Khurram S. Chaudhary

Cytoplasmic induction and over‐expression of cyclooxygenase‐2 in human prostate cancer: implications for prevention and treatment

Objective To assess the level and morphological distribution of cyclooxygenase (COX)‐1 and ‐2 in human prostates and to determine any association with the Gleason grade of prostate cancer.

Human MUC1 Mucin: A Potent Glandular Morphogen

Human MUC1 mucin is a high‐molecular‐weight transmembrane glycoprotein, which is apically expressed in the majority of glandular epithelia. During embryonic development, changes in the pattern of MUC1 mucin expression coincide with the onset of glandular differentiation. This mucin is also frequently overexpressed and aberrantly glycosylated in carcinomas. To investigate the potential role of MUC1 mucin in morphogenesis, a full length MUC1 cDNA was transfected into murine mammary adenocarcinoma (410.4) and Madin‐Darby canine kidney (MDCK) cells. This generated four clonal cell lines. Western blotting, FACS analysis, and immunohistochemistry confirmed expression of MUC1. All four MUC1‐expressing clones demonstrated altered morphogenesis when cultured in three‐dimensional type I collagen gels. While parental and vector control 410.4 cells formed compact spherical structures, the MUC1‐expressing clones formed complex branching structures. Similarly, while parental and vector control MDCK cells formed small circumscribed colonies with a central lumen, the MUC1‐expressing clones formed elongated tubules. MUC1 expression was also associated with reduced cellular cohesion and enhanced migration on type I collagen‐coated surfaces for all except one of the clones, which expressed only low levels of MUC1 on the cell surface. These results show that MUC1 expression stimulates morphogenetic changes in two distinct epithelial cell lines. Taken together with previous observations on MUC1 expression in embryonic development and carcinomas, this finding suggests that MUC1 may induce changes in tissue architecture in both normal development and cancer. Copyright

Differential expression of cell death regulators in response to thapsigargin and adriamycin in Bcl-2 transfected DU145 prostatic cancer cells.

Functional overexpression of Bcl‐2 has been reported to confer an anti‐apoptotic potential in a variety of cell types. The role of Bcl‐2 in epithelial cell‐cycle control and in interactions with other cell‐cycle regulators is not clearly understood. Its expression has been correlated with the hormono‐ and chemo‐resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl‐2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl‐2 encoding expression plasmid. Two Bcl‐2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl‐2 expression on cellular proliferation, cell death (± adriamycin or thapsigargin), and expression of cell‐cycle/death regulators (p53, PCNA, Bax, Bak, Bcl‐XL) were evaluated. Compared with controls, Bcl‐2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (∼two‐fold), an increase in Bax (∼two‐fold) and PCNA (∼three‐fold), and no change in the levels of Bcl‐XL and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025–5 µM) and thapsigargin (0.0025–5 µM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl‐2 and its heterodimeric partner Bax were elevated ∼two‐fold with no change in Bak in Bcl‐2 transfectants in contrast to controls, where Bak was increased (two‐fold). This is the first study to demonstrate that Bcl‐2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl‐2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin. Copyright

Ligand Activation of the Androgen Receptor Downregulates E-Cadherin-Mediated Cell Adhesion and Promotes Apoptosis of Prostatic Cancer Cells

Androgen independence is the major cause of endocrine therapy failure in advanced prostate cancer (PC). To examine the effects of human androgen receptor (AR) expression on growth of human PC cells, transfection of full-length AR cDNA in an androgen-insensitive human prostatic adenocarcinoma cell line (DU145) was performed. Transcriptional activity of AR was confirmed by the MMTV luciferase assay and AR expression was assessed by reverse transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry. Two stable transfectant cell lines expressing functional AR were established and passaged over 60 times. Under standard culture conditions, AR expression in transfected cells was predominantly cytoplasmic. Exposure to dihydrotestosterone (DHT; 60 pM-10 nM) resulted in a rapid (maximal at 30 minutes) translocation of AR to the nucleus. Treatment with DHT (5 nM) caused a significant reduction in cell-cell adhesion and aggregation accompanied by a decrease in E-cadherin expression. This was associated with up to 40% inhibition of proliferation and approximately two-fold increase in apoptosis. These results suggest that gene transfer-mediated AR expression in DU145 cells confers sensitivity to DHT, modulates cell-cell adhesion through E-cadherin, and suppresses cell growth by inhibiting proliferation and promoting apoptosis. This provides amodelfor studies ofAR-regulated cell signalling and identification of novel androgen-regulated genes in PC.

