Paul D. Phillips

Growth regulation of WI38 cells in a serum-free medium☆

Abstract A serum-free growth medium composed of MCDB-104 supplemented with platelet-derived growth factor (PDGF) (3 μg/ml), epidermal growth factor (EGF) (100 ng/ml), insulin (INS) (5 μg/ml), transferrin (TRS) (5 μg/ml), and dexamethasone (DEX) (55 ng/ml) supports the proliferation of WI38 cells at a rate and to a density similar to that of medium supplemented with 10% fetal bovine serum (FBS). EGF exerts its effect at moderate cell densities while PDGF appears to modulate cell proliferation at high densities. Cells seeded into EGF-, INS-, TRS-, and DEX-supplemented medium enter S phase approx. 3 h earlier than cells seeded into PDGF-, EGF-, INS-, TRS- and DEX-supplemented medium or FBS-supplemented medium.

EGF- and PDGF-stimulated phosphorylation in young and senescent WI-38 cells☆

We have examined the ability of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to stimulate cultures of young and senescent WI-38 cells to carry out tyrosine-specific phosphorylation of their respective membrane receptors. Previously we reported no reduction in EGF-stimulated phosphorylation in plasma membrane preparations of senescent cells. In this study we found no reduction in PDGF-stimulated phosphorylation in plasma membrane preparations from senescent cells. Furthermore, we found no differences in the EGF- or PDGF-stimulated phosphorylation of their respective receptors in intact cells. These data support the previous findings that although the EGF receptor autokinase activity becomes highly labile during extraction and immunoprecipitation of senescent cells, in situ loss of receptor tyrosine kinase activity is apparently not responsible for the age-associated loss of mitogenic responsiveness.

Growth factors as probes of cell aging

We present examples of four types of alterations which contribute to the senescence phenotype of WI-38 cells: a) in senescent cells there is an increased lability of the tyrosine autophosphorylation capacity of detergent isolated EGF receptor; b) following serum stimulation, the calmodulin protein level fails to increase in senescent cells, although the calmodulin mRNA level increases as expected; c) following heat shock at 43 degrees C, senescent cells produce both less RNA and less protein for the HSP70 and HSP90 genes; d) we find that membranes isolated in basic buffer from senescent or young cells increase the EGF proliferative response of senescing cells, in contrast to the finding by others that membranes isolated in neutral buffer inhibit cell proliferation (Pereira-Smith et al., Senescent and quiescent cell inhibitors of DNA synthesis Exp. Cell Res. 160, 297-306, 1985; Stein and Atkins, Membrane-associated inhibition of DNA synthesis in senescent human diploid fibroblasts: Characterization and comparison to quiescent cell inhibitor. Proc. Natl. Acad. Sci. USA 83 9030-9034, 1986). We conclude that senescence alterations are complex and occur at many levels, and that senescence changes are not identical to quiescence features.

Classification system based on the functional equivalency of mitogens that regulate WI-38 cell proliferation

Analysis of the proliferative response of WI-38 cells to nine mitogens, which in various specific combinations stimulate DNA synthesis in these cultures, delineated three classes of mitogens. Class I includes epidermal growth factor (EGF), fibroblasts growth factor (FGF), platelet-derived growth factor (PDGF), and thrombin (THR); Class II includes insulin-like growth factor I (IGF-I), multiplication stimulating activity (MSA) (the rat homolog of human IGF-II), and insulin; and Class III includes hydrocortisone (HC) or the synthetic analog dexamethasone (DEX). In cultures arrested at low density, members of each of the three classes act synergistically in stimulating DNA synthesis. Any Class I mitogen in combination with any Class II and either Class III mitogen stimulated DNA synthesis of levels observed in 10% serum-supplemented medium. At least some (EGF, FGF, PDGF) and possibly all (THR) of the Class I mitogens are known to act through separate receptor systems. Our experiments using blocking antibodies to the IGF-I receptor confirm that the Class II mitogens all act by binding to IGF-I receptors. Use of the inhibitory synthetic glucocorticoid analog RU 486 confirmed that the Class III mitogens act via the glucocorticoid receptor. Thus, growth factor-induced DNA synthesis in WI-38 cells is apparently mediated by the glucocorticoid receptor (Class III), the IGF-I receptor (Class II), and most interestingly any one of several Class I growth factor receptors.

