In BALB/c-3T3 cells, expression of the c-myc gene is stimulated by platelet-derived growth factor (PDGF). Using mouse mammary tumour virus promoter: c-myc recombinant plasmids, 3T3 sublines were constructed in which hydrocortisone was the primary determinant of myc mRNA content. The c-myc gene product is an intracellular mediator of the growth response to PDGF though probably not the only one. Both the human and the mouse c-myc genes stimulate clonal growth of 3T3 cells in PDGF-free medium suggesting new strategies for analysis of oncogenes which do not function in focus formation assays.

Results are reported showing that pituitary extracts stimulate growth of 3T3 cells, an established line of mouse fibroblast. Cells were cultured in medium containing an organic buffer (HEPES; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), which increases the serum requirement for cell growth. The pituitary growth-promoting activity is (a) not dialyzable; (b) thermolabile; (c) protease sensitive; and (d) enhanced by hydrocortisone (a steroid hormone). Activity was quantitatively assayed by measurement of stimulation of cell division or [3Hjthymidine uptake into DNA. The active species is probably different from pituitary protein hormones and it seems to be effective at concentrations observed for protein hormones in vivo. 3T3 cells transformed by simian virus 40 are not responsive to this activity.3T3 designates a series of established cell lines of mouse fibroblasts growing in monolayer (1). In these cells, growth stops at a defined position of G1 phase when confluency is reached, a phenomenon called "contact inhibition" (2). This phenomenon is assumed to be a manifestation of normal growth control in vivo, rendering this cell system an important experimental model. "Contact inhibition" might simply reflect the property of these cells to undergo the transition between a "resting state" and a "proliferative state" under very strict control of serum growth factors (3). Viruses abolish this cell property, transforming the cell to an uncontrolled proliferative state and leading to loss of response to the postulated serum growth factors. Experimental evidence indicates that survival, migration, and proliferation are cell functions under control of different serum factors (4-6). Pure factors are necessary probes to study control of growth and cell cycle. However, purification of factors from serum has proven to be very difficult, limiting the progress of these investigations (7 MATERIALS AND METHODSSTS Cells used were derived from cells available at Dr. Gordon Sato's laboratory. They showed morphology described for "contact-inhibited" 3T3 cells and mouse chromosomes ranging from 65-84 per cell (8). Stock cultures were kept in a standard growth medium where cells grow with a doubling time of 14-16 hr, being subcultured every 3-4 days from subconfluent plates. The plating efficiency (number of colonies developed per 100 cells, density 100-200 cells per plate) observed in the standard growth medium was 80-95.Growth Medium. The standard growth medium was 85% of Dulbecco's Modified Eagle's Medium from GIBCO (but with 1.2 g of sodium bicarbonate per liter), 12.5% horse serum (GIBCO), 2.5% fetal-calf serum (GIBCO), and 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (Calbiochem). Medium referred to as 10% calf serum (Flow Laboratories, Inc.) and 10% horse serum is 90% of modified Eagle's medium, 10% serum, and 15 mM HEPES.The organic buffer was used in order to avoid the pH variation normally observed with the bicarbonate buffer alone (9). All media were adjusted to give pH 7.2 after equilib...

The growth-inhibitory effects of the adrenocorticotropic hormone (ACTH) 1 on adrenal cells in vitro are well documented. ACTH-induced inhibition of cell proliferation has been observed in the Y1 mouse adrenocortical tumor cell line (1) as well as in normal adrenocortical cells isolated from a variety of species including rat, cow, and human (for review, see Ref.2). ACTH arrests dividing adrenal cells by interfering with progression through the G 1 phase of the cell cycle (3) and inhibits the initiation of DNA synthesis in G 1 -arrested cells following addition of serum or growth factors (4, 5). Several lines of evidence indicate that the growth-inhibitory effect of ACTH is mediated by cAMP with the most compelling data arising from studies of Y1 adrenal tumor cells harboring dominant inhibitory mutations in cAMP-dependent protein kinase (PKA) that specifically disrupt cAMP-dependent signaling pathways (6). These PKA mutants are resistant to the growth-inhibitory actions of ACTH and cAMP analogs (7,8), indicating that cAMP and PKA are obligatory components of this effect of ACTH on cell proliferation. The inhibition of proliferation seen in isolated adrenocortical cells contrasts sharply with the growth-promoting effects of ACTH on the adrenal gland in vivo and has led to the widely held view that ACTH serves as an indirect mitogen for the adrenal cortex in intact animals (2). Paradoxically, however, ACTH induces expression of genes often associated with enhanced cell proliferation such as ornithine decarboxylase (9) and fos and jun protooncogenes (10 -12) in isolated adrenocortical cells, raising the possibility of an underlying growth-promoting action of the hormone.The MAP kinase cascade, an important regulator of cell cycle progression, has been used recently as a biochemical marker to evaluate the status of hormones and growth factors as mitogens. Activation of the MAP kinase pathway is involved in the mitogenic effects of growth factors such as epidermal growth factor, platelet-derived growth factor, and FGF (13, 14), acting via receptor tyrosine kinases and also appears to mediate the mitogenic effects of thyrotropin on thyrocytes (15), angiotensin II on smooth muscle cells (16), and thrombin on fibroblasts (17), each acting through a G protein-coupled receptor. Conversely, inhibition of the MAP kinase cascade accompanies the growthinhibitory effects of cAMP observed in fibroblasts and other cell types (for review, see Ref. 18). In the present study, we examined the regulation of the MAP kinase pathway in Y1 mouse adrenocortical tumor cells to reconcile the growth-inhibiting effect of ACTH in vitro with the conflicting biochemical data that suggests an underlying mitogenic effect of the hormone. Although we expected that ACTH would inhibit MAP kinase activity in Y1 cells, consistent with the growth-inhibitory effects of the hormone, we find that ACTH activates the MAP kinase cascade via a signaling mechanism that is cAMP-independent. This effect of ACTH on MAP kinase prompted us to reexamine the effects of...

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