Classifications

• • 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
  • G01N33/57407—Specifically defined cancers
  • G01N33/57434—Specifically defined cancers of prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03002—Acid phosphatase (3.1.3.2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)

Definitions

• This invention relates to the fields of prostate cancer diagnosis and treatment.
• Prostate cancer has the highest incidence of male cancers in the U.S.
• the molecular mechanisms underlying prostate carcinogenesis that may lead to new therapeutic treatments remain enigmatic.
• Tyrosine phosphorylation signaling in prostate cell proliferation is mediated by PTPases.
• PAcP represents the major PTPase activity in prostate epitheliums (Lin and Clinton, 1987, Advances in Protein Phosphatases 4:199-228; Li et al . , 1984, Eur . J. Biochem. 138:45-51; Lin and Clinton, 1986, Biochem. J., 235:351-357). This notion is further supported by results from crystallographic studies (Schneider et al .
• PAcP protein has active "-SH" groups and can function as an authentic Mysteine" PTPase in those cells .
• PAcP is a classically known androgen-responsive enzyme (Lin and Clinton, 1987, Advances in Protein Phosphatases 4:199-228)
• the cellular form of PAcP may participate in androgen promotion of cell proliferation via a tyrosine phosphorylation pathway (Lin et al . , 1998, J. Biol. Chem. 273:5939-5947) .
• Development and maintenance of differentiated function of the normal prostate gland require androgen (Tenniswood, 1986, Prostate 9:375-385). Androgen has also been implicated in the carcinogenesis of prostate epithelium (Gyorkey, 1973, Methods Cancer Res.
• Hormone therapy is not curative, and disease relapse will inevitably occur, usually within 24 months (Gittes, 1991, N. Eng. J. Med. 324:236-245).
• the molecular mechanism(s) underlying this transition from androgen-responsive to androgen-unresponsive prostate cancer is not understood, slowing the development of effective treatments.
• the genes driven by the promoter will be expressed differentially in these cells, minimizing systemic toxicity.
• the promoter of the Prostate Specific Antigen gene is a possible candidate serving for that approach.
• the expression of the PSA gene is not specific only to the prostate. Its expression was observed in several breast tumors and endometrium (Shan et al., 1997, Endocrinology 138:3764-3770).
• PAcP has a long history of serving as a tumor marker of prostate cancer and has been proposed to have a tissue-specific manner of expression (Chu et al., 1982, in Biochemical Markers of Cancer, Chu, Ed., pp. 117-136, Dekker, New York; Lin and Clinton, 1987, Adv. Prot. Phosphatase 4:199-228). Nevertheless, controversial results exist (Yam et al . , 1982, Ann. New York Academy
• the present invention provides novel methods of using human cellular PAcP in prostate cancer diagnosis and therapy.
• the inventor has discovered that induced expression of cellular PAcP can increase the efficacy of androgen deprivation therapy by prolonging and/or restoring its effect.
• cellular PAcP itself is useful as a therapeutic because its expression concurs with a diminished growth rate of cancer cells.
• the cellular level of PAcP exhibits a consistent negative correlation with the growth of several human prostate cancer cell lines.
• the data indicate that cellular PAcP down-regulates prostate cell growth, apparently by dephosphorylating c-ErbB-2/neu.
• reduced cellular PAcP expression in cancer cells can lead to prostate tumor progression and, conversely, by increasing the cellular PAcP concentration, tumor progression should be impeded or prevented.
• cellular PAcP is used as a therapeutic agent for treatment of prostate cancer.
• the cellular PAcP protein is from a human.
• the cellular PAcP protein is administer in a liposome, preferably comprised of lipofectin.
• the cellular PAcP protein is coupled to a monoclonal antibody, preferably one that is immunologically specific to a human prostate cancer cell.
• the cellular PAcP protein is administered by administering a nucleic acid comprising a coding sequence of cellular PAcP and allowing the cellular PAcP coding sequence to be expressed in the prostate carcinoma.
• the nucleic acid administered is comprised of a pCMV-neo expression vector operably linked to the coding sequence of cellular PAcP protein, the coding sequence encodes Genbank Accession No. M34840, and the coding sequence is Genbank Accession No. M34840.
• the coding sequence of cellular PAcP is operably linked to a virus that is herpes simplex virus, cyto episcopovirus, murine leukemia virus, recombinant adeno-associated virus, a recombinant adenoviral vector, human immunodeficiency virus or feline immunodeficiency virus, and/or a promoter that is a cytomeglovirus promoter and a PAcP promoter.
• a kit to carry out the therapeutic method of the invention comprising instructions and a reagent in a container that is purified cellular PAcP protein and/or a nucleic acid encoding cellular PAcP.
• a method for using the cellular form of PAcP as a marker for androgen responsiveness of prostate cancer therapy.
• the diagnosis method of the invention comprises determining the expression of cellular PAcP protein in the prostate carcinoma, a decrease in the expression being indicative of the androgen-insensitive nature of the carcinoma.
• the expression is determined by quantifying the concentration of cellular PAcP protein in the prostate carcinoma, preferably with an antibody immunologically specific to cellular PAcP.
• the expression level of PAcP is determined by the activity of cellular PAcP in the prostate carcinoma, preferably by measuring acid phosphatase activity.
• the expression level of cellular PAcP is determined by quantifying the concentration of cellular PAcP in the prostate carcinoma, preferably by a method that is PCR, Northern or Southern and/or hybridization to a nucleic acid sequence that is SEQ ID NO: 3, SEQ ID NO: and/or at least 15 consecutive nucleotides of M34840.
• kit to diagnose androgen-insensitive prostate carcinomas which comprises instructions to carry out the diagnosis method of the invention and a container containing an antibody immunologically specific to cellular PAcP protein, at least one a nucleic acid that hybridizes at moderate stringency to Genbank Accession No. M34840 and/or a reagent to assay acid phosphatase activity.
• the promoter region of a gene encoding cellular PAcP is provided that is useful for prostate specific expression of a coding sequence.
• the promoter region is the regulatory regions of a PAcP gene, preferably a human PAcP gene. More preferably, the promoter region is at least 100 consecutive nucleic acids of Genbank Accession No. U07083 or Genbank Accession No. X74961, and most preferably is the -1356 to +87 nucleotide region, where nucleotide 1 is the transcription start site, of Genbank Accession No. U07083 or Genbank Accession No. X74961.
• a xenograft animal model that mimics clinical human prostate cancers in cellular PAcP expression during tumor progression comprising an athymic mammal hosting at least one transgenic human prostate carcinoma cell.
• the mammal is a mouse
• the human prostate carcinoma cell is derived from the LNCaP, PC-3 or DU145 cell lines
• the cells encode an exogenous nucleic acid sequence encoding cellular PAcP protein, preferably human cellular PAcP protein.
• Fig IB RT-PCR analyses.
• P PC-3 cells
• L LNCaP (clone 33) cells
• D DU145 cells.
• Fig. 1C Western blots.
• PC PC-3 cells
• DU DU 145 cells
• 33, 51, and 81 in LNCaP cells indicated clone 33, 51, and 81 cells, respectively.
• Figure 2 Androgen effect and AR expression in different LNCaP cells.
• Fig. 2A androgen effect on cell growth. Bar, the range of results from duplicate wells.
• Fig. 2B RT-PCR analyses on AR expression. 33, 51, and 81, clone 33, 51, and 81 LNCaP cells, m, ⁇ Hindlll and ⁇ xl74 Haelll digest DNA markers. Sizes from top to bottom are 2.3, 2.0, 1.3, 1.0, 0.87, 0.6, 0.56, 0.31, 0.28, 0.27, 0.19, 0.18, 0.12, and 0.11 kb.
• FIG. 3A DHT effects on PSA mRNA expression. 33, 51, and 81, clone 33, 51, and 81 of LNCaP cells, respectively.
• Fig. 3B DHT effect on cellular PAcP protein level. 33, 51, and 81, clone 33, 51, and 81 cells, respectively.
• FIG. 4A Protein phosphatase activity and growth rates of different LNCaP cells.
• Fig. 4A PAcP and serine/threonine protein phosphatase activities.
