Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Abstract

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four-to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.Malignant transformation of chicken and rat embryo fibroblasts by Rous sarcoma virus (RSV) results in a marked increase in the rate of hexose transport across the plasma membrane (11,14,15,17,26,27). In cells transformed by RSV mutants in which the transforming protein pp60j-fsrc is temperature sensitive, the increase in transport is also temperature sensitive, indicating that the activity of pp6Ovsrc is necessary for both establishing and maintaining the transport alteration (14,15,17). It is generally accepted that the enhancement of transport is due to an increased number of hexose transporter molecules at the cell membrane, rather than a modification of existing carriers, for the following reasons. (i) Kinetic evidence indicates that the Vmax for transport is increased (7,14,15,26,27 biosynthesis showed that there was little or no difference between normal and transformed cells (22).In contrast, results obtained by others using rodent cells indicate that src transformation induces increased glucose transport by elevating the level of the mRNA for the transporter (8). To resolve this apparent discrepancy, we measured the effects of src transformation on the levels of transporter mRNA, transporter biosynthesis, and transporter degradation in chicken embryo fibroblasts, rat-i cells, and primary cultures of fibroblasts explanted from a rat embryo. We concluded that src increases the level of transporter by mechanisms that differ between rat and chicken cells. In the former, src elevates transporter mRNA levels and biosynthesis, whereas in the latter this is not the case and src acts to inhibit transporter turnover. 2) and stored at -20°C. Lysates were cleared by centrifugati...

Expression of the v-Src oncoprotein in fibroblasts disrupts normal regulation of the CDK inhibitor p27 and inhibits quiescence

“…Expression of v-Src decreased the proportion of cells in the G 0 /G 1 phase Rat-1 ®broblasts expressing a temperature sensitive (ts) mutant of Rous sarcoma virus (ts LA 29 Rat-1) are transformed at the permissive temperature (358) and display normal morphology and behaviour at the restrictive temperature (39.58) (Welham et al, 1990;White and Weber, 1988). To study the e ect of v-Src on cell cycle control, we compared the growth characteristics of ts LA 29 Rat-1 with non transfected Rat-1 cells at both temperatures in medium containing 5% serum.…”

“…We report here a further examination of the mitogenic action of v-Src, exploiting the rapidly reversible activity of the temperature sensitive v-Src mutant, ts LA 29, in Rat-1 cells (Welham and Wyke, 1988;White and Weber, 1988). We found that v-Src transformed cells traversed G 1 more rapidly and did not exit cycle e ciently in response to inhibitory stimuli.…”

“…Rat-1 cells and ts LA 29 Rat-1 cells (White and Weber, 1988) were maintained in Dulbecco's modi®ed Eagle medium with 5% new born calf serum (NBCS), 1 mM Sodium pyruvate, 2 mM L-Glutamine, 50 mg/ml streptomyv-Src transformation disrupts regulation of p27 D Johnson et al cin, 50 units/ml penicillin at 358C. For growth rate determinations, 2610 5 cells seeded in 100 mm dishes were fed and counted daily for 4 days using a Coulter counter.…”

The mechanism of cell cycle regulation by v-Src

“…Cell culture ts LA 29 Rat-1 cells (White and Weber, 1988) were cultured as described previously (Johnson et al, 1998). To induce quiescence 5 to 10610 5 cells per 9 cm plate or 2.5 to 5610 6 cells per 15 cm plate were seeded at the permissive temperature (358C).…”

Tumor-specific gene expression using regulatory elements of the glucose transporter isoform 1 gene

“…12 Especially, the type 1 glucose transporter (GLUT1) gene is one of the early genes that are activated after transformation of cells with oncogenes such as src, ras or fps. [13][14][15][16][17][18][19][20] An increase of GLUT1 mRNA was found 4-6 h after induction of the p21 c-H-ras oncoprotein, whereas morphologic changes occurred 72-96 h later. 18,21 The facilitative glucose transporter GLUT1 has also been shown to be upregulated in human tumor tissues.…”