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 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.

[1]  G. Semenza,et al.  Oncogenic alterations of metabolism. , 1999, Trends in biochemical sciences.

[2]  J. Sherratt,et al.  Mathematical modelling of tumour acidity: regulation of intracellular pH. , 1999, Journal of theoretical biology.

[3]  S. Grinstein,et al.  Role of Intracellular pH in Proliferation, Transformation, and Apoptosis , 1997, Journal of bioenergetics and biomembranes.

[4]  J. Pouysségur,et al.  Molecular physiology of vertebrate Na+/H+ exchangers. , 1997, Physiological reviews.

[5]  A. Hatzigeorgiou,et al.  Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter , 1996, Molecular and cellular biology.

[6]  I. Tannock,et al.  The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects. , 1996, British Journal of Cancer.

[7]  A. Paradiso,et al.  Differential responsiveness of proliferation and cytokeratin release to stripped serum and oestrogen in the human breast cancer cell line, MCF-7. , 1996, European journal of cancer.

[8]  X. Breakefield,et al.  Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells , 1996, Journal of virology.

[9]  H. zur Hausen,et al.  Malignant progression of an HPV16-immortalized human keratinocyte cell line (HPKIA) in vitro. , 1995, Cancer genetics and cytogenetics.

[10]  A. Paradiso,et al.  Characterization of a h-3 GnRH method for the measurement of GnRH binding-sites in human breast-cancer and breast-cancer cell-lines. , 1995, International journal of oncology.

[11]  L. Crawford,et al.  Human Papillomavirus E6 and E7: Proteins which deregulate the cell cycle , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[12]  T. Kouzarides.  Transcriptional control by the retinoblastoma protein. , 1995, Seminars in cancer biology.

[13]  A. Eastman,et al.  Apoptosis in an Interleukin-2-dependent Cytotoxic T Lymphocyte Cell Line Is Associated with Intracellular Acidification , 1995, The Journal of Biological Chemistry.

[14]  J. Pouysségur,et al.  Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. , 1995, The American journal of physiology.

[15]  J L Pedraz,et al.  Hydrogen ion-dependent oncogenesis and parallel new avenues to cancer prevention and treatment using a H(+)-mediated unifying approach: pH-related and pH-unrelated mechanisms. , 1995, Critical reviews in oncogenesis.

[16]  M. Barbosa,et al.  In-vivo analysis of hpv e7 protein association with prb, p107 and p130. , 1995, International journal of oncology.

[17]  Bonnie F. Sloane,et al.  Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. , 1994, Cancer research.

[18]  L. Ng,et al.  Activity and density of the Na+/H+ antiporter in normal and transformed human lymphocytes and fibroblasts. , 1994, The American journal of physiology.

[19]  M. Montrose,et al.  Rapid computerized analysis of Na+/H+ exchange flux. , 1994, Biochimica et biophysica acta.

[20]  W. Boron,et al.  Long-term expression of c-H-ras stimulates Na-H and Na(+)-dependent Cl-HCO3 exchange in NIH-3T3 fibroblasts. , 1994, The Journal of biological chemistry.

[21]  E. Androphy,et al.  Cellular transformation by papilomavirus oncorproteins , 1993 .

[22]  A. Eastman,et al.  Etoposide-induced apoptosis in human HL-60 cells is associated with intracellular acidification. , 1993, Cancer research.

[23]  T. Crook,et al.  Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation , 1993, Journal of virology.

[24]  I. Tannock,et al.  Therapeutic potential of analogues of amiloride: inhibition of the regulation of intracellular pH as a possible mechanism of tumour selective therapy. , 1993, British Journal of Cancer.

[25]  E. Androphy,et al.  Cellular transformation by papillomavirus oncoproteins. , 1993, Biochimica et biophysica acta.

[26]  T. Hunt,et al.  HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A. , 1993, Oncogene.

[27]  K. Münger,et al.  Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein , 1992, Journal of virology.

[28]  R. Gillies,et al.  Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  K. Vousden,et al.  Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells. , 1990, Oncogene.

[30]  H. Land,et al.  Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. , 1990, Nucleic acids research.

[31]  K. Münger,et al.  The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. , 1989, Science.

[32]  S Grinstein,et al.  Requirement of the Na + / H + Exchanger for Tumor Growth 1 , 2006 .

[33]  R. Serrano,et al.  Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump , 1988, Nature.

[34]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[35]  S. Aaronson,et al.  Microinjection of ras p21 induces a rapid rise in intracellular pH , 1987, Molecular and cellular biology.

[36]  J. Pouysségur,et al.  Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series. , 1984, The Journal of biological chemistry.

[37]  R. Brandt.  [6] Determination of d-lactate in plasma , 1982 .

[38]  R. Brandt.  Determination of D-lactate in plasma. , 1982, Methods in enzymology.

[39]  P. Pedersen,et al.  Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. , 1981, The Journal of biological chemistry.

[40]  W. Boron,et al.  Intracellular pH. , 1981, Physiological reviews.

[41]  E. Racker,et al.  Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids. , 1979, Biochemistry.

[42]  W. Boron,et al.  Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors , 1976, The Journal of general physiology.