• PAcP activity represented the average of triplicate results
• Fig. 4B growth rates of different LNCaP cells. The data shown are the average of duplicate wells after normalization to day 0, indicating cell growth rates. Similar results were observed in three sets of independent experiments. Bar, the range of results from duplicate wells.
• PAcP cDNA-transfected clone 81 LNCaP cells PAcP activity.
• the A 410 represented the PAcP activity in 1 mg of total cell lysate proteins.
• Fig 5B effect of additional PAcP expression on the basal cell growth rate. Similar results were observed in two sets of independent experiments in duplicate wells.
• Fig. 5C the Tyr(P) level in total cellular proteins. Arrows indicate the respective positions of 185 and 150 kDa .
• Fig. 5D androgen effect on the cell growth rate. The growth stimulation by DHT was normalized to the control cells (as 100%) that were maintained in the absence of androgen. The data shown are the average of duplicate wells. Similar results were obtained in three sets of independent experiments with duplicate wells.
• FIG. 6A expression of functional AR in PC-3 cells. The percentage of conversion of [ 14 C] chloramphenicol to monoacetylated product was indicated.
• Fig. 6B expression of PAcP and the basal cell growth rate. The data shown are the average results of duplicate wells, and similar results were obtained from three independent experiments. Bar, the range of results from duplicate wells.
• Fig. 6C the Tyr(P) level in cellular proteins from PC-transfectants . Arrows, 185, 150, 70, and 55 kDa, respectively.
• Fig. 6D androgen effect on the growth of PC-411 and PC-416 cells.
• the cell number in each control well was defined as ratio 1.
• FIG. 7A LNCaP clone 33, clone 81, and LNCaP-34 cells;
• Fig. 7B PC-3 and PC-416 cells. Arrow, 185 kDa.
• FIG. 8A western blot analyses of PAcP and SHP- 1 protein levels in different prostate cancer cells.
• Fig. 8B the level of tyrosine phosphorylation (pY) of c- ErbB-2/neu protein and the cellular PAcP expression in different LNCaP cells, and PAcP cDNA-transfected C-81 LNCaP cells.
• Fig. 8C the expression of cellular PAcP and p-Tyr (pY) level of c-ErbB-2 protein in PAcP cDNA transfected PC-3 cells.
• Fig. 8D the phosphorylation level of ppl85 (i.e., c-ErbB-2) by in vi vo 32 P X labeling in low passage (p4), and high passage (p30) PC-416 cells.
• FIG. 9A Untransformed LNCaP clones. Each column represents the colony number in one dish. Similar results were observed in two sets of independent experiments.
• Fig. 9B PAcP cDNA transfected C-81 LNCaP cells, -23, -28 and -40. Similar results were obtained in two sets of independent experiments.
• Fig. 9C PC-416 cells which express cellular PAcP, and PC-18 and PC-CMV cells which lack cellular PAcP expression. Similar results were obtained in two sets of independent experiments .
• FIG. 10 The expression of cellular PAcP and the tumorigenicity of prostate cancer cells in xenograft athymic mice.
• Fig. 11A expression of PAcP mRNA in normal human tissue.
• the multiple tissue membrane a- spleen; b-thymus; c-prostate; d-testis; e-ovary; f-small intestine; g-colon; h-peripheral blood leukocyte.
• PAP is PAcP.
• Fig. 11B slot blot of nuclear run-on assay. L- low, and H- high density of LNCaP cells.
• FIG. 12 Promoter activity of the 1.4 kb fragment of PAcP 5 '-flanking region.
• LNCaP cells were transiently transfected with pCATPAP, pCATasPAP, pCATEPAP, and pCATPromoter vectors, as a reporter, respectively.
• the relative CAT activity were calculated from results of triplicate samples after normalizing to pCATasPAP. Similar results were obtained from four independent experiments. Bar represents standard deviation.
• FIG. 13 Serum effect on the PAcP promoter activity in LNCaP cells.
• the relative CAT activities were calculated as the mean of triplicates from three independent experiments. Cell growth was calculated based on total cellular protein. Bar represents standard deviation.
• isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
• the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote.
• An “isolated nucleic acid molecule” may also comprise a cDNA molecule.
• isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
• the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below) .
• isolated protein or peptide
• isolated and purified protein or peptide
• This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
• the term "immunologically specific” refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
• the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”).
• the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
• substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
• Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences.
• nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure, thermostability characteristics and/or biological activity of the protein) .
• nucleic acid sequences the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
• amino acid sequences refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
• percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program.
• Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein.
• percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
• promoter region refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3 ' side of the coding region, or within the coding region, or within introns .
• expression cassette comprises 5' and 3' regulatory regions operably linked to a coding sequence.
• the coding sequence may be in the sense or antisense orientation with respect to the 5' regulatory region.
• reporter gene refers to genetic sequences which may be operably linked to a promoter region forming a transgene, such that expression of the reporter gene coding region is regulated by the promoter and expression of the transgene is readily assayed.
• vector refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated.
• expression vector is a specialized vector that contains a gene with the necessary regulatory regions needed for expression in a host cell.
• selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
• operably linked means that the regulatory sequences necessary for expression of a particular coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other regulatory elements (e.g., enhancers or translation regulatory sequences) in an expression vector.
• biologically acceptable medium includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph.
• the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the antisense molecules to be administered, its use in the pharmaceutical preparation is contemplated.
• the invention provides several methods for using cellular prostatic acid phosphatase (PAcP) for the diagnosis and treatment of prostate cancer.
• diagnosis methods that use the presence of cellular PAcP activity, protein or nucleic acids to differentiate an early stage androgen-responsive prostate cancer from a late stage androgen-unresponsive prostate cancer.
• Therapeutic methods provided by the invention are based on the discovery that increasing cellular PAcP activity in prostatic tumor cells lines in vi tro and in xenograft mice decreases the growth rate of the tumor cells.
• therapeutic methods for increasing the activity of PAcP in tumor cells including injection of protein and/or nucleic acids and gene therapy.
• a promoter suitable for specific, androgen- responsive expression in the prostate the PAcP promoter.
• the invention provides a xenograft mammal model system for studying prostate cancer.
• the correlation between the expression of several genes related to cancer of the prostate and cell proliferation were examined in cell lines derived from human prostate carcinoma cells (see Example 1).
• the expression of the androgen receptor (AR) and two prostate-specific androgen-regulated antigens, PAcP and prostate specific antigen (PSA) was determined in the cell lines with and without transformation with a PAcP cDNA expression vector and before and after stimulation by the androgen 5 ⁇ - dihydrotestosterone (DHT) .
• LNCaP-33 human prostate carcinoma cell line
• LNCaP-51 moderate response
• LNCaP-81 low response
• the clone that showed a relatively low response to DHT, LNCaP-81 was considered to be a model for late stage androgen-unresponsive prostate cancer cells.
• Two androgen insensitive carcinoma cell lines, DU145 and PC-3 were used as well.
• the loss of androgen- regulated growth was found to be related to PAcP mRNA levels and not AR levels. While in all of the LNCaP cells the expression of AR was very similar and the expression of PSA cDNA was stimulated by DHT, in clone 81 the expression of PAcP was diminished. The degree of androgen response of LNCaP-81 cells was diminished with a parallel decrease in cellular PAcP. In the different LNCaP clones, the PAcP RNA levels decreased while the level of Ser/Thr protein dephosphorylation increased.
• PAcP expression vector cellular PAcP activity and protein increase and cell growth decreased to levels lower than the control LNCaP-81 cells.
• the expression of cellular PAcP correlated with decreased Tyr(P) levels in two cellular phosphoproteins .
• the expression of an exogenous PAcP gene in the LNCaP-81 cells also restored the androgen sensitivity of cell growth.
• the DU145 and PC-3 prostate carcinoma cells lines are also insensitive to androgen stimulation.
• DU145 did not have a detectable level of AR mRNA while PC-3 cells have a very low level of AR mRNA and protein.
• DU145 nor PC-3 cells have detectable levels of PAcP or PSA mRNA.
