Effects of 1alpha,25-dihydroxyvitamin D3 and growth hormone on apoptosis and proliferation in UMR 106 osteoblast-like cells.

Mechanisms maintaining a correct balance between bone-forming osteoblasts and bone-resorbing osteoclasts are essential for bone formation. Apoptosis has been proposed to play a key role in controlling osteoblast homeostasis. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and GH, which are important regulators of bone growth and bone metabolism, also play pivotal roles in regulation of mitogenesis, differentiation, and apoptosis. We have recently shown that 1,25(OH)2D3 prolongs GH signaling via the Janus kinase 2 (JAK2)/STAT5 (signal transducer and activator of transcription 5) pathway in UMR 106 osteoblast-like cells. In the present study, we have investigated the effects of GH and 1,25(OH)2D3 on proliferation and apoptosis in UMR 106 cells. We found that 1,25(OH)2D3 and GH, separate or in combination, inhibited apoptosis. GH also had profound effects on cell cycle distribution and proliferation. In addition, pretreatment of cells with 1,25(OH)2D3 was necessary to detect GH-induced MAPK activation. We hypothesize that these hormones separately regulate the processes of apoptosis and proliferation, which may be important for maintaining osteoblast cell number.

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells.

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.

A pro-apoptotic effect of the CDK inhibitor p57(Kip2) on staurosporine-induced apoptosis in HeLa cells.

Apoptosis, or programmed cell death, is involved in many biological events, including tumorigenesis. Recently, it has been reported that two members of the Cip/Kip family of CDK inhibitors, p21(Cip1) and p27(Kip1), are involved in the regulation of apoptosis. Here, we report that selective expression of the third member in this family, p57(Kip2), potentiated staurosporine-induced apoptosis in HeLa cells. This pro-apoptotic effect was associated with an increased caspase-3 activity. In contrast, glucocorticoid treatment, despite inducing p57(Kip2) expression in HeLa cells, was found to have an inhibitory effect on staurosporine-induced apoptosis. This anti-apoptotic effect of glucocorticoids could be explained by a concomitant increase in Bcl-x(L) expression. The results presented in this study show that p57(Kip2) has a stimulatory effect on apoptosis induced by staurosporine, suggesting a role for p57(Kip2) in the response of tumor cells to cytotoxic drugs.

Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2.

BCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFN gamma-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors.