Biomedical subjects

D M Peehl

Publications and source records attributed to D M Peehl.

At least 55 records · Page 3Linked to original sources

PubMed1997

Loss of response to epidermal growth factor and retinoic acid accompanies the transformation of human prostatic epithelial cells to tumorigenicity with v-Ki-ras.

Growth factor-independent proliferation and loss of response to differentiation factors are believed to be critical elements in carcinogenesis. We have developed an in vitro model of human prostatic carcinogenesis by the introduction of SV40 DNA into normal prostatic epithelial cells to create a transformed, immortal cell line, pRNS-1-1. This non-tumorigenic cell line responded similarly to normal prostatic epithelial cells to most growth- and differentiation-regulatory factors, with the notable exception of loss of response to the inhibitory factor 1,25-dihydroxyvitamin D3. In this study, we describe the introduction of the ras oncogene into pRNS-1-1 cells to create a tumorigenic cell line, pRNS-1-1/ras. In addition to an attenuated response to 1,25-dihydroxyvitamin D3, these cells also became unresponsive to retinoic acid and gained the ability to undergo clonal proliferation in the absence of epidermal growth factor (EGF). EGF-independent growth could not be linked to the production of autocrine transforming growth factor-alpha, but instead was likely due to sustained signaling by the ras oncogene, bypassing ligand-activation of the EGF receptor. Ligand-independent proliferation, coupled with the loss of response to the growth-inhibitory and differentiation agent retinoic acid, may be important elements in the conversion of human prostatic epithelial cells to tumorigenicity.

Animals↗

PubMed1997

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent.

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.

Analysis of Variance↗

PubMed1997

Decreased expression of Wilms' tumor gene WT-1 and elevated expression of insulin growth factor-II (IGF-II) and type 1 IGF receptor genes in prostatic stromal cells from patients with benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a common proliferative disorder of unknown etiology. We have previously documented that the insulin-like growth factor (IGF) axis is critical for prostate cell growth and is abnormal in BPH. The type 1 IGF receptor (IGF-1R) is constitutively expressed by most body tissues and plays a significant role in regulating cell proliferation, consistent with the role of its ligands (IGF-I and IGF-II) as important mitogenic factors. The Wilms' tumor gene product (WT-1) is a tumor suppressor that has been shown to be altered in rare kidney tumors and is known to regulate IGF-II and IGF-1R. We investigated the possibility that the expression of prostatic WT-1, IGF-1R, and IGF-II genes is altered in patients with BPH. We utilized primary cultures of prostatic stromal cells grown from normal (n = 9) and hyperplastic (n = 9) surgical specimens and analyzed WT-1, IGF-1R, and IGF-II messenger RNA levels. In all of the BPH cell strains, WT-1 expression (measured by RT-PCR and RNase protection assays) was strikingly lower than that found in normal strains (0-20% of normal, mean 14% of normal, P < 0.01). The expression of both the IGF-1R (300% of normal, P < 0.05) and IGF-II (1000% of normal, P < 0.01) messenger RNAs was higher in BPH strains as compared with normal strains. No changes were seen in stromal cell strains derived from prostatic adenocarcinoma. Thus, in cultured human prostatic stromal cell strains from patients with BPH, decreased WT-1 gene expression is associated with increases in the expression of the IGF-1R and IGF-II genes that are known transcriptional targets of WT-1. These findings indicate that reduced expression of the WT-1 tumor suppressor gene and elevated IGF-1R and IGF-II gene expression may be involved in the pathophysiology of prostatic hyperplasia, implying a new role for the Wilms' tumor gene in nonmalignant states.

Aged↗

PubMed1997

Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3 alpha/17 beta-HSD activity and cellular distribution.

