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Cyclic exposure to hypoxia and reoxygenation selects for tumor cells with defects in mitochondrial apoptotic pathways

Department of Radiation Oncology, University of Tübingen, Tübingen, Germany; and
^* Department of Radiation Oncology, Charite Berlin, Berlin, Germany

¹Correspondence: Department of Radiation Oncology, University of Tübingen, Hoppe-Seylerstrasse 3, Tübingen 72076, Germany. E-mail: claus.belka{at}uni-tuebingen.de

SPECIFIC AIMS

Tumor hypoxia is associated with unfavorable prognosis. Several findings support the assumption that hypoxia contributes to treatment failure by indirect mechanisms such as the selection of resistant tumor cells under repetitive hypoxia.

It is likely that cyclic hypoxia represents a bottleneck situation leading to cell death but it may also select for cells with enhanced hypoxia tolerance. To gain insight into death-signaling pathways targeted by hypoxic selection, the impact on paradigmatic triggers of mitochondrial apoptotic pathways was compared with the influence on death-receptor-mediated apoptosis. Since selection of p53-defective cells has been described as a consequence of cyclic hypoxia, the effect of hypoxic selection on radiation-induced p53-dependent activation of p21, Puma, and Bax was tested in parallel with a variety of other Bcl-2 family proteins.

PRINCIPAL FINDINGS

1. Hypoxia induces HIF-1 activation
The transcription factor HIF-1 could not be detected under normoxic conditions. Under hypoxic conditions chosen for all experiments, HIF-1 was stabilized and HIF-1 binding to the HRE-consensus sequences could be detected in an ELISA-based assay.

2. NCI H460 cells are susceptible to hypoxia-induced apoptosis
Apoptosis was quantified using fluorescence microcopy and CaspACE FITC-VAD-FMK in situ marker. NCI H460 cells were clearly susceptible to hypoxia-induced apoptosis. After 48 h, the apoptotic rate reached 20%–25%.

3. Hypoxic selection leads to increased hypoxia tolerance
Apoptosis after the first hypoxic period (P1) was compared with the apoptotic rate after ten cycles of hypoxia (P10). Unselected cells were sensitive to hypoxia-induced apoptosis, whereas exposure to ten cycles of hypoxia renders cells more resistant against hypoxic stress.

4. Hypoxic selection increases apoptotic resistance against ionizing radiation
To analyze whether hypoxic selection leads to cross-resistance against radiation-induced apoptosis, cells were irradiated after recovery from different numbers of hypoxic cycles under normoxia. Apoptosis was quantified after 24 h and compared with controls. As shown in Fig. 1 A, B, the rate of apoptosis induction by ionizing radiation decreases steadily with an increasing number of hypoxic cycles.

View larger version (16K): [in this window] [in a new window]   Figure 1. Radiation-induced apoptosis of cells exposed to increasing numbers of hypoxic cycles. A) Counting of apoptotic cells using Hoechst 33342. B) Caspase activation determined by the CaspAce® system.

5. Hypoxic selection increases apoptotic resistance against etoposide
Similar experiments were performed to evaluate whether hypoxic selection affects other triggers of the mitochondrial death pathway. Selected cells (P10) and control cells (P0) were incubated with 0–25 µg/mL etoposide. Control cells were sensitive to etoposide-induced apoptosis. [Apoptotic rates: 45% ± 8% (6.25 µg/mL etoposide), 63% ± 7% (25 µg/mL etoposide) (CaspAce® staining), (similar for _m breakdown)]. In contrast, NCI H 460 cells selected by cyclic hypoxia showed significantly reduced apoptosis 22% ± 4 (6.25 µg/mL etoposide, 44% ± 7% (25 µg/mL etoposide) (CaspAce® staining), (similar for _m breakdown).

6. Hypoxic selection does not affect p53-dependent activation of p21
To determine whether the hypoxia-tolerant phenotype is associated with selection of p53-deficient cells, radiation-induced p53-dependent activation of p21 was analyzed in p53wt-NCI H 460 cells before and after 10 cycles of hypoxia/reoxygenation. Western blots demonstrated a time-dependent increase of p21 protein after irradiation. The radiation-induced activation of p21 was not altered in the selected cells.

7. Expression of key regulator proteins of mitochondrial apoptosis is not altered after hypoxic selection
To examine in how far regulatory proteins of the mitochondrial pathway are affected by hypoxic selection, the expression of Bax, Bak, Bim, Puma, Bcl-xL, and Bcl-2 was analyzed before and after hypoxic selection by Western blot. Irradiation triggered an increased Bax and Puma expression. Although Bax and Puma are known to be involved in apoptosis regulation by DNA damage, the expression levels were not found be differentially regulated in cells before and after hypoxic selection. Moreover, hypoxic selection did not affect Bim, Bak, Bcl-2 or Bcl-xL expression.

