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 its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that...

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Bibliographic Details
Published in:The FASEB journal 2000-11, Vol.14 (14), p.2185-2197
Main Authors: Reshkin, Stephan J., Bellizzi, Antonia, Caldeira, Sandra, Albarani, Valentina, Malanchi, Ilaria, Poignee, Manuela, Alunni‐Fabbroni, Marianna, Casavola, Valeria, Tommasino, Massimo
Format: Article
Language:English
Subjects:
3T3 Cells
Amiloride - analogs & derivatives
Amiloride - pharmacology
Animals
Binding Sites
Binding, Competitive
Cell Division - drug effects
Cell Line
Cell Transformation, Neoplastic - genetics
Cell Transformation, Viral - genetics
Culture Media, Serum-Free - pharmacology
Cyclin E - drug effects
Cyclin E - metabolism
cytoplasmic alkalinization
Glycolysis
HPV16 E7 oncoprotein
Humans
Hydrogen-Ion Concentration
Keratinocytes - cytology
Keratinocytes - virology
Mice
Mice, Nude
Neoplasm Transplantation
Neoplasms, Experimental - pathology
Neoplasms, Experimental - prevention & control
NHE‐1
Oncogene Proteins, Viral - genetics
Oncogene Proteins, Viral - physiology
Papillomavirus E7 Proteins
Phenotype
S Phase
Sodium-Hydrogen Exchangers - antagonists & inhibitors
Sodium-Hydrogen Exchangers - metabolism
Sodium-Hydrogen Exchangers - physiology
Transplantation, Heterologous
Xenograft Model Antitumor Assays
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Summary:In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE‐1 proton regulatory site. Annulment of the E7‐induced cytoplasmic alkalinization by specific inhibition of the NHE‐1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum‐independent growth, anchorage‐independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F‐mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformationdependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE‐1, DMA, delayed the development of HPV16‐keratinocyte tumors. Our data confirm that activation of the NHE‐1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.—Reshkin, S. J., Bellizzi, A., Caldeira, S., Albarani, V., Malanchi, I., Poignee, M., Alunni‐Fabbroni, M., Casavola, V., Tommasino, M. 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.
ISSN:0892-6638
1530-6860
DOI:10.1096/fj.00-0029com