Carboxypeptidase A3 (CPA3): A Novel Gene Highly Induced by Histone Deacetylase Inhibitors during Differentiation of Prostate Epithelial Cancer Cells¹

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths in men in the United States. Within the prostate, androgens are capable of stimulating proliferation, as well as inhibiting the rate of epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate epithelium and androgen-dependent prostate cancer cells. Currently, the ablation of testosterone remains the most effective systemic therapy of advanced carcinoma of the prostate and is believed to induce apoptosis in these tumors (1). However, tumor cells that were formerly sensitive to androgen ablation therapy almost always emerge as androgen-independent tumors after 1–3 years of treatment (2). The development of resistance to androgen ablation therapy acquired by prostate tumor cells remains a severe obstacle to the effective treatment of metastatic prostate cancer (3, 4). Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway on androgen ablation; however, they do retain the cellular machinery necessary to activate the differentiation and programmed cell death cascade when these cellular processes are sufficiently initiated by injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones or growth/survival factors). Therefore, differentiation therapy or apoptosis therapy has been proposed as a method for the treatment of advanced prostate cancer (5, 6, 7).

Butyrate, a four-carbon short-chain fatty acid, is naturally produced by bacterial fiber fermentation within the colon and is found in the plasma of mammals (8). The biological significance of this compound is its ability to regulate cell growth and differentiation. Several studies have established that this agent has the ability to induce in vitro differentiation of prostate cancer, breast cancer, pancreatic cancer, and hematopoietic cells (9, 10, 11, 12). The concentrations of butyrate that cause growth inhibition in vitro are similar to those measured within the mammalian colon (13), and it has been found to inhibit the growth of colon carcinoma cells in vivo (14). Although the molecular mechanisms by which butyrate mediates its effects are not well understood, it is known to induce a variety of changes within the nucleus, including histone hyperacetylation and the changes are secondary to hyper- or hypo-methylation of DNA (15, 16). Histone acetylation has been shown to have a permissive effect on mRNA transcription (17), possibly by relaxing specific segments of tightly coiled DNA and thereby facilitating the binding of transcription factors to selectively activate expression of genes (18, 19). Numerous epidemiological and experimental studies have found an association between a high-fiber diet and a decreased incidence and growth of colon cancer (20). The molecular link between a high-fiber diet and the arrest of colon carcinogenesis has been investigated, and it has been established that butyrate mediates growth inhibition of colon cancer cells by inducing p21^WAF1/CIP1 expression through histone hyperacetylation (21). Recently, the product of certain oncogenes was shown to suppress transcription of their target genes by recruiting histone deacetylase (22, 23, 24), which cleaves acetyl groups from histones and blocks their ability to induce DNA conformational changes. This transcriptional block can be overcome by agents that inhibited histone deacetylase, and, clinically, transcription targeting therapy for leukemia has been achieved by using butyrate to inhibit histone deacetylases to relieve the transcriptional repression caused by certain oncogenes (25).

Butyrate and its structural analogues recently entered clinical trials for prostate cancer at the National Cancer Institute (26). In the present study, we sought to investigate the molecular mechanisms by which butyrate mediates growth arrest and differentiation of androgen-independent prostate cancer cells and to identify potential markers for the diagnosis and treatment of androgen-independent prostate cancer. We first treated the androgen-independent prostate cancer cell line PC-3 with NaBu³ and confirmed that they underwent typical differentiation and apoptosis. We then used the DD-PCR technique to identify a new gene, designated CPA3, which was up-regulated in PC-3 cells induced by NaBu. A homology search indicated that CPA3 belongs to the CP family that includes prostate-specific membrane antigen. We also demonstrate that the induction of CPA3 is due to the histone hyperacetylation pathway.

The prostate cancer cell lines PC-3, DU145, and LNCaP and the pancreatic cancer cell lines AsPC-1, BXPC-3, Capan-1, HS.766, PANC-1, and SU.86 were purchased from the American Type Culture Collection (Manassas, VA). Dr. Donald J. Tindall (Department of Urologic Research, Mayo Foundation, Rochester, MN) kindly provided the BPH1 benign prostatic hyperplasia cell line. All cells were maintained at 37°C as monolayers in a humidified atmosphere containing 5% CO₂. Cells were passaged at confluence by trypsinization. Tissue culture medium for each cell line was as follows. PC-3, DU145 and LNCaP were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin; BPH1 was cultured in RPMI 1640 containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Chemical treatments were performed on cells that were 60–80% confluent. Cells were treated with various concentrations of NaBu or TSA as indicated. Some cells were pretreated with actinomycin D (4 μm) or concomitantly treated with CHX (10 μg/ml). All these chemicals mentioned were obtained from Sigma Chemical Co. (St. Louis, MO).

