Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
1999-06-03
2002-03-12
Gambel, Philip (Department: 1644)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S004000, C435S006120, C435S007100, C435S007210, C435S007230, C435S007240, C435S325000, C435S363000, C435S366000, C435S372000, C435S372100, C435S372200, C435S372300, C435S375000
Reexamination Certificate
active
06355410
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to treatment of patients with leukemia. In particular, the present invention relates to the apoptosis of leukaemic cells.
BACKGROUND OF THE INVENTION
The ubiquitous, amiloride-sensitive, growth factor activatable sodium/hydrogen exchanger isoform 1 (NHE-1) represents one of the primary mechanisms by which cells regulate intracellular pH (pH
i
) (1, 2). The human NHE-1 is an 815 amino acid membrane protein transporter produced from the 70 kb long APNH gene on chromosome 1 (3). The protein has 12 membrane spanning segments containing the amiloride-binding site and the highly conserved Na
+
/H
+
binding site. The cytoplasmic domain contains the pH sensor and maintenance sites (2). It also contains regions which activate the antiporter when growth factors, mitogens or non-mitogenic signals act on the cell (2). Activation of NHE-1 results in a 1:1 efflux of H
+
and influx of Na
+
ions, with a concomitant increase in pH
i
. Increased pH
i
is associated with cell stimulation and proliferation (1). Using the interleukin-3 (IL-3)-dependent stem cell line, FDCP-mix, Whetton et al (4) demonstrated that IL-3 activated the NHE and that the resulting intracellular alkalinization was a signal for proliferation of these cells. When granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to stimulate macrophage proliferation, activation of the NHE was detected (5). Removal of growth factors such as IL-2 from IL-2 dependent cytotoxic T cells resulted in a decrease in pH
i
and the onset of apoptosis (6, 7). We have demonstrated that murine bone marrow cells could be stimulated by interaction of the &agr;
4
integrin subunit with fibronectin, in the absence of growth factors, causing activation of NHE-1, an increase in pH
i
and increased colony formation of hemopoietic stem and progenitor cells (8). Several reports using different leukemic cell lines demonstrated that if the Na
+
/H
+
exchanger was inhibited by amiloride analogues, acidification of the cells occurred with induction of apoptosis (9-11). We hypothesize that cells maintaining a high rate of proliferation should exhibit a sustained increase in pH
i
relative to normal cells as a result of activation of the sodium/hydrogen exchanger. Here we show that leukemia cell lines and primary patient leukemic samples exhibit a greater pH
i
than normal cells, that pH
i
is correlated with cell cycle status and that inhibition of NHE-1 in leukemic patient samples results in a decrease in pH
i
and increase in apoptosis. We therefore demonstrate that an inhibitor of NHE-1 has anti-leukemic activity.
SUMMARY OF THE INVENTION
A method is provided to sort cells by flow cytometry into subpopulations of proliferating and non-proliferating cells and to induce apoptosis in proliferating hemopoietic and leukemic cells by inhibiting the Na
+
/H
+
exchanger, thereby lowering the internal pH.
REFERENCES:
patent: 5523286 (1996-06-01), McGlave et al.
Noguchi 1991. Use of Flow Cytometry for DNA Analysis. In Current Protocols in Immunology (J.E. Colligan et al, eds) pp 5.7.1-5.7.6. Greene Publishing and Wiley-Interscience, New York.*
Reynolds et al. Cytometry 25:349-357 1996.*
van Erp et al. Cytometry 12;127-132 1991.*
Shapiro 1995. In Practical Flow Cytometry. Published by Wiley-Liss, New York. Chpt 6 p 217; Chpt 7 pp 229-230, 244-245, 326, 345-348; Chpt 10 pp 367, 388-391.*
“Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ”; JA Thomas, RN Buchsbaum, A. Zimniak, E. Racker; Biochemistry 18, 2210-2218; 1979.
“A specific mutation abolishing Na+/H+antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH”; J. Pouyssegur, C. Sardet, A. Franchi, G. L'Allemain, S. Paris; Proc. Natl. Acad. Sci. U.S.A. 81, 4833-4837; 1984.
“Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+exchange without inositol lipid hydrolysis”; AD Whetton et al.; Biochem. J. 256, 585-592; 1988.
“Amiloride and its analogs as tools in the study of ion transport”; TR Kleyman, EJ Cragoe; J. Membrane Biol. 105, 1-21; 1988.
“Requirement of the Na+/H+exchanger for tumor growth”; D. Rotin, D. Steele-Norwood, S. Grinstein, I. Tannock; Cancer Res. 49, 205-211; 1989.
“Cloning of the human genomic amiloride-sensitive Na+/H+ antiporter gene, identification of genetic polymorphisms, and localization on the genetic map of chromosome 1p”; RP Lifton, C. Sardet, J. Pouyssegur, JM Lalouel; Genomics 7, 131-135; 1990.
“Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+antiport. Evidence for two distinct roles for Na+/H+ antiport activation”; SJ Vallance, CP Downes, EJ Cragoe, AD Whetton; Biochem. J. 265, 359-364; 1990.
“The Na+/H+antiporter in oncology in the light of the spontaneous regression of cancer and cell metabolism”; S. Harguindey, EJ Cragoe; Med. Hypotheses 39, 229-237; 1992.
“The Na+/H+exchanger: an update on structure, regulation and cardiac physiology”; L. Fliegel, O. Frohlich; Biochem. J. 296, 273-285; 1993.
“Antitumor activity of nigericin and 5-(N-ethyl-N-isopropyl)amiloride: an approach to therapy based on cellular acidification and the inhibition of regulation of intracellular pH”, K. Hasuda, C. Lee, IF Tannock; Oncol. Res. 6, 259-268; 1994.
“Apoptosis in an interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification. Role of the Na(+)/H(+)-antiporter”; J. Li, A. Eastman, J. Biol. Chem. 270, 3203-3211; 1995.
“Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of the pH-dependent endonuclease”; D. Perez-Sala, D. Collado-Escobar, F. Mollinedo; J. Biol. Chem. 270, 6235-6242; 1995.
“Apoptosis induced by IL-2 withdrawal is associated with an intracellular acidification”; A. Rebollo et al.; Exp. Cell. Res. 218, 581-585; 1995.
“Growth pattern and clinical correlation of subcutaneously inoculated human primary acture leukemias in severe combined immunodeficiency mice”; Y. Yan et al.; Blood 88, 3137-3146; 1996.
“The relationship between thermosensitively and intracellular pH in cells deficient in Na+/H+antiport function”; FF Liu, D. Diep, RP Hill; Radiother. Oncol. 40, 75-78; 1996.
“Activation of the Na(+)/H(+) antiporter, Na+/HCO3(-)/CO3(2−-) cotransporter, or Cl(−-)/HCO3(−-) exchanger in spontaneous thymocyte apoptosis”; N. Tsao, HY Lei; J. Immunol. 157, 1107-1116; 1996.
“Molecular physiology of vertebrate Na+/H+exchangers”; S. Wakabayashi, M. Shigekawa, J. Pouyssegur; Physiol. Rev. 77, 51-74; 1997.
“Acid pH in tumors and its potential for therapeutic exploitation”; IF Tannock, D. Rotin; Cancer Res. 49, 4373-4384; 1997.
“Tumor cell proliferation is abolished by inhibitors of Na+/H+and HCO3/Cl exchange”; B. Horvat, S. Taheri, A. Salihagic; Eur. J. Cancer 1992 29 A, 132-137; 1997.
“Role of acid/base homestasis in the suppression of apoptosis in haemopoietic cells by v-Abl protein tyrosine kinase”; Q. Chen et al.; J. Cell Sci. 110, 379-387; 1997.
“Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation”; IN Rich, I. Brackman, D. Worthington-White, MJ Dewey; J. Cell. Physiol. 177, 109-122; 1998.
Gambel Philip
Roark Jessica H.
Womble Carlyle Sandridge & Rice PLLC
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