Altered cell cycle responses to insulin-like growth factor I, but not platelet-derived growth factor and epidermal growth factor, in senescing human fibroblasts

Human diploid fibroblasts (HDF) were used to study aging-related changes in the proliferative response to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor I (IGF-I, somatomedin-C) in serum-free, chemically defined culture medium. Cell cycle kinetic parameters were determined by using 5-bromodeoxyuridine incorporation and flow cytometric analysis with the DNA stain Hoechst 33258. This allowed analysis of the growth factor response to be focussed exclusively upon of the cycling faction of cells within the culture, even in senescent cell cultures which contained predominantly nondividing cells. PDGF and EGF exert their primary effect upon regulation of the proportion of cycling cells in the culture. The doses of PDGF and EGF that produced a half-maximal cycling fraction, analogous to Km, showed no large or consistent difference between young- and old-passage cells. In contrast, IGF-I primarily affects the rate of transition of cells from G1 into S phase, and the dose of IGF-I which produced a half-maximal rate of G1 exit increased up to 130-fold in older-passage cells. Unexpectedly, supraphysiologic concentrations of IGF-I were found to increase the G1 exit rate of the dividing subpopulation of cells in older-passage cultures to rates higher than those seen in young cultures. In summary, among cells capable of cycling in aging cultures, there were few changes in the regulation of the growth fraction by PDGF and EGF, but there was a greatly increased dependence on IGF-I for regulation of the rate of entry into S phase. The slower growth of the dividing population of cells in aging cultures may be related to a requirement for IGF-I at levels which are greatly above those usually supplied.