To determine whether DNA fragmentation occurred after treatment with NaBu, all cells were collected and the DNA was extracted by the method described by Borner et al. (27). Attached cells were detached from the culture dishes with 5 mm EDTA, pooled with detached cells, spun down, and lysed in 5 mm Tris (pH 7.4), 5 mm EDTA, and 0.5% Triton X-100 for 2 h on ice. The lysate was centrifuged at 27,000 × g for 20 min. The supernatant was incubated with 200 μg/ml proteinase K for 1 h at 50°C and extracted with phenol/chloroform; then the DNA was precipitated overnight at −20°C in 2 volumes of ethanol and 0.13 M NaCl with 20 μg glycogen. Nucleic acids were treated with 1 mg/ml boiled bovine pancreatic RNase A for 1 h at 50°C, then the DNA was loaded onto a 2% (w/v) agarose gel containing 0.3 μg/ml ethidium bromide, and run in 1 x TBE buffer at 2.5 V/cm.

The DD-PCR technique was used to identify genes up-regulated or down-regulated by butyrate treatment (28, 29). Total RNA was extracted from PC-3 cells exposed to NaBu (10 mm) for different periods of time using Trizol (Life Technologies, Inc.) and treated with RNase-free DNase I (Life Technologies, Inc.) to eliminate genomic DNA contamination. mRNA DD-PCR was performed using the RNAimage Kit (GenHunter, Corp., Nashville, TN). After isolation of potentially interesting cDNA fragments from the differential display gel, each fragment was reamplified and cloned into the pGEM-T vector (Promega, Madison, WI). Cloned cDNAs were then sequenced, followed by database analysis, Northern blot hybridization, and RACE.

To obtain the full-length cDNA sequence of CPA3, the 5′-RACE procedure was used. cDNA was synthesized from poly(A)⁺ RNA isolated from NaBu-treated PC-3 cells. Adaptor ligation and PCR were performed using the Marathon cDNA Amplification Kit following the manufacturer’s recommendations (Clontech, Palo Alto, CA).

Total cellular RNA (10–15 μg) was applied to and run on 1.2% denaturing formaldehyde-agarose gels and transferred onto positively charged nylon membranes. Filters were hybridized first with [³²P]dCTP-labeled target cDNA (CPA3, p21, and Actin) and, after stripping, rehybridized with [³²P]dCTP-labeled GAPDH and 18S rRNA as controls. Human multiple tissue Northern blots were purchased from Clontech (numbers 7759–1 and 7760–1). According to the manufacturer’s information, each lane was loaded with ∼2 μg of poly(A)⁺ RNA prepared from different normal human tissues. The premade blots were hybridized with the CPA3 full-length cDNA probe and, after stripping, rehybridized with a human actin cDNA probe as a control.

On the basis of the p21^WAF1/CIP1 cDNA sequence (GenBank number U03106), two primers were designed to amplify a 530-bp fragment of p21^WAF1/CIP1 (from base 66–595). A BamHI (Promega) cut site (underlined) was inserted into the forward primer AGGAGGATCCATGTCAGAA, and a HindIII (Promega) cut site (underlined) was inserted into the reverse primer GGACTGCAAGCTTCCTGTGG. The PCR product was digested with both BamHI and HindIII, and a 517-bp cDNA fragment from the 5′ end of the p21^WAF1/CIP1 coding region that includes the start ATG codon was isolated, then subcloned into the cloning sites of the mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA) and transformed into Escherichia coli DH5α (Life Technologies, Inc.). Mini-preparations of ampicillin-resistant clones were sequenced and analyzed for the orientation of the inserts. Transient transfections were accomplished by using the LipofectAmine Plus system (Life Technologies, Inc.). To examine the effects of p21^WAF1/CIP1 transactivation on CPA3 mRNA induction by NaBu, PC-3 cells were transfected with a p21^WAF1/CIP1 antisense expression plasmid before treatment with NaBu. For controls, cells were similarly transfected with pcDNA3.1(+). Exponentially growing cells in 100-mm culture dishes were washed with serum-free medium, then a mixture of 5 μg of plasmid, 30 μl of LipofectAmine, and 20 μl of Plus was added. After a 3-h incubation, we added complete medium with serum, and then the cells were incubated at 37°C; 24 h after the start of transfection, cells were treated with or without 10 mm NaBu for 12 h, cells were harvested, and total RNA was isolated. Northern blot analysis was performed using p21^WAF1/CIP1 and CPA3 cDNAs as probes.

