Primary Human Prostate Epithelial Cells Research Articles

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5899.

A first-in-class HBO1 inhibitor WM-3835 inhibits castration-resistant prostate cancer cell growth in vitro and in vivo

The prognosis and overall survival of castration-resistant prostate cancer (CRPC) patients are poor. The search for novel and efficient anti-CRPC agents is therefore extremely important. WM-3835 is a cell-permeable, potent and first-in-class HBO1 (KAT7 or MYST2) inhibitor. Here in primary human prostate cancer cells-derived from CRPC patients, WM-3835 potently inhibited cell viability, proliferation, cell cycle progression and in vitro cell migration. The HBO1 inhibitor provoked apoptosis in the prostate cancer cells. It failed to induce significant cytotoxicity and apoptosis in primary human prostate epithelial cells. shRNA-induced silencing of HBO1 resulted in robust anti-prostate cancer cell activity as well, and adding WM-3835 failed to induce further cytotoxicity in the primary prostate cancer cells. Conversely, ectopic overexpression of HBO1 further augmented primary prostate cancer cell proliferation and migration. WM-3835 inhibited H3-H4 acetylation and downregulated several pro-cancerous genes (CCR2, MYLK, VEGFR2, and OCIAD2) in primary CRPC cells. Importantly, HBO1 mRNA and protein levels are significantly elevated in CRPC tissues and cells. In vivo, daily intraperitoneal injection of WM-3835 potently inhibited pPC-1 xenograft growth in nude mice, and no apparent toxicities detected. Moreover, intratumoral injection of HBO1 shRNA adeno-associated virus (AAV) suppressed the growth of primary prostate cancer xenografts in nude mice. H3-H4 histone acetylation and HBO1-dependent genes (CCR2, MYLK, VEGFR2, and OCIAD2) were remarkably decreased in WM-3835-treated or HBO1-silenced xenograft tissues. Together, targeting HBO1 by WM-3835 robustly inhibits CRPC cell growth.

BPA-induced prostatic hyperplasia in vitro is correlated with the unbalanced gene expression of AR and ER in the epithelium and stroma

As a typical environmental endocrine disruptor (EED), bisphenol A (BPA) can induce pathological hyperplasia of the prostatic epithelium and stroma. This study concentrates mainly on the effect and underlying mechanisms of BPA on prostatic hyperplasia, which is based on the culture of primary human prostate epithelial cells (HPEpiC) and human prostate fibroblasts (HPrF). In an effect to screen the optimal pro-survival BPA levels, HPEpiC and HPrF were, respectively, exposed to concentration gradients of BPA (10-12M-10-4M) solution diluted with two corresponding medium and incubated for 72h at 37°C. CCK-8 assay showed that 10-9 M-10-5M BPA could facilitate the proliferation of HPEpiC, while similar proliferative effect of HPrF only needed 10-11 M-10-7M BPA. HPrF were more sensitive to BPA than HPEpiC. The qualification of PCNA gene expression measured using quantitative real-time polymerase chain reaction (qRT-PCR) also mirrored the BPA-induced cell proliferation. Additionally, our results considered that androgen receptor (AR), estrogen receptor (ERα, ERβ), and NFKB1 gene expressions exhibited up-regulation in HPEpiC treated with 10-9M BPA for 72h. However, in HPrF, the identical BPA treatment could activate ERα, ERβ, and NFKB1 gene expressions and down-regulated the expression of AR levels. It is further confirmed that low-dose BPA can indeed promote the proliferation of human prostate cells in vitro, and the mechanisms of BPA for prostatic epithelial and stromal hyperplasia may not be consistent.

Altered expression of genes identified in rats with prostatic chronic inflammation in a prostate spheroid model treated by estradiol/testosterone.

Rats are the standard model for male reproductive toxicity testing. Rat prostates are physiologically and anatomically different from those of humans. Drug and chemical toxicity testing would benefit from an in vitro model of human prostate cells. Recently, spheroids derived by three-dimensional culture of human cell lines have been used for assessing drug and chemical toxicity in vitro as they mimic in vivo environments more closely than two-dimensional culture. However, forming consistently sized, uniform spheroids is technically challenging for toxicity testing. The purpose of this study was to identify potential genetic markers for assessing prostatic toxicity in spheroids. We formed prostate spheroids using agarose-coated plates seeded with human primary prostate epithelial cells. Prostate spheroids were treated with either 17β-estradiol (E2) or testosterone (T) on days 2-7 of culture. Samples were harvested on culture day 7. qPCR was used to examine gene expression levels previously identified in rats with chronic inflammation exposed to estradiol benzoate, E2 and/or T. Changes in some gene expression levels were observed in the spheroids treated with E2 or T. We found that treatment with 1 nM E2 and/or 10 μM T significantly altered spheroid proliferation and viability, as well as the expression levels of genes including Nanog homeobox (NANOG), C-C motif chemokine ligand 2 (CCL2) and bone morphogenetic protein receptor type 2 (BMPR2). Further studies using biologically active molecules with prostatic toxicity are needed to verify the results and to determine whether gene expression changes in the spheroid are specific to E2 or T treatment.

Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase

BackgroundImmortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown.Materials and methodsHere, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene.ResultsUsing this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin.ConclusionsUse of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Prostate cancer remains the second leading cause of cancerassociated deaths of men in the United States...

Genome-Wide Analysis of Low Dose Bisphenol-A (BPA) Exposure in Human Prostate Cells.

Endocrine disrupting compounds (EDCs) have the potential to cause adverse effects on wild-life and human health. Two important EDCs are the synthetic estrogen 17α-ethynylestradiol (EE2) and bisphenol-A (BPA) both of which are xenoestrogens (XEs) as they bind the estrogen receptor and dis-rupt estrogen physiology in mammals and other vertebrates. In the recent years the influence of XEs on oncogenes, specifically in relation to breast and prostate cancer has been the subject of considerable study.Methodology: In this study, healthy primary human prostate epithelial cells (PrECs) were exposed to environmentally relevant concentrations of BPA (5nM and 25nM BPA) and interrogated using a whole genome microarray.Results: Exposure to 5 and 25nM BPA resulted in 7,182 and 7,650 differentially expressed (DE) genes, respectively in treated PrECs. Exposure to EE2 had the greatest effect on the PrEC transcriptome (8,891 DE genes).Conclusion: We dissected and investigated the nature of the non-estrogenic gene signature associated with BPA with a focus on transcripts relevant to epigenetic modifications. The expression of transcripts encoding nuclear hormone receptors as well as histone and DNA methylation, modifying enzymes were significantly perturbed by exposure to BPA.

Assessing the Advantages, Limitations and Potential of Human Primary Prostate Epithelial Cells as a Pre-clinical Model for Prostate Cancer Research.

Choosing an appropriate cell model(s) is the first decision to be made before starting a new project or programme of study. Here, we address the rationale that can be behind this decision and we summarize the current cell models that are used to study prostate cancer. Researchers face the challenge of choosing a model that recapitulates the complexity and heterogeneity of prostate cancer. The use of primary prostate epithelial cells cultured from patient tissue is discussed, and the necessity for close clinical-academic collaboration in order to do this is highlighted. Finally, a novel quantitative phase imaging technique is described, along with the potential for cell characterization to not only include gene expression and protein markers but also morphological features, cell behaviour and kinetic activity.

Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells

Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca2+ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca2+ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca2+ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca2+ increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca2+ stores, store-operated Ca2+ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca2+ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca2+ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.

Abstract 538: Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform

Abstract Prostate cancer is the most frequent cancer diagnosis among men and the second leading cause of cancer-related death. Despite the widespread use of digital rectal exam (DRE) and blood-based screening of prostate-specific antigen (PSA) for prostate cancer screening, there are significant limitations in their specificity and prognostic value. Biomarkers which distinguish i) PSA-low prostate cancer from benign prostatic hyperplasia (BPH), and ii) indolent versus aggressive disease course represent unmet clinical needs. Experimentally, a panel of prostate cancer cell lines and non-tumorigenic, human primary cells were exposed to in vitro conditions designed to simulate poor oxygenation, low pH, diminished nutrient microenvironments, and metabolic perturbations (24-48 h) followed by iTRAQ proteomic analysis of cell lysates. Using the Berg Interrogative Biology™ platform, proteomic data were then subjected to Bayesian network learning to map molecular interactions, with cytoskeletal and scaffolding proteins Filamin A (FLNA), Filamin B (FLNB), and Keratin 19 (KRT19) identified as candidate prostate cancer biomarkers. To validate biomarker expression, mRNA and protein was quantified in panel of primary human prostate epithelial cells (HPrEC) and androgen-sensitive (LnCAP) or refractory (DU145, PC-3) prostate cancer cells, and each was differentially detected in one or more prostate cancer cell lines compared to HPrEC. Using proteomic analysis, peptides from FLNA, FLNB, and KRT19 were also detected cell culture media conditioned by prostate cancer cells (24 h), indicating they can be secreted. Importantly, unlike PSA expression, global regulation of FLNA, FLNB, and KRT19 expression remained unaltered after treatment with multiple prostate-cancer relevant stimuli (e.g., hypoxia, androgens, and inflammatory stimuli). In vivo validation was next conducted in sera from men (N = 447) with confirmed prostate cancer, benign prostate tumors, or BPH using LDT ELISA assays in a CLIA-certified laboratory. To assess the sensitivity and specificity of FLNA, FLNB, and KRT19 compared to PSA, ROC curve analysis was performed. The individual predictive power of each biomarker alone was comparable to that of PSA. However, the combination of age, levels of FLNA, FLNB, and KRT19, and PSA out-performed PSA alone in identification of patients with prostate cancer stratified compared to benign status, gleason scores and incidence of BPH. Together, these data validate the use of the Berg Interrogative Biology™ platform for biomarker discovery and indicate that FLNA, FLNB, and KRT19 can be used in conjunction with PSA for more sensitive and specific prostate cancer screening, a critical unmet need in the field. Citation Format: Niven R. Narain, Anne Diers, Rakibou Ouro-Djobo, Joyce Chan, Leonardo O. Rodrigues, Vivek K. Vishnudas, Eleftherios P. Diamandis, Viatcheslav R. Akmaev, Rangaprasad Sarangarajan. Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2015-538

Icrac in Human Primary Prostate Epithelial Cells

Expression levels of membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. Rapid androgen response in prostate cancer can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Thus mAR have been proposed as targets for therapeutic strategies. However, the molecular identity and downstream signaling pathways of mAR are still elusive. In primary cultures of human prostate epithelial cells from non-tumorous tissue (hPEC), we identified a rapid 5alpha-dihydrotestosterone (DHT) induced activation of store-operated Ca2+ entry.