• PC-3 cells exhibited a modest but detectable response to the synthetic androgen R1881.
• Transfection with an exogenous PAcP expression vector yielded cells that expressed the exogenous PAcP and had a slower growth rate and decreased Tyr(P) level in cellular proteins, but also had significant androgen stimulation of cell growth. In these cells as well, androgen-stimulation of cell growth correlated with an increased Tyr(P) level of ppl85.
• Example 2 the significance of cellular PAcP expression on prostate tumor development and progression is shown in a xenograft athymic model mouse system.
• LNCaP cells that expressed higher cellular PAcP yielded xenograft tumors in male athymic mice that developed more slowly and were smaller than LNCaP cells that expressed lower levels of the enzyme.
• Male athymic mice inoculated with PC-3 cells expressing exogenous PAcP developed tumors more slowly than control cells. Progression of xenograft tumor growth correlated with a decreased expression of cellular PAcP in isolated tumors.
• PAcP expression on tumor development was more pronounced and no detectable tumors where found in many of the mice grafted with LNCaP cells expressing exogenous PAcP at 100 days after inoculation.
• cellular PAcP is involved in androgen promotion of prostate cell growth. Therefore, the male xenograft mouse model supports the growth of transgenic human prostate carcinomas, and can be used to develop treatments for prostate cancer. Furthermore, ectopic expression of cellular PAcP correlates with a delayed tumor induction and decreased tumor size in a mouse xenograft model.
• the promoter of PAcP gene has been characterized (Example 3).
• a 1.4 kb DNA fragment of the promoter region of the PAcP gene (-1356/+87) was cloned and its characteristic as an exogenous promoter determined.
• the promoter was PCR amplified from LNCaP cells using primers designed from DNA sequences previously reported.
• Northern analysis of mRNA populations from normal human tissue indicates that PAcP mRNA is found only in human prostate, and not in the spleen, thymus, testis, ovary, small intestine, colon or peripheral blood leukocyte. Nuclear run-on experiments indicated that in LNCaP cells, PAcP expression was induced on the transcriptional level by DHT treatment.
• the PAcP promoter region -1356/+87 drove expression of CAT activity that was approximately 2.5 times higher than the equivalent antisense construct.
• the addition of a SV40 enhancer to the PAcP promoter region gave a three-fold induction of expression as compared to the antisense construct.
• a CAT coding sequence driven by an SV40 promoter resulted in approximately 5.5-fold activity. Therefore the PAcP promoter was able to drive a low but significant level of expression in prostate carcinoma cells.
• CAT activity increased in transfected LNCaP cells grown in steroid- free media simultaneously with decreased cell growth indicating that the exogenous PAcP promoter activity is very similar to that of the native PAcP promoter.
• Presented with the present invention is a therapeutic method to slow or prevent the growth of prostate tumor cells in mammals.
• Examples 1 and 2 the ability of increased cellular PAcP activity to decrease the growth of both androgen-sensitive and androgen-insensitive prostate cancer cells is shown.
• This therapeutic method is particularly useful because it may be used to effectively treat androgen-insensitive prostate carcinomas which currently do not have an effective method of treatment.
• This method comprises the step of increasing the level of cellular PAcP activity in the carcinoma cell.
• the method of the invention for treating mammalian prostate carcinomas comprises administering a therapeutically effective amount of PAcP protein to the target cells.
• the administration of the PAcP protein can be accomplished via several methods, including exposing the target cells, the prostate carcinoma, to cellular PAcP protein, or exposing the target cell to a nucleic acid construct that expresses an appropriate cellular PAcP coding sequence. Any method of administration of cellular PAcP protein is appropriate as long as it results in increased levels of cellular PAcP protein within the target cells.
• Target cells may be removed from the patient and treated ex vivo, and then reintroduced to the patient. Additionally, the treatment may be used in cell cultures or animal model systems for experimental purposes, as illustrated in Example 1 and Example 2.
• the target cells comprise prostate carcinomas.
• the administration of cellular PAcP protein to target cells can be accomplished by exposing the target cell to cellular PAcP protein.
• the target cells are tumor cells within an animal, it is preferred that the protein is administered in a protected form to increase the stability in cells.
• One strategy of accomplishing this is to use liposomes.
• Liposomes are water-filled vesicles composed of several phospholipid layers surrounding an aqueous core with an outer shell capable of providing direction to specific target cells.
• liposomes are composed of some combination of phosphatidylcholine, cholesterol, phosphatidylglycerol or other glycolipids or phospholipids (Hudson and Black, 1993, American Pharmacy NS33 (5) : 23-24 ) .
• Insoluble polymers composed of polyethylene may also be used to form a protective layer around the protein, inhibiting degradation while traveling to the target cell (Hudson and Black, 1993, American Pharmacy NS33 (5) : 23-24 ) .
• Lipofectin a commercially available cationic liposome, has previously been shown effective in introducing active purified PAcP protein into a human prostate carcinoma cell line (Lin et al . , 1993, Biochem. Biophys. Res. Commun. 192:413-419; incorporated by reference herein).
• Another way to deliver cellular PAcP protein to target cells is to couple the protein to a target cell-specific monoclonal antibody. This approach allows the protein to be specifically delivered to the target cell and minimizes toxic effects on non-target cells (Houston, 1993, Current Opinion in Biotechnology 4:739-744).
• the cellular PAcP protein is administered to the target cell through the use of exogenous nucleic acids that will cause the protein to be synthesized within the target cell.
• nucleic acids can be temporary residents in the target cell, such as temporary expression plasmids, or permanent resident nucleic acids that will replicate with the target cell.
• Expression plasmids are particularly appropriate for experimental work with cell cultures, as illustrated in Example 1 and Example 2. The construction of such plasmids and the transformation of target cells with them in vi tro is well known to those of skill in the art of cell biology.
• Expression vectors suitable for cellular PAcP expression in mammalian cells are commercially available (Gene Therapy Systems, San Diego) .
• a pCMV-neo expression vector is used (Lin et al., 1992, Cancer Res 52:4600-4607). Naked DNA and plasmids may be delivered to the target cells by several known means. The naked DNA may be transferred directly into the genetic material of the cells (Wolff et al . , 1990, Science 247:1465-1468), the cellular PAcP-encoding DNA may be delivered in liposomes (Ledley, 1987, J. Pediatrics 110:1) or proteoliposomes that contain viral envelope receptor proteins (Nicolau et al, 1983, Proc. Natl. Acad. Sci . U.S.A.
• the cellular PAcP-encoding DNA may be coupled to a polylysine-glycoprotein carrier complex.
• gene therapy using viral vectors is preferred.
• retroviral vectors such as the herpes simplex virus (U.S. Patent 5,288,641, incorporated herein by reference) , Cytomegalovirus, murine leukemia virus (Blaese et al., 1995, Science 270:475-479) and similar as described by Miller (Miller, 1992, Curr. Top. Microbiol. Immunol. 158:1).
• Recombinant adeno-associated virus such as those described by U.S. Patent No. 5,139,941 (which is incorporated herein by reference) , and recombinant adenoviral vectors (He et al . , 1998, PNAS 95:2509-2514, incorporated by reference herein) are particularly preferred.
• recombinant lentivirus vectors such as a recombinant Human Immunodeficiency Virus (U.S. Patent No. 5,885,805; Blaese et al . , 1995, Science 270:475-479; Onodera et al . , 1998, J.
• viral vectors that are replication competent may be used to improve the efficacy of the treatment of solid tumors (Wildner et al . , 1999, Gene Ther. 6:57-62).
• the recombinant vector of the invention will comprise a nucleic acid construct comprising a sequence encoding the cellular PAcP protein operably linked to an appropriate promoter.
• a strong constitutive promoters such as a cytomeglovirus promoter, a viral LTR, RSV or SV40 promoter are preferred.
• a cytomegalovirus promoter is used.
• promoters associated with genes that are expressed at high levels in mammalian cells such as elongation factor-1 and actin are also contemplated. It is most advantageous to use a promoter region that will express the PAcP protein specifically in prostate cells.
• a PAcP promoter is used.
• the PAcP promoter is isolated from humans.
