Human Fibroblast Commitment to a Senescence-Like State in Response to Histone Deacetylase Inhibitors Is Cell Cycle Dependent

Abstract

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...

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