|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1427-1434
NEOPLASIA
From the Division of Transplantation Medicine, South Carolina Cancer
Center, Palmetto Richland Memorial Hospital, Columbia, SC.
The Na+/H+ exchanger isoform 1 (NHE1) is
primarily responsible for the regulation of intracellular pH
(pHi). It is a ubiquitous, amiloride-sensitive, growth
factor-activatable exchanger whose role has been implicated in
cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate
that leukemic cell lines and peripheral blood from primary patient
leukemic samples exhibit a constitutively and statistically higher
pHi than normal hematopoietic tissue. We then show that a
direct correlation exists between pHi and cell-cycle status
of normal hematopoietic and leukemic cells. Advantage was taken of this
relationship by treating leukemic cells with the
Na+/H+ exchanger inhibitor, 5-(N,
N-hexamethylene)-amiloride (HMA), which decreases the pHi
and induces apoptosis. By incubating patient leukemic cells in vitro
with pharmacologic doses of HMA for up to 5 hours, we show, using flow
cytometry and fluorescent ratio imaging microscopy, that when the
pHi decreases, apoptosis
The role of intracellular pH (pHi)
regulation in hematopoiesis is an area of research that has received
little attention. Yet the concern for maintaining pH as an important
parameter in all in vitro work is well known. By keeping the
extracellular pH (pHe) constant, we assume that the cells
will be able to regulate their own pHi. This is indeed the
case because cells are equipped with several different exchangers to
help regulate pHi and to counteract acidification either by
efflux of H+ ions or influx of
HCO3 The ubiquitous Na+/H+ exchanger isoform 1 (NHE1) is the primary membrane transporter used by cells to regulate
pHi and cell volume.1,2 The human NHE1 is an
815-amino acid membrane protein transporter produced from the 70-kb
long APNH gene located on chromosome 1p35-36.1.5,6
The 1p36 region has been shown to contain the tumor suppressor
p73,7,8 deletion of which has been implicated in the
development of neuroblastoma, breast cancer, colon cancer, melanoma,
and Wilms tumor,7 and other reports have documented loss of
heterozygosity in this region to be associated with chronic myelogenous
leukemia.9,10
The NHE1 protein has 12 membrane-spanning segments. The extracellular
region contains a highly conserved
Na+/H+-binding site and an amiloride-binding
site. Amiloride is a potassium-sparing diuretic used to treat
hypokalemia and manage edema, and it is an adjunct in hypertension.
Many amiloride analogs have been produced with variable potency and
specificity11-13 and, as described below, have been used in
various applications with hematopoietic cells. The cytoplasmic domain
of the NHE1 contains the pH sensor and maintenance sites.2
However, it also contains regions that activate the exchanger when
growth factors, mitogens, or nonmitogenic signals act on the
cell.2 For this reason, the NHE1 has been called a
"growth factor-activatable" exchanger. Activation of NHE1
results in a 1:1 efflux of H+ and influx of Na+
ions, with a concomitant increase in pHi. All the growth
factors tested have been shown to increase pHi with an
associated activation of cell stimulation and
proliferation.1 For example, using the interleukin-3
(IL-3)-dependent stem-cell line, FDCP-mix, Whetton et al14
demonstrated that IL-3 activates the NHE and that the resultant
intracellular alkalinization was a signal for proliferation of these
cells. When granulocyte-macrophage colony-stimulating factor was used
to stimulate macrophage proliferation, activation of the NHE was
detected.15 Removal of growth factors such as IL-2 from
IL-2-dependent cytotoxic T cells resulted in a decrease in
pHi and the onset of apoptosis.16,17
Growth factors are not the only stimuli to activate the NHE1. Phorbol
esters activate the exchanger nonspecifically within 5 minutes.18 In addition, cell-cell interactions can also
activate the NHE1. For example, many members of the integrin family can activate the exchanger.19 Adherence of cells to fibronectin or specific fibronectin-binding sequences results in localization of
the exchanger together with integrins and growth factor receptors in a
so-called focal adhesion complex.20 Our laboratory has demonstrated that the NHE1 is involved in cell-cell interactions of
normal murine hematopoietic cells.21 When murine bone
marrow cells are brought together at high cell density by
centrifugation, hematopoietic stem and progenitor cells can be
stimulated to produce colonies in vitro in the absence of growth
factors. The stimulation results from interaction of the
In contrast to activation of the NHE1 and the concomitant increase in
pHi by various stimuli, a decrease in pHi can
result in apoptosis of the cell. Several reports using different
leukemic cell lines demonstrated that if the
Na+/H+ exchanger was inhibited by amiloride
analogs, acidification of the cells occurred with a concomitant
induction of apoptosis.22-24 We hypothesized that cells
maintaining a high rate of proliferation should exhibit a sustained
increase in pHi relative to normal cells as a result of
activation of the NHE1. Here we show that leukemic cell lines and
primary patient leukemic samples exhibit a greater pHi than
normal cells, that pHi is correlated with cell-cycle status, and that inhibition of NHE1 in patient leukemic cells results
in a decrease in pHi and an increase in apoptosis.
