p21WAF1 mRNA Research Articles

Infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins.

It has been demonstrated that infection of primary human cells with adeno-associated viruses (AAV) leads to a decrease in cellular proliferation and to growth arrest. We analyzed the molecular basis of this phenomenon and observed that infection with AAV type 2 (AAV2) had an effect on several factors engaged in the control of the mammalian cell cycle. In particular, all of the pRB family members, pRB, p107, and p130, which are involved in G1 cell cycle checkpoint control, were affected. After infection, a shift from hyper- to hypophosphorylated forms was observed. Cyclins A and B1, which are required for G1/S transition and progression into mitosis, respectively, were downregulated at the transcriptional level as well as at the protein level, whereas the G1 cyclins D1 and E remained unaffected. In addition, the steady-state levels of cyclin-dependent kinases CDK1 and CDK2 and of transcription factor E2F-1 were diminished. Of all the factors known to be involved in phosphorylation of pRB family proteins, only the CDK inhibitor p21WAF1 exhibited a response to AAV2 infection. p21WAF1 mRNA was quickly and progressively upregulated in a p53-independent manner over at least 72 h. Consistent with the increased p21WAF1 protein levels, cyclin E- and cyclin A-dependent kinase activities declined to low levels and E2F-p130-cyclin-CDK2 complexes were disrupted. From these data, we conclude that the major effect of AAV2 infection on primary human fibroblasts appears to be upregulation of p21WAF1 gene expression and thus cell cycle arrest by the suppression of pRB family protein phosphorylation.

Resistance to p53-mediated growth arrest and apoptosis in Hep 3B hepatoma cells.

Mutations in the tumor suppressor p53 are a common event in hepatocellular carcinoma (HCC). Because HCCs typically occur in livers with chronic injury and impaired function, we have explored the role of wild-type p53 in regulating the growth and differentiation of Hep 3B hepatoma cells, a p53-negative line derived from a liver cancer. Stable Hep 3B cell lines were generated in which inducible p53 was introduced using either a temperature-sensitive mutant (p53val135) or a tamoxifen-regulated p53-estrogen receptor chimera (p53-mERtm-pBabepuro). In both cell lines, induction of transcriptionally active p53 was confirmed by assessing several p53 targets: Mdm2 protein, p21waf1 mRNA and protein, and the cyclin G promoter. Despite marked induction of p21waf1, cells with active p53 failed to undergo growth arrest, which is probably due to the presence of a non-functional retinoblastoma protein (pRb) in these cells. Apoptosis also was not observed, even after prolonged (48 h) serum starvation or exposure to cisplatinum. Lack of apoptosis was correlated with unchanged bax mRNA levels following p53 induction. Additionally, albumin mRNA levels remained unchanged, and there was no change in basal transactivation of a reporter containing the promoter of the haptoglobin gene, encoding an acute phase protein. This suggests that growth arrest may be required to promote liver-specific gene expression. Overall, our data demonstrate that introduction of transcriptionally active p53 does not alter the malignant, dedifferentiated phenotype of Hep 3B hepatoma cells. Hence, not all cancer cells are equally responsive to the re-activation of wild-type 53. The ability of a cancer cell to undergo p53-mediated phenotypic alterations may depend on the retention of functional downstream effector pathways.

P21WAF1 expression during spermatogenesis of the normal and X-irradiated rat

The cyclin dependent kinase inhibitor p21 WAF1 has been shown to be upregulated during differentiation and after DNA damage in somatic cells. We examined the expression of p21WAF1 mRNA during the differentiation of germ cells in normal and X-irradiated rat testis by in situ hybridization and Northern blotting. p21 WAF1 was normally expressed in primary spermatocytes of the pachytene phase, but could also be detected in round spermatids. In preparations of defined segments of the seminiferous tubules, the strongest hybridization signals were detected in the segments containing stages VII-VIII and IX-XII of the seminiferous epithelium. Ionizing radiation (1-12 Gy) induced the expression of p21 WAF1 in a dose-dependent manner and the lowest dose that showed a clear increase in mRNA levels was WAF1 3 Gy. The p21 mRNA levels peaked after 3-4 hours, but remained high compared with the control levels during the 24-h follow-up. No change in the in situ hybridization pattern was seen when comparing unirradiated and irradiated tissue. Thus, it appears that X-irradiation induces p21 WAF1 in the pachytene spermatocytes. Since p21 WAF1 mRNA was found in pachytene spermatocytes and in round spermatids in normal testis, the protein may take part in the regulation of meiosis and in the 'terminal' differentiation of the male germ cells.

The Cyclin-Dependent Kinase Inhibitor p21WAF1 Is Required for Survival of Differentiating Neuroblastoma Cells

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.