Attenuation of genotoxicity under adhesion-restrictive conditions through modulation of p53,γ H2AX and nuclear DNA organization

Interactions between tumor cells and their substratum influence cancer progression by modulating cell proliferation and survival. We now investigated whether signaling responses to UV irradiation differ on adhesion-permissive or restrictive substrates. The latter conditions diminished spreading and proliferation of neo 6.3/C8161 melanoma in which metastasis is suppressed by introduction of neo-tagged chromosome 6, but permitted proliferation of human metastatic C8161 melanoma. Apoptosis-associated PARP cleavage and DNA fragmentation induced by UV irrradiation were diminished on the restrictive substrate in C8161 melanoma. Genotoxic responses to UV irradiation like persistent increases in the phosphorylation of histone H2AX, induction of the tumor suppressor p53 protein and greater binding of this protein to its DNA consensus sequence, were all decreased on the restrictive substrate. The latter also promoted a 2 fold increase of DNA condensation in chromatin and enhanced activation of the survival - and invasion-associated MMP-9 gelatinase B, preferentially in metastatic C81261 melanoma. Our data suggest that adaptation to restrictive substrates in metastatic C8161 melanoma decreases UV-induced apoptosis, partly through attenuation of DNA damage signaling responses and changes in genomic organization. Mary Strasberg Rieber and Manuel Rieber: This research was supported by the Terry Fox Run for Cancer Research (CIC-CCA) to MR.     

Regulation of cyclin-dependent kinase inhibitor p21<Superscript>WAF1/CIP1</Superscript> by protein kinase Cδ-mediated phosphorylation

Cyclin-dependent kinase (CDK) inhibitor p21^{WAF1/CIP1(-/-)}-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21^WAF1/CIP1 is unstable in HeLa cells treated with siRNA duplexes that target PKCδ. PKCδ phosphorylates p21^WAF1/CIP1 at a serine residue (¹⁴⁶Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21^WAF1/CIP1 and its ¹⁴⁶Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism (SNP) at ¹⁴⁹Asp found in certain cancer patients, strongly compromises PKCδ-mediated phosphorylation at ¹⁴⁶Ser and results in cells that are relatively resistant to TNFα-induced apoptosis. Thus, post-translational phosphorylation of p21^WAF1/CIP1 is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer.     

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21(WAF1/CIP1) expression

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.     

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.