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. 1998 May;18(5):2845-54.
doi: 10.1128/MCB.18.5.2845.

Regulation of proliferation-survival decisions during tumor cell hypoxia

C Schmaltz  1 , P H Hardenbergh, A Wells, D E Fisher
Affiliations

Regulation of proliferation-survival decisions during tumor cell hypoxia

C Schmaltz et al. Mol Cell Biol. 1998 May.

Abstract

Hypoxia may influence tumor biology in paradoxically opposing ways: it is lethal as a direct stress trigger, yet hypoxic zones in solid tumors harbor viable cells which are particularly resistant to treatment and contribute importantly to disease relapse. To examine mechanisms underlying growth-survival decisions during hypoxia, we have compared genetically related transformed and untransformed fibroblast cells in vitro for proliferation, survival, clonogenicity, cell cycle, and p53 expression. Hypoxia induces G0/G1 arrest in primary fibroblasts but triggers apoptosis in oncogene-transformed derivatives. Unexpectedly, the mechanism of apoptosis is seen to require accumulated acidosis and is rescued by enhanced buffering. The direct effect of hypoxia under nonacidotic conditions is unique to transformed cells in that they override the hypoxic G0/G1 arrest of primary cells. Moreover, when uncoupled from acidosis, hypoxia enhances tumor cell viability and clonogenicity relative to normoxia. p53 is correspondingly upregulated in response to hypoxia-induced acidosis but downregulated during hypoxia without acidosis. Hypoxia may thus produce both treatment resistance and a growth advantage. Given strong evidence that hypoxic regions in solid tumors are often nonacidotic (G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, Nat. Med. 3:177-182, 1997), this behavior may influence relapse and implicates such cells as potentially important therapeutic targets.

