Characterization of PacMetUT1, a recently isolated human prostate cancer cell line

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.     

Characterzation of a newly established human urinary bladder cancer cell line (BT-1)

Summary A new human transitonal cell carcinoma cell line has been established in long term tissue culture. The BT-1 cells were derived from a poorly differentiated human bladder cancer. The tumor cell line produces tumors in nude mice. BT-1 has been characterized by cytogenetic and flow cytometric analysis and by isoenzyme typing. As in direct preparations of human bladder tumors, an isochromosome 5p is a consistent marker of the newly established line. The BT-1 cells produce transforming growth factors but do not respond to exogeneous EGF.     

mRNA expression of the five membrane-type matrix metalloproteinases MT1-MT5 in human prostatic cell lines and their down-regulation in human malignant prostatic tissue

BACKGROUND: The aim of this study was to assess the expression of membrane-type matrix metalloproteinases (MT-MMPs) 1-5 in the human prostatic cell lines BPH-1, LNCaP, DU 145, PC-3, in malignant and non-malignant prostatic tissue samples, and in epithelial cells cultured from these tissue samples. METHODS: Matched malignant and non-malignant tissue specimens were obtained from 12 men with untreated prostate carcinoma after radical prostatectomy. Expression of mRNA for the five MT-MMPs was quantified by real-time PCR technique and normalized to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The expression of the five MT-MMPs was distinctly different not only between the prostate cell lines but also varied in the same cell line. There was a general higher expression of all MT-MMPs except for MT3-MMP in the androgen-insensitive cells DU 145 and PC-3 compared with that in the androgen-sensitive LNCaP cells. Their relatively high expression in the benign prostatic cell line BPH-1 and also in the primary cell cultures from malignant and non-malignant tissue samples argues against a simple association between MT-MMP expression and invasiveness. In malignant tissue samples and their corresponding cell cultures, the expression of most MT-MMPs was down-regulated in comparison to the normal counterparts. There was no correlation between tumor classification data and the MT-MMP expression results. CONCLUSIONS: In contrast to other carcinoma, the down-regulation of most MT-MMPs is typical for prostate carcinoma. It seems to occur mainly in epithelial cells and has to be examined as special characteristic of this tumor entity in further studies.     

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

BACKGROUND: The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. METHODS: Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. RESULTS: The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. CONCLUSIONS: The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression.     

Rational basis for Trk inhibition therapy for prostate cancer

BACKGROUND: Prostatic cancer cells are lethal because they acquire the ability to activate survival pathways that do not require androgenic stimulation. As a rational approach to developing effective therapy for these devastating cells, specific signal transduction pathways uniquely required for the survival of these nonandrogen-dependent prostate cancer cells must be identified. Previous studies suggested that the neurotrophin/trk signal transduction axis may regulate such unique survival pathways. In the present study, the changes in expression of the neurotrophins (NGF, BDNF, and NT-3) and their cognate receptors (i.e., trk and p75NTR) during the progression of normal prostatic epithelial cells to malignancy were documented. Additionally, the consequences of inhibiting these trk signaling pathways on the in vitro survival of prostate cancer cells was tested. METHODS: Immmunocytochemistry, RT-PCR, and ELISA assays were used to characterize the changes in the neurotrophin ligands (i.e., NGF, BDNF, and NT-3) and their cognate high-affinity (i.e., trk A, B, and C) and low-affinity neurotrophin (i.e., p75 NTR) receptors in normal vs. malignant human prostatic tissues. CEP-751 is an indolocarbazole compound specifically designed to inhibit the initiation of these neurotrophin/trk signal transductions. The consequence of CEP-751 inhibition of trk signaling for in vitro clonogenic survival of a series of human prostatic cancer lines was also tested. RESULTS: These studies demonstrated that normal prostatic tissue from patients without prostate cancer contains substantial levels of nerve growth factor (NGF), which is produced in a paracrine manner by stromal cells. These stromal cells lack both trk and p75NTR receptors. In contrast, normal prostatic epithelial cells from patients without prostate cancer do not secrete detectable levels of neurotrophins, but do express trk A and p75 NTR. While the NGF/trkA/p75 NTR axis is present in the normal prostate, normal prostatic epithelial cells do not depend on this axis for their survival. In contrast, malignant prostate epithelial cells directly secrete a series of neurotrophins (i.e., NGF, BDNF, and/or NT-3) and express at least one if not more of the trk receptor proteins (i.e., trk A, B, and/or C), while no longer expressing the p75NTR receptors. In addition, inhibition of autocrine trk signaling via CEP-751 treatment induces the apoptotic death of these malignant cells. CONCLUSIONS: Prostate carcinogenesis involves molecular changes leading to the paracrine and/or autocrine production of a series of neurotrophins. This is coupled to the ectopic expression of trk B and trk C, as well as to the continued expression of trk A, and the loss of expression of p75NTR receptors. These changes result in the acquisition by malignant prostate cells of a unique requirement for trk signaling pathways for survival. Based on these findings, trk inhibition is a novel, rational approach for prostate cancer therapy.     

