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Adenocarcinoma of the prostate is the most common type of cancer, excluding skin cancer, and the second leading cause of cancer death in adult men in the United States. The lifetime risk for developing symptomatic prostate cancer is one in five for an American man. A pivotal step in carcinogenesis is a shift in the balance between proliferation, differentiation, and apoptosis that favors cell proliferation. Transforming growth factor-beta (TGF-beta) is a key negative growth regulator in the normal prostate. Although TGF-beta) inhibits the proliferation of normal prostate cells and functions as a tumor suppressor in early tumorigenesis, it acts as a tumor promoter in later stages of tumor progression. Elevated expression of TGF-beta in prostate cancer cells is associated with poor clinical outcome. Over-expression of TGF-beta aids tumorigenesis by not only stimulating angiogenesis and suppressing the immune system, but also by acting directly on the prostate tumor cells. While prostate cancer cells become resistant to TGF-beta-induced growth inhibition and apoptosis, they retain other TGF-beta-induced responses that enhance tumorgenicity. such as induction of extracellular matrix proteins, cell adhesion proteins and proteases. These direct tumor effects are mediated primarily through Smad signaling. This review addresses the mechanisms by which prostate cancer cells may acquire TGF-beta resistance and promote tumorgenicity. Understanding the mechanisms underlying TGF-beta resistance is important for the identification and development of better diagnostic markers and more effective strategies for treating prostate cancer.

The development of prostate cancer through a multistep process of carcinogenesis may have a long latent period of 20-30 years. It is possible that progression to a malignant state could be blocked or reversed during this time. This study focuses on the ability of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide (4-HPR), to reverse changes associated with malignant transformation and tumor progression, towards a normal phenotype. To examine the responsiveness of cells at different steps of prostate carcinogenesis, three immortalized, but non-tumorigenic (RWPE-1, WPE1-7 and WPE1-10), and one human prostate carcinoma cell line (DU-145), were used. The effects of 4-HPR on cell proliferation, expression of intermediate filament proteins cytokeratin 18 and vimentin, and tumor suppressor proteins p53 and pRb were examined by immunostaining and compared. Results show that 4-HPR caused inhibition of growth in all cell lines in a dose-dependent manner. 4-HPR induced an increase in staining for cytokeratin 18, a marker of differentiation for prostate epithelial cells. While all cell lines showed strong immunostaining for vimentin, treatment with 4-HPR for 8 days caused a marked decrease in staining for vimentin in all cell lines. In an in vitro assay, 4-HPR also caused inhibition of invasion by DU-145 cells in a dose-dependent manner. Furthermore, 4-HPR treatment was effective in significantly decreasing the abnormal nuclear staining for the tumor suppressor proteins p53 and pRb. Because 4-HPR decreased invasion-associated vimentin expression, inhibited invasion, and normalized p53 and pRb immunostaining, we propose that 4-HPR may be an effective agent for secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

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.

Background: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis., Methods: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression., Results: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway., Conclusions: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis., (Copyright 2001 Wiley-Liss, Inc.)

A long latent period of 20 to 30 years may be involved in the multistep process of carcinogenesis represented by prostatic intraepithelial neoplasia (PIN) in the prostate. It is, therefore, possible that progression to a malignant state could be blocked or reversed during this time. Retinoids not only have the ability to block steps in the process of carcinogenesis but they may also modulate or reverse some malignant characteristics of cancer cells. This study focuses on the ability of N-(4-hydroxyphenyl)-retinamide (4-HPR), a synthetic retinoid, to reverse malignant characteristics towards a normal phenotype, using the human prostate carcinoma cell line DU-145. These malignant characteristics include abnormal cell proliferation, intermediate filament expression, motility, invasion, and cell survival. Results show that 1 microM and 10 microM 4-HPR caused 31% and 96% inhibition of growth, while all-trains retinoic acid (ATRA) produced similar effects at 10 and 100 microM, making 4-HPR ten times more effective than ATRA. While DU-145 cells show strong immunostaining for vimentin, treatment with 1 microM 4-HPR for eight days caused a marked decrease in vimentin staining. This was accompanied by a change from an elongated to an epithelial cell morphology. Densitometric analysis of Western blots for vimentin showed a 53% decrease in vimentin expression in 1 microM 4-HPR treated cells. Concomitant with the decrease in vimentin expression, cell motility and invasive ability also decreased by 32% and 52%, respectively. Growth inhibition was accompanied by DNA fragmentation and apoptosis. Exposure of cells to 1 microM 4-HPR caused a marked upregulation of nuclear retinoid receptors RARalpha and a detectable expression of RARgamma. These results suggest that inhibition of growth and vimentin expression, and induction of apoptosis by 4-HPR in prostate cancer cells may occur via a receptor-mediated mechanism involving transrepression of AP-1 by retinoid receptors. We propose that vimentin may serve as a useful intermediate marker for early detection of prostate cancer in biopsy specimens and that 4-HPR may be effective in blocking several steps in prostate carcinogenesis as well as the progression of PIN to invasive carcinoma.

Objective: To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells., Study Design: Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer., Results: The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes., Conclusion: The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.