WO2000033067A1 - Methode de diagnostic de la neoplasie - Google Patents

Methode de diagnostic de la neoplasie Download PDF

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Publication number
WO2000033067A1
WO2000033067A1 PCT/US1999/028099 US9928099W WO0033067A1 WO 2000033067 A1 WO2000033067 A1 WO 2000033067A1 US 9928099 W US9928099 W US 9928099W WO 0033067 A1 WO0033067 A1 WO 0033067A1
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cgmp
antibody
neoplasia
sample
specific
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PCT/US1999/028099
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Li Liu
Bing Zhu
Han Li
W. Joseph Thompson
Gary A. Piazza
Rifat Pamukcu
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Cell Pathways, Inc.
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Priority to EP99961832A priority Critical patent/EP1133689A4/fr
Priority to AU18330/00A priority patent/AU1833000A/en
Priority to JP2000585653A priority patent/JP2002531826A/ja
Publication of WO2000033067A1 publication Critical patent/WO2000033067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • 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.
  • cGMP cyclic GMP
  • PDE phosphodiesterases
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibodies preferably are immunoreactive with at least the PDE2-like enzymes described herein and PDE5.
  • Antibodies specific for cGMP-specific PDEs 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.
  • immunoassay methods such as EIAs, ELISAs, or RIAs
  • PDE protein in the neoplastic tissue is indicative that the neoplasia is likely to respond to treatment with a cGMP-specific PDE inhibitor.
  • 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 ⁇ -catenin protein, in a neoplastic tissue sample.
  • 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 600 ⁇ M; lanes 3, 4, 5) and E4021 (0.1 , 1 and 1 O ⁇ M, 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 600 ⁇ M: lanes 3, 4, 5) and E4021 (0.1 , 1 and l O ⁇ M, lanes 6, 7, 8).
  • Figure 7 is a bar graph of the results of Western blot experiments of the effects of exisulind on ⁇ -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-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • SRB assay 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.
  • 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.
  • cGMP-specific PDE inhibitors were tested on a number of neoplastic cell lines.
  • 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 IC 50 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
  • sensitivity of a neoplastic tissue to treatment with a cGMP-specific PDE inhibitor is tested with an apoptosis assay.
  • 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.
  • necrosis and apoptosis 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.
  • 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.
  • 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.
  • apoptosis is measured in the following fashion.
  • the sample (cell-lysate) is placed into a streptavidin-coated microtiter plate (MTP).
  • MTP streptavidin-coated microtiter plate
  • a mixture of anti-histone-biotin and anti-DNA peroxidase conjugate are added and incubated for two hours.
  • 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.
  • 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.
  • Increases in apoptosis are indicative that the neoplasia in question is sensitive to treatment with a cGMP-specific PDE inhibitor.
  • FIG. 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
  • FIG. 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.
  • 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
  • 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 ⁇ g streptomycin) and kept on ice for less than 1 hour until transfer to the pathology department.
  • transfer media 500 ml RPMI 1640 containing 50 ml fetal calf serum, 5x10 units penicillin G, and 5x10 ⁇ g streptomycin
  • 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.
  • tissue samples 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 (H 2 0 2 ) 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
  • 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.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • CYCLIC NUCLEOTIDE MEASUREMENTS 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.
  • 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 (Ha ⁇ er, J., Brooker, G., Advances in Nucleotide Research, l_0:l-33, 1979, which is incorporated herein by reference).
  • 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 -
  • 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 ⁇ -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 inco ⁇ orated herein by reference.)
  • Anti-PKG l ⁇ and anti- ⁇ -catenin antibodies can be purchased from StressGen Biotechnologies Co ⁇ , BC, Canada and Upstate Biotechnology, NY, respectively.
  • 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.
  • 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.
  • a carrier examples include keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • 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.
  • 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.
  • 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.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies
  • the animal 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.
  • affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent No. 4,196,265, inco ⁇ orated herein by reference.
  • 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 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.
  • adjuvant such as Freund's complete or incomplete adjuvant.
  • Booster injections with the same antigen are made at approximately two week intervals.
  • somatic cells with the potential for producing antibodies specifically B lymphocytes (B cells)
  • B cells B lymphocytes
  • 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.
  • PEG polyethylene glycol
  • the use of electrically-induced fusion methods is also appropriate.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine 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.
  • HPRT hypoxanthine phosphoribosyl transferase
