Defining cellular senescence in IMR-90 cells: a flow cytometric analysis.

Abstract

Using multiparameter flow cytometric analysis, we find that senescent cells accumulate in a unique cell-cycle compartment characterized in cell-cycle arrest in G, and a significantly reduced nucleocytoplasmic ratio (genome size/cell mass) relative to cycling cells. With respect to gross cellular phenotype, the quiescent state of senescent cells differs from quiescence induced by density inhibition; the former is associated with a reduction in the nucleocytoplasmic ratio, while the latter is associated with an increase in the nucleocytoplasmic ratio. Senescent cells were present at all passages examined. The frequency of senescent cells was low in earlypassage cultures and increased with passage number. Senescence of populations of IMR-90 cells reflects change in the relative frequency of these cells. The frequency of cells with karyotypic changes increased with the progressive accumulation of out-of-cycle cells.Human diploid fibroblast cells display the phenomenon of cellular senescence and have frequently been used as a model system to study in vitro cellular aging (1, 2). Senescence is defined as the loss ofproliferative capacity ofthe cultures and results in the inability of the population to increase in cell number typically after cultures have been passaged 50 or more times. Senescence is associated with a number of gross cellular changes including cell-cycle arrest (3, 4), increase in cell size and size heterogeneity (5-8), and increase in the frequency of cells with chromosomal aberrations, including polyploidy (9-12). However, molecular data indicate that young and senescent cells do not consistently differ in the pattern of regulation of genes that are sensitive to serum stimulation (13) and several aspects of DNA replication have been reported not to change with culture age (14,15). Thus, while senescent cells may retain normal DNA synthetic capacity, they fail to initiate DNA synthesis and the decline and finally loss of proliferative capacity involves alterations in some process(es) necessary for the initiation of DNA synthesis and/or cell-cycle progression.We have examined the nature of the cell-cycle changes and correlated changes in the frequency of cells with karyotypic abnormalities occurring in IMR-90 cells (16) carried from passage 25 (young cells) to passage 53 (senescent cells). Using multiparameter flow cytometric analysis, we find an age-related increase in the frequency of cells that, based on DNA content, are blocked at the G1/S (diploid and tetraploid) boundary but that become very large (>2 times the population mean) in size relative to cycling cells in the same cell-cycle phase. These large cells fail to label when grown in the presence of bromodeoxyuridine (BrdU) for up to 12 hr while small cells, even in late-passage cultures, do label. Senescent cells thus occupy a unique cell-cycle compartment definable on the basis of altered cell-cycle progression and nucleocytoplasmic ratio. Karyotypic changes associated with cellular aging induced an increase in the frequency of aneupl...

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