Salmaan Quader

Immortalized and tumorigenic adult human prostatic epithelial cell lines : Characteristics and applications Part 2. Tumorigenic cell lines

This is Part 2 of a three‐part review and deals with tumorigenic cell lines. Several immortalized and malignant adult human prostatic epithelial cell lines have been recently developed. The three most widely used carcinoma cell lines—DU‐145, PC‐3, and LNCaP—developed between 1977 and 1980, have greatly contributed to our current understanding of prostate cancer. Before a cell line can be accepted as having prostatic epithelial origin, some basic characteristics must be established. Expression of specific cytokeratins but absence of desmin and factor VIII should be first determined to establish epithelial origin. Responsiveness to androgens and expression of androgen receptor and prostate‐specific antigen should be examined under stringent culture conditions to establish prostatic epithelial origin. Response to growth factors and expression of their receptors facilitates further characterization of cell behavior. Cell lines immortalized by human papillomaviruses (HPVs) are of special interest because HPVs are involved in a variety of anogenital cancers and may also play a role in prostate carcinogenesis. Malignant transformation of HPV‐18 immortalized cells with the ras oncogene provides cell systems for investigating the multistep process of carcinogenesis. Each cell line has some unique characteristics, whether it arose directly from a carcinoma or resulted from immortalization with Simian virus 40 (SV40) or HPV, or was transformed in vitro by oncogenes. Comparisons of these characteristics should facilitate elucidation of the mechanisms involved in the initiation, promotion, and progression of prostate cancer. These cell lines will further serve as useful models for investigating tumor progression, invasion, metastasis, new therapeutic strategies, drug resistance, and its reversal and chemoprevention. The nontumorigenic cell lines were discussed in Part 1 [1]. This review summarizes the characteristics of several currently available tumorigenic, adult human prostatic epithelial cell lines. Prostate 30:58–64, 1997

Immortalized and tumorigenic adult human prostatic epithelial cell lines: Characteristics and applications part I. Cell markers and immortalized nontumorigenic cell lines

Several immortalized and malignant adult human prostatic epithelial cell lines have recently been developed. The three most widely used carcinoma cell lines, DU‐145, PC‐3, and LNCaP, developed between 1977 and 1980, have greatly contributed to our present understanding of prostate cancer. Before a cell line can be accepted as having prostatic epithelial origin, some basic characteristics must be established. Expression of specific cytokeratins, but absence of desmin and factor VIII, should be first determined to establish epithelial origin. Responsiveness to androgens and expression of androgen receptor and prostate specific antigen should be examined under stringent culture conditions to establish prostatic epithelial origin. Response to growth factors and expression of their receptors facilitates further characterization of cell behavior. Cell lines immortalized by human papillomaviruses (HPVs) are of special interest because HPVs are involved in a variety of anogenital cancers and may also play a role in prostate carcinogenesis. Malignant transformation of HPV‐18 immortalized cells with the ras oncogene provides cell systems for investigating the multistep process of carcinogenesis. Each cell line has some unique characteristics, whether it arose directly from a carcinoma or resulted from immortalization with simian virus 40 (SV40) or HPV or was transformed in vitro by oncogenes. Comparisons of these characteristics should facilitate elucidation of the mechanisms involved in initiation, promotion, and progression of prostate cancer. These cell lines will further serve as useful models for investigating tumor progression, invasion, metastasis, new therapeutic strategies, drug resistance, and its reversal and chemoprevention. This review will be published in three parts and will summarize cell markers necessary for characterization, as well as the characteristics and some applications of the immortalized as well as malignant adult human prostatic epithelial cell lines. Part 1 deals with cell markers and the immortalized, nontumorigenic cell lines.

Immortalized and tumorigenic adult human prostatic epithelial cell lines: Characteristics and applications. Part 3. Oncogenes, suppressor genes, and applications