  • 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).
  • 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.
  • 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.
  • 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.”
  • 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.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
  • Preferred secondary binding ligands are biotin and avidin or streptavidin compounds.
  • the present invention concerns immunoassays for binding, purifying, quantifying and otherwise generally detecting PDE protein components.
  • 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.
  • ELISAs enzyme linked immunoadsorbent assays
  • RIA radioimmunoassays
  • 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 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.
  • 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.
  • 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.
  • 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.
  • an enzyme linked immunoadsorbent assays is a type of binding assay.
  • 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.
  • 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.
  • 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.
  • the immune complexes may be detected directly.
  • 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.
  • 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.
  • RIA 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.
  • the percent of either the bound or free labeled antigen or both is determined.
  • a suitable detector such as a gamma or beta radiation detector
  • 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.
  • 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.
  • GST glutathione-S-transferase
  • the first antibody designated PDE5(1), was made using a short peptide of 17 amino acids as a hapten.
  • 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,
  • PDE5(2) 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 (lOO ⁇ M) 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 ⁇ L containing 5 mM sodium phosphate buffer (pH 6.8), 1 mM EDTA and 0.25 mg/ml BSA and H 3 -cGMP (5.8 Ci/mmol, NEN).
  • the purified soluble GST-PDE5 binding domain fusion protein (5 to 50 ⁇ g/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 H 2 O and 6 ml of Ready Safe liquid scintillation cocktail was added and the samples were counted on a Beckman LS 6500 scintillation counter. A 3 H- cGMP saturation binding curve at 25 °C was generated.
  • the GST-cGMP binding domain fusion protein displays one high affinity binding site for cGMP.
  • K d 0.5 ⁇ M
  • 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 lOO ⁇ g 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.
  • 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.
  • FIGS. 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.
  • 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).
  • 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.)
  • 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).
  • poly-A RNA polyadenylated RNA
  • the mRNA can be converted into complementary DNA (cDNA).
  • cDNA complementary DNA
  • 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.
  • 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.
  • 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.
  • 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 inco ⁇ orated 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 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.
  • 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 ⁇ l reaction using 2 ⁇ g 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
  • 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
  • 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.
  • 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 ⁇ and anti- ⁇ - 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.
  • 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.
  • 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).
  • 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 cm 2 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 MgAc 2 , 0.1 mM EDTA, 0.8% Triton-100, lO ⁇ M benzamidine, 1 O ⁇ M TLCK, 2000 U/mL aprotinin, 2 ⁇ M leupeptin, 2 ⁇ M pepstatin A) and homogenized on an ice bath using a polytron tissumizer (three times, 20 seconds/pulse).
  • homogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc 2 , 0.1 mM EDTA, 0.8% Triton-100, lO ⁇ M benzamidine, 1 O ⁇ M TLCK, 2000 U/mL aprotinin, 2 ⁇ M leupeptin, 2 ⁇
  • 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.
  • 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.
  • peak A is PDE5
  • 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.
  • the reaction was in 400 ⁇ l containing Tris-HCl (40mM; pH 8.0), MgCl 2 (5mM), 2- mercaptoethanol (4 mM), bovine serum albumin (30 ⁇ g), cGMP (0.25 ⁇ M-5 ⁇ M) 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 (50 ⁇ g) 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.
  • 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.
  • homogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc 2 , 0.1 mM EDTA,
  • 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 ⁇ -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 ⁇ M cAMP substrate and showed a two-fold activation by 5 ⁇ M cGMP (see Figure 13).
  • the selective PDE2 inhibitor EHNA inhibited 2 ⁇ M cGMP PDE activity in this peak B with an IC 50 of 1.6 ⁇ M and inhibited 2.0 ⁇ M cAMP PDE activity in peak B with an IC 50 of 3.8 ⁇ M (and IC 50 of 2.5 ⁇ M with addition of 10 ⁇ M rolipram).
  • peaks A and B constitute cGMP-specific PDE activities but not classic or previously known PDE1, PDE2, PDE3 or PDE4 activities.
  • 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.
  • cGS cGMP- stimulated cAMP hydrolysis
  • PEAK A IS A CLASSIC PDE5, BUT THE NOVEL PEAK B-
  • Peak A showed typical "PDE5" characteristics.
  • the K m of the enzyme for cGMP was 1.07 ⁇ M, and Vmax was 0.16 nmol/min/mg.
  • sildenafil inhibited activity of peak A.
  • 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.
  • Compound E is defined as (Z)-5-Fluoro-2- methyl-l-(3,4,5-trimethoxybenzylidene)-3-indenylacetamide, N-benzyl.
