Pituitary Extracts and Steroid Hormones in the Control of 3T3 Cell Growth

Abstract

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...

Mitogenic hormone-induced intracellular message: assay and partial characterization of an activator of DNA replication induced by epidermal growth factor.

“…The concentration of EGF required for half-maximal induction of the activator substance (Fig. 3) is very similar to that required for half-maximal mitogenic response in 3T3 and other animal cells (12,(18)(19)(20) (14). We are therefore interested in developing an improved preparative procedure that will yield a 3T3 nuclear preparation more susceptible to the stimulatory effects of EGF-treated cell extract.…”

Steroid hormones mediate reversible phenotypic transition between transformed and untransformed states in mouse fibroblasts.

“…Glucocorticoids stimulate the growth of "normal" 3T3 cells (1,12), a somewhat intriguing fact because normal fibroblasts are inhibited by glucocorticoids (13,14). Therefore it is not surprising that ST1 cells are inhibited by glucocorticoids although the inhibitory activity could not be perfectly correlated with glucocorticoid activity.…”

Biomedical advances from tissue culture

“…However, purified LH obtained from another source was ineffective on the ovarian cells. At the same time a post-doctoral fellow in the lab found that NIH-LH was growth stimulatory for 3T3 cells, which suggested a more general activity of a contaminating substance (Armelin 1973). Pursuing this observation, the contaminating activity in NIH-LH was shown to be the novel pituitary-derived growth factor basic FGF (Gospodarowicz et al 1974).…”