In androgen target tissues, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5alpha-dihydrotestosterone (5alpha-DHT) (a potent androgen) and 3alpha-androstanediol (a weak androgen). In this study, a 3alpha-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library. The human prostatic 3alpha-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3alpha-HSD and human type 1, type 2, and type 3 3alpha-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3alpha-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3alpha-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3alpha-androstanediol oxidation over 5alpha-DHT reduction. Using [14C]-5alpha-DHT as substrate, TLC analysis confirmed that the reaction product was [14C]-3alpha-androstanediol. However, in the reverse reaction, [3H]-3alpha-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3alpha- and 17beta-HSD activities. The 17beta-HSD activity accounted for the higher catalytic efficiency observed with 3alpha-androstanediol. These findings indicate that, in the prostate, type 2 3alpha-HSD does not interconvert 5alpha-DHT and 3alpha-androstanediol but inactivates 5alpha-DHT through its 3-ketosteroid reductase activity. Levels of 3alpha-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3alpha-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues. Expression of 3alpha-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney. This study is the first complete characterization of recombinant type 2 3alpha-HSD demonstrating dual activity and cellular distribution in the human prostate.

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Role of prostatic basal cells in the regulation and suppression of human prostate cancer cells.

Cytokeratin expression in normal and malignant prostatic tissue indicates a loss of basal epithelial cells in cancer. We investigated the ability of basal-like prostatic epithelial cells to inhibit the growth of prostatic cancer cells. Human prostate LNCaP cells were grown in medium with or without 10 nM dihydrotestosterone (DHT) on plastic culture dishes or on extracellular matrix derived from basal-like epithelial cells (primary cultures derived from normal peripheral zone of the prostate) that were grown with or without 10 nM DHT. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to assess the growth of LNCaP cells. On plastic dishes, growth of LNCaP cells was increased 5-10% by the presence of DHT in the medium. On matrix derived from basal-like cells that were grown in the absence of DHT, growth of LNCaP cells with or without DHT was similar to that on plastic. However, on matrix derived from basal-like cells that were grown with DHT, growth of LNCaP cells was suppressed when compared to all other culture conditions (P < 0.01). To determine whether basal-like cells could alter the function of LNCaP cells, we measured prostate-specific antigen (PSA) mRNA expression with the use of comparative RT-PCR. We found a significant decrease in the mature PSA transcript in cells grown on matrix derived from basal-like cells that were grown with DHT. The expression of PSA transcript was not altered in LNCaP cells that were grown on matrix derived from basal-like cells that were grown in the absence of DHT. Furthermore, using differential display of mRNA, we demonstrated that there were induction and suppression of multiple unique transcripts in the LNCaP cells when grown on the various culture conditions. To determine a possible mechanism for these observations. We used a dot blot immunoassay for several known inhibitory factors. We determined that DHT can induce the basal-like cells to secrete transforming growth factor-beta (TGF-beta 1), and that TGF-beta 1 can inhibit the proliferation of LNCaP cells in a dose dependent manner. We conclude that basal-like epithelial cells, in the presence of DHT, secrete an extracellular matrix o matrix associated factor(s), e.g. TGF-beta 1, that suppresses proliferation and function of prostate cancer cells. Our data suggest that the disappearance of the basal cell layer may be a prerequisite for the progression of prostatic neoplasia.

Base Sequence↗

PubMed1996

Parathyroid hormone--related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells.

Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

Adenocarcinoma↗

PubMed1996

Keratin 19 in the adult human prostate: tissue and cell culture studies.

Keratin 19 is found primarily in simple epithelia. In mammary epithelia, keratin 19 was localized to a subset of luminal cells, suggesting that keratin 19-negative cells may be the proliferative compartment of the secretory cell lineage. The structural and functional similarities of prostate and breast led us to examine keratin 19 expression in the prostate. Immunohistochemical studies revealed that keratin 19 expression was heterogeneous and frequently occurred in basal as well as in luminal cells of normal, dysplastic, and benign hyperplastic tissues. Keratin 19 was observed in cancer, but usually in a minority of cells. This was in dramatic contrast to invasive breast cancers, which are reportedly uniformly positive for keratin 19. Prostatic epithelial cells cultured from tissues of all histologies expressed keratin 19. In summary, keratin 19 does not obviously correlate with any epithelial cell lineage or phenotype in the prostate.

