is washed three times for ten minutes each with TBST. The nitrocellulose membrane is incubated in a solution containing a secondary POD conjugated rabbit anti-goat antibody for 1 hour at room temperature with agitation. . The nitrocellulose membrane is washed three times for ten minutes each time with TBST. The detection is performed by using Boehringer

Mannheim BM blue POD substrate.

As graphically illustrated in Figure 7, exisulind causes the drop of β-catenin and the increase of PKG, which data were obtained by Western blot. SW480 cells were treated with exisulind or vehicle (0.1 % DMSO) for 48 hours. 50 μg supernatant of each cell lysates were loaded to 10%) SDS-gel and blotted to nitrocellulose membrane, and the membrane was probed with rabbit-anti- β-catenin and rabbit anti- PKG antibodies.

E. SAANDS REDUCE β-CATENIN LEVELS IN NEOPLASTIC CELLS

This observation was made by culturing SW480 cells with either 200, 400 or600 μM exisulind or vehicle (0.1 % DMSO). The cells are harvested 48hours post treatment and processed for immunoblotting.. Immuno-reactive protein can be detected by Western blot. Western blot analysis demonstrated that expression of β- catenin was reduced by 50 %> in the exisulind-treated cells as compared to control.

These results indicate that β-catenin is reduced by SAANDs treatment. Together with the results above establishing PKG activity increases with such treatment and the results below establishing that β-catenin is phosphorylated by PKG, these results indicate that the reduction of β-catenin in neoplastic cells is initiated by activation of PKG. Thus, using PKG activity as a diagnostic tool to determine whether neoplasias would be responsive to treatment with anti-neoplasties is useful.

F. THE PHOSPHORYLATION OF β-CATENrN BY PKG In vitro, PKG phosphorylates β-catenin. The experiment that established this involves immunoprecipitating the β-catenin-containing complex from SW480 cells

(not treated with any drug) in the manner described below under "β-catenin immunoprecipitation " The immunoprecitated complex, while still trapped on the solid phase (i.e., beads) is mixed with ³²P-γ-ATP and pure PKG (100 units). Corresponding controls with out added PKG are prepared. The protein is released from the solid phase by SDS buffer, and the protein- containing mixture is run on a 7.5%>SDS-page gel. The running of the mixture on the gel removes excess ³²P-γ-ATP from the mixture. Any ³²P-γ-ATP detected in the 93Kd β-catenin band, therefore, is due to the phosphorylation of the β-catenin. Any increase in P-γ-ATP detected in the 93 Kd β-catenin band treated with extra PKG relative to the control without extra PKG, is due to the phosphorylation of the β- catenin in the treated band by the extra PKG.

The results we obtained were that there was a noticeable increase in phosphorylation in the band treated with PKG as compared to the control, which exhibited minimal, virtually undetectable phosphorylation. This result indicates that β-catenin can be phosphorylated by PKG.

G. THE PHOSPHORYLATION OF MUTANT β-CATENIN BY PKG The same procedure described in the immediately preceding section was performed with HCT1 16 cells, which contain no APC mutation, but contain a β- catenin mutation. The results of those experiments also indicate that mutant β-catenin is phosphorylated by PKG.

Thus, for the puφoses of the present invention, we refer to the phosphorylation of β-catenin in the claims to refer to the phosphorylation of wild type and/or mutant forms of that protein. H. β-CATENIN PRECIPITATES WITH PKG Supernatants of both SW480 and HCT1 16 cell lysates are prepared in the same way described above in the Western Blot experiments. The cell lysate are pre- cleared by adding 150 μl of protein A Sepharose bead slurry (50%>) per 500 μg of cell lysate and incubating at 4°C for 10 minutes on a tube shaker. The protein A beads are removed by centrifugation at 14,000 x g at 4°C for 10 minutes. The supernatant are transferred to a fresh centrifuge tube. 10 μg of the rabbit polyclonal anti-β-catenin antibody (Upstate Biotechnology, Lake Placid, New York) are added to 500 μg of cell lysate. The cell lysate/antibody mixture is gently mixed for 2 hours at 4°C on a tube shaker. The immunocomplex is captured by adding 150 μl protein A Sepharose bead slurry (75 μl packed beads) and by gently rocking the mixture on a tube shaker for overnight at 4°C. The Sepharose beads are collected by pulse centrifugation (5 seconds in the microcentrifuge at 14,000 φm). The supernatant fraction is discarded, and the beads are washed 3 times with 800 μl ice-cold PBS buffer. The Sepharose beads are resuspended in 150 μl 2 x sample buffer and mixed gently. The Sepharose beads are boiled for 5 minutes to dissociate the immunocomplexes from the beads. The beads are collected by centrifugation and SDS-PAGE is performed on the supernatant.