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Figures

FIG. 1
FIG. 1
(a) Hypoxia-induced growth arrest in untransformed REF. Cells were plated at a density of 2 × 105/60-mm-diameter dish, and exposure to hypoxia was started approximately 16 h later (time zero). At 0, 24, and 48 h, cells were harvested and the number of viable (trypan blue-excluding) cells per 60-mm-diameter dish was counted. Results of experiments in triplicate are expressed as means ± standard deviations. (b) Hypoxia-induced G1-arrest in untransformed REF. The DNA content of fixed and PI-stained cells from the same experiment described for panel a was measured by flow cytometry, and cell cycle analysis was performed. Percentages of cells in each phase of the cell cycle are given for each diagram. (c) Absolute changes in distribution of cells in the cell cycle. Percentages immediately before exposure to hypoxia are taken as the starting point (0); absolute increases and decreases in the percentages of cells in G1/G0, S, and G2/M after 24 and 48 h are plotted. All studies are representative of at least three independent experiments.
FIG. 2
FIG. 2
Viability of cells under hypoxic conditions. (a and b) Cells plated at a density of 5 × 105 cells/60-mm-diameter dish were kept in a hypoxic environment for different time periods, and their viability was quantitated by exclusion of trypan blue. Values for normoxic control plates were measured at the same time points; the viability of these cells reflects a small degree of background apoptosis but does not change over the course of the experiment. Results of experiments in triplicate are expressed as means ± standard deviations. (a) REF and c-Myc/Ras-transformed REF. (b) E1a/Ras-transformed MEF (p53+/+ and p53−/−). (c and d) Phosphatidylserine exposure as a quantitative measure of apoptosis. Untransformed REF or Myc/Ras-transformed REF (c) and E1a/Ras-transformed MEF (p53+/+ or p53−/−) (d) were plated at 5 × 105 cells/60-mm-diameter dish and rendered hypoxic for the time periods indicated. Flow cytometry for Annexin V staining (indicating phosphatidylserine flippage) produced histograms which demonstrate apoptotic death in hypoxic transformed cells (fluorescence intensity for Annexin V is plotted on the x axis, and cell number is plotted on the y axis). Phosphatidylserine flippage precedes trypan blue permeability (a and b).
FIG. 3
FIG. 3
(a) Internucleosomal DNA cleavage as a marker of hypoxia-induced apoptosis. Genomic DNA from normoxic or hypoxic cells (5 × 105 cells/60-mm-diameter plate) was electrophoretically resolved and revealed laddering in hypoxic cells but not in normoxic controls. M, markers. (b) Apoptotic morphology of cells exposed to 48 h of hypoxia at high density. Hypoxic E1a/Ras-transformed MEF (p53+/+) were fixed and stained for nuclear morphology with DAPI. Fluorescence microscopy demonstrates nuclear condensation and fragmentation (including apoptotic bodies) in the hypoxic cells compared to oxic controls (identical magnifications and exposures were used). (c) PARP cleavage activity in hypoxia-triggered apoptosis. Equal amounts of cytoplasmic extracts from hypoxic and normoxic E1a/Ras-transformed MEF (p53+/+) were incubated with a recombinant N-terminal PARP fragment. Immunoblotting shows PARP cleavage activity in the hypoxic extract but no PARP cleavage activity in equivalent extracts from normoxic cells.
FIG. 4
FIG. 4
Viability of transformed cells under hypoxia at different densities. Cells plated at different densities were incubated in a hypoxic environment for a fixed time, and viability was determined by exclusion of trypan blue. Oxic controls of both cell lines were viable at all times of the experiment. Results of experiments in triplicate are expressed as means ± standard deviations. (a) REF and c-Myc/Ras-transformed REF, examined after 24 h of treatment. ▵, REF, normoxia; □, REF, hypoxia; ○, Myc/Ras REF, normoxia; ◊, Myc/Ras REF, hypoxia. (b) E1a/Ras-transformed MEF (p53+/+ and p53−/−), examined after 53 h of treatment. ○, p53+/+, normoxia; ▵, p53−/−, normoxia; •, p53−/−, hypoxia; □, p53+/+, hypoxia.
FIG. 5
FIG. 5
(a) Decline of medium pH during hypoxia. E1a/Ras-transformed MEF (p53+/+) were plated at densities of 1 × 105, 5 × 105, and 2 × 106 cells/60-mm-diameter dish and incubated under hypoxic or normoxic conditions for the time periods indicated. (b and c) Rescue from hypoxia-triggered cell death in E1a/Ras-transformed p53+/+ fibroblasts through increased buffer capacity of the medium. Viability was assessed by exclusion of trypan blue. Results of experiments in triplicate are expressed as means ± standard deviations. (b) Additional buffer. Cells were plated at 2 × 106/60-mm-diameter dish and exposed to hypoxia for 22 h in 1.7 ml of either HEPES-free or 25 mM HEPES-containing medium (see Materials and Methods). (c) Increased volume of degassed medium. Cells were plated at 2 × 106/60-mm-diameter dish and exposed to hypoxia for 17 h in the presence of either 1.7 or 5 ml of previously degassed medium. (d) pH of medium correlates with viability. The pH of the medium following incubation under the conditions used for panel c is plotted. (e) Preacidification of medium triggers apoptosis in the absence of hypoxia. Medium was acidified to pH 6.5 (matching the pH of high-density hypoxic p53+/+ apoptotic cells) by using HCl or acidified to 6.5 and then reneutralized with NaOH. Myc/Ras-transformed REF or E1a/Ras-transformed MEF (p53+/+) were exposed to these media at a low cell density (105/60-mm-diameter plate) for 5 h, followed by Annexin V staining and flow cytometric quantitation of apoptosis.
FIG. 6
FIG. 6
Effects of hypoxia at various cell densities (a and b) E1a/Ras-transformed MEF (p53+/+ and p53−/−) were plated at a low density (1 × 105/60-mm-diameter dish; <5% confluence) or at a higher density (2 × 106/60-mm-diameter dish; ∼50% confluence), incubated under hypoxia for 30 h, trypsinized, and assessed for viability by exclusion of trypan blue, (a) or reseeded for clonogenic assays (b). Colonies containing more than 50 cells were scored after 7 days. Results of experiments in triplicate are expressed as means ± standard deviations. (c) Increase in S phase in low-density hypoxic cultures. E1a/Ras-transformed MEF (p53+/+) at a low density (105/60-mm-diameter dish) were BrdU pulse-labeled for the last 30 min of 30-h hypoxic incubations. S phase was determined by flow cytometry for BrdU uptake and PI staining. Unlike primary fibroblasts, which undergo cell cycle arrest (Fig. 1), transformed fibroblasts fail to arrest and even show a modest but consistent increase in S phase. The results shown are representative of three independent experiments.
FIG. 7
FIG. 7
p53 protein levels rise following hypoxia at high density (with acidosis), but fall following hypoxia at low density (without acidosis). p53 protein levels were determined by immunoblotting of lysates from cells exposed to hypoxia for the times indicated at 2 × 106 or 1 × 105 cells/60-mm-diameter dish and compared to those for oxic controls and cells treated with 5 Gy of ionizing radiation (IR) (measurements were made 2 h after irradiation). p53 levels rise at 2 × 106 cells/60-mm-diameter dish, although these cells eventually lose viability, whereas p53 levels fall at the low density (in the absence of acidosis), although these cells display enhanced viability (Fig. 6). Antitubulin (α-tubulin) reblotting (loading control) is shown for the low-density blot.
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