Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes

In this study we investigate the mechanism of intracellular pH change and i ts role in malignant transformation using the E7 oncogene of human papillom avirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruse s expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transf ormation can be turned on and followed by induction of the HPV16 E7 oncogen e expression, we demonstrate that cytoplasmic alkalinization is an early ev ent and was driven by stimulation of Na+/H+ exchanger activity via an incre ase in the affinity of the intracellular NHE-1 proton regulatory site. Annu lment of the E7-induced cytoplasmic alkalinization by specific inhibition o f the NHE-1, acidification of culture medium, or clamping the pHi to nontra nsformed levels prevented the development of later transformed phenotypes s uch as increased growth rate, serum-independent growth, anchorage-independe nt growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 a nd HPKIA cells show that alkalinization acts on pathways that are independe nt of the E2F-mediated transcriptional activation of cell cycle regulator g enes. Moreover, we show that the transformation-dependent increase in proli feration is independent of the concomitant stimulation of glycolysis. Final ly, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delay ed the development of HPV16-keratinocyte tumors. Our data confirm that acti vation of the NHE-1 and resulting cellular alkalinization is a key mechanis m in oncogenic transformation and is necessary for the development and main tenance of the transformed phenotype.