Tsutomu Shirahama

Overexpression of bax sensitizes human breast cancer MCF‐7 cells to radiation‐induced apoptosis

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl‐2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF‐7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation‐induced apoptosis. bax overexpression in MCF‐7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF‐7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAFI about 5‐ to 10‐fold and downregulated bcl‐2 and bcl‐XL by 80–90% at 6 hr in both parent and bax stably transfected MCF‐7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF‐7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Relation between cyclooxygenase‐2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder

Expression of the inducible form of cyclooxygenase (COX)‐2 is known to correlate with development of transitional cell carcinoma (TCC) of the human urinary bladder. However, the clinical significance of COX‐2 expression with respect to clinicopathologic findings and patient survival is unknown.

Suppression of Bcl-2 gene expression by sphingosine in the apoptosis of human leukemic HL-60 cells during phorbol ester-induced terminal differentiation

Our recent studies have shown that intracellular levels of sphingosine, an endogenous PKC inhibitor, increase during apoptosis resulting from phorbol ester (PMA)‐induced terminal differentiation of human myeloid leukemic HL‐60 cells, and have suggested that sphingosine may function as an endogenous mediator of apoptosis in these cells [Ohta, et al. (1995) Cancer Res. 55, 691–697]. We report here that apoptosis induced by PMA, sphingosine, and N,N‐dimethylsphingosine (DMS) was accompanied by a concomitant decrease of bcl‐2 expression in both RNA and protein levels in HL‐60 cells, while expression of bcl‐XL and bax mRNA did not change, and neither sphingosine nor DMS induced differentiation of HL‐60 cells. In contrast, in apoptotic cells induced by pharmaceutical PKC inhibitors H7 or staurosporine, expression of bcl‐2 did not change nor did the intracellular sphingosine concentration. These results suggest that sphingosine may function as an endogenous mediator of apoptotic signaling in PMA‐induced terminal differentiation of HL‐60 cells through bcl‐2 down‐regulation, probably independent from PKC inhibition.

Expression of a carbohydrate signal, sialyl dimeric Lex antigen, is associated with metastatic potential of transitional cell carcinoma of the human urinary bladder

Sialyl dimeric Le(x) antigen was expressed in significant portion of transitional cell carcinoma of the human urinary bladder, but not in the normal uroepithelial tissue. Primary tumors with weak or no expression of the antigen scarcely metastasized to lymph nodes, whereas tumors with high levels of antigen expression metastasized frequently. Metastatic lymph nodes expressed the antigen in most cases. Sialyl dimeric Le(x) antigen was mainly located on 60 and 42 KDa glycoproteins. Since a group of cell adhesion molecules, called LECCAMs, recognize a portion of the antigen, the above results strongly suggest that a LECCAM on the surface of host cells recognizes the carbohydrate structure on the glycoprotein, leading to promotion of metastasis.

Sphingosine induces apoptosis in androgen‐independent human prostatic carcinoma DU‐145 cells by suppression of bcl‐XL gene expression

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL‐60 cells [Ohta et al. Cancer Res. 1995;55:691–697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down‐regulation of bcl‐2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177–180]. In this study, we examined the sphingosine‐induced apoptosis of the androgen‐independent human prostatic carcinoma cell line DU‐145, which expresses bcl‐XL and Bax but not bcl‐2, and found that treatment of DU‐145 cells with sphingosine suppressed bcl‐XL in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl‐XL or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1‐phosphate, failed to induce apoptosis. These results indicate that, in DU‐145 cells, sphingosine, but not its metabolites, induces apoptosis through down‐regulation of bcl‐XL, independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl‐2 or bcl‐XL activity in these cells.

Expression of the multidrug transporter, P‐glycoprotein, in renal and transitional cell carcinomas

Background. Renal cell carcinomas (RCC) respond poorly to anthracyclines, Vinca alkaloids, and other agents. P‐glycoprotein is overproduced in multidrug‐resistant cells and thought to function as an energy‐dependent drug efflux pump. The authors thus examined the expression level of P‐glycoprotein in RCC and transitional cell carcinomas (TCC).

Overexpression of Bax Sensitizes Breast Cancer MCF-7 Cells to Cisplatin and Etoposide

Bax, one of thebcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weakbax gene expression, were stably transfected withpCX2neo bax, encoding humanbax; and two unique clones,MCF-7/bax-1 andMCF-7/bax-2, that expressed different levels ofbax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level ofbax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenousbax-α overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.

Inhibition of 5‐lipoxygenase pathway suppresses the growth of bladder cancer cells

Aim: Many stimuli, including growth factors and cytokines, activate arachidonic acid (AA) metabolic pathways, which are involved in cancer development and progression. We examined the effects of a series of pharmacological inhibitors of AA metabolic enzymes on bladder cancer cells to determine the role of AA pathway in this malignancy.

OVEREXPRESSION OF BAX ENHANCES THE RADIATION SENSITIVITY IN HUMAN BREAST CANCER CELLS

Bax-a, a splice variant ofbax which promotes apoptosis, is expressed in many kinds of untransformed cell lines and breast tissue, whereas only weak or no expression could be detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weakbax gene expression, were stably transfected with pCX2neobax encoding humanbax and neomycin-resistant genes, and two unique clones (MCF-7/bax-1 and MCF-7/bax-2) were thus generated which expressed different levels ofbax-α. Sensitivity to ionizing radiation (IR) was examined and each was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level ofbax, and IR was found to induce intranucleosomal DNA fragmentation in stable transfectant but not in parent cells, thus suggesting that this sensitization is due to apoptosis. We suggest that exogenousbax-α expression might therefore be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may potentially have a therapeutic application by enhancing radiation sensitivity in breast cancer cells.

Expression of SSEA-1 Carbohydrate Antigen Correlates with Stage, Grade and Metastatic Potential of Transitional Cell Carcinoma of the Bladder

Expression of stage-specific embryonic antigen-1 (SSEA-1) was immunohistochemically analyzed in primary cancerous regions from transitional cell carcinoma of the bladder. Lymph node metastasis occurred in 14 (50%) of 28 patients with high expression of SSEA-1 antigen, but in only one (4%) of 23 patients with low or no expression. Of 36 invasive bladder carcinoma patients with no metastasis at operation, 8 (57%) of 14 with high expression of SSEA-1 antigen died of cancer or had metastasis, whereas 4 (18%) of 22 with low or no expression had progression of the disease. In addition, stage pT3b-4 tumors more frequently reacted with SSEA-1 MAb than stage pT1 tumors (p less than 0.01). Grade 2 or 3 tumors were also more reactive than grade 1 tumors, which were all unreactive except for one (p less than 0.01, respectively). Our data suggest that SSEA-1, which is a Le(x) antigen, correlates with stage and grade as well as metastatic phenotype of transitional cell carcinoma of the bladder.