ICRAC controls the rapid androgen response in human primary prostate epithelial cells and is altered in prostate cancer

Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.

Abstract IA15: Context-specific oncogenesis by ETS-family transcription factors

Abstract Recurrent translocation involving ETS-family transcription factors including ERG and ETV1, ETV4, and ETV5 occur in over half of prostate cancers, implicating them as driver oncogenes in prostate tumorigenesis. Two observations pose important questions. 1) The same ETS factors aberrantly expressed in prostate cancer can be endogenously highly expressed in other tissues to modulate normal physiology (e.g., ERG expression in endothelial cells and ETV1 expression in neurons). 2) Forced overexpression in prostate model systems such as immortalized human primary prostate epithelial cells and mouse prostate yields minimal phenotype. These observations indicate that ETS mediated oncogenesis is highly dependent on the cellular context, which is mediated by the cell/tissue type and cooperating lesions. We asked how cellular context affects the DNA binding, transcriptional output, and phenotype of ETS proteins. We compared human ETS dependent tumor cell lines from two lineages, prostate cancer and gastrointestinal stromal tumor and show that ETS binding is largely tissue specific. Further, we have generated genetically engineered mouse models that express ETS in different tissues, within different compartments of the prostate gland, and with different cooperating lesions. We show that ERG binding to genomic DNA is largely similar in PTEN intact and PTEN null mouse prostates. Yet, ERG can only mediate a transcriptional program that mediates an aggressive phenotype in PTEN null prostates. These data suggests that cellular context is critical for ETS transcription factor function. Citation Format: Yu Chen, Shipra Shukla, Ping Chi, Brett Carver, Charles L. Sawyers. Context-specific oncogenesis by ETS-family transcription factors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr IA15.

Purification and direct transformation of epithelial progenitor cells from primary human prostate

Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8-16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.

LHRH-conjugated lytic peptides directly target prostate cancer cells

Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone–releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC 50 concentrations for LNCaP cells, 4.4 and 9.1 μM; for DU-145 cells, 4.8 and 5.7 μM; and for PC-3 cells, 4.4 and 8.2 μM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3 h of exposure to either peptide; after 6 h, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24 h to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer.

P16INK4adownregulation is involved in immortalization of primary human prostate epithelial cells induced by telomerase

Prostate cancer is a major cause of cancer death among male population. Therefore, development of appropriate model systems is critical for understanding the molecular basis of prostate cancer progression. In this study, introduction of human telomerase (hTERT) into normal human prostate epithelial cells (PrECs) renders them higher telomerase activity, elongated telomere length and an extended proliferative lifespan. The immortal mass culture of PrEC-hTERT cell line with stabilized telomere length has been established using hTERT transfection. However, activation of hTERT alone appears to be insufficient for immortalization of PrEC cells because methylation of p16(INK4a) promoter has been found to be involved in the immortalization process. p53 was functionally intact and no mutations of p53 gene were identified in the immortalized PrECs. In addition, the immortal PrECs show a near diploid complement of chromosomes albeit a few reciprocal and non-reciprocal translocations are identified. They are anchorage dependent and do not form tumors in immunosuppressed host animals. Therefore, premalignantly transformed human PrECs provide a valuable model for prostate cancer research.

Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement

Cellular DNA damage triggers the DNA damage response pathway and leads to enforcement of cell cycle checkpoints, which are essential for the maintenance of genomic integrity and are activated in early stages of tumorigenesis. A special feature of prostate cancer is its high incidence and multifocality. To address the functionality of DNA damage checkpoints in the prostate, we analyzed the responses of human primary prostate epithelial cells (HPECs) and freshly isolated human prostate tissues to gamma-irradiation. We find that gamma-irradiation activates the ataxia telangiectasia mutated-associated DNA damage response pathway in the HPECs but that the clearance of phosphorylated histone H2AX (gammaH2AX) foci is delayed. Surprisingly, gamma-irradiated HPECs were unable to enforce cell cycle checkpoint arrest and had sustained cyclin-dependent kinase 2 (Cdk2)-associated kinase activity because of a lack of inhibitory Cdk phosphorylation by Wee1A tyrosine kinase. We further show that HPECs express low levels of Wee1A and that ectopic Wee1A efficiently rescues the checkpoints. We recapitulate the absence of checkpoint responses in epithelium of ex vivo irradiated human prostate tissue despite robust induction of gammaH2AX. The findings show that prostate epithelium has a surprising inability to control checkpoint arrest, the lack of which may predispose to accrual of DNA lesions.