Human samples
Leukemic cell lines
Growth and expansion of primary leukemic cells in NOD-SCID mice The nonobese diabetic, severe combined immunodeficient (NOD/LtSz scid/scid, NOD-SCID; Jackson Laboratories, Bar Harbor, MA) provides an excellent animal model for growing and expanding primary human leukemic cells,25,26 which are normally difficult to grow in culture. The method described by Yan et al27 for growing leukemic cells as subcutaneous nodules provided us with a method of expanding the original leukemic sample containing 1-5 × 107 cells to more than 5 × 108 cells. Because relatively large numbers of cells were required for flow cytometric analyses (see below), especially those involving both pHi and cell-cycle measurements, cells derived from the nodules were used. The NOD-SCID mice were kept under stringent sterile conditions in a Hepa-filtered mouse rack. Mice were injected subcutaneously with 1 × 107 cells suspended in 100 µL Matrigel (Becton Dickinson, San Jose, CA) into untreated NOD-SCID mice as described previously by Yan et al.27 Nodules were usually observed between 4 and 8 weeks after subcutaneous injection and were excised after a maximum of 12 weeks. A single cell suspension was prepared after collagenase/dispase digestion for 45 minutes at 37°C and repeated pipetting. After isolation, the cells were analyzed phenotypically and cytogenetically to determine whether any changes had occurred from the primary inoculum. Phenotypic characterization was performed using the same panel of monoclonal antibodies used to define the original sample. In addition, to differentiate and determine the proportion of mouse-to-human cells present in the nodule, the cells were stained with mouse CD45-fluorescien isothiocyanate (FITC) and human CD45-phycoerythrin (both obtained from Pharmingen, San Diego, CA) and were analyzed by flow cytometry. Figure 1 shows a phenotypic analysis of the primary inoculum cells obtained from a patient with ALL and the phenotype of the cells obtained after 3 months growth in a NOD-SCID mouse. With the exception of the loss of the small populations of CD4 and CD8 cells from the primary inoculum to nodule cells, little change has occurred in the phenotype of the leukemic cells. In addition, in this particular sample, human CD45+ cells comprised more than 85% of the sample (Figure 1B). No further purification was performed.