• the -1365/+87 region of the human PAcP gene is used.
• the -1365/+87 region of the gene whose sequence is Genbank Accession No. X74961 or Genbank Accession No. U07083.
• the amino acid sequence of cellular PAcP protein on which to base the nucleic acid construct is ideally from the gene that is endogenous to the species which is being treated.
• Homo sapi ens is being treated and the nucleic acid construct encodes Genbank Accession No. M34840.
• the nucleic acid sequence is Genbank Accession No. M34840.
• Other variants of PAcP protein exist in Homo sapi ens and other mammalian species and the sequences of these variants are also contemplated for use with the invention.
• a recombinant adenoviral vector is used to deliver the PAcP-expressing construct to the target cells.
• adenoviral vectors for gene therapy is well known in the art (El-Deiry et al . , 1993, Cell 75:817; Blogosklonny and El-Deiry, 1996, Int. J. Cancer 67:386-395; Prabhu et al., 1996, Clin Cancer Res. 2:1221-1230; Zeng et al . , 1997, Int. J. Oncol. 11:221-226; Mitchell and El-Deiry, 1999, Cell Growth and Diff. 10:223-230; Meng et al . , 1998, Clin. Cancer Res.
• an adenovirus vector has been used successfully to deliver p53 to target cells to treat lung cancer in human patients (Roth et al . , 1996, Nature Med. 2:974 incorporated herein by reference; and U.S. Patent 5,747,469 incorporated herein by reference) . It is contemplated that these protocols with simple variation, that will be well known to those in the art, can be used to administer the PAcP protein to target cells in the invention. In a most preferred embodiment, therapeutically effective amounts of the viral vector are delivered to the prostate carcinoma by direct injection.
• kits that may be used to carry out the therapeutic method of the invention are contemplated.
• the kits comprise purified cellular PAcP protein in a container and instructions for using the protein in the therapeutic method to slow or prevent the growth of prostate tumor cells in mammals described above.
• the PAcP protein is human cellular PAcP.
• the kit comprises isolated nucleic acids that encode PAcP cellular protein in a container and instructions for using the nucleic acids in the therapeutic method to slow or prevent the growth of prostate tumor cells in mammals described above.
• the nucleic acids encode the human cellular PAcP.
• the nucleic acids comprise the human PAcP promoter of the invention operably linked to a nucleic acid encoding the human cellular PAcP.
• the therapeutic method of the invention includes any manner of modifying the tyrosine signaling pathway in mammalian prostate cancer cells that is analogous to increasing cellular PAcP activity. These methods include increasing or decreasing the activity of a component of the tyrosine signaling pathway that will result in increasing the expression of the PAcP gene, or increasing or decreasing the activity of a component of the tyrosine signaling pathway that will mimic the effect of increasing cellular PAcP activity.
• Also presented with the invention is a method to diagnose androgen-insensitive prostate carcinomas.
• This method provides a non-invasive method of determining the androgen sensitivity of a diagnosed prostate carcinoma, and thereby enabling the selection of the most appropriate therapy method.
• Present methods rely on the presence of a prostate carcinoma specific antigen to signal the presence of a prostate carcinoma in patients. While the presence of the PSA is routinely used to detect prostate carcinomas, it cannot provide the critical information of whether the cancer is androgen- insensitive. Information as the tumor's androgen sensitivity is critical for determining if the tumor may be controlled by the standard anti-androgen therapies.
• the diagnosis method of the invention determines whether the diagnosed carcinoma is an early stage androgen- sensitive or later stage androgen-insensitive prostate carcinoma .
• the diagnosis method present comprises the step of determining the expression of cellular PAcP in the prostate tumor.
• This step may be accomplished by several approaches.
• a preferred approach is to assay a biopsy sample from the tumor for nucleic acids encoding cellular PAcP, as detailed in Example 1. Standard and established methods for determining the presence and amount of nucleic acids may be used, which will be well known to those in the art of molecular biology.
• PAcP nucleic acids may be extracted from the biopsy samples and detected by virtue of their specific hybridization to a nucleic acid encoding the PAcP protein.
• quantization of nucleotides hybridizing to PAcP nucleic acids is by PCR, Northern blot or Southern blot analysis.
• the level of cellular PAcP protein and activity can also be used to diagnose an androgen-insensitive prostate carcinoma.
• the PAcP protein is quantified in the biopsy tissue using antibodies immunologically specific to cellular PAcP.
• monoclonal antibodies are used.
• the activity of cellular PAcP is quantified in biopsy tissue.
• the activity of PAcP in the tissue can be quantified directly using assays to measure the acid phosphatase activity, such as using p-nitrophenyl phosphate as the substrate, as detailed in Example 1.
• PAcP activity may also be quantified by determining the phosphorylation level of the natural substrates of PAcP in the biopsy tissue. For example, the a decreased phosphorylation of Tyr(P) on the native polypeptide ppl85 will indicate a higher PAcP activity in the biopsy tissue (see Example 1) .
• kits that may be used to carry out the diagnosis method of the invention are contemplated.
• the kits comprise an antibody immunologically specific to the cellular PAcP protein in a container, and instructions for performing the method to diagnose androgen-insensitive prostate carcinomas described above.
• the antibody is immunologically specific to the human cellular PAcP protein.
• the antibody is monoclonal.
• the kit comprises nucleic acids that hybridize at moderate stringency (high stringency, more preferred; very high stringency, most preferred) to the PAcP coding sequence and instructions for performing the method to diagnose androgen-insensitive prostate carcinomas described above.
• the nucleic acids hybridize at moderate stringency (high stringency, more preferred; very high stringency, most preferred) to the coding sequence of the human PAcP gene.
• the nucleic acids are PCR primers.
• the promoter region of the gene encoding human cellular PAcP Since PAcP is tissue-specifically expressed, this promoter is expected to have great utility for gene targeting, followed by tissue-specific expression in prostate epithelial cells of protein therapeutic agents for treatment of prostate carcinoma. Targeted expression of such molecules eliminates side toxicities, thereby facilitating the therapeutic effect of such agents.
• PAcP promoter of the invention retains the androgen regulation of the native PAcP gene
• a PAcP promoter reporter gene such as the one illustrated in Example 3, is suitable for studying the mechanism of androgen action in prostate.
• the expression of PAcP gene in prostate epithelium is regulated by a complicated process, and a PAcP promoter reporter gene will enable the study of the regulatory mechanisms of PAcP expression at the molecular level.
• the PAcP promoter is the regulator regions of a mammalian PAcP gene.
• the PAcP promoter is the regulatory regions from the mammalian PAcP, Genbank Accession No. X74961 or U07083.
• the PAcP promoter is the -1356 to +87 nucleotide region, where nucleotide 1 is the transcription start site.
• a xenograft animal model that mimics clinical human prostate cancers in cellular PAcP expression during tumor progression. The correlation between the cellular activity of PAcP and the progression of prostate cancer was demonstrated in this animal model in Example 2.
• the present invention provides a xenograft mammal model system that hosts transformed xenograft human prostate tumor cells.
• Example 2 illustrates that surprisingly, transgenic human prostate cell lines expressing transgenes are stable in a mouse system.
• xenograft mammal systems are well known to those in the act of cell biology (see, for example, Xenotransplantation: the transplantation of organs and tissues between species, 1997, D.K.C. Cooper et al . , eds . , 2nd ed., Berlin, Springer) . While athymic mice are illustrated in Example 2, the model system can be used to advantage in any mammal, including but not limited to, rat, cat, dog, sheep, pig and rabbit. While the use of athymic animals is used here, any animal with a compromised immune system may also be used.
• Example 2 illustrates the use of the xenograft mouse model to host tumors from the LNCaP cell line
• prostate tumor cells lines may be used, such as PC-3, DU145, and cell lines derived from these.
• prostate biopsy cells may be used to seed tumors in the xenograft mammal model system.
• the model system utilizes transgenic human prostate cells to seed the tumor.
• the system utilized athymic mice to host the xenograft cells.
• the transgenic human prostate cells express the human PAcP protein.