Involvement of HGF/SF-Met signaling in prostate adenocarcinoma cells: evidence for alternative mechanisms leading to a metastatic phenotype in Pr-14c

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) facilitates intercellular communication between the epithelial carcinoma and its surrounding stromal tissue during metastatic invasion through interaction with its proto-oncogenic receptor, Met, found on carcinoma cells. This study utilizes the C31/Tag transgenic mouse prostate cancer cell line model in an attempt to characterize the interaction between HGF/SF and Met on the metastatic potential of prostate cancer. METHODS: Exogenous HGF was supplied to the prostate adenocarcinoma cell line (Pr-14) and metastatic cell line (Pr-14c) to evaluate mitogenicity by proliferation assays, morphological characteristics on an extracellular matrix substrate, and motogenic properties using the scatter assay, invasion chambers, and zymogram studies to analyze secretory enzymes produced by the cell lines. RESULTS: RNA and protein analyses show that the cell lines express similar amounts of Met. Pr-14 cells have an increased growth rate following HGF/SF treatment, whereas the metastatic Pr-14c cells show little change. Morphological studies of Pr-14c cells on extracellular matrix demonstrate negligible changes when compared to the tubular formation of Pr-14 cells after HGF/SF stimulation. Motility studies of the metastatic cells following HGF/SF treatment reveal a potentially faulty signaling pathway downstream of Met activation in the metastatic prostate cells. CONCLUSIONS: Our studies suggest that proliferation, invasion, and cell morphological characteristics may be induced independently from the HGF/SF-Met pathway in C31/Tag metastatic prostate cancer cells.     

Bombesin modulates the association of Src with a nuclear 110-kd protein expressed in dividing prostate cells

Polyclonal antibodies produced against a peptide derived from the Fer tyrosine kinase sequence also specifically recognized a 110-kd protein (p110) up-regulated in dividing versus resting dog prostate epithelial cells in vitro. In vivo in the dog prostate, p110 expression was detected when basal cell metaplasia was induced by estrogens after castration but not when renewing the differentiated epithelium with androgens. It was also detected in extracts from the human prostatic carcinoma cell lines LNCaP, DU145, and PC-3, and from 6 out of 11 human prostate cancer tissues analyzed, but not from normal or hyperplastic glands. The tyrosine kinase Src was shown by coimmunoprecipitation to associate with p110, and this interaction was positively modulated by bombesin stimulation of PC-3 cells. However, p110 was not tyrosine phosphorylated. Moreover, it was mainly distributed in the nuclear fraction. This nuclear p110 protein, expressed in dividing prostate epithelial cells and in human prostate cancer cells and tissues, could thus be a downstream mediator of bombesin-signaling pathways, acting via its association with Src.     

Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression

BACKGROUND: The study of prostate carcinogenesis and tumor progression is made difficult by the lack of appropriate in vitro and in vivo models. High prevalence of prostatic intra-epithelial neoplasia and latent prostatic carcinoma, representing multiple steps in carcinogenesis to invasive carcinoma, are relevant targets for cancer prevention. From the RWPE-1, immortalized, non-tumorigenic, human prostate epithelial cell line, we have derived four tumorigenic cell lines with progressive malignant characteristics. METHODS: Cell lines were derived by exposure of RWPE-1 to N-methyl-N-nitrosourea (MNU), selected and cloned in vivo and in vitro, and characterized by prostatic epithelial and differentiation markers, karyotype analysis, anchorage-independent growth, invasiveness, tumorigenicity, and pathology of the derived tumors. RESULTS: Cytokeratins 8 and 18, androgen receptor, and prostate-specific antigen expression in response to androgen, confirm prostatic epithelial origin. RWPE-1 cells do not grow in agar and are not tumorigenic in mice, but the growth, tumorigenicity, and tumor pathology of the MNU cell lines correlate with their invasive ability. The WPE1-NA22 (least malignant) form small, well-differentiated, and WPE1-NB26 cells (most malignant) form large, poorly differentiated, invasive tumors. Overall, loss of heterozygosity for chromosomes 7q, 13q, 18q, and 22, and gain of 5, 9q, 11q, and 20, was observed. The MNU cell lines, in order of increasing malignancy are; WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26. CONCLUSIONS: This family of cell lines with a common lineage represents a unique and relevant model which mimics stages in prostatic intra-epithelial neoplasia (PIN) and progression to invasive cancer, and can be used to study carcinogenesis, progression, intervention, and chemoprevention.     

