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

Abstract

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.

E2F3a Is Critically Involved in Epidermal Growth Factor Receptor–Directed Proliferation in Ovarian Cancer

“…On the other hand, VDR is not a known component of the JAK/STAT1/3 pathway, and furthermore, treatment of ovarian cancer cell lines with 1α, 25-dihydroxyvitamin D 3 , the most active metabolite of vitamin D 3 , did not affect E2F3a mRNA levels. 6 The effect of a balanced ratio between the two mutually antagonistic transcription factors IRF-1 and IRF-2 was shown earlier (28)(29)(30). Whereas overexpression of IRF-2 induced oncogenic transformation of NIH3T3 cells, stable coexpression of IRF-1 caused these cells to revert to a nontransformed phenotype (28).…”

“…Although its selective targeting poses an appealing new approach in cancer treatment, the functional complexity of the various EGFR downstream pathways in oncogenesis and cancer progression is far from fully elucidated at the molecular level. EGF and other growth factors are known to affect key transition points in the cell cycle in terms of exit from quiescence and entry into G 1 -phase or G 1 -S checkpoint transition (5,6). In this context, the E2F family of transcription factors and its governor, the retinoblastoma tumor suppressor protein, are crucially involved in regulating these transition points during the cell cycle (7)(8)(9)(10).…”

Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts.

“…3). Conversely, addition of supraphysiologic concentrations of IGFs in the face of high IGFBP-3 levels could even lead to enhanced cell replication, possibly accounting for recent observations in the residual cycling fraction of senescent HDFs (12).…”

“…A prominent characteristic of senescent HDFs, whether derived from normal donors or from subjects with WS, is unresponsiveness to the action of various polypeptide mitogens present in fetal bovine serum (FBS) including insulinlike growth factor I (IGF-I), platelet-derived growth factor, and epidermal growth factor (7)(8)(9)(10)(11)(12). However, no significant alterations have been detected in the number of binding sites or in the binding affinity of IGF-I and these other factors after correction for the increased surface area of enlarged senescent HDFs (ref.…”

Epidermal growth factor receptor-mediated motility in fibroblasts

“…It is well established that primary fibroblasts and keratinocytes have limited proliferative capacity in vitro, and that the extent of doublings is inversely correlated to the age of the donor (Bruce and Deamond, 1991). In addition, higher population doublings (PDL) of human diploid fibroblasts (HDF) demonstrate decreased basal and EGF-and PDGF-induced proliferation, independent of growth factor concentration (Carlin et al, 1983;Chen and Rabinovitch, 1990;Tang et al, 1994). Senescent human umbilical vein endothelial cells do not respond to FGF (Garfinkel et al, 1996).…”