A H+-suicide technique based on the reversibility of Na+/H+ antiport was developed for the selection of mutants deficient in this membrane-bound activity. The strategy was to use the Na+/H+ antiporter as a H+-vector killing device. Chinese hamster lung fibroblasts (CCL39) were loaded with LiCl and incubated in Na+-, Li+-free choline Cl saline solution (pH 5.5). Under these conditions, intracellular pH dropped in 5 min from 7.1 to 4.8, leading to a rapid loss of cell viability (less than 0.1% survival after 30 min). Cytoplasmic acidification and cell death were prevented by treatment with 5-N,N-dimethylamiloride, a potent inhibitor of Na+/H+ antiport. Of the H+-suicide resistant clones that survived two cycles of selection, 90% were found deficient in Na+/H+ antiport activity. One class of mutants (PS10, PS12) fully resistant to the H+-suicide test, does not acidify the cell interior in response to an outward-directed Li+ gradient and has no detectable amiloride-sensitive Na+ influx measured either in Li+- or H+-loaded cells. Growth of these fibroblast clones lacking Na+/H+ antiport was found to be pH conditional in HCO3(-)-free medium. Whereas wild-type cells can grow over a wide range of external pHs (6.6-8.2), PS mutants cannot grow at neutral and acidic pHs (pH less than 7.2); their optimal growth occurs at alkaline pH values (pH 8-8.3). These findings strongly suggest that the Na+/H+ antiport activity through regulation of intracellular pH plays a crucial role in growth control.

https://www.pnas.org/content/pnas/81/15/4833.full.pdf

A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.

Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport.

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series.

There is increasing evidence that at least some of the cellular homologues to retroviral oncogenes (c-onc or proto-oncogenes) are directly linked to the control of cell growth (for a review see ref. 1). Among these, c-myc, the cellular homologue to the avian myelocytomatosis virus (MC29) oncogene, has been shown to express high levels of mRNA during early G0/G1 phase after mitogenic stimulation of T lymphocytes by concanavalin A or of fibroblasts by platelet-derived growth factor (PDGF) or serum. An attractive model proposed for this regulation is that the c-myc gene is strongly repressed in cells arrested in the G0 phase of the cell cycle by a growth factor-sensitive repressor. We have investigated an alternative model of post-transcriptional regulation. This latter model leads to two testable predictions. First, that c-myc mRNA should be unusually unstable, which we have confirmed. And second, that there would be a high level of constitutive expression, a situation opposite to that implied by the repressor model. Here we report that c-myc gene is indeed transcribed at a high rate in G0-arrested chinese hamster lung fibroblasts, although the level of mature c-myc mRNA is barely detectable. The early and dramatic increase in c-myc mRNA levels when these resting cells are stimulated by growth factors is not accompanied by any appreciable change in the transcription rate of c-myc gene. Taken together these findings support a model of post-transcriptional regulation of c-myc expression at the level of mRNA degradation.

c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors.

The primary action of a family of mitogens including bombesin1,2, bradykinin3, vasopressin3,4 and α-thrombin5–7 is to activate the hydrolysis of polyphosphoinositides8. Hydrolysis of phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C is mediated through coupling of surface receptors to a GTP-binding protein9,10 (Gp protein) which, in some cells6,11,12, is inactivated by the toxin of Bordetella pertussis13. It is not known whether this signalling pathway is involved in initiating DNA replication, whereas it has been firmly established that reinitiation of DNA synthesis can be triggered without activation of PtdIns(4,5)P2 hydrolysis by, for example, EGF (epidermal growth factor) 14,15, FGF (fibroblast growth factor)16 and insulin/IGF-I (insulin-like growth factor-I)14, members of a class of mitogens known to activate receptor tyrosine kinases17,18. Taking advantage of the fact that Chinese hamster lung fibroblasts respond to either class of mitogens and that their Gp protein appears to be sensitive to pertussis toxin6, we have now analysed the toxin's effect on reinitiation of DNA synthesis and find that it inhibits up to 95% of thrombin-induced mitogenicity without affecting EGF- or FGF-induced DNA synthesis and proliferation. These findings strongly suggest that activation of PtdIns(4,5)P2-phospholipase C has a determinant function in growth control, and confirm the existence of alternative growth factor-signalling pathways independent of polyphosphoinositide breakdown.

Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin.

Reports from several laboratories have suggested increased rates of glycolysis play an essential part in the initiation of DNA synthesis. This is based on observations that aerobic glycolysis: (1) occurs at low rate in resting mammalian cells and at very high rate in tumour cells; (2) increases rapidly after DNA synthesis is initiated by addition of serum or purified growth factors, and (3) correlates with the expression of the transformed phenotype. Also, specific inhibitors of aerobic glycolysis prevent the initiation of DNA synthesis. To determine whether the rapid activation of phosphofructokinase--and therefore glycolysis--by purified growth factors is necessary for the initiation of cell proliferation, we have isolated and studied two classes of glycolytic mutants. The first, isolated from Chinese hamster fibroblasts, has a total block in the glycolytic pathway. The second, from hamster and Fisher rat fibroblasts maintains a permanent high rate of glycolysis. We have found that both classes of mutants retain normal control of DNA synthesis in response to serum. This dissociation indicates that growth-factor-stimulated glycolysis is not involved in the control of initiation of DNA synthesis and cell proliferation.

P. Silvestre

Papers

Relationship between increased aerobic glycolysis and DNA synthesis initiation studied using glycolytic mutant fibroblasts.

"Carrier activation" and glucose transport in Chinese hamster fibroblasts.