Adenocarcinoma↗

PubMed1996

Prostate specific antigen cleaves parathyroid hormone-related protein in the PTH-like domain: inactivation of PTHrP-stimulated cAMP accumulation in mouse osteoblasts.

PURPOSE: To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA. MATERIALS AND METHODS: Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production. RESULTS: Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production. CONCLUSIONS: Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.

Amino Acid Sequence↗

PubMed1996

7S nerve growth factor is an insulin-like growth factor-binding protein protease.

Insulin-like growth factor (IGF)-binding protein (IGFBP) proteases modulate IGF action by cleaving IGFBPs into fragments with lower affinity to IGFs, thereby increasing the levels of free IGFs. We have previously documented that prostate-specific antigen (PSA), a serine protease of the kallikrein family, is an IGFBP-3 protease. In this study, we characterized the potential IGFBP proteolytic activity of nerve growth factor (NGF gamma-subunit), which shares high sequence homology with PSA. [125I]IGFBP-3 was cleaved by NGF (but not other kallikreins) at a 3-fold lower concentration than that of PSA, thus proving NGF to be a more potent IGFBP protease than PSA. NGF-generated, lower mol wt IGFBP-3 fragments, detected by immunoblotting and cross-linking to IGFs, had a lower affinity to IGFs than intact IGFBP-3. Unlike PSA, which cleaves primarily IGFBP-3 and -5, NGF also displayed potent proteolytic activity against IGFBP-4 and -6. These data suggest that NGF may be involved in the growth of cells by more than one mechanism. In addition to binding to its receptors, NGF is capable of cleaving IGFBPs and, thus, enhancing IGF action. This synergistic action between NGF and IGF may have important implications on cell growth, development, and repair in the brain and other tissues.

Blotting, Western↗

PubMed1996

Cellular biology of prostatic growth factors.

The etiology of prostate cancer or of benign prostatic hyperplasia (BPH) is essentially not understood. It is becoming clear, however, that major determinants of the malignant or hyperplastic phenotype are various growth-stimulatory or -inhibitory factors and their receptors, whose inappropriate expression or loss disrupts normal regulation of cell proliferation and differentiation. Cell culture has been a versatile tool for studying the expression and interaction of growth factors in prostatic cells. Immunohistochemistry and in situ hybridization have provided a view of growth factor expression coupled with histopathology. The eventual definition of autocrine, paracrine, and endocrine pathways of growth regulation in the human prostate will facilitate the design of new preventative, diagnostic, and therapeutic strategies.

Animals↗

PubMed1996

KGF and EGF differentially regulate the phenotype of prostatic epithelial cells.

Previous studies indicate that keratinocyte growth factor (KGF) acts as a paracrine factor in the prostatic epithelium and epidermal growth factor (EGF) acts as an autocrine factor. In serum-free medium, KGF or EGF promoted similar growth of human prostatic epithelial cells. Response to two growth-inhibitory factors (suramin and transforming growth factor-beta), and expression of keratins and prostate-specific antigen (PSA), were similar with either mitogen. However, colonies in medium with KGF were very compact with extensive intercellular bonds, whereas colonies with EGF consisted of widely-separated cells. Growth was decreased to a greater extent by deletion of growth factors from medium with KGF versus EGF, and retinoic acid was 10-fold more potent at inducing growth inhibition and differentiation-associated keratin with KGF compared with EGF. We conclude that regulation of growth and differentiation in the prostate might vary depending on the availability of KGF versus EGF.

Cell Division↗

PubMed1995

Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents.

The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.

Adenocarcinoma↗

PubMed1995

Vitamin D and prostate cancer.