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

BACKGROUND: Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. METHODS: Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. RESULTS: Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. CONCLUSIONS: Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.     

Secretion of prostatic specific antigen, proliferative activity and androgen response in epithelial-stromal co-cultures from human prostate carcinoma

We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer.     

Xenografted small cell undifferentiated cancer of prostate: possible common origin with prostatic adenocarcinoma

The first xenograft line of small cell undifferentiated carcinoma of the prostate (UCRU-PR-2) has been established and characterized. The donor tumor and the xenograft share the common morphological and ultrastructural features of small cell undifferentiated carcinoma (including neurosecretory granules) but also elaborate epithelial membrane antigen and carcinoembryonic antigen, in addition to neurone-specific enolase. The line expresses a diploid DNA complement. Androgen and estrogen receptors are not expressed, although prostatic acid phosphatase is present in sera from tumor-bearing mice in low levels. From these studies, we postulate a possible common stem cell origin for adenocarcinoma and small cell undifferentiated carcinoma of the prostate; further studies of a cell line derived from this tumor may clarify the issue.     

Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues

BACKGROUND: Over expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. METHODS: RT-PCR and Western blot analyses were used to analyze the expression of MUC18 mRNA and protein in four human prostate cancer cell lines, cultured primary normal prostate epithelial cells, normal prostate and malignant prostate tissues. Immunohistochemistry was used to determine the expression of MUC18 antigen in prostatic tissues at different stages of malignancy. RESULTS: Human MUC18 mRNA and protein was expressed in three different prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one prostate cancer cell line (LNCaP.FGC). HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from either cultured primary normal prostatic epithelial cells or the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas and in cells of a lymph node metastasis compared to that in normal or benign hyperplastic epithelium (BPH). CONCLUSIONS: We therefore conclude that MUC18 is expressed at higher levels in pre-malignant and malignant prostatic epithelium, including metastasis. We suggest that over-expression of MUC18 may be a new marker of human prostate cancer and also implicates its possible role in development and progression of prostate cancer.     

Anti-tumor effects and lack of side effects in mice of an immunotoxin directed against human and mouse prostate-specific membrane antigen

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature in humans, but not on the vasculature of normal tissues. Expression of low levels of PSMA has also been found on non-vascular cells in several normal tissues, most prominently on the brain and kidney in humans. PSMA is an excellent candidate for targeting prostate cancer or targeting tumor vasculature of various solid tumors. The high potential clinical benefit of these agents has prompted the search for an animal model in which to assess the efficacy and safety of anti-PSMA monoclonal antibody (mAb)-based therapies. METHODS: A rat monoclonal antibody, E6 that recognizes both mouse and human PSMA was generated using conventional hybridoma techniques. The antibody was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. An immunotoxin composed of E6, antibody and deglycosylated ricin A-chain (dgA) was prepared chemically. The anti-tumor effects of the immunotoxin were determined in vitro and in mice bearing subcutaneous LnCaP human prostate tumors, which express PSMA on the tumor cell surface. RESULTS: E6 recognizes the extracellular domain of both human and mouse PSMA in ELISA, immunoblot and by immunohistochemistry. E6 strongly stained the vascular endothelium of tumors from humans but not from mice. E6 stained proximal tubules in mouse and human kidneys, and neurons in the mouse and human hippocampus but, unlike the human, did not detectably stain epithelial cells in mouse prostate or small intestine. An E6-dgA immunoconjugate strongly inhibited the growth of LnCaP tumor xenografts without causing apparent toxicity to the mice. Histological observation indicated that the anti-tumor effects were mediated through direct cytotoxic effects on the tumor cells. CONCLUSIONS: We have generated and characterized a rat mAb (E6) that reacts specifically with both human and mouse PSMA and have demonstrated that an immunotoxin constructed from E6 is safe and effective against human prostatic carcinoma cells growing subcutaneously in nude mice.