• nucleic acids having the appropriate sequence homology with a Homo sapi ens PAcP synthetic nucleic acid molecule may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al . (1989, supra ) , using a hybridization solution comprising: 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours.
• filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
• T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
• [N+] [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T m is 57°C.
• the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
• targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.
• the stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25°C below the calculated T m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20°C below the T m of the hybrid.
• a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt' s solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42 °C, and wash in 2X SSC and 0.5% SDS at 55°C for 15 minutes.
• a high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt' s solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42 °C, and wash in IX SSC and 0.5% SDS at 65°C for 15 minutes.
• a very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt' s solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42 °C, and wash in 0. IX SSC and 0.5% SDS at 65°C for 15 minutes.
• the nucleic acids of the PAcP gene may be maintained as DNA in any convenient cloning vector.
• clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA) , which is propagated in a suitable E. coli host cell.
• PAcP protein The availability of amino acid sequence information, such as the full length sequence in Genbank Accession No. M34840 enables the preparation of a synthetic gene that can be used to synthesize the cellular PAcP protein in standard in vi vo expression systems or to make viral vectors expressing the cellular PAcP protein.
• the sequence encoding cellular PAcP from isolated native nucleic acid molecules such as Genbank Accession No. M34840 can be utilized.
• an isolated nucleic acid that encodes the amino acid sequence of the invention can be prepared by oligonucleotide synthesis.
• Codon usage tables can be used to design a synthetic sequence that encodes the protein of the invention.
• the codon usage table has been derived from the organism in which the synthetic nucleic acid will be expressed.
• the codon usage for E. coli would be used to design an expression DNA construct to produce the cellular PAcP in E. coli .
• Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant oligonucleotide may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
• HPLC high performance liquid chromatography
• PAcP may be isolated from 'appropriate species using methods well known in the art.
• Native nucleic acid sequences may be isolated by screening mammalian or other cDNA or genomic libraries with oligonucleotides preferably designed to match the Homo sapi ens coding sequence of PAcP (Genbank Accession No. M34840) .
• Oligonucleotides designed to match any of these sequences or to match regions of high homology between these sequences may also be used to screen for mammalian PAcP- encoding nucleotides.
• nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
• a neutral base such as inosine may be used.
• the strategy of oligonucleotide design is well known in the art (see also Sambrook et al . , Molecular Cloning, 1989, Cold Spring Harbor Press, Cold Spring Harbor NY) .
• PCR (polymerase chain reaction) primers may be designed by the above method to match a known coding sequence of PAcP, and these primers used to amplify the native nucleic acids from isolated mammalian cDNA or genomic DNA.
• the protein may be produced by expression in a suitable expression system.
• a DNA molecule such as a DNA encoding the amino acid sequence in Genbank Accession No. M34840, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli , or a eukaryotic cell, such as Saccharomyces cerevi siae or other yeast.
• Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell.
• Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
• the PAcP protein produced by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art.
• a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
• an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or fusion proteins such as His tags. Such methods are commonly used by skilled practitioners.
• PAcP protein can be purified from cells that express endogenous PAcP genes. Methods to purify PAcP from human cells are well known to those who study prostate cancer (see, for example, Lin et al . , 1993, Biochem Biophys Res Commun 192:413-419; Lin et al . , 1883, Biochemistry 22:1055-1062; both are incorporated by reference herein) . Purification methods may be used to isolated the PAcP protein from any cell expressing it. Cells contemplated as sources of native PAcP protein include LNCaP and PC-3 cells, among others.
• Antibodies immunologically specific to cellular PAcP protein may be prepared utilizing purified cellular PAcP protein.
• Polyclonal antibodies may be prepared according to standard methods.
• monoclonal antibodies are prepared, which immunologically specific to various epitopes of the protein.
• Monoclonal antibodies may be prepared according to general methods of K ⁇ hler and Milstein, following - 35 - standard protocols.
• Polyclonal or monoclonal antibodies that are immunologically specific to cellular PAcP protein can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they are immunologically specific or to quantify the protein.
• Human Prostatic Acid Phosphatase is a Marker for
• FBS and RPMI 1640 medium were purchased from Life Technologies, Inc.
• the steroid-reduced medium consisted of RPMI 1640 medium supplemented with 2 or 5% (v/v) heat-inactivated dialyzed FBS.
• the final concentration of testosterone was less than 4 pM (Lin et al., 1993, Arch. Biochem. Biophys. 300:384-390).
• PC-411 and PC-416 cells were subclones of PC-3 parent cells transfected with a full-length human PAcP cDNA, as described in our previous publications (Lin et al . , 1992, Cancer Res. 52, 4600-4607; Lin et al . , 1994, Differentiation 57:143-149) .
• the expression of PAcP cDNA was driven by a pCMV-neo expression vector (Lin et al . , 1992, Cancer Res. 52, 4600-4607; Lin et al . , 1994, Differentiation 57:143-149).
• PC-CMV cells were a subcloned cell line of PC-3 that was transfected with the expression vector alone (Lin et al .
• PC-411 and PC-416 cells have been characterized and shown to express a putative cellular form of exogenous PAcP (Lin et al . , 1992, Cancer Res. 52, 4600-4607; Lin et al., 1994, Differentiation 57:143-149).
• LNCaP-CMV cells were a subline of clone 81 LNCaP that was transfected with the expression vector alone.
• Cells were plated at a density of approximately 1 X 10 4 cells/cm 2 in RPMI 1640 medium containing 7% FBS and maintained in a 37°C incubator (5% CO 2 ) for 3 days (Lin et al., 1992, Cancer Res. 52, 4600-4607; Lin et al . , 1993, Arch. Biochem. Biophys. 300:384-390; both are incorporated by reference herein) .
• Fig. 1A the total cell number was counted at day 3 for PC-3 and DU145 cells and at day 4 fro LNCaP cells.
• Fig. 2A cells were treated with 10 nM DHT. Total cell numbers were counted at days 2, 4, and 7, while 3 ml/well fresh medium with or with out DHT was added to the remaining cultures at days 2 and 4. Ratios of DHT stimulation were calculated from cell numbers in wells with DHT divided by wells without DHT. The data shown are the average of duplicate wells. Similar results were observed in three sets of independent experiments .
• Protein Determination For biochemical experiments, cells were harvested by scraping, rinsing, and pelleting in 20 mM Hepes, 0.9% NaCl, pH 7.0. Cell pellets were lysed in 20 mM Hepes, pH 7.0, containing 0.5% Nonidet P-40, 0.5 mM dithiothreitol, and various protease inhibitors (Lin et al . , 1986, Mol. Cell. Biol. 6:4753-4757; Lin and Clinton, 1988, Mol. Cell. Biol. 8:5477-5485). The protein concentration in cell lysates was quantified by the Bio-Rad dye protein assay using bovine serum albumin as a standard.
• L- (+) -tartrate is a conventional inhibitor of PAcP (Lin and Clinton, 1987, Adv. Prot. Phosphatases 4:199-228).
• LNCaP cells greater than 90% of L- (+) -tartrate-sensitive AcP activity is precipitated by anti-PAcP Ab (Lin et al .
• L- (+) -tartrate-sensitive AcP activity is taken to represent PAcP activity (Lin and Clinton, 1987, Adv. Prot. Phosphatases 4:199-228; Lin et al . , 1986, Mol. Cell. Biol. 6:4753-4757).
• Protein Phosphatase Activity Assay The activity of serine/threonine protein phosphatase was determined as described previously (Cohen, 1991, Annu. Rev. Biochem. 201:389-398). Briefly, cells were homogenized in 10 mM Tris (pH 7.4) containing a mixture of various protease inhibitors, and centrifuged at 15,000 X g for 15 min at 4°C. The protein concentration of each supernatant was determined and adjusted to 1 mg/ml . Serial dilutions of the cellular lysates were incubated in buffer containing 32 P-phosphorylase a as the substrate (1.0 ⁇ g/ ⁇ l in a 30 ⁇ l total reaction volume) for 15 min at room temperature.
• the reaction was stopped by the addition of 100 ⁇ l of trichloroacetic acid (10% solution) , and the released radioactivity was determined by scintillation counting.