Our findings demonstrate the presence of VDR in various human prostate cancer cell lines and in primary cultures derived from normal, BPH and prostate cancer. In addition, 1,25-D induced several bioresponses in these cells including growth inhibition and PSA stimulation. Based on examples in many different malignant cells as well as our data in prostate cells, that vitamin D is anti-proliferative and promotes cellular maturation, it seem clear that vitamin D must be viewed as an important cellular modulator of growth and differentiation if addition to its classical role as regulator of calcium homeostasis. In this respect, vitamin D has the potential to have beneficial actions on various malignancies including prostate cancer. Its ultimate role in prostate cancer remains to be determined, but 1,25-D may prove useful in chemoprevention and/or differentiation therapy. We believe the data currently available provide the basis for an optimistic view on the possible use of vitamin D to treat prostate cancer in patients and that further investigation is clearly warranted to better define its potential therapeutic utility.

Calcitriol↗

PubMed1995

The insulin-like growth factor system in the prostate.

The insulin-like growth factor (IGF) system is involved in the regulation of cell growth. The system involves a network of molecules that includes the IGFs themselves (IGF-I and -II), IGF receptors (types I and II), IGF-binding proteins (IGFBP-1 through -6), and IGFBP proteases. Characterization of this complex system in the prostate has recently been initiated. Prostatic cell lines as well as primary cultures of prostatic epithelial and stromal cells have been analyzed for expression of IGFs, receptors, and IGFBPs. Prostatic epithelial cells and, quite likely, stromal cells as well respond to the mitogenic activity of IGFs via the type I IGF receptor. Prostatic stromal cells synthesize and secrete IGF-II; there is evidence that prostatic cell lines also synthesize IGFs, but this has not been confirmed in primary cultures of prostatic epithelial cells. Prostatic stromal and epithelial cells secrete a number of IGFBPs. The biological impact of some of these IGFBPs on the growth of prostatic cells has been examined, and proteolytic cleavage of IGFBP-3 by prostate-specific antigen (PSA) has been demonstrated. Aberrations in several elements of the IGF system have been observed in stromal cells derived from benign prostatic hyperplasia (BPH). The IGF system may therefore have a part in the etiology of BPH as well as in normal and malignant processes in the prostate.

Animals↗

PubMed1995

Keratinocyte growth factor: an androgen-regulated mediator of stromal-epithelial interactions in the prostate.

Members of the fibroblast growth factor (FGF) family are important growth-regulatory elements. Of the FGFs, keratinocyte growth factor (KGF) appears to have unique properties that implicate it as a paracrine factor in the prostate. Two KGF transcripts (approximately to 2.4 and 5.0 kb) encode a protein of approximately 22 kDa. In contrast to several other members of the FGF family, KGF has a signal peptide and is actively secreted. Cellular response to KGF is mediated by a specific receptor that is transcribed from an alternately spliced variant of the FGF type 2 receptor (FGFR-2). KGF transcripts have been detected in prostatic tissues and in stromal cells cultured from rat and human prostates as well as in a variety of stromal cells derived from other organs. Prostatic epithelial cells and numerous other types of epithelial cells are targets of KGF's mitogenic activity. Several factors involved in wound healing regulate the expression of KGF, but androgen regulation of KGF is of greatest relevance to the role of KGF in the prostate. Current efforts to localize and manipulate KGF activity in vivo should reveal the significance of KGF expression and function in the prostate and in other organs.

Androgens↗

PubMed1995

Actions of vitamin D3, analogs on human prostate cancer cell lines: comparison with 1,25-dihydroxyvitamin D3.

