We have previously reported that expression of periostin mRNA is markedly reduced in a variety of human cancer cell lines, suggesting that downregulation of periostin mRNA expression is correlated with the development of human cancers. In our study, to clarify the role of the periostin in human bladder carcinogenesis, we examined the expression of periostin mRNA in normal bladder tissues, bladder cancer tissues and bladder cancer cell lines by Northern blot analysis and RT-PCR analysis. Although the expression of periostin mRNA was detected in 100% (5/5) of normal bladder tissues, it was not detected in 3 human bladder cancer cell lines examined. It was also detected in 81.8% (9/11) of grade 1, 40.0% (4/10) of grade 2 and 33.3% (4/12) of grade 3 bladder cancer tissues, indicating that downregulation of periostin mRNA is significantly related to higher grade bladder cancer (p<0.05). To assess the tumor suppressor function of periostin, we investigated the ability of periostin gene to suppress malignant phenotypes of a bladder cancer cell line, SBT31A. Ectopic expression of periostin gene by a retrovirus vector suppressed in vitro cell invasiveness of the bladder cancer cells without affecting cell proliferation and tumor growth in nude mice. Periostin also suppressed in vivo lung metastasis of the mouse melanoma cell line, B16-F10. Mutational analysis revealed that the C-terminal region of periostin was sufficient to suppress cell invasiveness and metastasis of the cancer cells. Periostin may play a role as a suppressor of invasion and metastasis in the progression of human bladder cancers. ' 2005 Wiley-Liss, Inc.

The increased risk of several types of cancer in Klinefelter syndrome (47XXY) suggests that the extra X chromosome may be involved in the tumorigenesis associated with this syndrome. Here, we show that cancer cells (PSK-1) derived from a patient with Klinefelter syndrome (47XXY) showing loss of an inactive X chromosome subsequently gained active X chromosomes. We found that this abnormal X chromosome composition in PSK-1 is caused by a loss of an inactive X chromosome followed by multiplication of identical active X chromosomes, not by reactivation of an inactive X chromosome. Furthermore, we extended the characterization of loss-of-inactive X in a series of 22 female-derived cancer cell lines (eight breast cancer cell lines, seven ovarian cancer cell lines, and seven cervical cancer cell lines). The data demonstrate that lossof-inactive X in the female-derived cancer cells is mainly achieved by loss of an inactive X chromosomes followed by multiplication of an identical active X chromosomes. However, distinctive pathways, including reactivation of an inactive X chromosome, are also involved in the mechanisms for loss-of-inactive X and gain-of-active X in female-derived cancer cells. The biological significance of the loss-of-inactive X and gain-of-active X in the oncogenesis of Klinefelter syndrome and female-derived cancer cells are discussed.

Abstract.We have previously reported that the expression of periostin mRNA is significantly repressed in human bladder cancer tissues, and that periostin plays a role as a suppressive factor for invasion and metastasis in the progression of human bladder cancers. In this study, to clarify the role of alternative splicing of periostin in human bladder carcinogenesis, we examined the expression of wild-type (WT) and spliced variants of periostin mRNA in normal bladder and bladder cancer tissues. Although both WT and spliced periostin mRNA were expressed in all normal bladder tissues examined, no WT periostin mRNA was detected in the examined transitional cell carcinomas (TCCs) of the bladder (0/23) or in bladder cancer cell lines (0/6). Spliced variants of periostin were detected in 48% (11/23) of TCC tissues and 33% (2/6) of bladder cancer cell lines. Two types of spliced periostin (Variants I and II) were successfully isolated from bladder cancer tissues, but Variant I, which is predominantly expressed in bladder cancer tissues, did not show suppressor activity on in vitro invasiveness and in vivo metastasis of cancer cells. Immunohistochemical analysis indicated that strong belt-like expression of periostin protein was observed in the stroma just beneath the normal bladder epithelium, while it was mostly attenuated in bladder cancer tissues. These results indicate that the loss of WT periostin by down-regulation and/or alternative splicing, which produces Variant I, is closely correlated with the development of bladder cancer.

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The human cyclin D1 gene generates two major isoforms, cyclin D1a and cyclin D1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclin D1b siRNA, we investigated the anti-oncogenic effects of cyclin D1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclin D1b mRNA. Knockdown of cyclin D1b by specific siRNA significantly suppressed cell proliferation, in vitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclin D1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclin D1b siRNA showed that cyclin D1b siRNA induced apoptosis. In T24 cells, knockdown of cyclin D1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclin D1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclin D1b siRNA. Taken together, these results indicate that knockdown of cyclin D1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclin D1b siRNA will be a novel therapy for cyclin D1b-expressing bladder cancers.