• the ratio of PP-1 to PP-2A activity was determined by the inclusion of okadaic acid (5 nM to inhibit only PP-2A) and calyculin-A (1 ⁇ M to inhibit both PP-1 and PP-2A) in the in vi tro dephosphorylation reaction.
• PP-2A is the activity that is inhibited by okadaic acid
• PP-1 is the activity that is sensitive to calyculin A subtracted from the activity that is blocked by okadaic acid (although PAcP could dephosphorylate Ser (P) /Thr (P) in proteins, PAcP activity is not affected by 20 nM okadaic acid (Lin and Meng, 1996, Biochem. Biophys. Res. Commun.
• the filter was incubated with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 at 24°C for 60 min, followed by rabbit polyclonal anti-human AR Ab (PharMingen, San Diego, CA) or rabbit polyclonal anti-human PAcP Ab (Lin et al . , 1994, Differentiation 57:143-149; Lin and Meng, 1996, Biochem. Biophys. Res. Commun. 226:206-213) for 2 h. After rinsing, the filter was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG Ab (Life
• RNA and approximately equal amounts of RNA per lane were stained with EtBr, visualized to ensure the quality of RNA and approximately equal amounts of RNA per lane, and then blotted to Zeta-Probe GT membranes (Bio-Rad) by standard techniques (Garcia-Arenas et al . , 1995, Mol. Cell. Endocrinol. 111:29-37; Brown, 1993 in Current Protocols in Molecular Biology (Ausubel et al . , eds), pp. 4.9.1-4.9.14, Greene/Wiley-Interscience, New York) . Filters were hybridized and washed under stringent conditions as described previously (Lin et al . , 1992, Cancer Res.
• cDNA probes were labeled with [ [ ] -32P] dCTP using random oligonucleotide-primed synthesis (Feinberg and Vogelstein, 1983, Anal. Biochem. 132:6-13) with a commercial system from Life Technologies. Both PAcP (0.29 kb) and GAPDH (0.78 kb) cDNA probes were prepared as described previously (Lin et al., 1993, Arch. Biochem. Biophys. 300:384-390; Garcia-Arenas et al . , 1995, Mol. Cell. Endocrinol.
• PSA cDNA probe (0.214 kb) was a RT-PCR product (Garcia-Arenas et al . , 1995, Mol. Cell. Endocrinol. 111:29-37) and is described under "Polymerase Chain Reaction.”
• the PAcP and PSA mRNA bands were visualized by autoradiography followed by densitometric scanning for quantitation.
• cDNA was synthesized in an RT reaction mixture with a total volume of 20 ⁇ l including PCR buffer (10 mM Tris, pH 8.3, containing 50 mM KC1), 5 mM MgCl 2 , 1 mM each of deoxynucleotides (dCTP, dGTP, dTTP, and dATP from Perkin-Elmer) , 1 unit/ ⁇ l RNase inhibitor (Boehringer Mannheim), 2.5 units/ ⁇ l Moloney murine leukemia virus RT (Life Technologies), 2.5 ⁇ M random primers (Life
• the PCR reaction mixture contained 2 mM MgCl2, a 0 . 4 mM concentration each of dCTP, dGTP, dTTP, and dATP, 2.5 units of Taq polymerase (Perkin Elmer) , and a 1 ⁇ M concentration each of specific primers in PCR buffer along with the cDNA synthesis reaction mixture (20 ⁇ l) .
• the total reaction volume was 100 ⁇ l .
• the PCR reaction was carried out in a Perkin-Elmer apparatus by denaturation at 94°C for 30 s, annealing at 54°C for 1 min, and extension at 72 °C for 2 min for 30 cycles and subsequently at 72°C for 10 min and then soaking at 4°C (Garcia-Arenas et al . , 1995, Mol. Cell. Endocrinol. 111:29-37).
• a 28-cycle amplification was performed, since preliminary results demonstrated that the PCR, under described conditions with a 28-cycle amplification, followed a linear relationship.
• primers used for AR, PAcP, PSA, and actin in PCR reactions were synthesized and prepared as described previously (Lin et al . , 1992, Cancer Res. 52, 4600-4607; Garcia-Arenas et al . , 1995, Mol. Cell. Endocrinol. 111:29-37) .
• sequences of primers A and B for AR cDNA that were specific to the ligand-binding domain were as in a previous report (Garcia-Arenas et al . , 1995, Mol. Cell.
• RT-PCR analyses demonstrate that clone 33 LNCaP cells expressed specific mRNAs of PAcP and PSA, while PC-3 and DU 145 cells did not have a detectable level of mRNAs of these two antigens (Fig. IB) .
• PC-3 cells expressed a low level of AR but not PAcP or PSA, while its growth rate was not responsive to androgen stimulation. Androgen Sensitivity of Different LNCaP Cells.
• LNCaP cells expressed PSA mRNA (Fig. 3, A and C) , and its level was up-regulated by DHT as indicated by RT-PCR and Northern blot analyses (Fig. 3A) .
• DHT RT-PCR
• Fig. 3A Northern blot analyses
• the growth rate of different LNCaP cells in steroid-reduced medium was determined to avoid serum androgen effect.
• the growth rates of clone 81 LNCaP cells increased, higher than clone 33 cells.
• the doubling time of clone 81, 51, and 33 cells was approximately 29, 48, and 110 h, respectively. Therefore, a decreased expression of PAcP correlated with an increased growth rate.
• PC-3 cells as controls, there was no significant effect on the growth rate with increasing passage levels (Lin et al., 1992, Cancer Res. 52:4600-4607; Lin et al . , 1994, Differentiation 57:143-149).
• FIG. 1, B and C A transient expression assay was used to investigate whether AR in PC-3 cells could have an androgen action.
• R1881 a synthetic androgen, reproducibly had approximately a 2-fold stimulation of the CAT activity in PC-3 cell lysate proteins.
• R1881 had approximately a 9-fold stimulation of the CAT activity in human AR cDNA-transfected PC-3 cells, as in LNCaP cells (Fig. 6A) .
• As a control R1881 did not have an effect on the CAT activity in DU 145 cells, since no AR expression was detected in those cells.
• PC-3 cells expressed a functional AR, although the level was lower.
• PC-411 and PC-416 cells that express an exogenous, cellular form of PAcP (Lin et al . , 1992, Cancer Res. 52:4600-4607; Lin et al., 1994, Differentiation 57:143-149).
• PC-416 and PC-411 cells expressed low levels of AR mRNA, as in PC-3 parent cells (Fig. IB) .
• PC-416 and PC-411 cells expressed an exogenous cellular PAcP and had a slow growth rate (Fig. 6B) as well as a decreased Tyr(P) level in cellular proteins including ppl85 and ppl50 (Fig. 6C) . Nevertheless, the Tyr(P) level of two other phosphoproteins with a molecular size of approximately 70 and 55 kDa, respectively, was also changed notably (Fig. 6C) . Furthermore, the growth of PC-411 and PC-416 cells was stimulated significantly by DHT (p ⁇ 0.05) (Fig. 6D) . Thus, in PC-416 and PC-411 cells that express an endogenous AR, the expression of an exogenous, cellular PAcP correlated with androgen stimulation of cellular growth.
• PAcP Since cellular PAcP expression correlates with androgen responsiveness of cellular growth, it is imperative to understand the possible molecular mechanism by which cellular PAcP is involved. Utilizing phosphomonoesters as substrates, PAcP has classically been classified as a "histidine” acid phosphatase without known function (Lin and Clinton, 1987, Adv. Prot. Phosphatases 4:199-228). Recently, several lines of evidence support the notion that the cellular form of
• PAcP could indeed function as a neutral "cysteine" PTPase in prostate epithelial cells (Young et al., 1991, Cancer Res. 51:3748-3752; Lin and Clinton, 1988, Mol. Cell. Biol. 8:5477-5485; Lin and Meng, 1996, Biochem. Biophys. Res. Commun. 226:206-213; Schneider et al . , 1993, EMBO J. 122:2609-2614; Ostanin et al . , 1994, J. Biol. Chem. 269:8971-8978).