  • 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.
  • 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.
  • FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mg acetate, 0.1 mM EDTA, pH 7.5
  • 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.
  • ⁇ -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 ⁇ -catenin.
  • This phosphorylation of ⁇ -catenin by PKG is important in neoplastic cells because it circumvents the effect of the APC and ⁇ -catenin mutations.
  • the mutated APC protein affects the binding of the ⁇ -catenin bound to the mutant APC protein, which change in binding has heretofore been thought to prevent the phosphorylation of ⁇ -catenin by GSK-3b kinase.
  • an elevation of PKG activity also allows the mutant ⁇ -catenin to be phosphorylated.
  • 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).
  • GST glutathione S-transferase
  • GSH glutathione
  • 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.
  • 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.
  • GST pGEX-5X-3 glutathione-S-transferase
  • 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.
  • 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 2.7 ⁇ M and Vmax is 2.8 ⁇ M, while the K m of BPDEtide phosphorylation is 68 ⁇ M.
  • the phosphorylation by PKG shows one molecular phosphate inco ⁇ orated into one GST-cGB-PDE5 protein ratio.
  • the sample and the solid phase are mixed with phosphorylation buffer containing 32 P- ⁇ -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- ⁇ -ATP.
  • PBS phosphate-buffered saline
  • the solid phase can then be tested directly (e.g., by liquid scintillation counter) to ascertain whether P is inco ⁇ orated. 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 for 32 P inco ⁇ oration. 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 for 32 P inco ⁇ oration.
  • 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 for 32 P inco ⁇ oration.
  • 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 32 P phosphorylation of the PDE5 portion of the fusion protein by PKG can be measured by X-ray film exposure to the gel. If 32 P 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.
  • PKI protein kinase inhibitor
  • PKA protein kinase A
  • PKI protein kinase inhibitor
  • Cell lysis buffer 50 mM Tris-HCl, 1 % NP-40, 150 mM NaCl, 1 mM EDTA, ImM Na 3 VO 4 , 1 mM NaF, 500 ⁇ M IBMX, proteinase inhibitors.
  • Protein kinase G solid phase substrate recombinant GST-cGB-PDE5 bound Sepharose 4B (50%> slurry).
  • 2x Phosphorylation buffer 32 P- ⁇ -ATP (3000 mCi/mmol, 5-10 ⁇ Ci/assay), 10 mM KH 2 PO 4 , 10 mM K 2 HPO 4 , 200 ⁇ M ATP, 5 mM MgCl 2.
  • 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.
  • 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:
  • 3 - 10cm dishes 200 ⁇ M, 400 ⁇ M. 600 ⁇ M exisulind, and 3 - 10cm dishes — E4021 ; 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M.
  • the dishes are incubated for 48 hrs at 37°C in 5% CO 2 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 ⁇ L cell lysis buffer (i.e., 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, ImM Na 3 VO , 1 mM NaF, 500 ⁇ M IBMX with proteinase inhibitors) is added to each dish.
  • cell lysis buffer i.e., 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, ImM Na 3 VO , 1 mM NaF, 500 ⁇ M IBMX with proteinase inhibitors
  • 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).
  • PKG assays 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.
  • 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.
  • 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.
  • an anti-neoplastic drug elevates PKG activity relative to control
  • 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.
  • parallel samples with and without added cGMP are prepared, 50 ⁇ L of each cell lysate is added to 20 ⁇ L of the PDE5/GST solid phase substrate slurry described above.
  • the reaction is started by adding phosphorylation buffer containing 10 ⁇ Ci 32 P- ⁇ -ATP solution (200 ⁇ M ATP, 4.5 mM MgCl; 5 mM KH 2 PO 4 ; 5 mM K 2 HPO 4 ;) 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 ⁇ L cold PBS.
  • Laemmli sample buffer Bio-Rad
  • 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.
  • 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.
  • 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.
  • 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.
  • HCT1 16 colon cancer cells were employed.
  • HCT1 16 is known to contain the ⁇ -catenin mutation, but is known not to contain the APC mutation.
  • pu ⁇ oses of the present invention we refer to "reducing ⁇ - catenin" in the claims to refer to wild type and/or mutant forms of that protein.
  • 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 ⁇ g 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
  • exisulind causes the drop of ⁇ -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 ⁇ g supernatant of each cell lysates were loaded to 10%) SDS-gel and blotted to nitrocellulose membrane, and the membrane was probed with rabbit-anti- ⁇ -catenin and rabbit anti- PKG antibodies.
  • H. ⁇ -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 ⁇ l of protein A Sepharose bead slurry (50%>) per 500 ⁇ g 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 ⁇ g of the rabbit polyclonal anti- ⁇ -catenin antibody (Upstate Biotechnology, Lake Placid, New York) are added to 500 ⁇ g 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 ⁇ l protein A Sepharose bead slurry (75 ⁇ l 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 ⁇ m).
  • the supernatant fraction is discarded, and the beads are washed 3 times with 800 ⁇ l ice-cold PBS buffer.
  • the Sepharose beads are resuspended in 150 ⁇ l 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 ⁇ -catenin antibody. Then the membrane is washed 3 times for 10 minutes each with TBST to remove excess anti ⁇ -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 ⁇ -catenin with an HRPO substrate. In this experiment, we could clearly visualize the presence of ⁇ -catenin.
  • the anti- ⁇ -catenin antibody conjugate is first stripped from the membrane with a 62 mM tris-HCl buffer (pH 7.6) with 2 % SDS and 100 ⁇ M 2 ⁇ -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.

