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Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts.

ABSTRACT: Early-passage human diploid fibroblasts (HDFs) undergo senescence-like growth arrest in response to sublethal concentrations of H(2)O(2) [Chen and Ames (1994) Proc. Natl. Acad. Sci. USA. 95, 4130-4134]. We determine here whether H(2)O(2) can cause apoptosis in HDFs and the molecular changes that differ between apoptosis and senescence-like growth arrest. When exponentially growing early-passage IMR-90 cells were treated for 2 h with 50-200 microM (or 0.25-1 pmol/cell) H(2)O(2), a fraction of cells detached at 16-32 h after the treatment. The cells remaining attached were growth-arrested and developed features of senescence in 1 week. The detached cells showed caspase-3 activation and typical morphological changes associated with apoptosis. Caspase-3 activation was H(2)O(2) dose-dependent and preceded nuclear condensation or plasma membrane leakage. Apoptotic cells were mainly distributed in the S-phase of the cell cycle, while growth-arrested cells exhibited predominantly G1- and G2/M-phase distributions. H(2)O(2) pretreatment induced G1 arrest and prohibited induction of apoptosis by a subsequent H(2)O(2) challenge. The p53 protein showed an average 6.1-fold elevation in apoptotic cells and a 3.5-fold elevation in growth-arrested cells. Reduction of p53 levels with human papillomavirus E6 protein prohibited the activation of caspase-3 and decreased the proportion of apoptotic cells. Growth-arrested cells had elevated p21, while p21 was absent in apoptotic cells. Bcl-2 was elevated in both growth-arrested and apoptotic cells. Finally, although the overall level of bax did not change in growth-arrested or apoptotic cells, the solubility of bax protein increased in apoptotic cells. Our data suggest that in contrast with growth-arrested cells, apoptotic cells show an S-phase cell cycle distribution, a higher degree of p53 elevation, an absence of p21 protein and increased solubility of bax protein.

SUBMITTER: Chen QM

PROVIDER: S-EPMC1220988 | biostudies-other | 2000 Apr

REPOSITORIES: biostudies-other

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Project description:Human diploid fibroblasts lose the capacity to proliferate and enter a state termed replicative senescence after a finite number of cell divisions in culture. When treated with sub-lethal concentrations of H2O2, pre-senescent human fibroblasts enter long-term growth arrest resembling replicative senescence. To understand the molecular basis for the H2O2-induced growth arrest, we determined the cell cycle distribution, levels of p53 tumour suppressor and p21 cyclin-dependent kinase inhibitor proteins, and the status of Rb phosphorylation in H2O2-treated cells. A 2-h pulse of H2O2 arrested the growth of IMR-90 fetal lung fibroblasts for at least 15 days. The arrested cells showed a G1 DNA content. The level of p53 protein increased 2- to 3-fold within 1.5 h after H2O2 exposure but returned to the control level by 48 h. The induction of p53 protein was dose dependent, beginning at 50-75 microM and reaching a maximum at 100-250 microM. The induction of p53 did not appear to correlate with the level of DNA damage as measured by the formation of 8-oxo-2'-deoxyguanosine in DNA. The level of p21 protein increased about 18 h after H2O2 exposure and remained elevated for at least 21 days. During this period, Rb remained underphosphorylated. The induction of p53 by H2O2 was abolished by the iron chelator deferoxamine and the protein synthesis inhibitor cycloheximide. The human papillomavirus protein E6, when introduced into the cells, abolished the induction of p53, reduced the induction of p21 to a minimal level and allowed Rb phosphorylation and entry of the cells into S-phase. The human papillomavirus protein E7 reduced the overall level of Rb and also abolished H2O2-induced G1 arrest. Inactivating G1 arrest by E6, E7 or both did not restore the replicative ability of H2O2-treated cells. Thus H2O2-treated cells show a transient elevation of p53, high level of p21, lack of Rb phosphorylation, G1 arrest and inability to replicate when G1 arrest is inactivated.

Project description:The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

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Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts.

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