Inverse correlation between high level expression of cyclin E and proliferation index in transitional cell carcinoma of the bladder

Background/Aims: Overexpression of the G1 cyclins, D1 and E, and/or downregulation of p27Kip1 allow uncontrolled tumour cell proliferation. This study investigated the relation between these three cell cycle proteins and tumour proliferation in bladder cancer. Method: Nuclear expression of cyclin D1, cyclin E, and p27Kip1 was determined immunohistochemically in 52 primary transitional cell carcinomas, and the Ki-67 proliferation marker was also assessed. For each protein, the percentage of positive tumour cell nuclei was determined and analysed as a continuous variable. Results: Advancing tumour grade and pathological stage were accompanied by increasing proliferation indices, but decreasing p27Kip1 and cyclin D1 expression, with no significant change in cyclin E expression. Overall, cyclin D1 and E expression did not correlate with proliferation. However, in cyclin D1 overexpressing tumours (⩾ 5% nuclei positive), the level of cyclin D1 expression positively correlated with proliferation. The correlation between cyclin E expression and proliferation changed from positive to negative with increasing levels of cyclin E expression, accompanied by a coordinate increase in p27Kip1 expression. Overall, there was an inverse association between p27Kip1 expression and proliferation. However, a subset of tumours displayed high proliferation indices despite high p27Kip1 expression. The G1 cyclin index (sum of the level of expression of cyclins D1 and E) correlated positively with proliferation in superficial but not muscle invasive tumours. This correlation was stronger when the G1 cyclin index was adjusted for p27Kip1 expression. Conclusion: These findings support a role for these proteins in the proliferation, differentiation, and progression of bladder transitional cell carcinomas.

Differential effects of toremifene on doxorubicin, vinblastine and Tc-99m-sestamibi in P-glycoprotein-expressing breast and head and neck cancer cell lines

The effect of toremifene on P-glycoprotein-mediated multidrug resistance (MDR) in breast and head and neck cancer cell lines was measured in vitro and in vivo. Pgp expression was low and high, respectively, in drug-sensitive (MCF7-S, KB) and drug-resistant (MCF7-R, MCF7-R1, KBV1) cell lines. Toremifene (7.5 microM) significantly enhanced cytoplasmic and nuclear accumulation of doxorubicin in drug-resistant cells. Toremifene (10 microM) increased the in vitro cytotoxicity of doxorubicin in drug-resistant breast cancer cells (13-fold and 21-fold for MCF7-R and MCF7-R1, respectively) without affecting the sensitivity of MCF7-S cells. Similarly, toremifene (10 microM) caused a 12-fold increase in the sensitivity of KBV1 cells to vinblastine. In contrast, toremifene (5 microM) reduced the net uptake of the radiolabelled Pgp substrate, Tc-99m-sestamibi, in the Pgp-overexpressing cell lines by factors of 0.32 and 0.42 for MCF7-R1 and KBV1 cells, respectively (p < 0.01), and, to a lesser extent, by corresponding factors of 0.89 and 0.86 in the drug-sensitive cell lines (p < 0.05 and p > 0.05, respectively). In nude mice bearing both KB and KBV1 xenograft tumours, significantly higher tumour levels of Tc-99m-sestamibi were recorded in KB tumours compared with KBV1 tumours. After 3 days of treatment with intraperitoneal toremifene (25 mg/kg), tumour levels of Tc-99m-sestamibi were reduced in KB and KBV1 tumours but only statistically significantly for KB tumours. Toremifene is a potent MDR modulating agent with respect to chemotherapeutic agents but has the opposite effect with respect to Tc-99m-sestamibi. This finding is of importance in view of the widespread use of Tc-99m-sestamibi as an imaging surrogate for a chemotherapeutic agent.