This is Part 3 of a three‐part review. It deals with the possible role of oncogenes and suppressor genes in human prostate carcinoma as well applications of nontumorigenic and tumorigenic human prostate cell lines described in Parts 1 and 2 [1,2]. Several immortalized and malignant adult human prostatic epithelial cell lines have recently been developed. The three most widely used carcinoma cell lines, DU‐145, PC‐3, and LNCaP, developed between 1977 and 1980, have greatly contributed to our present understanding of prostate cancer. Before a cell line can be accepted as having prostatic epithelial origin, some basic characteristics must be established. Expression of specific cytokeratins but absence of desmin and factor VIII should be first determined to establish epithelial origin. Responsiveness to androgens and expression of androgen receptor and prostate‐specific antigen should be examined under stringent culture conditions to establish prostatic epithelial origin. Response to growth factors and expression of their receptors facilitates further characterization of cell behavior. Cell lines immortalized by human papillomaviruses (HPVs) are of special interest because HPVs are involved in a variety of anogenital cancers and may also play a role in prostate carcinogenesis. Malignant transformation of HPV‐18 immortalized cells with the ras oncogene provides cell systems for investigating the multistep process of carcinogenesis. Each cell line has some unique characteristics, whether it arose directly from a carcinoma or resulted from immortalization with simian virus 40 (SV40) or HPV, or was transformed in vitro by oncogenes. Comparisons of these characteristics should facilitate elucidation of the mechanisms involved in initiation, promotion and progression of prostate cancer. These cell lines will further serve as useful models for investigating tumor progression, invasion, metastasis, new therapeutic strategies, drug resistance and its reversal and chemoprevention. This review summarizes some applications of the currently available immortalized, non‐turmorigenic as well as the tumorigenic adult human prostatic epithelial cell lines. Prostate 30:136–142, 1997.

Intercellular Communication and Human Prostate Carcinogenesis

Abstract: Gap‐junction‐mediated intercellular communication (GJIC) is required for completion of embryonic development, tissue homeostasis, and regulation of cell proliferation and death. Although, as emphasized in several reports, defects or disruption of GJIC may be important in carcinogenesis, the potential role of GJIC in the onset and progression of human prostate cancer remains ill‐defined. The gap junction channel‐forming connexins (Cx) comprise a multigene family of highly conserved proteins that are differentially expressed in a tissue‐ and development‐specific manner; changes in connexin expression are also commonly seen during cellular differentiation. However, when multiple connexins are concurrently expressed, gap junction channels may consist of more than one connexin species. This is important, because only certain pairings give rise to functional channels. In our studies, we investigated GJIC in a panel of both nontumorigenic (RWPE‐1) and malignant (RWPE‐2, LNCaP, DU‐145) human prostate epithelial cells, compared to a normal rat liver epithelial F344 (WB‐1) cell line, as it was found to be junctionally proficient. In addition, expression and regulation of Cx43 and Cx32 were also inspected using western blot analysis. The ability of hormones, antihormones, and the antihypertensive drug forskolin to restore GJIC in nontumorigenic and malignant human prostate epithelial cells was examined by the scrape‐loading/dye transfer (SL/DT) or fluorescence recovery after photobleaching (FRAP) methods using an Ultima laser cytometer. Results from both assays showed that neither nontumorigenic nor malignant prostate cells have functional GJIC. However, both estrone (E1) and forskolin (FK) induced a significant increase (4.4‐ and 2.8‐fold, respectively) in cell‐cell communication only in the RWPE‐1 cells. Interestingly, the use of Matrigel, a solubilized basement membrane, as substrate for cell attachment and growth resulted in the rescue of GJIC activity in RWPE‐1 cells, as revealed by the SL/DT method. Furthermore, E1 induced a twofold increase in connexin 43 (Cx43), whereas forskolin caused a 50% reduction in Cx32 expression in RWPE‐1 cells. These data suggest that agents that increase Cx43:Cx32 ratio may restore GJIC in junctionally deficient cells, providing a basis for the development of new strategies for the prevention and treatment of human prostate cancer.

N-(4-hydroxyphenyl)retinamide (4-HPR) decreases neoplastic properties of human prostate cells: An agent for prevention

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.

Interleukin-6 and epidermal growth factor promote anchorage-independent growth of immortalized human prostatic epithelial cells treated with N-methyl-N-nitrosourea.

Epidermal growth factor (EGF) and interleukin (IL)‐6 are implicated in the growth of benign and malignant prostatic epithelial cells. We investigated the role of EGF and IL‐6 during the process of prostate carcinogenesis.

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model

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.

Connexin Expression in Nonneoplastic Human Prostate Epithelial Cells

Abstract: Expression of gap‐junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE‐1) and tumorigenic (RWPE‐2) human prostate epithelial cells as well as in two cell clones (WPEI‐7 and WPEI‐10) originating from the RWPE‐1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE‐1 and WPEI‐10 cells, whereas Cx32 is expressed predominantly in RWPE‐2 and WPEI‐7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI‐10 cells, with no apparent effect on WPEI‐7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI‐7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI‐10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE‐1 and WPEI‐10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE‐2 and WPEI‐7 cells. We recently reported that junctional communication could be rescued in RWPE‐1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.