Cellular Senescence: Factors Modulating Cell Proliferation In Vitro

In our view, two major areas of the investigation of the aging process have been most fruitful over the past few years: namely, the genetic and hormonal strategies aimed at the understanding of in vitro cellular senescence. The genetic studies have primarily utilized cell fusion techniques and viral probes. Along with cell cycle studies involving the induction of thymidine kinase activity and TTP synthesis, the cell fusion studies are most consistent with a late G1 block in senescent cells. This effect would appear to be distinct from the G0 arrest of density-inhibited or mitogen-restricted cell populations. The hormonal studies which have centered on the regulation of cell proliferation have recently focused on peptide hormones. EGF has been of particular interest since it is so well characterized. This receptor system remains largely unchanged throughout the lifespan with the notable exception of the purified receptor-associated, autocatalytic, tyrosine-specific kinase activity, which decreases with age. The functional significance of this decrease in enzyme activity is unknown, although its growth regulatory importance is implicated in several systems, and may well represent a critical early G0/G1 event which is absent in senescent cells.

The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells.

Addition of 0.14 microM dexamethasone (DEX) to young log-phase WI38 cultures seeded at various densities in serum-free medium containing 4.1 nM epidermal growth factor (EGF) resulted in a synergistic increase in proliferation and final cell density. The action of DEX plus EGF was stimulatory but not synergistic in young confluent cultures. DEX plus EGF had no synergistic effect on senescent cells either during log phase or at confluence. Analysis of the effect of DEX on [125I]EGF binding revealed no statistically significant changes in either the number of binding sites or the apparent dissociation constant of the EGF-receptor complex.

A procedure for the serum-free growth of normal human diploid fibroblasts

We have developed a method for the growth of WI-38 cells in a serum-free medium. Basal medium MCDB-104 is supplemented with platelet-derived growth factor (30 µg/ml), epidermal growth factor (100 ng/ml), insulin (5 µg/ml), transferrin (5 µg/ml), and dexamethasone (55 ng/ml). With this medium cells will grow at a rate and to an extent similar to that produced by medium containing 10% serum. During one growth cycle the cultures can accomplish up to seven population doublings.

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.

Growth Factors and Cell Aging

Although the fundamental mechanisms of in vitro cell aging are still undetermined, certain prominent features have emerged that serve to delineate or focus the search for these mechanisms. In Table 3.1 we present a selected list of changes that accompany senescence in culture. As cells get older they get bigger (Cristofalo & Kritchevsky, 1969) in general, although the synthesis rate of all macromolecules goes down (Houck, Sharma, & Hayflick, 1971; Macieira-Coelho, Ponten, & Phillipson, 1966), the cellular content of macromolecules other than deoxyribonucleic acid (DNA) goes up (Cristofalo & Kritchevsky, 1969; Cristofalo et al., 1970). This is a situation reminiscent of the phenomenon of unbalanced growth in bacteria, which was described more than 30 years ago (Cohen & Barner, 1954). In this situation all processes except the ability to synthesize DNA proceed, albeit not at the normal rate. It appears that DNA synthesis becomes uncoupled from other macromolecular syntheses, and there is a general disintegration of regulation as the cells proceed through the cell cycle.

Effects of tumor necrosis factor and interferon-β on proliferation and epidermal growth factor binding in young and senescent WI-38 cells

SummaryTumor necrosis factor-α (TNF) and various interferons (IFN) have potent cytostatic or cytotoxic effects on a variety of human tumor-derived cell lines. Their effects on normal cells are more controversial. We have examined the effects of TNF and IFN-β on the proliferation of WI-38 cells in a serum-free, growth factor-supplemented medium and in serum-containing medium. These cells respond to the combination of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dexamethasone by DNA synthesis at a rate and extent equivalent to serum-stimulated cells. TNF has no effect on this growth factor-stimulated proliferation. However, it is stimulatory in serum-containing medium. IFN-β inhibits DNA synthesis 60 to 70% in both young and senescent cells. TNF and IFN-β together have a synergistic effect and completely inhibit growth factor-stimulated DNA synthesis in young cells. No synergism was observed with senescent cells. TNF stimulated an increase in the number of EGF specific binding sites two- to threefold in 24 h in both young and senescent cells. This seems to result from a proportional increase in a very high affinity binding site. IFN-β has little or no effect on EGF binding either alone or in combination with TNF.