A Western blot is run on the supernatant, and the membrane is then probed with an rabbit anti β-catenin antibody. Then the membrane is washed 3 times for 10 minutes each with TBST to remove excess anti β-catenin antibody. A goat, anti- rabbit antibody conjugated to horseradish peroxidase is added, followed by 1 hour incubation at room temperature. When that is done, one can visualize the presence of β-catenin with an HRPO substrate. In this experiment, we could clearly visualize the presence of β-catenin.

To detect PKG on the same membrane, the anti-β-catenin antibody conjugate is first stripped from the membrane with a 62 mM tris-HCl buffer (pH 7.6) with 2 % SDS and 100 μM 2 β-mercaptoethanol in 55°C water bath for 0.5 hour. The stripped membrane is then blocked in TBST with 5% non-fat dried milk for one hour at room temperature while agitating the membrane. The blocked, stripped membrane is then probed with rabbit polyclonal anti-PKG antibody (Calbiochem, LaJolla, CA), that is detected with goat, anti-rabbit second antibody conjugated to HRPO. The presence of PKG on the blot membrane is visualized with an HRPO substrate. In this experiment, the PKG was, in fact, visualized. Given that the only proteins on the membrane are those that immunoprecipitated with β-catenin in the cell supernatants, this result clearly establishes that PKG was physically linked to the protein complex containing the β-catenin in the cell supernatants.

The same Western blot membrane was also probed after stripping with anti- GSK3-β antibody to ascertain whether it also co-precipitated with β-catenin. In that experiment, we also detected GSK3-β on the membrane, indicating that the GSK3-β precipitated with the GSK3-β and PKG, suggesting that the three proteins may be part of the same complex. Since GSK3-β and β-catenin form part of the APC complex in normal cells, this that PKG may be part of the same complex, and may be involved in the phosphorylation of β-catenin as part of that complex.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

WE CLAIM:

1. A method of determining whether a patient has a neoplasia, comprising obtaining a biological specimen from the patient, and evaluating whether the specimen exhibits elevated cGMP PDE activity relative to a normal specimen, wherein elevated cGMP PDE activity in the specimen obtained is indicative that the patient has a neoplasia.

2. The method of claim 1 where the cGMP PDE activity is PDE5 activity.

3. The method of claim 1 where the cGMP PDE activity is PDE2 activity.

4. The method of claim 1 where the cGMP PDE activity is the activity of a cGMP-specific PDE characterized by: (a) cGMP specificity over cAMP;

(b) positive cooperative kinetic behavior in the presence of cGMP substrate;

(c) submicromolar affinity for cGMP; and

(d) insensitivity to incubation with purified cGMP-dependent protein kinase.

5. A method for identifying neoplasias responsive to treatment with compounds that selectively inhibit neoplasia, comprising exposing a sample of the neoplasia to a compound that has cGMP-specific PDE inhibition activity and determining whether the compound inhibits the neoplasia.

6. The method of claim 5 wherein the determination of neoplasia inhibition comprises determining whether the compound inhibits neoplastic cell growth in a culture.

7. The method of claim 5 wherein the determination of neoplasia inhibition comprises determining whether the compound induces apoptosis of tumor cells.

8. A method for identifying neoplasias responsive to treatment with a cGMP-specific PDE inhibitor comprising determining the level of cGMP-specific PDEs in a sample of neoplastic tissue, wherein an elevated level of cGMP-specific PDEs in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

9. The method of claim 8 wherein the determination of the level of cGMP-specific PDEs in the neoplastic tissue comprises determining the amount of cGMP-specific PDE protein in the neoplastic tissue sample.

10. The method of claim 8 wherein the determination of the level of cGMP-specific PDEs in the neoplastic tissue comprises determining the amount of mRNA encoding for GMP-specific PDEs in the neoplastic tissue sample.

1 1. The method of claim 8 wherein the determination of the level of cGMP-specific PDEs in the neoplastic tissue comprises determining the cGMP hydrolytic activity of GMP-specific PDEs in the neoplastic tissue sample.

12. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of: (a) obtaining a suspected neoplastic tissue sample from the patient;

(b) contacting the sample with an antibody that is immunoreactive with cGMP-specific PDEs under conditions effective to allow the formation of immune complexes; and

(c) detecting the complexes thus formed, wherein an elevated amount of cGMP-specific PDEs in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

13. The method of claim 12, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with cGMP-specific PDEs and an immunodetection reagent.

14. The method of claim 13, wherein the immunodetection reagent is selected from the group consisting of urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, and glucose oxidase.

15. The method of claim 12, wherein the method is carried out using a kit comprising:

(a) a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with cGMP-specific PDEs;

(b) a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and cGMP-specific PDEs, and is linked to a detectable label;

(c) a washing buffer used to remove non-specifically bound immune complexes; and

(d) reagents necessary for detecting the amount of detectable label.

16. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with cGMP-specific PDEs, under conditions effective to allow the formation of immune complexes;

(c) washing the solid phase to remove non-specifically bound immune complexes; (d) exposing the solid phase to a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and cGMP-specific PDEs, and is linked to a detectable label;

(e) washing the solid phase to remove non-specifically bound second antibody; and

(f) detecting the amount of detectable label to ascertain the level of cGMP-specific PDE protein, wherein an elevated amount of cGMP-specific PDE protein in the neoplastic tissue, relative to the amount in normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

17. A method for identifying neoplasias from a patient responsive to treatment with compounds that inhibit cGMP-specific PDEs comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient; (b) isolating nucleic acids from the suspected neoplastic tissue sample;

(c) contacting nucleic acids isolated from the tissue sample with an isolated cGMP-specific PDE nucleic acid segment under conditions effective to allow hybridization of substantially complementary nucleic acids; and

(d) detecting the hybridized complementary nucleic acids thus formed, wherein an elevated amount of nucleic acid encoding for cGMP-specific PDEs in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

18. The method of claim 17, wherein the method is carried out using a kit comprising:

(a) reagents for isolating nucleic acids from a tissue sample; and

(b) an isolated cGMP-specific PDE nucleic acid segment.

19. A method for identifying neoplasias from a patient responsive to treatment with compounds that inhibit cGMP-specific PDEs comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient; (b) isolating nucleic acids from the sample;

(c) contacting the nucleic acids isolated from the tissue sample with a pair of nucleic acid primers that hybridize to distant sequences of a cGMP-specific PDE, the primers being capable of amplifying a nucleic acid segment of a cGMP-specific PDE;

(d) conducting a polymerase chain reaction to create amplification products; and

(e) detecting and characterizing the amplification products thus formed, whereby if the amplification products contain sequence coding for cGMP- specific PDEs, it is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

20. The method of claim 19, wherein the method is carried out using a kit comprising: (a) reagents for isolating nucleic acids from a tissue sample;

(b) a pair of nucleic acid primers that hybridize to distant sequences of a cGMP-specific PDE, the primers being capable of amplifying a nucleic acid segment of a cGMP-specific PDE; and

(c) reagents for conducting a polymerase chain reaction.

21. A method for identifying neoplasias responsive to treatment with compounds that selectively inhibit neoplasia, comprising exposing a sample of the neoplasia to a compound that inhibits the activity of PDE5, and determining whether the compound inhibits the neoplasia.

22. The method of claim 21 wherein the determination of neoplasia inhibition comprises determining whether the compound inhibits neoplastic cell growth in a culture.

23. The method of claim 21 wherein the determination of neoplasia inhibition comprises determining whether the compound induces apoptosis of tumor cells.

24. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a sample of suspected neoplastic tissue from the patient;

(b) contacting the sample with an antibody that is immunoreactive with PDE5 under conditions effective to allow the formation of immune complexes; and (c) detecting the complexes thus formed, wherein an elevated amount of said PDE in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP-specific PDE inhibitor.

25. The method of claim 24, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with PDE5 and an immunodetection reagent.

26. The method of claim 25, wherein the immunodetection reagent is selected from the group consisting of urease, alkaline phosphatase, (horseradish) hydrogen peroxidase. and glucose oxidase.

27. The method of claim 24, wherein the method is carried out using a kit comprising: (a) a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with PDE5;

(b) a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and

PDE5, and is linked to a detectable label; (c) a washing buffer used to remove non-specifically bound immune complexes; and (d) reagents necessary for detecting the amount of detectable label.

