Toshinori Ide

Steps Involved in Immortalization and Tumorigenesis in Human B-Lymphoblastoid Cell Lines Transformed by Epstein-Barr Virus

Epstein-Barr virus (EBV) is closely associated with the generation of various tumors, including Burkitt’s lymphoma. Human resting B cells from peripheral blood are easily transformed by EBV to actively proliferating B-lymphoblastoid cell lines (LCLs). These LCLs with normal diploid karyotypes have been believed to be “immortal”, without becoming tumorigenic. A series of recent studies, however, indicate that this initial, simple concept needs extensive reconsideration. Most LCLs from normal individuals are mortal because their telomeres shorten. Some LCLs are truly immortalized by developing strong telomerase activity and aneuploidy, accompanied by various other changes: down-regulation of p16/Rb; mutation of the p53 gene; modulation of apoptosis; and sensitivity to various chemical agents. Some post-immortal LCLs additionally develop the ability to form colonies in agarose and even become tumorigenic by developing the ability to grow in nude mice. The genetic background of LCLs markedly affects the frequency of immortalization. In summary, changes of B cells after infection by EBV are roughly divided into two steps: (a) transformation of B cells into LCLs caused by EBV proteins; and (b) immortalization and tumorigenesis of LCLs mainly regulated by the factors of host cells in cooperation with EBV proteins. The new concept as reviewed here is essential for the future study of tumorigenesis by EBV.

Differential regulation of human RecQ family helicases in cell transformation and cell cycle

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5–40u2009h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5β, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell.

Expression of an interferon inducible gene 6-16, G1P3, increases not only in type I interferon-treated cells but also in human senescent fibroblasts. However, the function of 6-16 protein is unknown. Here we report that 6-16 is 34xa0kDa glycosylated protein and localized at mitochondria. Interestingly, 6-16 is expressed at high levels in gastric cancer cell lines and tissues. One of exceptional gastric cancer cell line, TMK-1, which do not express detectable 6-16, is sensitive to apoptosis induced by cycloheximide (CHX), 5-fluorouracil (5-FU) and serum-deprivation. Ectopic expression of 6-16 gene restored the induction of apoptosis and inhibited caspase-3 activity in TMK-1 cells. Thus 6-16 protein has anti-apoptotic function through inhibiting caspas-3. This anti-apoptotic function is expressed through inhibition of the depolarization of mitochondrial membrane potential and release of cytochrome c. By two-hybrid screening, we found that 6-16 protein interacts with calcium and integrin binding protein, CIB/KIP/Calmyrin (CIB), which interacts with presenilin 2, a protein involved in Alzheimer’s disease. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyses are need. These results overall indicate that 6-16 protein may have function as a cell survival protein by inhibiting mitochondrial-mediated apoptosis.

Clonal chromosomal aberrations accompanied by strong telomerase activity in immortalization of human B-lymphoblastoid cell lines transformed by Epstein-Barr virus

Human B-lymphoblastoid cell lines transformed by Epstein-Barr (EBV-LCLs) are considered to be immortalized, although most of them show a normal diploid karyotype. Recently, we and others have shown that only part of EBV-LCLs is immortalized by developing strong telomerase activity that stabilizes the telomeres. In this study, we investigated the change in karyotypes during immortalization. All the eight immortalized cell lines developed clonal chromosomal aberrations accompanied by the development of strong telomerase activity. Interestingly, abnormal chromosomes were not shared among the immortalized cell lines. These results strongly suggest that chromosomal rearrangements and induction of strong telomerase activity are two events that take place in parallel in the process of immortalization of EBV-LCLs, and indicate that EBV-LCLs are clearly divided into two distinct groups, pre-immortal cell lines mostly with a normal diploid karyotype and post-immortal cell lines with a clonally abnormal karyotype.

Growth Inhibition of Human Glioma Cells by Transfection-induced P21 and its Effects on Telomerase Activity

The aim of this study is to investigate the effect of the p21 gene transfection on the growth of cultured human glioma cell lines, and analyze the telomerase activity, and detection of telomerase components in p21 transfectant.The p21 gene was transfected into human glioma cell lines, U251MG and T98G with our novel liposome. The cell growth was assessed by counting the number of trypan blue-excluding cells in a hemocytometer and flow cytometry analysis. The expression of P21 protein and its mRNA were examined by Western and Northern blot analysis. The telomerase activity was assayed by TRAP (telomerase repeat amplification protocol)/TRAP–HPA (hybridization protection assay) method qualitatively and quantitatively. The length of telomere was measured by Southern blot analysis. The expression of telomerase components (hTERT, hTERC and TEP1) were examined by RT-PCR (reverse transcriptase-polymerase chain reaction).The p21 transfectant demonstrated the expression of P21 protein and its mRNA. The p21 transfection of human glioma cells results in growth inhibition and G0/G1 arrest. The p21 transfectant revealed a decrease of telomerase activity and hTERT expression as compared with control cells.These results suggest that p21 transfection induces G0/G1 arrest in human glioma cells which associates with the reduction in the telomerase activity and hTERT expression.