The androgen-independent prostate cancer cell PC-3 began to show dramatic morphology changes after exposure to 10 mm NaBu for 24 h. The treated cells demonstrated a differentiation phenotype of cellular spreading and flattening and extension of pseudopodia (data not shown). Simultaneously, DNA fragmentation indicative of apoptosis was detected by agarose gel electrophoresis analysis (Fig. 1,A). DD-PCR was used to isolate cDNAs up-regulated and down-regulated after NaBu treatment of the PC-3 cells. In the course of this work, one 330-bp fragment was identified using DD-PCR, which was induced by NaBu treatment (Fig. 1,B). As is shown in Fig. 1,B, expression of the fragment corresponding to CPA3 was very highly induced by the treatment with NaBu (more so than any of the other fragments detected by DD-PCR). Therefore, we selected this fragment for further characterization. The differential expression of CPA3 between parental and differentiated cells was further confirmed by Northern blot hybridization using the 330-bp fragment as a probe (Fig. 1,C). The 330-bp fragment hybridized to a 3.0-kb message on these Northern blots. Sequence analysis and database searching revealed that this cDNA belonged to a novel gene. To clone the full length of this gene, we isolated poly(A)⁺ mRNA from the PC-3 cells after they were treated with NaBu for 2 days. cDNA was synthesized for 5′-RACE. From RACE experiments, we obtained an additional 2.5-kb cDNA fragment for this gene. These two clones collectively define a 2795-bp full-length cDNA sequence. The nucleotide sequence and the corresponding amino acid sequence of the protein are shown in Fig. 2. The analyzed full-length cDNA contained a very short 5′-UTR, a large 3′-untranslated region, a poly(A) tail, and an open reading frame of 1266 bp capable of encoding a 421-amino acid protein. Computer-based analysis predicted that the putative protein encoded by this gene contained two zinc-binding signature domains of metallocarboxypeptidases (Fig. 2). Similar to some of the other CP mRNAs (30), the 5′ UTR is only several bp in length (7 bp). However, distinct from other CP mRNAs, the 3′ UTR of CPA3 mRNA is quite long and has a 1.5-kb segment containing the consensus polyadenylation signal sequence AATAAA, located 17 nucleotides upstream from the poly(A) tail. By using TMAP (software for the identification of transmembrane segments on a protein sequence; EMBL-Heidelberg) analysis, we found a 16-amino acid signal peptide present at the NH₂ terminus of the putative protein encoded by this cDNA (Fig. 2).

Computer-assisted homology comparison revealed that although the CPA3 cDNA sequence shares very low homology with other genes in the database (data not shown), the CPA3 amino acid sequence shares significant homology with metallocarboxypeptidases in different species from human to E. coli (Table 1). The overall homology of the putative CPA3 protein is highest to human and rat CPA2, with 63% and 61% amino acid identity, respectively. CPA3 also shows very high amino acid similarity with bovine CPA, human CPA1, and rat CPA1. Both CPA1 and CPA2 exist as procarboxypeptidases with a structure containing an NH₂-terminal signal peptide, a COOH-terminal CP domain, and an activation segment in the middle. As shown in Fig. 3, CPA3 shares significant structural similarity with CPA2 and CPA1. Residues known to be involved in Zn²⁺ binding (His⁶⁹, Glu⁷², and His¹⁹⁶, using the CPA1 numbering system), substrate anchoring and positioning (Arg⁷¹, Arg¹²⁷, Asn¹⁴⁴, Arg¹⁴⁵, and Tyr²⁴⁸), and catalysis (Arg¹²⁷ and Glu²⁷⁰) are present in comparable positions in CPA3 in its COOH-terminal enzyme domain (Figs. 2 and 3). On the basis of this data, we named this gene CPA3.