Data from epidemiological studies has suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. We have previously demonstrated the presence of vitamin D receptors (VDR) in three human prostate carcinoma cell lines (LNCaP, PC-3, and DU-145) as well as in primary cultures of stromal and epithelial cells derived from normal and malignant prostate tissues. We have also shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can elicit an antiproliferative action in these cells. In the present study we compared the biological actions of 1,25-(OH)2D3 to those of a series of natural vitamin D3 metabolites and several synthetic analogs of vitamin D3 known to exhibit less hypercalcemic activity in vivo. In ligand binding competition experiments, we demonstrated the following order of potency in displacing [3H]1,25-(OH)2D3 from VDR: EB-1089 > 1,25-(OH)2D3 > MC-903 > 1,24,25-(OH)3D3 > 22-oxacalcitriol (OCT) > 1 alpha,25-dihydroxy-16-enecholecalciferol (Ro24-2637) > 25-hydroxyvitamin D3, with EB-1089 being approximately 2-fold more potent than the native hormone. No competitive activity was found for 25-hydroxy-16,23-diene-cholecalciferol. When compared for ability to inhibit proliferation of LNCaP cells, MC-903, EB-1089, OCT, and Ro24-2637 exhibited 4-, 3-, and 2-fold greater inhibitory activity than 1,25-(OH)2D3. Interestingly, although OCT and Ro24-2637 exhibit, respectively, 10 and 14 times lower affinity for VDR than 1,25-(OH)2D3, both compounds inhibited the proliferation of LNCaP cells with a potency greater than that of the native hormone. The relative potency of vitamin D3 metabolites and analogs to inhibit cell proliferation correlated well with the ability of these compounds to stimulate prostate-specific antigen secretion by LNCaP cells as well as with their potency to induce the 25-hydroxyvitamin D3-24-hydroxylase messenger RNA transcript in PC-3 cells. In conclusion, these results demonstrate that synthetic analogs of vitamin D3, known to exhibit reduced calcemic activity, can elicit antiproliferative effects and other biological actions in LNCaP and PC-3 cell lines. It is noteworthy that although binding to VDR is critical for 1,25-(OH)2D3 action, the analog data indicate that additional factors significantly contribute to the magnitude of the biological response. Finally, the strong antiproliferative effects of several synthetic analogs known to exhibit less calcemic activity than 1,25-(OH)2D3 suggest that these compounds potentially may be useful as an additional therapeutic option for the treatment of prostate cancer.

Binding Sites↗

PubMed1994

Lipofection-mediated immortalization of human prostatic epithelial cells of normal and malignant origin using human papillomavirus type 18 DNA.

Human papillomavirus (HPV) type 18 DNA was introduced into epithelial cell strains derived from normal and cancer tissues of human prostatectomy specimens by the lipofection transfection method. Two cell lines were established: PZ-HPV-7 (transfected normal cell) and CA-HPV-10 (transfected cancer-derived cell). These lines have been maintained for over 100 passages. Incorporation of HPV type 18 DNA was confirmed by polymerase chain reaction. Immunocytochemical analysis showed expression of keratins 5 and 8, similar to the cells of origin, and the early region 6 oncoprotein of HPV. PZ-HPV-7, derived from normal diploid cells, had a modal chromosome number of 46 in early passages but became tetraploid later. CA-HPV-10 cells were aneuploid, and some retained the double minute chromosomes that were noted in the cancer-derived cells of origin. The cell lines showed a typical transformed morphology and were nontumorigenic in nude mice. We conclude that human prostatic epithelial cells derived from both normal and cancer tissues have been successfully transformed to immortality with HPV type 18 DNA. The establishment of these cell lines provides an opportunity for further development of an in vitro model of carcinogenesis for prostate cancer.

Animals↗

PubMed1994

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells.

Cultures of adult human prostatic epithelial and fibroblastic cells were established from normal, benign hyperplastic, and malignant tissues. Vitamin D receptors were detected by ligand binding of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cytosolic extracts prepared from all types of cell cultures as well as from fresh prostatic tissues. Vitamin D receptor transcripts were demonstrated by Northern blot analysis. 1,25-(OH)2D3 inhibited the growth of epithelial cells with half-maximal inhibition at approximately 1 nM. The growth of fibroblasts was also inhibited by 1,25(OH)2D3 but to a lesser extent. This is consistent with the apparently lower level of vitamin D receptors in fibroblasts compared to epithelial cells determined by ligand binding and Northern analysis of RNA transcripts. The growth inhibition of epithelial cells by 1,25(OH)2D3 was irreversible even after a short 2-h exposure, but morphology and keratin expression were not appreciably altered by long-term exposure to the hormone. A physiological role for 1,25(OH)2D3 in the prostate is postulated, and the inhibitory effect of 1,25(OH)2D3 on cancer-derived prostate cells may provide a basis for new preventive or therapeutic strategies.

Adult↗