Treatment of normal peripheral blood with phorbol ester Normal PBMC were incubated for 5 minutes with 10-6 mol/L phorbol-12-myristate-13-acetate (Sigma Chemical, St. Louis, MO) to activate the NHE1 nonspecifically. Thereafter the cells were washed with phosphate-buffered saline (PBS).Measurement of pHi by flow cytometry and fluorescence ratio imaging microscopy Measurement of pHi for hematopoietic cells has been described in detail elsewhere.21 Briefly, intracellular pH was measured by both flow cytometry and fluorescence ratio imaging microscopy (FRIM) by incubating 1 × 106 cells with a final concentration of 10 µmol/L carboxy SemiNaphthoRhodaFluor-1 acetoxymethyl ester, acetate (carboxy SNARF-1 AM; Molecular Probes, Eugene, OR) for 15 minutes at 37°C. After incubation, the cells were washed twice with PBS. Because the addition of bicarbonate can affect pHi, all incubations and cell washes were performed in bicarbonate-free buffer. SNARF is excited at 488 nm and emits at 580 nm and 640 nm. The ratio of the emission wavelengths 640/580 nm was used to estimate the pHi from a calibration curve.21 The calibration curve was performed using the "nigericin clamp technique."28 After SNARF labeling, aliquots of cell suspensions were resuspended in a high K+-containing buffer at a specific pH (usually 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8 for flow cytometry or 6.6, 7.0, 7.4, and 7.8 for FRIM). The cell suspensions were then "clamped" at the specific pH value by the addition of 0.03 µmol/L nigericin (Molecular Probes). Nigericin is an ionophore that allows the exchange of H+ for K+ ions by abolishing the pH gradient across the cell membrane. Thus, when the internal and external K+ concentrations are approximately the same, the pH rapidly equilibrates to the pH of the bathing solution. For flow cytometry, 50 000 events were acquired on a FACSort flow cytometer (Becton Dickinson). Using FRIM, measurements and images were acquired using 2-image intensifying charged-couple device cameras (for 580 nm and 640 nm) attached to an Axiovert 150 microscope (Zeiss, Oberkochen, Germany). Attofluor Ratio Vision software (Atto Instruments, Rockville, MD) was used to acquire and analyze the data. Flow cytometric results were analyzed using WinList Software (Verity Software House, Topsham, ME) to obtain fluorescence ratio measurements. For flow cytometry and FRIM analysis, the fluorescent ratio values obtained for each pH of the calibration reagents were obtained, and a calibration curve was obtained using Table Curve software (SPSS, Chicago, IL), from which the sample pHi values were determined.Measurement of cell cycle The proportion of cells in the S-phase of the cell cycle was determined using the propidium iodide technique (CycleTEST Plus DNA reagent kit; Becton Dickinson) on 1 × 106 cells/sample according to the manufacturer's instructions. Data were acquired by flow cytometry and analyzed using the ModFit LT software program (Verity Software House).Measurement of apoptosis by annexin-V and TUNEL Two methods were used to determine whether reduction in pHi induced apoptosis. The first was annexin-V conjugated to fluorescein isothiocyanate to determine the translocation of phosphatidylserine from the inside to the outside of the plasma membrane. Cells were counterstained with propidium iodide to determine the proportion of necrotic cells. Cell staining was performed according to the manufacturer's instructions. The second technique used terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA. Fluorochrome (FITC)-labeling of DNA (Boehringer-Mannheim, Mannheim, Germany) was performed according to the manufacturer's instructions. Data acquisition was performed by flow cytometry and analyzed using WinList software (Verity Software House).Treatment of cells with ammonium chloride or 5-(N, N-hexamethylene)-amiloride To induce intracellular acidosis, cells were incubated in 20 mmol/L ammonium chloride (NH4Cl) solution for varying periods of time. Thereafter, the cells were washed twice in PBS and labeled with SNARF, and the pHi was measured by FRIM. To inhibit the NHE1, the amiloride analogue, 5-(N, N-hexamethylene)-amiloride HMA (Sigma Chemical) was used. A stock solution of 10 2
mol/L was prepared by dissolving the solid in dimethyl sulfoxide (DMSO). Further dilutions were prepared in DMEM. Cells were
incubated at concentrations ranging from 106 mol/L
to 103 mol/L for varying periods of time specified
in "Results." After incubation, cells were washed and sectioned
into aliquots according to the type of measurement (pHi or
apoptosis) performed. Controls included cells not treated with HMA.
Previous studies demonstrated that the solvent vehicle (DMSO), at the
concentrations of HMA added, had no adverse effect on
pHi measurements or apoptosis.
Statistics The number (n) of experiments performed is given in each figure legend. All results are given as the mean ± SEM for the given number of experiments stated in the figure legends. Statistical significance was determined either using the 2-way, unpaired t test or 1-way analysis of variance.