28. A method for identifying neoplasias from a patient responsive to treatment with an anti-neoplastic cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with PDE5 under conditions effective to allow the formation of immune complexes;

(c) washing the solid phase to remove non-specifically bound immune complexes;

(d) exposing the solid phase to a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and said cGMP-specific PDE, and is linked to a detectable label;

(f) detecting the amount of detectable label to ascertain the level of said PDE protein, wherein an elevated amount of PDE5 protein in the neoplastic tissue, relative to the amount in normal tissue, is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP-specific PDE inhibitor.

29. A method for identifying neoplasias responsive to treatment with compounds that selectively inhibit neoplasia, comprising exposing a sample of the neoplasia to a compound that inhibits the activity of a PDE2, and determining whether the compound inhibits the neoplasia.

30. The method of claim 29 wherein the determination of neoplasia inhibition comprises determining whether the compound inhibits neoplastic cell growth in a culture.

31. The method of claim 29 wherein the determination of neoplasia inhibition comprises determining whether the compound induces apoptosis of tumor cells.

32. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a sample of suspected neoplastic tissue from the patient;

(b) contacting the sample with an antibody that is immunoreactive with a PDE2 under conditions effective to allow the formation of immune complexes; and

(c) detecting the complexes thus formed, wherein an elevated amount of said PDE in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP-specific PDE inhibitor.

33. The method of claim 32, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with PDE2 and an immunodetection reagent.

34. The method of claim 33, wherein the immunodetection reagent is selected from the group consisting of urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, and glucose oxidase.

35. The method of claim 32, wherein the method is carried out using a kit comprising:

(a) a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with PDE2;

(b) a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and PDE2, and is linked to a detectable label;

(c) a washing buffer used to remove non-specifically bound immune complexes; and (d) reagents necessary for detecting the amount of detectable label.

36. A method for identifying neoplasias from a patient responsive to treatment with an anti-neoplastic cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with PDE2 under conditions effective to allow the formation of immune complexes;

(c) washing the solid phase to remove non-specifically bound immune complexes;

(f) detecting the amount of detectable label to ascertain the level of said PDE protein, wherein an elevated amount of PDE2 protein in the neoplastic tissue, relative to the amount in normal tissue, is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP-specific PDE inhibitor.

37. A method for ifentifying neoplasias responsive to treatment with compounds that selectively inhibit neoplasia, comprising exposing a sample of the neoplasia to a compound that inhibits the activity of a cGMP-specific PDE characterized by:

(a) cGMP specificity over cAMP;

(b) positive cooperative kinetic behavior in the presence of cGMP substrate;

(c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase, and determining whether the compound inhibits the neoplasia.

38. The method of claim 37 wherein the determination of neoplasia inhibition comprises determining whether the compound inhibits neoplastic cell growth in a culture.

39. The method of claim 37 wherein the determination of neoplasia inhibition comprises determining whether the compound induces apoptosis of tumor cells.

40. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of: (a) obtaining a sample of suspected neoplastic tissue from the patient;

(b) contacting the sample with an antibody that is immunoreactive with a cGMP-specific PDE characterized by:

( 1 ) cGMP specificity over cAMP;

(2) positive cooperative kinetic behavior in the presence of cGMP substrate;

(3) submicromolar affinity for cGMP; and

(4) insensitivity to incubation with purified cGMP-dependent protein kinase, under conditions effective to allow the formation of immune complexes; and (c) detecting the complexes thus formed, wherein an elevated amount of said cGMP-specific PDE in the neoplastic tissue, relative to normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

41. The method of claim 40, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with said cGMP-specific PDE and an immunodetection reagent.

42. The method of claim 41 , wherein the immunodetection reagent is selected from the group consisting of urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, and glucose oxidase.

43. The method of claim 40, wherein the method is carried out using a kit comprising:

(a) a first antibody, the first antibody being immobilized onto a solid . phase, wherein the first antibody is immunoreactive with said cGMP-specific PDE;

(b) second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and said cGMP-specific PDE, and is linked to a detectable label;

(d) reagents necessary for detecting the amount of detectable label.

