Walker Wharton

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells☆

Abstract The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.

Induction of alkaline phosphatase activity in hela cells: Inhibition by xanthine derivatives and thermostability studies

Abstract The induction of alkaline phosphate activity in HeLa cells by 5-iodo-2′-deoxyuridine (IUdR) or hydrocortisone was inhibited in a dose-dependent manner by the addition of the xanthine derivatives caffeine, theophylline or 3-isobutyl-1-methylxanthine to the culture medium during the 72 hr of the induction. Pretreatment with theophylline from −24 to 0 hr or treatment from 0 to 24 hr with any of the xanthine derivatives was ineffective in inhibiting alkaline phosphatase induction produced by treatment with IUdR from 0 to 72 hr. The induction of alkaline phosphatase activity produced by treatment with hydrocortisone from 0 to 72 hr was inhibited by pretreatment with theophylline from −24 to 0 hr, although this inhibition was only about 60 per cent as great as that seen with treatment from 0 to 72 hr. As judged by heat inactivation studies, IUdR predominantly increased the heat-labile form of alkaline phosphatase activity, while hydrocortisone predominantly increased the heat-stable form. Regardless of the inducer used, the xanthine derivatives mainly decreased the heat-stable form of alkaline phosphatase activity. Treatment with imidazole over a 72-hr period produced over a 2-fold induction of alkaline phosphatase activity, which could be inhibited completely by concurrent treatment with theophylline.

Isolation of cell membrane for epidermal growth factor receptor studies

The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the purified cell membranes. The membrane purification procedure has been adapted by others in assessing EGF kinase activity and has been used for the purification of cell membranes from other types of cultured cells.

Antagonism of Etonitazene's effects in rats and pigeons☆

The effects of etonitazene were studied in the pigeon under a mult FR FI schedule of food presentation and in the rat under a continuous avoidance-escape schedule. A low dose of etonitazene increased rates of responding by pigeons under the FI component of the multiple schedule, whereas higher doses produced dose-related decreases in rates of responding under both components of the multiple schedule. These effects were blocked by cyclazocine. Under the continuous avoidance-escape schedule, etonitazene produced only dose-related decreases in rates of responding by rats, and these decreases were blocked by naloxone.

Regulation of uridine kinase activity in BLAB/C-3T3 cells by serum components

SummaryAfter the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels of uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce an induction of thymidine kinase activity in late G1.

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate.

SummaryThe induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline > caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2′-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, from of the activity.

Karyotypic Evolution during Neoplastic Progression in Nude Mice

When tumorigenic cultured cell populations are inoculated into nude mice, the tumorigenic process generally requires further progression and selection in vivo. This in vivo progression should be reflected in the altered properties of the tumor cells, as compared to the cells implanted. Karyotypic instability was studied during this process. 6 refs., 5 figs.

Mammalian Cell Proliferation is Regulated by the Synergistic Actions of Multiple Growth Factors

Normal mammalian fibroblasts require serum for growth in culture. When serum is withdrawn from the culture medium or when a high cell density is reached, normal fibroblastic cells become growth arrested in a distinct region (Go) of the cell cycle with a Gl content of DNA. When serum is added to quiescent growth-arrested fibroblasts, DNA synthesis begins after a 12 hour lag. Viral transformation of fibroblasts lessens their serum requirement and can alter the ability of the cells to enter the normal growth arrested state.

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells

SummaryPlatelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine whole blood. Bovine platelet-poor plasma had properties similar to those previously decribed for human platelet-poor plasma; e. g., it would (a) support the growth of virally transformed but not normal BALB/ c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.