Steady-state pHi of peripheral blood and bone marrow mononuclear cells Using flow cytometry and the pH-sensitive fluorescent indicator SNARF, we first determined the pHi of normal human PBMC and BMMC. No significant difference (P > .05) in pHi occurred between PBMC (pHi = 6.99 ± 0.037; n = 25) and BMMC (pHi = 7.06 ± 0.08; n = 10). These results are seen in Figure 2. It should be noted that although the pHi of specific cell subpopulations can be measured, data are presented here for whole populations. This is because specific subpopulations measured in normal cells may not exist in leukemic cell lines or primary leukemic patient samples. Therefore, to compare different samples under the same conditions, measurements were performed on whole populations.
Intracellular pH of leukemic cell lines and primary leukemic cells Having obtained baseline pHi values for normal cells, we then found that various leukemic cell lines exhibited a higher pHi (P < .01) than normal PBMC and BMMC (Figure 2). These observations were concordant with those of previous reports describing intracellular alkalinization of continuously proliferating cell lines,17,22,29,30 but they prompted review of the behavior of primary leukemic samples.
Correlation between pHi and cell-cycle status
Effect of NHE1 inhibition on pHi and
apoptosis of normal peripheral blood mononuclear cells
Effect of NHE1 inhibition on pHi and
apoptosis of leukemic cell lines
Effect of NHE1 inhibition on pHi and apoptosis of
primary leukemic cells
The results presented here demonstrate a novel method by which
primary leukemic cells can be induced into apoptosis by inhibiting the
Na+/H+ exchanger protein, a product of a
housekeeping gene. They also implicate the NHE1 as playing an integral
role in normal and abnormal hematopoiesis. We have previously
demonstrated that normal murine bone marrow cells can be stimulated by
the interaction of extracellular matrix proteins with the
Submitted July 19, 1999; accepted October 21, 1999.
Supported by the Department of Defense Sponsored Program 18 190-F103,
the South Carolina Cancer Center, and Palmetto Richland Memorial
Hospital, Columbia, SC.
Reprints: Ivan N. Rich, Division of Transplantation Medicine,
South Carolina Cancer Center, Palmetto Richland Memorial Hospital, 7 Richland Medical Park, Columbia, SC 29203; e-mail: ivan.rich{at}rmh.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
1.
Fliegel L, Frohlich O.
The Na+/H+ exchanger: an update on structure, regulation and cardiac physiology.
Biochem J.
1993;296:273.
2.
Wakabayashi S, Shigekawa M, Pouyssegur J.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol Rev.
1997;77:51
3.
Ludt J, Sandvig K, Olsnes S.
Rapid increase in pH set-point of the Na(+)-independent chloride/bicarbonate antiporter in Vero cells exposed to heat shock.
J Membr Biol.
1993;134:143[Medline]
[Order article via Infotrieve].
4.
Orlowski J, Grinstein S.
Na+/H+ exchangers of mammalian cells.
J Biol Chem.
1997;272:22,373
5.
Mattei MG, Sardet C, Franchi A, Pouyssegur J.
The human amiloride-sensitive Na+/H+ antiporter: localization to chromosome 1 by in situ hybridization.
Cytogenet Cell Genet.
1988;48:6[Medline]
[Order article via Infotrieve].
6.
Lifton RP, Sardet C, Pouyssegur J, Lalouel JM.
Cloning of the human genomic amiloride-sensitive Na+/H+ antiporter gene, identification of genetic polymorphisms, and localization on the genetic map of chromosome 1p.
Genomics.
1990;7:131[Medline]
[Order article via Infotrieve].
7.
Kaghad M, Bonnet H, Yang A, et al.
Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.
Cell.
1997;90:809[Medline]
[Order article via Infotrieve].
8.
Jost CA, Marin MC, Kaellin WG Jr.
p73 is a human p53-related protein that can induce apoptosis.
Nature.
1997;389:191[Medline]
[Order article via Infotrieve].
9.
Mori N, Morosetti R, Lee S, et al.
Allelotype analysis in the evolution of chronic myelocytic leukemia.
Blood.
1997;90:2010
10.
Mori N, Morosetti R, Spira S, et al.
Chromosome band 1p36 contains a putative tumor suppressor gene important in the evolution of chronic myelocytic leukemia.
Blood.
1998;92:3405
11.
Zhuang Y, Cragoe EJ, Shaikewitz T, Glaser L, Cassel D.