We analyzed expression of CPA3 mRNA in 16 human normal tissues by using Clontech’s Multiple Tissue Northern blots #1 and #2. However, we did not detect any Northern hybridization signal in tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood lymphocytes. However, we did detect very strong hybridization signals on the blots made from prostate cell lines in the same hybridization experiment (Fig. 4). Hybridization with an actin cDNA demonstrated that the quality of the mRNA on the commercial multiple tissue Northern blots was good. We, therefore, analyzed the expression status of CPA3 in different human tissues using RT-PCR analysis. Using RT-PCR, we did observe low expression of CPA3 in some of aforementioned tissues, including normal prostate, as compared with the expression of GAPDH (data not shown). There are very few ESTs homologous to CPA3 cDNA in the database, and all these ESTs were obtained from normalized cDNA libraries made from fetal brain, melanocyte, and pregnant uterus, respectively. Thus, it seems that CPA3 mRNA could be an extremely rare transcript in normal human cells. Because the two homologous CPs CPA1 and CPA2 are of pancreatic origin, we also analyzed expression of CPA3 mRNA in pancreatic cells. In normal pancreatic tissues, no expression of CPA3 mRNA was observed by Northern hybridization, although low expression was detected by RT-PCR analysis (data not shown). Similar very low expression levels of CPA3 were observed in seven pancreatic cancer cell lines (data not shown).

Previous data indicated that CPA3 mRNA was preferentially expressed in specific types of tissues or cells. We then sought to establish whether CPA3 mRNA was inducible by NaBu in other prostate cell lines. CPA3 induction by NaBu was found to be dosage-dependent in PC-3 cells (Fig. 5,A). It is considered that the half-life of NaBu is quite short, and no cytotoxic effect was found after cells were treated with 20 mm NaBu. Therefore, we selected to use 10 mm NaBu to treat PC-3, DU145, LNCaP, and BPH1 cells for the time-course study. In PC-3 cells, CPA3 can be induced as early as 3 h after NaBu treatment and was highly induced from 12 h to 48 h (Fig. 5,B). In DU145 cells, CPA3 expression was slightly higher after 6 h of NaBu treatment and extremely high after 48 h of treatment (Fig. 5,C). Induction of CPA3 mRNA by NaBu was detected in the BPH1 cell line, although the induction pattern of this gene was slightly different from PC-3 and DU145 (Fig. 5 D). CPA3 could not be induced in the androgen-sensitive cell line LNCaP (data not shown). We also tested the inducibility of CPA3 in several pancreatic cancer cell lines (see “Materials and Methods”). No induction of CPA3 mRNA by NaBu was detected in any of the seven tested cell lines (data not shown).

Because butyrate is known to be a histone deacetylase inhibitor, we wonder if another histone hyperacetylating agent could induce the CPA3 gene. TSA is a potent and specific inhibitor of histone deacetylase and, like butyrate, has been shown to cause G₁ cell cycle arrest and differentiation of various cell types (31, 32). We found that in PC-3 cells, TSA induces CPA3 expression in a dosage-dependent manner, with maximal effects occurring at 0.6 μm and 1.2 μm (Fig. 6,A). Similar to NaBu, the effects of TSA on CPA3 occur as early at 3 h, but distinct from NaBu, the expression level of CPA3 comes down by 48 h of TSA treatment (Fig. 6,B). To investigate whether CPA3 induction by either NaBu or TSA was due to an increase in the rate of CPA3 mRNA synthesis or an enhancement of its stability, the transcriptional inhibitor ActD was used. Pretreatment with 4 μm ActD for 30 min completely abolished CPA3 induction by NaBu and TSA (Fig. 6, C and D), thus, indicating that induction of CPA3 is dependent on transcriptional activity of cells. Because induction of CPA3 occurs at 3 h by treatment of NaBu and TSA (Figs. 5,B and 6,B), we tested whether CPA3 was induced by an early drug response mechanism. As shown in Fig. 6 E, the induction of CPA3 was inhibited by simultaneous treatment with CHX, indicating that other proteins mediate CPA3 induction by NaBu or TSA.

Induction of p21^WAF1/CIP1 by NaBu and TSA in the colorectal cancer cell line HT-29 cannot be blocked by the protein synthesis inhibitor CHX. Instead, the mRNA expression level of p21^WAF1/CIP1 increases slightly, indicating that p21^WAF1/CIP1 was induced by these chemicals as an immediate-early gene (21). We obtained the same result when the PC-3 cell line was simultaneously treated with NaBu or TSA and CHX (data not shown). This result suggests that p21^WAF1/CIP1 also acts as an immediate-early gene in the in vitro differentiation of PC-3 cells due to treatment with NaBu or TSA. Because the induction of CPA3 was mediated by a late response mechanism, we investigated whether there was any effect of p21^WAF1/CIP1 transactivation on CPA3 induction by NaBu. An antisense p21^WAF1/CIP1 expression vector was transiently transfected into PC-3 cells, and the cells were treated with NaBu. As is shown in Fig. 7, expression of antisense p21^WAF1/CIP1 not only completely blocked expression of p21^WAF1/CIP1, but also almost completely inhibited induction of CPA3 by NaBu. For a control, PC-3 cells were transfected with only the expression vector pcDNA3.1. Similar to the PC-3 cells, both p21^WAF1/CIP1 and CPA3 can be highly induced in these cells by NaBu treatment (Fig. 7). Therefore, induction of CPA3 by NaBu requires the transcription activity of p21^WAF1/CIP1.