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Abstract

L'invention concerne une méthode qui permet de diagnostiquer une néoplasie chez un patient.
PCT/US1999/028099 1998-11-25 1999-11-24 Methode de diagnostic de la neoplasie WO2000033067A1 (fr)

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EP99961832A EP1133689A4 (fr) 1998-11-25 1999-11-24 Methode de diagnostic de la neoplasie
AU18330/00A AU1833000A (en) 1998-11-25 1999-11-24 Method for diagnosing neoplasia
JP2000585653A JP2002531826A (ja) 1998-11-25 1999-11-24 新形成診断法

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EP1178115A2 (fr) * 2000-06-29 2002-02-06 Pfizer Limited Phosphodiestérase
US6794192B2 (en) 2000-06-29 2004-09-21 Pfizer Inc. Target

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US5652131A (en) * 1993-05-27 1997-07-29 Icos Corporation Cyclic GMP-binding, cyclic GMP-specific phosphodiesterase materials and methods
US5858694A (en) * 1997-05-30 1999-01-12 Cell Pathways, Inc. Method for identifying compounds for inhibition of cancerous lesions

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IL132366A0 (en) * 1998-10-15 2001-03-19 Cell Pathways Inc Methods for identifying compounds for inhibition of neoplastic lesions and pharmaceutical compositions containing such compounds

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US5401774A (en) * 1991-03-08 1995-03-28 University Of Arizona Method for treating patients with precancerous lesions by administering substituted sulfonyl idenyl acetic and propionic acids and esters to patients with lesions sensitive to such compounds
US5652131A (en) * 1993-05-27 1997-07-29 Icos Corporation Cyclic GMP-binding, cyclic GMP-specific phosphodiesterase materials and methods
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SKEHAN, P.; STORENG, R.; SCUDIERO, D.; MONKS, A.; MCMAHON, J.; VISTICA, D.; WARREN, J.T.; BOKESCH, H.; KENNEY, S.; BOYD, M.R.: "New Colorimetric Assay For Anticancer-Drug Screening", J. NATL. CANCER INST., vol. 82, 1990, pages 1107 - 1112
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1178115A2 (fr) * 2000-06-29 2002-02-06 Pfizer Limited Phosphodiestérase
EP1178115A3 (fr) * 2000-06-29 2002-08-07 Pfizer Limited Phosphodiestérase
US6794192B2 (en) 2000-06-29 2004-09-21 Pfizer Inc. Target

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