Effects of Hypophysectomy and Subsequent Growth Hormone Replacement in the Rat on the Ability of Serum to Stimulate Proliferation of Human and Mouse (BALB/c 3T3) Fibroblasts

Summary

The ability of cultured human fibroblasts to synthesize and secrete a mitogenic, somatomedin C-like peptide suggests that human fibroblasts unlike certain other cell lines may not require exogenous somatomedin for cell proliferation. The aim of this study was to examine the effects of hypophysectomy and subsequent growth hormone (GH) replacement on the ability of rat serum to stimulate proliferation and DNA synthesis in cultured human fetal skin fibroblasts. For comparison we have also examined the effects of serum from hypophysectomized (hypox) and GH-replaced hypox rats on mouse BALB/c 3T3 fibroblasts, a cell line which has been shown to require exogenous somatomedin (SM) for DNA synthesis and cell proliferation.

Human fibroblast proliferation and DNA synthesis (measured as thymidine incorporation) were significantly enhanced (P < 0.025) when these cells were cultured in low concentrations of hypox rat serum compared to normal rat serum. When thymidine incorporation into DNA was expressed in terms of a normal rat serum pool (arbitrarily assigned a potency of 1 unit/ml), serum from hypox rats was significantly more potent than serum from normal rats (1.52 ± 0.16 (SEM) vs. 0.99 ± 0.10 units/ml, P < 0.0125).

Replacement treatment of hypox rats with human GH, 0.2 IU/100 g body weight/day or 0.4 IU/100 g body weight/day decreased the potency of the rat serum in terms of stimulation of thymidine incorporation from 1.52 ± 0.16 to 1.30 ± 0.14 and 1.08 ± 0.06 units/ml respectively. In contrast serum from untreated hypox rats was significantly less potent than normal rat serum in stimulating BALB/c 3T3 DNA synthesis (0.36 ± 0.06 vs. 1.04 ± 0.16 units/ml, P < 0.0025). When hypox and normal rat serum were mixed in various ratios and tested for potency in stimulating thymidine incorporation into DNA of quiescent human fibroblasts, and BALB/c 3T3 cells, values intermediate between normal and hypox rat serum were obtained. These data are consistent with the presence of an inhibitor of human fibroblast DNA synthesis in serum from both normal and GH-treated hypox rats. The effect of Multiplication Stimulation Activity (MSA), a SM analog, on thymidine incorporation into DNA of human fibroblasts in the presence of normal or hypox rat serum was examined. Significantly more thymidine incorporation occurred at each concentration of MSA in the presence of hypox rat serum compared to normal rat serum consistent with enhanced inhibitory activity in normal rat serum.

From these observations we conclude that under the conditions described here human fibroblast DNA synthesis is not dependent on the serum somatomedin concentration. In addition inhibitory activity present in normal rat serum may in part be GH-dependent.

Key-Words:

Somatomedins - Growth Factors - DNA Synthesis - Mitogenesis