Characterization of potent Na+/H+ exchange inhibitors from the amiloride series.
Biochemistry.
1984;23:4481[Medline]
[Order article via Infotrieve].
12.
Benos DJ.
Amiloride: chemistry, kinetics, and structure-activity relationships. In:
Grinstein S, ed.
Na+/H+ Exchange. Boca Raton, FL: CRC Press; 1988:121.
13.
Kleyman TR, Cragoe EJ.
Amiloride and its analogs as tools in the study of ion transport.
J Membr Biol.
1988;105:1[Medline]
[Order article via Infotrieve].
14.
Whetton AD, Vallance SJ, Monk PN, Cragoe EJ, Dexter TM, Heyworth CM.
Interleukin-3-stimulated haemopoietic stem cell proliferation: evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.
Biochem. J.
1988;256:585[Medline]
[Order article via Infotrieve].
15.
Vallance SJ, Downes CP, Cragoe EJ, Whetton AD.
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.
Biochem J.
1990;265:359[Medline]
[Order article via Infotrieve].
16.
Li J, Eastman A.
Apoptosis in an interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification: role of the Na(+)/H(+)-antiport.
J Biol Chem.
1995;270:3203
17.
Rebollo A, Gomez J, Martinez de Aragon A, Lastres P, Silva A, Perez-Sala D.
Apoptosis induced by IL-2 withdrawal is associated with an intracellular acidification.
Exp Cell Res.
1995;218:581[Medline]
[Order article via Infotrieve].
18.
Owen PJ, Musk P, Evans CA, Whetton AD.
Identification of cellular signalling events stimulated by a conditional v-abl mutant in an IL-3-dependent cell line.
J Biol Chem.
1993;268:15,696
19.
Schwartz MA, Ingber DE, Lawrence M, Springer TA, Lechene C.
Multiple integrins share the ability to induce elevation of intracellular pH.
Exp Cell Res.
1991;195:533[Medline]
[Order article via Infotrieve].
20.
Plopper GE, McNamee HP, Dike LE, Bojanowski K, Ingber DE.
Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.
Mol Biol Cell.
1995;6:1349[Abstract].
21.
Rich IN, Brackmann I, Worthington-White D, Dewey MJ.
Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation.
J Cell Physiol.
1998;177:109[Medline]
[Order article via Infotrieve].
22.
Perez-Sala D, Collado-Escobar D, Mollinedo F.
Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.
J Biol Chem.
1995;270:6235
23.
Chen Q, Benson RS, Whetton AD, et al.
Role of acid/base homeostasis in the suppression of apoptosis in haemopoietic cells by v-Abl protein tyrosine kinase.
J Cell Sci.
1997;110:379[Abstract].
24.
Tsao N, Lei HY.
Activation of the Na(+)/H(+) antiporter, Na+/HCO3(
25.
Lapidot T, Pflumio F, Dick JE.
Modeling human hematopoiesis in immunodeficient mice.
Lab Anim Sci.
1993;43:147[Medline]
[Order article via Infotrieve].
26.
Sirard C, Lapidot T, Vormoor J, et al.
Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis.
Blood.
1996;87:1539
27.
Yan Y, Salomon O, McGuirk J, et al.
Growth pattern and clinical correlation of subcutaneously inoculated human primary acute leukemias in severe combined immunodeficiency mice.
Blood.
1996;88:3137
28.
Thomas JA, Buchsbaum RN, Zimniak A, Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry.
1979;18:2210[Medline]
[Order article via Infotrieve].
29.
Bischof G, Cosentini E, Hamilton G, et al.
Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line.
Biochim Biophys Acta.
1996;1282:131[Medline]
[Order article via Infotrieve].
30.
Larsson R, Nygren P, Forsbeck K, Gylfe E, Nilsson K.
Early ionic events associated with phorbol ester induced differentiation and inhibition of cell growth in hematopoietic tumor cell lines.
Anticancer Res.
1989;9:1[Medline]
[Order article via Infotrieve].
31.
Uchida N, He DP, Friera AM, et al.
The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood.
Blood.
1997;89:465
32.
Look AT, Melvin SL, Williams DL, et al.
