Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research最新文献

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.

As cell cycle regulators whose activity is frequently altered in human cancers, cyclin-dependent kinases (cdks) are novel targets for therapeutic intervention. cdk inhibition is an emerging strategy for the treatment of non-small cell lung carcinomas (NSCLCs) because most derived cell lines express functional retinoblastoma protein (Rb) but appear to bypass its function with inappropriate cdk activity. Elevated cdk4/cdk6 activity in NSCLC cells is often due to inactivation of the p16Ink4a cdk inhibitor. To model the effects of cdk4/cdk6 inhibition, we have expressed p16Ink4a in a Rb-positive NSCLC cell line that lacks endogenous p16Ink4a expression. Whereas cdk4/cdk6 inhibition and Rb dephosphorylation are expected on p16Ink4a expression, we have also observed indirect cdk2 inhibition. cdk2 inactivation by the redistribution of other cdk inhibitors may be required for p16Ink4a-mediated growth suppression of Rb-positive cells. The implications of such a requirement on the use of chemical cdk inhibitors to treat human cancers will be discussed.

The Nm23 protein has been implicated in a wide variety of biological processes, including suppression of metastasis, phytochrome responses in plants, and regulation of differentiation. Here we examine whether Nm23 is involved in Xenopus laevis oocyte maturation. We found that Nm23 is present in oocytes, indicating that it has the potential to be a regulator of maturation. Furthermore, modest overexpression of Nm23 inhibited progesterone-induced oocyte maturation. This maturation-inhibitory activity was shared by both the acidic Nm23-H1 isoform and the basic Nm23-H2 isoform and by Nm23 mutants that lack nucleoside diphosphate kinase activity (Nm23-H1 H118F and Nm23-H2 H118F). Expression of Nm23 proteins delayed the accumulation of Mos and the activation of p42 mitogen-activated protein kinase (MAPK) in progesterone-treated oocytes but had no discernible effect on Mos-induced p42 MAPK activation. Therefore, Nm23 appears to act upstream of the Mos/mitogen-activated protein/extracellular signal-regulated kinase kinase/p42 MAPK cascade. These findings suggest a novel biological role for Nm23.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.

Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.

The K562 cell line undergoes megakaryocytic differentiation in response to phorbol 12-myristate 13-acetate (PMA) stimulation. This event correlates with mitogen-activated protein kinase activation, cell cycle arrest, and expression of the Egr-1 transcription factor. We have isolated K562 cells that are resistant to the growth-inhibitory action of PMA. Molecular characterization demonstrates that PMA resistance is downstream from PMA-induced activation of the mitogen-activated protein kinase pathway. Although the levels of Egr-1 expression and cyclic AMP-responsive element-binding protein phosphorylation are comparable in wild-type and PMA-resistant clones in response to PMA, the expression of megakaryocytic cell surface marker CD41 is detected only in the wild-type cells. The lack of differentiation of the PMA-resistant clones correlates with a failure of the PMA-treated cells to induce dephosphorylation and down-regulation of the retinoblastoma protein. These cells may provide a useful model system to distinguish those events that are connected to cell cycle arrest from those involved in the differentiation program initiated by PMA.

p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose expression in transfected cells retards proliferation. Interestingly, the reduced levels of p202 in fibroblasts (in consequence of the expression of antisense to 202 RNA), under reduced serum conditions, increase the susceptibility of cells to apoptosis. To identify the functional role of p202 in cell growth regulation, we tested whether serum growth factor levels in the culture medium affect p202 levels. Here we report that, under reduced serum conditions, the p202 levels were increased in fibroblasts, and the increase was seen at both the mRNA and protein levels. Moreover, an increase in p202 levels was correlated with cell growth arrest in the G1 phase of the cell cycle. Interestingly, the presence of platelet-derived growth factor AB, basic fibroblast growth factor, or transforming growth factor beta1 in the culture medium abrogated the increase in p202 levels seen under reduced serum conditions. We found that the increase in p202 levels was accompanied by an increase in JunD/activation protein 1(AP-1) levels, and transfection of a JunD-encoding plasmid along with a reporter plasmid in which transcription of the reporter gene (luciferase) was driven by the 5'-regulatory region of the 202 gene resulted in an increase in the activity of luciferase. Additionally, stable overexpression of JunD in cells, under reduced serum conditions, also resulted in an increase in p202 levels. Interestingly, one of the AP-1-like DNA-binding sequences present in the 5'-regulatory region of the 202 gene could selectively bind to the JunD/AP-1 transcription factor. Taken together, our observations reported herein suggest that in fibroblasts, under reduced serum conditions, the increased levels of JunD/AP-1 contribute to the transcriptional up-regulation of p202 levels, which may be important for the regulation of apoptosis.

The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.

Previously, we found that vorozole (Vz), a nonsteroidal aromatase inhibitor, suppresses the development and progression of mammary tumors in rats. Here we evaluated for the first time the expression of cell death-related proteins Bcl-2 and Bax in hyperplastic, premalignant (carcinoma in situ), or malignant (carcinoma) lesions of mammary carcinogenesis; we also assessed whether these proteins are involved in mediating Vz-induced cell death in tumors. We found that Bcl-2 and Bax were equally expressed in epithelial cells of terminal end buds, ducts, and alveoli. However, in myoepithelial cells, the level of Bax expression was much higher than the level of Bcl-2 expression. Bcl-2 and Bax levels in hyperplastic lesions were similar to those of normal mammary epithelial cells but lower in most carcinomas in situ and carcinomas. In animals with established mammary tumors, Vz induced apoptotic cell death, which was primarily associated with a decrease in Bcl-2 and, to a lesser extent, with a decrease in Bax. These data support the hypothesis that Bcl-2 loss is more potent than Bax gain in regulating apoptotic cell death in mammary tumors.

RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.