8. Hypoxic selection affects radiation-induced conformational changes of Bax
The effect of hypoxic selection on radiation-induced conformational changes of Bax has been evaluated. Selected and unselected NCI H 460 cells have been irradiated with 10 Gy. Changes of the Bax conformation were analyzed using a conformation-specific antibody. Conformational changes of Bax were not detectable in nonirradiated controls, whereas antibody staining increased significantly after irradiation. Compared to controls, the proportion of radiation-induced Bax-conformation changes was significantly decreased after hypoxic selection.

9. Hypoxic selection does not influence TRAIL-induced apoptosis
To evaluate the specificity of hypoxic selection, the effect on mitochondrial triggers was compared with the effects on TRAIL-induced apoptosis. Therefore, radiation- and TRAIL-induced apoptosis in unselected cells (P0) were compared with apoptosis in cells exposed to ten cycles of hypoxia (P10). As shown in Fig. 2 A, B, cyclic hypoxia leads to an increased resistance against radiation-induced apoptosis, whereas the efficacy of TRAIL-induced apoptosis was not significantly influenced.

View larger version (23K): [in this window] [in a new window]   Figure 2. Hypoxic selection affects radiation-induced but not TRAIL-induced apoptosis. A) Radiation-induced apoptosis (10 Gy). Upper panel: apoptotic rate determined by CaspAce® staining; lower panel: Hoechst staining: P0: 32% ± 3.8%, P10: 12% ± 3%. B) TRAIL-induced apoptosis. Upper panel: apoptotic rate determined by CaspAce® staining; lower panel: Hoechst staining.

CONCLUSIONS AND SIGNIFICANCE

The key observation is that this selection pressure mainly affects mitochondrial death pathways whereas TRAIL-receptor death signaling is not significantly impaired. Hypoxic selection was associated with a global cross resistance affecting other triggers of mitochondrial apoptosis. Graeber et al. demonstrated that exposure of mixed p53 wt and p53–/– cells to cyclic hypoxia was associated with and increased resistance to hypoxia and a selection p53 negative cells. Using a much defined but somewhat artificial system, the findings by Graeber et al. delivered the initial ideas for the generation of a concept of hypoxic selection as an important mechanism of increasing tumor resistance.

Up to now it has not been shown that hypoxic selection indeed affects the death inducing capabilities of anticancer treatment modalities. In contrast to Graeber et al. who started with a defined genetic heterogeneity (quantitatively defined mixture of p53–/– and p53 +/+ cells on a Ras transformed cellular background) we have shown that even using a standard tumor cell system the process of hypoxic selection is functional and leads to cross-resistance toward other death triggers.

Most data support a p53-dependent hypoxic selection process. To clarify whether increased stress tolerance after hypoxic selection is associated with altered p53-function, radiation-induced p53-dependent downstream activation of p21 was analyzed before and after hypoxic selection. No alterations of p21 activation were observed, indicating that p53 functions are retained after hypoxic selection in this model system.

Members of the Bcl-2 family, main regulators of apoptosis on the level of mitochondria, are also potential targets for a selection process. We analyzed the effect of hypoxic selection on key antiapoptotic (Bcl-2 and Bcl-xL) and proapoptotic proteins (Bax, Bak, Bim, Puma). Radiation triggered the up-regulation of Puma and Bax in both, selected and unselected cells. Although this finding indicates a functional p53 in both situations, it does not explain the cross resistance induced by hypoxic selection. However, the analysis of conformational changes of Bax clearly points to the fact that functional aspects of the Bax system may targeted by hypoxic selection. Hypoxia-induced cell death is mainly triggered by Bcl-2 inhibitable mitochondrial death pathways. Combining the knowledge of the mechanisms of hypoxia-induced apoptosis with the effects exerted by hypoxic selection, leads to the conclusion that hypoxia renders tumors cells more resistant to therapy via selection of cells with mitochondrial apoptosis defects.

This is corroborated by our finding that TRAIL-induced apoptosis is only marginally affected by the hypoxic selection process. Receptor-mediated apoptosis is mainly initiated by a FADD-mediated activation of caspase-8. Via cleavage of BID and release of cytochrome c, mitochondrial death pathways are shown to be involved in receptor-mediated apoptosis. However, in case of optimal receptor stimulation the activation of mitochondrial death pathways is not mandatory for adequate execution of apoptosis. In accordance, hypoxic selection leads to cell clones with a insignificant reduction of TRAIL-induced apoptosis after 24 h indicative of a disturbed mitochondrial feedback loop. These findings indicate that hypoxic selection leads to an increased resistance toward mitochondrial death triggers.

View larger version (49K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1918fje;

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