In this study, we describe the isolation and characterization of a novel CP, CPA3, that shares significant homology with the CPA subfamily of metalloprecarboxypeptidases. CPA3 was highly induced by NaBu treatment of several prostate cancer cell lines, and it was also induced by TSA, which is a potent and specific inhibitor of histone deacetylase. We also demonstrated that the specific induction of CPA3 by NaBu was specifically inhibited by antisense p21^WAF1.

GenBank searches indicated that CPA3 shares significant homology to the members of the CPA and CPB subfamily of metallocarboxypeptidases. As shown in Table 1, CPA3 not only has 37–63% amino acid identity with other zinc CPs from different mammalian species, but also shares 27–43% of amino acid similarity with zinc CPs from a number of nonmammalian species. CPA3 shows the highest homology with CPA1 and CPA2, indicating that it is a new member of the CPA subfamily. CPA1 and CPA2 are also known as pancreatic CPs, the digestive enzymes involved in the hydrolysis of alimentary proteins, which are expressed and secreted into the pancreatic juices. We, therefore, examined the expression of CPA3, but did not find expression of CPA3 in either normal pancreas or pancreatic tumor cells by Northern analysis. We previously showed that several pancreatic cancer cell lines also undergo differentiation and apoptosis on exposure to NaBu (11), but, in this study, we did not detect any induction of CPA3 in seven pancreatic cancer cell lines after exposure to NaBu for more than 2 days. We detected extremely low levels of CPA3 expression in normal prostate tissue by RT-PCR analysis. However, expression of CPA3 was easily detectable in untreated prostate cancer cell lines by Northern hybridization analysis (Fig. 4), and CPA3 mRNA was highly induced by NaBu in these cancer cell lines (Fig. 5). These data indicate that CPA3 is a nondigestive pancreatic-like CPA.

Although, generally, similarities in primary structure between digestive and nondigestive CPs are quite low (33), sequence alignments clearly show that key catalytic residues are common to these enzymes, His⁶⁹, Glu⁷², and His¹⁹⁶ (using the CPA1 numbering system) for the Zn²⁺ binding; Arg⁷¹, Arg¹²⁷, Asn¹⁴⁴, Arg¹⁴⁵, and Tyr²⁴⁸ for substrate anchoring and positioning; and Arg¹²⁷ and Glu²⁷⁰ for catalytic activity (30, 33, 34). All of the residues essential for the coordination of the Zn²⁺ active site, substrate peptide anchoring, and CP activity are also preserved in comparable positions in the putative CPA3 protein (Figs. 2 and 3). The NH₂-terminal domains of most metallocarboxypeptidases contain a signal peptide critical for the proper targeting of the protein. Motif analysis predicts that CPA3 contains an NH₂-terminal sequence of 16 amino acids that resembles the signal peptide consensus sequence (35) and, thus, CPA3 is similar to other CP family members. Further analysis shows that similar to other metallocarboxypeptidases (including CPA1, CPA2, CPB, CPA-MC, pCPB, and CPE), CPA3 contains a pro-peptide between the NH₂-terminal signal peptide sequence and COOH-terminal CP moiety (Fig. 3). Thus, CPA3 is a proprecarboxypeptidase.

A number of nondigestive pancreatic-like CPs have been reported in the recent literature. AEBP1 has CP activity and has also been shown to be a new type of transcription factor (36). In addition to a CP domain, CPZ and ACLP contain the frizzled (fz) and discoidin-like domains, respectively, indicating that these two proteins might have other functions distinct from CP activity (34, 37). CPs catalyze the removal of the COOH-terminal basic amino acids arginine or lysine from peptides or proteins. The natural substrates of these enzymes seem to be peptide hormones including kinins, enkephalin hexapeptides, and anaphylatoxins, or proteins such as creatinine kinase (38). The removal of the COOH-terminal arginine or lysine results in modulation or inactivation of peptide hormone activity, and this might play an essential role in cell growth and differentiation. Induction of ACLP message and protein was observed as Monc-1 cells differentiate into smooth muscle cells, indicating that ACLP may play a role in the differentiation of vascular smooth muscle cells (37). Expression of CP M is associated with monocyte to macrophage differentiation (38). In this study, we found that induction of CPA3 mRNA expression was associated with in vitro differentiation of prostate epithelial cells.