Aneuploidy and percentage of S-phase cells determined by flow cytometry correlate with cell phenotype in childhood acute leukemia.
Blood.
1982;60:959
33.
Holdrinet RS, Pennings A, Drenthe-Schonk AM, van Egmond J, Wessels JM, Haanen C.
Flow cytometric determination of the S-phase compartment in adult acute leukemia.
Acta Haematol.
1983;70:369[Medline]
[Order article via Infotrieve].
34.
Khochbin S, Chabanas A, Albert P, Albert J, Lawrence JJ.
Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle.
Cytometry
1988;9:499[Medline]
[Order article via Infotrieve].
35.
Smets LA, Slater R, van Wering ER, et al.
DNA index and %S-phase cells determined in acute lymphoblastic leukemia of children: a report from studies ALL V, ALL VI, and ALL VII (1979-1991) of the Dutch Childhood Leukemia Study Group and The Netherlands Workgroup on Cancer Genetics and Cytogenetics.
Med Pediatr Oncol.
1995;25:437[Medline]
[Order article via Infotrieve].
36.
Sasaki K, Murakami T, Ogino T, Takahashi M.
Flow cytometric dual-parameter analysis of human leukemic cells using monoclonal anti-BrdUrd antibody.
Gan No Rinsho.
1985;31:549[Medline]
[Order article via Infotrieve].
37.
Rotin D, Steele-Norwood D, Grinstein S, Tannock I.
Requirement of the Na+/H+ exchanger for tumor growth.
Cancer Res.
1989;49:205
38.
Nwankwo JO.
Mechanism of chemical carcinogenesis: an alternative hypothesis.
Med Hypotheses.
1991;35:330[Medline]
[Order article via Infotrieve].
39.
Beech JA.
Carcinogenesis and initiation of cell cycling by charge-induced membrane clusters may be due to mitogen receptors and Na+/H+ antiports.
Med Hypotheses.
1994;42:385[Medline]
[Order article via Infotrieve].
40. Garden OA, Musk P, Worthington-White DA, Dewey MJ, Rich IN. Sequence
comparison of the coding region of human sodium/hydrogen
exchanger isoform-1 cDNA in peripheral blood mononuclear cells of
healthy volunteers and leukemic patients. Cancer Genet Cytogenet. In
press.
41.
Pouyssegur J, Sardet C, Franchi A, L'Allemain G, Paris S.
A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.
Proc Natl Acad Sci U S A.
1984;81:4833
42.
Park HJ, Makepeace CM, Lyons JC, Song CW.
Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells [abstract].
Eur J Cancer.
1996;32A:540.
43.
Harguindey S, Cragoe EJ Jr.
The Na+/H+ antiporter in oncology in the light of the spontaneous regression of cancer and cell metabolism.
Med Hypotheses.
1992;39:229[Medline]
[Order article via Infotrieve].
44.
Horvat B, Taheri S, Salihagic A.
Tumour cell proliferation is abolished by inhibitors of Na+/H+ and HCO3
45.
Hasuda K, Lee C, Tannock IF.
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.
Oncol Res.
1994;6:259[Medline]
[Order article via Infotrieve].
46.
Tannock IF, Rotin D.
Acid pH in tumors and its potential for therapeutic exploitation.
Cancer Res.
1989;49:4373
47.
Liu FF, Diep K, Hill RP.
The relationship between thermosensitivity and intracellular pH in cells deficient in Na+/H+ antiport function.
Radiother Oncol.
1996;40:75[Medline]
[Order article via Infotrieve].
48.
Bortner CD, Hughes FM Jr, Cidlowski JA.
A primary role for K+ and Na+ efflux in the activation of apoptosis.
J Biol Chem.
1997;272:32,436
49.
Tanaka S, Koike T.
Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na(+)-dependent mechanism in cultured rat sympathetic neurons.
Int J Dev Neurosci.
1997;15:15[Medline]
[Order article via Infotrieve].
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
measured by annexin-V and TUNEL
methodologies
4 integrin subunit with fibronectin, causing activation
of NHE1, an increase in pHi, and increased hematopoietic
colony formation.
)/CO3(2
