Mari C. S. Armelin

Serum and hormonal regulation of the "resting-proliferative" transition in a variant of 3T3 mouse cells.

3T3 CELLS are embryonic mouse fibroblasts established in culture1. Serum depletion or “step-down” causes them to enter a “resting” state, which they leave on restoration of serum or “step-up”, entering the “proliferative” state2–4. This transition between resting (also called G0, A or R) and proliferating states seems to be the critical step in cell growth control5–7. Its study has been aided recently by several observations on extracellular growth regulators for 3T3 cells, for example the discovery of a new pituitary growth factor4,8, growth stimulation by glucocorticoids4,9 and interactions between pituitary factor and classical hormones10–12. Further studies would be greatly helped if variants or mutants of 3T3 with different growth responses towards hormones could be isolated. Here we report our finding of the variant ST1 which is inhibited by glucocorticoids, unlike 3T3 cells whose growth is stimulated by glucocorticoids4,9 (growth of fibroblasts is normally inhibited by glucocorticoids13). In this paper we discuss the value of this new cell type for studies of the resting–proliferative transition; elsewhere we shall report how the adhesion of this variant to the substrate is greatly affected by both the removal of serum and the addition of cyclic AMP.

Regulation of fibroblast growth in culture.

Summary A protein factor extracted from pituitary tissue stimulated cell growth of human and mouse 3T3 fibroblasts and its activity was enhanced by hydrocortisone and insulin at physiological concentrations; such synergistic effects were observed in a serum free medium. Cells stimulated by the hormones in serum-free medium traverse G1 and S but accumulate at G2, the “termination” of cell division being serum dependent.

Mapping of polyomavirus middle T domain that is responsible for AP-1 activation

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factors-induced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a specific P13K inhibitor) leads to decreased AP-1 activity. Super-shift and Western blot analysis with specific antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results confirm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be sufficient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.

Cell proliferation induced by 8-oxoguanosine and 8-methylguanosine, two adducts produced by free radical attack on ribonucleosides and RNA.

The ability of C8-substituted guanine (Gua) ribonucleosides to induce B cell proliferation has been well documented in the literature. These compounds are analogues of adducts formed from free radical attack on ribonucleosides and RNA. Here we examined the proliferative properties of two of these radical adducts, 8-methylguanosine (8-MeG) and 8-oxo-7,8-dihydroguanosine (8-OxoG) and compared them with those of the well studied B cell mitogen, 8-bromoguanosine (8-BrG). 8-MeG and 8-OxoG were synthesized in the considerable yields of 28, and 55%, respectively, and were characterized by UV, NMR and CG-MS. Their effects upon [3H]thymidine uptake into DNA by Swiss mouse splenocytes, mouse embryo 3T3 fibroblasts (A31) and mouse B16F10 melanoma were examined. Both guanosine (G) radical adducts were shown to increase [3H]thymidine uptake by mouse splenocytes but displayed selectivity in respect to continuous cell lines. 8-MeG acted upon 3T3 fibroblasts whereas 8-OxoG acted upon B16F10 melanoma. The non-physiological analogue 8-BrG acted upon all tested cells. Parallel experiments of cell counting, cytotoxicity,and cell sorting indicated that DNA synthesis induced by the C8-substituted Gs reflected cell growth. It is proposed that the compounds act intracellularly because their proliferative effects were blocked in the presence of a nucleoside transport inhibitor but were not inhibited by an antagonist of the A2 purine receptor. The present results, taken together with data from the literature, suggest that in the case of 3T3 fibroblasts and mouse splenocytes the proliferative effects of the compounds are not dependent on metabolism through purine salvage pathways. In the case of melanoma, however, the compounds are likely to become part of the purine nucleoside pool. The demonstration that adducts produced by free radical attack on ribonucleosides and RNA are able to induce cell proliferation opens new perspectives for the understanding of free radical mediated carcinogenesis.