Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O 2 ) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H 2 O 2 represents a critical signal mediating replicative senescence.

Human histone deacetylases I (HDAC1) and II (HDAC2) are homologous proteins (84% identity) that catalyze release of acetyl groups from modified N-terminal lysines of core histones. Histone deacetylation is correlated with both transient and persistent states of transcriptional inactivity (i.e. silencing) in many eukaryotes. In this study, we analyzed complexes containing HDAC1 and HDAC2 to identify the proteins most stably associated with these deacetylases. Complex cI (9.5 S) contained transcriptional corepressor CoREST/ kiaa0071 and a protein homologous to FAD-dependent oxidoreductases, kiaa0601. Complex cII (15 S) contained >15 proteins, including CHD3/4 (Mi-2), Mta-L1, RbAp48/ 46, and MBD3, characteristic of vertebrate nucleosomeremodeling complexes. Under native conditions, cI and cII may contain HDAC1, HDAC2 or both; these can be dissociated to cI and cII core complexes containing only HDAC1 or HDAC2. The m CpG-binding protein MBD2 was associated only with the HDAC1 cII core complex. A model is proposed in which HDAC1 core complexes can be targeted to methylated DNA via MBD2 with recruitment of HDAC2 occurring through formation of HDAC1/2 cII dimers. We note that the cI component CoREST/kiaa0071 and the cII component Mta-L1 share a region of homology that includes a SANT domain; this domain may play a role in complex assembly.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G 1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.Cellular senescence in human diploid fibroblasts (HDF) and other human cell types has been extensively studied (17,26,35,53,73,85) yet remains incompletely understood. On the basis of current knowledge, it is not unreasonable to suppose that multiple mechanisms contribute to the senescence phenotype and that the relative contributions of such mechanisms may vary for different cell types and conditions of cell propagation. At least two senescence-associated changes, increased oxidative stress (74, 75) and telomere shortening (33,34,55,87), have been intensively studied and are widely viewed as important components of in vitro aging. Less well studied is a third aspect of senescence, namely, its apparent relatedness to differentiation (4,16,26,48,57). Although these two processes are not necessarily synonymous (60, 88), in many respects senescence resembles a partial or aberrant form of terminal differentiation, with cells appearing to acquire a phenotype that is suboptimal for tissue function and maintenance. HDF proliferative potential is dependent primarily on the number of rounds of DNA synthesis completed rather than the cumulative time in culture (17,19,27); thus, to the extent that senescence is akin to terminal differentiation, it most closely resembles model systems (e.g., murine erythroleukemia or promyelocytic leukemia cells) where a requirement for passage through the cell cycle has been demonstrated (7,45,46).Our attention to the differentiative aspects of senescence was first prompted by experiments designed to compare cell cycle arrest mechanisms in senescent and quiescent cells. Transient expression of simian virus 40 (SV40) T antigen is strongly mitogenic, i.e., is dominant over cell cycle arrest mechanisms, in both senescent cells and serum-deprived quiescent fibroblasts (29,39,80). We and others observed that in senescent HDF, but not quiescent cells, the domain in T antigen which mediates binding to retinoblastoma (Rb) family proteins is required for efficient stimulation of DNA synthesis (8a, 65). This...