The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones

Abstract

Epidermal growth factor (EGF) added in a single dose (between 10(-16) and 1.7 X 10(-9)M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (3H)thymidine labeling and autoradiography and by a 4-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10(-11)M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10(-14)M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10(-14)M) mixture of the two pancreatic hormones or EGF by itself at 10(-14)M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.

Arrest of epidermal growth factor-dependent growth in fetal hepatocytes after ethanol exposure.

“…The principal intent of this report is to document the effects of ethanol directly on fetal cells and on the growth factor interactions required for replication of these cells. These studies indicate that (a) exposure to moderate concentrations of ethanol blocks EGF-dependent replication of rat fetal hepa- (8,28). Our data confirm this and further show that exposure to ethanol blocks this growth.…”

Differential responsiveness of cultured suckling and adult rat hepatocytes to growth‐promoting factors: Entry into s phase and mitosis

“…The present results demonstrate a wide difference in the replicative activity of suckling versus adult rat hepatocytes in response to EGF, insulin, and glucagon A number of studies have been reported on the replicative activity of hepatocytes from 4-day-old rats, in response to these hormonal factors (Andreis and Armato, 1981;Draghi et al, 1980). However in those studies, hepatocytes represented only 40% of the isolated liver cell preparations and were stimulated after 4 days of culture in the presence of serum; such culture conditions are very different from those used to study the hormone response of adult rat hepatocytes (e.g., Laishes and Williams, 1976a;McGowan et al, 1981;Michalopoulos et al, 1982).…”

Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system.

“…Although EGF appears to have a growth-promoting effect on hepatocytes (17) and hepatocytes have been shown to contain saturable EGF receptors (18), a high-capacity uptake system with the ability to translocate immunoactive EGF across the hepatocyte has, to the best of our knowledge, not been demonstrated previously.…”

“…Obviously, the growth-promoting capabilities of EGF are not expressed in normal liver because liver cells divide at an exceedingly slow rate. However, EGF treatment has been shown under special circumstances to induce hepatic hypertrophy and hyperplasia in vivo (16), to induce DNA synthesis in rat hepatocytes in vitro (15,17), and to increase neonatal hepatocyte numbers in vitro (17). Based on these studies as well as those of Earp (20)(21)(22)(23)(24)(25).…”

“…In adult rat hepatocytes in vivo or in primary culture, EGF stimulates DNA synthesis (15) and has been reported to induce hepatic hypertrophy and hyperplasia (16). In newborn rats, EGF induces thymidine incorporation into developing liver cells and enhances mitosis (15,17). EGF receptors have been found on hepatocyte membranes (18), and an EGF-receptor complex has been isolated from hepatocytes by using a glutaraldehyde/sodium borohydride crosslinking technique (19).…”