• LNCaP cells including clone-33 (i.e., passages 20-30), -51 (passages 45-60), and -81 (passages 85-120) were described previously (Lin et al., 1998, J. Biol. Chem. 273:5939-5947).
• Clone-33 LNCaP cells represented the LNCaP parental cells, and expressed a high level of endogenous PAcP (Lin et al . , 1998, J. Biol. Chem. 273:5939-5947) .
• the expression of PAcP was decreased in clone-51 cells, and further diminished in clone-81 cells.
• LNCaP-23, -28, -34, and -40 cells were subcloned cells from clone-81 LNCaP cells that transfected with a full length human PAcP cDNA driven by a pCMV-neo expression vector followed by G418 selection (Lin et al . , 1998, J. Biol. Chem. 273:5939-5947). These PAcP cDNA transfected LNCaP cells expressed a low level of endogenous cellular PAcP as well as an exogenous cellular PAcP (Lin et al . , 1998, J. Biol. Chem. 273:5939- 5947) .
• LNCaP-CMV cell line was a subline of clone-81 LNCaP transfected with the vector alone, and expressed a low level of endogenous PAcP (Lin et al . , 1998, J. Biol. Chem. 273:5939-5947).
• PC-411, -412 and -416 cells were subclones of PC-3 cells transfected with the same PAcP cDNA expression vector, and express an exogenous, cellular form of PAcP (Lin et al . , 1992 Cancer Res., 52:4600-4607; 15).
• PC-18 cells Another subclone of PAcP cDNA transfectant, express only the secretory form of exogenous PAcP which has no growth effect (Lin et al . , 1992 Cancer Res., 52:4600-4607).
• PC-CMV cells were a subline of PC-3 cells transfected with the pCMV-neo vector alone, and did not express PAcP (Lin et al . , 1992 Cancer Res., 52:4600-4607). All cells were maintained in RPMI-1640 medium supplemented with 5% FBS, 1% glutamine, and 0.5% Gentamicin (Lin et al . , 1992 Cancer Res., 52:4600-4607; Lin et al . , 1998, J.
• Immunoprecipitation and Immunoblotting For immunoprecipitation and blotting, subconfluent cells were harvested, pelleted and rinsed with ice-cold 20 mM Hepes- buffered saline, pH 7.0, then lysed in ice-cold lysis buffer containing a battery of protease and phosphatase inhibitors (Lin et al., 1998, J. Biol. Chem. 273:5939- 5947; Meng and Lin, 1998, J. Biol. Chem., 273:22096- 22104). After being spun at 2,500 x g for 10 min at 4°C, the supernatants were quantified for protein amount using the Bio-Rad protein assay kit.
• Anchorage-independent growth Anchorage-independent growth of prostate cancer cells was performed by soft agar analysis (Weiner et al . , 1989, Oncogene 4:1175-1183; Horoszewicz et al . , 1983, Cancer Res. 43:1809-1818). Briefly, cells were seeded in 0.25% agarose on the top of a base layer containing 0.6% agarose. One day after seeding, cell clumps containing more than one cell were excluded and the colony number was counted after 3 weeks of incubation.
• Xenograft animal experiments The protocol for the subcutaneous xenograft animal model in athymic mice was essentially as that described in Lin et al., 1995 (The Prostate 26:194-204).
• 1 x 10 6 cells were suspended in 0.1 ml medium, mixed with 0.1 ml matrigel (Collaborative Biomedical, MA), and injected subcutaneously in the hind flank of male mice.
• 5 x 10 5 cells in matrigel were injected into female mice.
• 1 x 10 6 cells in 0.2 ml medium suspension without matrigel were injected into male mice.
• Sections and cells were fixed in methanol, rinsed briefly with PBS, rehydrated in PBS, and then treated with 1% sodium borohydride to block endogenous autofluorescence, followed by rinsing with PBS. After being permeabilized with Triton X-100, both cells and tumor sections were treated with rabbit anti- PAcP serum or preimmune rabbit serum after blocking nonspecific binding sites (Lin et al . , 1995, The Prostate 26:194-204). Cells were then incubated with fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG. The intensity and the subcellular localization of PAcP were visualized by epifluorescence microscopy and photographed with a same exposure time.
• the cellular level of PAcP in several human prostate cancer cell lines with different growth rates (the growth rate: DU145 ⁇ PC-3>clone-81>clone-51>clone-33 LNCaP cells) (Lin et al . , 1992 Cancer Res., 52:4600-4607; Lin et al . , 1998, J. Biol. Chem. 273:5939-5947) were analyzed initially. Western blotting clearly showed that the intracellular level of PAcP inversely correlates with the proliferation rate of these cells (Fig. 8A) .
• the PAcP enzyme activity corresponded to its protein level (Lin et al . , 1992 Cancer Res., 52:4600-4607; Lin et al .
• SHP-1 SHP-1 protein varies inconsistently with the rate of cell proliferation (Fig. 8A) .
• SHP-1 activity may be regulated by other mechanisms, only the level of cellular PAcP activity inversely correlates with the growth rate of these prostate cancer cells, suggesting a direct role of cellular PAcP involving in the control of proliferation rates in these cells.
• clone-81 cells In clone-81 cells, the cellular PAcP level was low, while the tyrosine phosphorylation of c-ErbB-2/neu was high (Fig. 8B) .
• cDNA transfection in clone-81 cells i.e., LNCaP-23, 28, 34, and 40 cells
• elevated PAcP expression was coincident with decreased p-Tyr of c-ErbB-2/neu (Fig. 8B) .
• PAcP cDNA-transfected PC-3 cells PC-411, -412, and -416 cells
• the ectopic expression of an exogenous cellular PAcP resulted in decreased tyrosine phosphorylation of the endogenous c- ErbB-2/neu (Fig. 8C)
• the PAcP activity decreased (Lin et al . , 1992 Cancer Res., 52:4600-4607; Lin et al . , 1994, Differentiation, 57:143-149) and the tyrosine phosphorylation of c-ErbB-2/neu was restored, as in clone-81 LNCaP cells (Fig. 8B) .
• PAcP acts as a dual-specificity protein phosphatase biochemically (Lin and Clinton, 1987, Advances in Protein Phosphatases 4:199-228; Lin and Clinton, 1986, Biochem. J., 235 : 351-357), cells were metabolically labeled with 32 Pi, and 32 P-phosphoproteins were resolved by SDS-gel electrophoresis (Lin and Meng, 1996, Biochem.
• anchorage- independent growth was dramatically diminished by more than 100 folds in cells that express an elevated level of cellular PAcP by a cDNA expression vector in LNCaP cells, including LNCaP-23, -28, -40 cells (Fig. 9B) .
• ectopic expression of cellular PAcP in PC-416 cells concurred with a diminished anchorage-independent growth (Fig. 9C) .
• the anchorage-independent growth of PC-18 cells was not significantly altered by the expression of the secretory form of PAcP protein which has no effect on c-ErbB-2/neu phosphorylation (Figs. ID & 2C) .
• LNCaP cells that express higher cellular PAcP (clone-33 cells, and LNCaP-23 and -34 cells) developed measurable xenograft tumors more slowly than cells that express lower level of the enzyme (clone-81 cells, Fig. 10A; and LNCaP-CMV cells) .
• PAcP-expressing cells developed a smaller tumor than PAcP-lacking cells. For example, 28 days after inoculation, the size of tumors that lack cellular PAcP expression was about twice of the tumors that express cellular PAcP. The averaged tumor size was 241, 93, 143, and 115 mm 3 for C-81 LNCaP, C-33 LNCaP, LN-23, and LN-34 cells, respectively.
• the delayed development of xenograft tumors by PAcP-expressing cells is at least in part caused directly by the expression of cellular PAcP, which down-regulates their growth rates by dephosphorylating c-ErbB-2/neu in xenograft animals.
• cellular PAcP protein is decreased (Fig. 11), similar to the observation in human prostate archival specimens (Sakai et al . , 1993, J. Urol. 149:1020-1023; Sinha et al . , 1998, The Prostate, 13 : 1-15) .