44. A method for identifying neoplasias from a patient responsive to treatment with a cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with a cGMP-specific PDE characterized by:

( 1 ) cGMP specificity over cAMP;

(2) positive cooperative kinetic behavior in the presence of cGMP substrate;

(3) submicromolar affinity for cGMP; and (4) insensitivity to incubation with purified cGMP-dependent protein kinase, under conditions effective to allow the formation of immune complexes;

(c) washing the solid phase to remove non-specifically bound immune complexes;

(f) detecting the amount of detectable label to ascertain the level of said cGMP-specific PDE protein, wherein an elevated amount of said cGMP-specific PDE protein in the neoplastic tissue, relative to the amount in normal tissue, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

45. A method for identifying neoplasias responsive to treatment with a cGMP-specific PDE inhibitor comprising:

(a) exposing a sample of the neoplasia to a 10 μM concentration of a compound that has cGMP-specific PDE inhibition activity; and

(b) determining the ratio of the concentrations of intracellular cyclic GMP to cyclic AMP of the sample, both before and after exposure to the compound, wherein at least a three-fold increase in said ratio after exposure, compared to the ratio before exposure, is indicative that the neoplasia has potential for being treated by a cGMP-specific PDE inhibitor.

46. A method for identifying neoplasias from a patient responsive to treatment with an anti-neoplastic cGMP-specific PDE inhibitor comprising the steps of:

(a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to an anti-neoplastic cGMP PDE inhibitor; and (c) evaluating whether PKG activity increases in said sample upon exposure to said inhibitor; wherein elevated PKG activity upon exposure to said inhibitor is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP PDE inhibitor.

47. The method of claim 46, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with PKG and an immunodetection reagent.

48. The method of claim 46, wherein the method is carried out using a kit comprising:

(a) a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with PKG; (b) a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and PKG, and is linked to a detectable label;

49. A method for identifying neoplasias from a patient responsive to treatment with an anti-neoplastic cGMP-specific PDE inhibitor comprising the steps of: (a) obtaining a suspected neoplastic tissue sample from the patient;

(b) exposing the suspected neoplastic tissue sample to an anti-neoplastic cGMP PDE inhibitor; and

(c) evaluating whether β-catenin levels in said sample decrease upon exposure to said inhibitor; wherein a reduction in β-catenin levels upon exposure to saioVinhibitor is indicative that the neoplasia has potential for being treated by an anti-neoplastic cGMP PDE inhibitor.

50. The method of claim 49, wherein the method is carried out using a kit comprising an antibody that is immunoreactive with β-catenin and an immunodetection reagent.

51. The method of claim 49, wherein the method is carried out using a kit comprising:

(a) a first antibody, the first antibody being immobilized onto a solid phase, wherein the first antibody is immunoreactive with β-catenin;

(b) a second antibody, wherein the second antibody is immunoreactive with at least one member of the complex formed between the first antibody and β- catenin, and is linked to a detectable label;

(d) reagents necessary for detecting the amount of detectable label.

52. A kit for the assay of PKG activity in a sample, comprising: (a) cell lysis buffer;

(b) a solid phase substrate having bound thereto at least the cGMP binding domain and the phosphorylation site of PDE5;

(c) phosphorylation agent containing P-γ-ATP; and

(d) PKA protein kinase I inhibitor; whereby a sample can be assayed for PKG activity by evaluating with said phosphorylation agent whether phosphorylation of the PDE5 sequence by PKG occurs when a sample suspected of containing PKG is exposed to said solid phase.

53. The kit of claim 52 wherein the cGMP binding domain and the phosphorylation site of PDE5 are bound to the solid phase by expressing that sequence as a fusion protein with one member of an amino acid binding pair, and chemically linking the other member of that amino acid binding pair to a solid phase, and introducing the fusion protein to the solid phase whereby said sequence will be bound to the solid phase.

54. The kit of claim 53 wherein said amino acid binding pair comprises glutathione S-transferase and glutathione.

55. A method to assay PKG activity in a sample, comprising:

(a) introducing into said sample a solid phase substrate having bound thereto at least the cGMP binding domain and the phosphorylation site of PDE5;

(b) adding phosphorylation agent containing ³²P-γ-ATP to said sample and solid phase mixture;

(c) separating said solid phase from said mixture; and

(c) evaluating whether said solid phase contains ³²P-γ-ATP, whereby if said separated solid phase contains P-γ-ATP, it is indicative of

PKG activity in said sample.

56. The method of claim 55 wherein said solid phase is evaluated for ³²P-γ-ATP by eluting said PDE5-containing protein from the solid phase, and assaying the eluent for ³²P incoφoration, whereby if ³²P is incorporated in said eluent,

PKG activity is indicated in the sample.

57. The method of claim 55 wherein after the solid phase is separated, the solid phase is tested directly to ascertain whether P is mcoφorated in order to assess whether PKG activity is present in the sample.