Butyrate and its structural analogues are known cell growth and differentiation regulators and are currently under clinical consideration as a tool for the management of prostate cancer (5, 26). However, the molecular link between butyrate treatment and prostate cell differentiation is not well understood. By investigating mechanisms by which induction CPA3 mRNA is mediated in NaBu-induced in vitro differentiation of prostate cancer cells, this study strongly supports the existence of a link between histone deacetylase activity and butyrate-mediated differentiation in prostate cancer cells.

Differentiation and apoptosis induced by NaBu was observed in three androgen-independent prostate cells including PC-3 (Fig. 1,A), DU145, and BPH1 (data not shown). We, therefore, believe that this is an ideal model to study gene expression and regulation during differentiation and apoptosis of androgen-independent prostate cells. CPA3 expression was highly up-regulated during NaBu-induced differentiation of PC-3 cells (Fig. 1C). Similar up-regulation was also observed in two other androgen-independent prostate cancer cell lines (Fig. 5), suggesting that a common signal pathway seems to be involved in induction of CPA3 mRNA. Butyrate is known to induce general histone acetylation through a noncompetitive inhibition of the histone deacetylation enzyme. This action likely occurs in vivo because rats fed a high-fiber diet have high butyrate levels, which were found to be associated with histone hyperacetylation in colon epithelial cells (39). Further investigation showed that CPA3 mRNA expression was also induced by TSA in a time- and dosage-dependent manner (Fig. 6). TSA is a potent and specific histone deacetylase inhibitor. Thus, our data indicates that CPA3 mRNA induction is mediated by the mechanism of histone hyperacetylation.

Induction of CPA3 mRNA expression by histone inhibitors can be blocked by CHX, indicating that CPA3 is a downstream gene in response to the hyperacetylating activity of histones. The mechanism by which CPA3 is induced by NaBu and TSA was further investigated in this study. Hyperacetylation of histones neutralizes their positive charge, disrupting their ionic interaction with DNA, and thereby allowing transcription factors to access and activate specific genes (17). During in vitro differentiation of colon cancer cells, it was shown that p21^WAF1/CIP1 mRNA is consistently induced by NaBu and TSA in an immediate-early manner through a mechanism involving histone hyperacetylation (21). We observed the same results in the differentiation of prostate cancer cells. By performing transient transfection assays with antisense p21^WAF1/CIP1, we discovered that antisense expression of p21^WAF1/CIP1 completely inhibits the induction of CPA3 by butyrate (Fig. 7). We do not know how NaBu and TSA induce expression of CPA3, but a plausible model is that butyrate and TSA inhibit histone deacetylase at the level of the p21^WAF1/CIP1 gene. This leads to changes in the chromatin that allow transcriptional activation of this gene (21), and transactivation of the p21^WAF1/CIP1 gene further mediates induction of CPA3 mRNA. Whether p21^WAF1/CIP1 directly interacts with CPA3 through the binding to its promoter or through other proteins is still unknown and needs to be determined.

Proliferation and differentiation of prostate cancer cells is hormone-related. Several studies have suggested that various factors including androgen and peptide growth factors, play a major role in the pathogenesis as well as in the promotion of prostate cancer (40, 41, 42). Prostate-specific membrane antigen was originally identified as a marker of prostate cancer (43). Recently, it was found to function as a glutamate CP (42), indicating that like androgen, peptide hormones such as neuropeptides might also be involved in the cell growth and differentiation of prostate epithelial cells. Of the five ESTs that were found to be homologous to the CPA3 cDNA in the DNA sequence databases, one was derived from a pregnant mouse uterus library, and another was derived from a pregnant human uterus library. We have identified the mouse CPA3, and its expression pattern was different between pregnant and nonpregnant mouse uterus (data not shown). Therefore, it seems that expression of CPA3 is associated with hormone-regulated tissues. The removal of the COOH-terminal amino acid results in modulation or inactivation of peptide hormone activity that could play an essential role in cell growth and differentiation. The structural similarity of CPA3 with other CPs suggests that it might modulate the function of peptide hormones that play an essential role in the growth and/or differentiation of prostate epithelial cells. However, the exact substrate of CPA3, and its function within the cell, is presently unknown.