Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

Abstract

Genomic instability is a hallmark of cancer development and progression, and characterizing the stresses that create and the mechanisms by which cells respond to genomic perturbations is essential. Here we demonstrate that antiapoptotic BCL-2 family proteins promoted tumor formation of transformed baby mouse kidney (BMK) epithelial cells by antagonizing BAX- and BAK-dependent apoptosis. Cell death in vivo correlated with hypoxia and induction of PUMA (p53 up-regulated modulator of apoptosis). Strikingly, carcinomas formed by transformed BMK cells in which apoptosis was blocked by aberrant BCL-2 family protein function displayed prevalent, highly polyploid, tumor giant cells. Examination of the transformed BMK cells in vivo revealed aberrant metaphases and ploidy changes in tumors as early as 9 d after implantation, which progressed in magnitude during the tumorigenic process. An in vitro ischemia system mimicked the tumor microenvironment, and gain of BCL-2 or loss of BAX and BAK was sufficient to confer resistance to apoptosis and to allow for accumulation of polyploid cells in vitro. These data suggest that in vivo, even in cells in which p53 function is compromised, apoptosis is an essential response to hypoxia and ischemia in the tumor microenvironment and that abrogation of this response allows the survival of cells with abnormal genomes and promotes tumorigenesis.

Figure 1.

Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. (A) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 (left top panel) or E1B 19K (right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. (B) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. (C) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

Figure 2.

Loss of BAX and BAK or gain of BCL-2 blocks cell death in nude mice. Transformed BMK cells expressing red fluorescent protein (RFP) were injected subcutaneously into nude mice. Mice were ear-tagged and individual mice were monitored over time using a whole-body fluorescence imaging system to follow injected BMK cells. One representative animal of each cell line is shown. Note the progressive loss of RFP signal in the control transformed BMK cells (W2.3.1–5), which was largely prevented by the loss of BAX and BAK (D3.zeo-2) or the gain of BCL-2 (W2.Bcl2–3).

Figure 3.

BMK cell death is accompanied by hypoxia in mice. Histology of the injected BMK cells reveals extensive cell death in large necrotic centers. Hematoxylin and eosin (H&E column)-stained sections of BMK cell masses excised from animals on days 2 (top panels) and 9 (bottom panels) after injection are shown at 200×. Necrotic regions are indicated (N). Insets show W2.3.1–5 cells dying with apoptotic morphology, evident as multiple compartments of condensed chromatin in the dying cells, as well as necrotic morphology, evident as a single large patch of condensed chromatin in a dying cell. In contrast, dying W2.Bcl2–3 and D3.zeo-2 cells show only necrotic morphology (1000×). White rectangles in each panel denote areas enlarged in insets. Arrows indicate scattered patches of surviving W2.3.1–5 cells on day 9 after injection. Sections adjacent to the H&E-stained sections were stained with antibodies specific to adenovirus E1A (E1A column) by immunohistochemistry to distinguish transformed BMK cells from host tissue (E1A reactive cells are stained brown). Sections adjacent to the E1A immunohistochemistry were developed by immunohistochemistry to hypoxyprobe adducts (hypoxyprobe column) to reveal hypoxic areas of the tumors (stained brown). Nearly adjacent sections were also developed using antibodies specific to active caspase 3 (active caspase 3 column) to reveal cells undergoing apoptosis (stained brown). Insets show active caspase 3 reactive apoptotic cells in W2.3.1–5 cell masses at 600×, and white rectangles indicate areas enlarged in the insets.

Figure 4.

BMK cells subjected to hypoxia induce HIF-1α and PUMA in vivo. Extracts of transformed BMK cells excised from animals were subjected to Western blotting for HIF-1α (A) or PUMA and BIM (B). For all cell lines, C indicates control extracts made from the same cell lines prior to injection into animals, whereas 2 and 9 indicate extracts made from BMK cell masses excised from animals on days 2 and 9 after injection, respectively. In A, lanes labeled N and H indicate extracts made from cells subjected to normal oxygen culture and in vitro hypoxic culture for 12 h, respectively. The bottom panels represent actin levels on each blot to demonstrate equivalent protein loads. (C) PUMA causes BAX- and BAK-dependent cell death. W2 cells, which contain BAX and BAK, and D2 cells, which are deficient for both BAX and BAK, were transfected with plasmids directing expression of PUMA, PUMA with a deletion of the BH3 domain (PUMA-BH3), or pCEP-4 vector control (C). Twentyfour hours later, viabilities were determined as described in Materials and Methods. Note the BH3-dependent killing of the W2 cells that is completely blocked by the absence of BAX and BAK (D2 cells).

Figure 5.

Formation of tumor giant cells in tumors defective for apoptosis. (A) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. (B) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

Figure 6.

Defective apoptosis confers resistance to ischemia-induced cell death and allows for the accumulation of polyploid cells in response to ischemia in vitro. (A) Gain of BCL-2 or loss of BAX and BAK blocks ischemia-induced cell death. Transformed BMK cell lines were subjected to in vitro ischemic culture conditions and viabilities were determined on the indicated days. Results of triplicates are plotted for each time point. (B) PUMA is induced by ischemia in vitro. Cell extracts made from cells cultured for 24 h in normal control conditions (C), or in ischemic culture conditions (I), were immunoblotted for PUMA, BIM, and actin. Note the induction of PUMA, but not BIM, in ischemic culture conditions. (C,D) Gain of BCL-2 or loss of BAX and BAK allows for accumulation of polyploid cells in response to ischemia. Transformed BMK cell lines were subjected to ischemic culture conditions for 2 d and then returned to normal culture conditions in complete media for 3 d. (C) Cells were collected prior to ischemia, following 2 d of ischemia, and after ischemia with an additional 3 d of recovery on return to normal culture conditions and analyzed by flow cytometry. Insets illustrate typical nuclear morphology in each condition visualized by DAPI staining of cells on coverslips. Arrows indicate typical multi-/giant-nuclei in W2.Bcl2–3 and D3.zeo-2 cells, and the arrowhead indicates a W2.3.1–5 cell dying in hypoxia. (D) Representative polyploid cells stained with DAPI were also imaged using confocal laser scanning microscopy. In each panel, a nucleus of normal size cell is included in the picture for comparison. Ratios of the total DNA content of the polyploid cells to the total DNA content of the control cells are included in the insets for each panel.

Figure 7.

Model for tumor selection and progression. See text for explanation.