• cellular PAcP is higher in carcinomas of early stages than that of late stages, i.e., the more aggressive disease is associated with the higher histological grade and lower cellular PAcP levels (Sakai et al . , 1993, J. Urol. 149:1020-1023; Sinha et al., 1998, The Prostate, 13 : 1-15).
• Cell culture medium fetal bovine serum (FBS) , gentamicin and Lipofectin reagent were obtained from Life Technologies, Inc.
• the MasterAmp PCR Optimization kit was from Epicentre Technologies Corp.
• Zero Blunt PCR cloning kit, and pCR-Blunt vector were obtained from Invitrogen Corp.
• pCATBasic, pCATEnchancer, pCATPromoter, pSV-, 6-galactosidase vectors and CAT assay kit were purchased from Promega Corp. DNA manipulations of plasmids were performed by conventional molecular biology techniques (Sambrook et al . , 1989 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.) .
• LNCaP cells were routinely maintained in RPMI1640 medium supplemented with 5% FBS, 1% glutamine, and 0.5% gentamicin.
• a steroid-reduced medium i.e., phenol red-free RPMI-1640 medium containing 5% heat-inactivated steroid-reduced FBS (SR-FBS) (Lin et al . , 1993, Arch. Biochem. Biophys. 300:384-390; Lin et al . , 1993, Cell. Mol. Biol. Res. 39:739-750) .
• SR-FBS heat-inactivated steroid-reduced FBS
• Cultured LNCaP cells were transfected with a pSV-, 8-galactosidase vector, containing, 6-galactosidase gene driven by a SV 40 promoter. After 24 h cells were rinsed twice with phosphate-buffered saline (PBS), pH 7.3, and fixed for 5 min in 2% formaldehyde plus 0.2% glutaraldehyde in PBS.
• PBS phosphate-buffered saline
• the cells were washed with PBS, overlaid with 1 ml per well of histochemical reaction mixture, containing 1 mg/ml 4-Cl-5-Br-3-indolyl-, 6-galactosidase (X-gal), and incubated at 37 °C for 18 hours to obtain visible staining (Sanes et al . , 1986, EMBO J 5:3133-3142).
• Northern blot Multiple tissue membrane was purchased from Clontech Laboratories, Inc. Northern blot hybridization was performed as described previously (Lin et al., 1993, Arch. Biochem. Biophys. 300:384-390; Lin et al., 1993, Cell. Mol. Biol. Res. 39:739-750).
• Nuclear run-on experiment Nuclear extracts and run-on assay was carried out essentially as described (Ausubel et al . ,1993, Current Protocols in Molecular Biology, Wiley, New York; Linial et al.,1985, Science 230:1126-1132). Briefly, nuclear extracts were prepared from LNCaP cells grown in the presence or absence of 5a- dihydrotestosterone (DHT) in a steroid-reduced medium as described (Lin et al . , 1993, Arch. Biochem. Biophys. 300:384-390; Lin et al . , 1993, Cell. Mol. Biol. Res. 39:739-750). Transcription was continued in the presence of "P-labeled UTP (Amersham. Life Science Inc.). The radioactive RNA was hybridized with a slot blot membrane containing the full length of PAcP cDNA.
• DHT dihydrotestosterone
• the promoter fragment of PAcP gene was obtained by a polymerase chain reaction (PCR) amplification using genomic DNA isolated from LNCaP cells as the template. PCR reaction was conducted in a volume of 100 ⁇ l in the presence of Pfu DNA polymerase (Stratagene) , and Buffer F from the MasterAmp PCR Optimization kit utilizing a Perkin-Elmer GeneAmp PCR System 2400 (Perkin-Elmer) . Two oligonucleotide primers were utilized: 5'TTG TAG GTT TGG GCT TTT TGC 3' (SEQ ID NO: 9) and TATT CTT AAT CTG TTG GGA GTC 3' (SEQ ID NO: 10).
• PCR polymerase chain reaction
• PCR mixture was first denatured by heating at 95°C for 5 min. The amplification was performed for 30 cycles using following conditions: 30 sec at 94°C, 1 min at 64.7°C, 1 min 30 sec at 72°C. A DNA fragment of 1.4 kb was obtained and cloned into the pCR-Blunt vector. The obtained DNA insert was sequenced and compared with reported sequences to ensure the accuracy of PCR product (Virkkunen et al . , 1994, Biochem. Biophys. Res. Commun. 202:49-57; Banas et al . , 1994, Biochim. Biophys. Acta 1217:188-194; Sharief and Li, 1994, Biochem. Mol. Biol. Int. 33:561-565).
• Plasmid constructs To assess the promoter activity, a Hindlll/ Xbal fragment of PAcP promoter from pCR-Blunt vector was cloned into pCATBasic and pCATEnchancer plasmids. Resulting plasmids, pCATPAP and pCATEPAP, contained a 1.4 kb promoter fragment of the PAcP gene covering the region -1356 to +87 in the sense orientation. A plasmid pCATasPAP containing the same 1.4 kb fragment in the antisense orientation was constructed as a control.
• LNCaP cells were routinely plated 2.5 X 10 5 cells per well in a 6-well plate in RPMI 1640 medium containing 5% FBS. To examine steroid effect on the promoter activity, cells were plated in a steroid-reduced medium. Five ⁇ g plasmid DNA were introduced into LNCaP cells by complexing with the Lipofectin reagent as described previously (Lin et al., 1992, Cancer Res,
• Quantitative CAT assays were performed with the same amount of total cell lysates in a reaction volume of 125 ⁇ l in the presence of 14 C-chloramphenicol (Amersham Life Science Inc.) as described in the Promega CAT-assay manual accompanying with the assay kit. Samples were incubated overnight followed by a single extraction with 300 ⁇ l xylene. The 250 ⁇ l organic phase was transferred to scintillation vials containing 2 ml EdoLume scintillation fluid (ICN Corp.) and counted by Beckman LS 1801 scintillation counter. All experiments were repeated four times in triplicates.
• the -1356/+87 fragment of the 5 '-flanking region could drive the expression of CAT activity which was approximately 2.5-fold higher compared to the vector containing the same DNA fragment in the antisense orientation (Fig. 12) .
• Approximately 3-fold induction of activity was observed when LNCaP cells were transfected with a vector containing the -1356/+87 PAcP gene fragment and a SV40 enhancer.
• Transfection with a pCATPromoter vector containing the CAT gene driven by a SV40 promoter resulted in an approximately 5.5-fold activity of the control vector. This 1.4 kb of 5' flanking region therefore contains the basic promoter activity although the activity is low.
• PAcP has been reported to be a useful marker of differentiated prostate epithelium cells and may play an important role in regulating the growth of those cells (Lin et al . , 1993, Arch. Biochem. Biophys. 300:384-390; Lin et al . , 1992, Cancer Res, 52:4600-4607; Lin et al . , 1994, Differentiation 57:143-149).
• tissue-specific expression in normal prostate cells has not been investigated at molecular level.
• a tissue-specific expression of PAcP in normal prostate epithelium by Northern blot analyses at the mRNA level.
• LNCaP cells also exhibit a low transcriptional activity which is indicated by a very slow growth rate (Lin et al . , 1992, Cancer Res, 52:4600- 4607) .
• This hypothesis is supported by the observations that a SV40 promoter displays a low level of ⁇ -galactosidase and CAT activity (Fig. 12).
• the low transcriptional activity of the PAcP promoter is also demonstrated by the weak hybridization bands in nuclear run-on experiments. The low promoter activity could be due to a low amount of basic transcription factors essential for the promoter activity in those cells.
• the cloned promoter part of PAcP gene lacks a specific enhancer element.
• the data taken together provide us with an explanation regarding the low PAcP promoter activity in LNCaP cells, consistent with a recent report (Shan et al . , 1997, Endocrinology 138:3764-3770).
• the promoter activity is increased even under non-permissive conditions of cell growth (Fig. 13), indicating that the transcriptional factors for PAcP expression are actively functioning despite suppression of the growth machinery.
• the results are consistent with previous observations that cellular PAcP activity as well as its protein level is inversely correlated with the growth rate of LNCaP cells (Lin et al., 1993, Arch. Biochem.

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