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[Cancer Research 64, 4180-4189, June 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Hypoxia Enhances Metastatic Efficiency by Up-Regulating Mdm2 in KHT Cells and Increasing Resistance to Apoptosis

Li Zhang1,2 and Richard P. Hill1,2,3

1 Research Division, Ontario Cancer Institute/Princess Margaret Hospital, 2 Department of Medical Biophysics, and 3 Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites. Both clinical and experimental studies have suggested a positive correlation between tumor hypoxia and increased metastatic efficiency; however, the mechanisms are not understood. In this study, the mechanisms of hypoxia-enhanced metastasis have been investigated in murine KHT fibrosarcoma and SCC VII cells. We have observed that hypoxia-pretreated KHT-C cells have a higher survival rate than control KHT-C cells after being arrested in mouse lungs. cDNA microarray analysis revealed many hypoxia-regulated genes, most of which have been reported to be involved in cell survival and growth. Among these genes, we have confirmed the up-regulation of Mdm2 by hypoxia and have demonstrated that this up-regulation is p53 independent. The up-regulation of Mdm2 by hypoxia is associated with decreased p53 protein and inhibition of the transactivation of p53 downstream proapoptotic genes. Overexpression of Mdm2 or suppression of p53 by transient transfection increased metastatic efficiency in KHT-C cells. These data suggest that hypoxia can increase tumor cell metastatic efficiency by rendering the tumor cells less sensitive to stress-induced cell death, e.g., through modifying the levels of Mdm2 and p53.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia in tumors is a consequence of a structurally and functionally disturbed microcirculation and the deterioration of oxygen diffusion capacity because of uncontrolled growth of tumor cells. Tumor hypoxia, which is a common feature of solid tumors, appears to be strongly associated with tumor growth, resistance to therapy, and malignant progression, and it has become a central issue in tumor physiology and cancer treatment. Numerous studies have shown a positive correlation between tumor hypoxia and increased metastasis (1 , 2) . A number of clinical studies suggest that one of the main reasons for the poor outcome of hypoxic tumors is increased tumor cell metastatic potential, e.g., in cervix cancer and soft tissue sarcoma (3, 4, 5, 6, 7) . In addition to clinical studies, laboratory experiments also found that hypoxia-treated tumor cells such as the murine cell lines KHT (fibrosarcoma), SCC VII (squamous cell carcinoma), and B16F10 (melanoma) were more metastatic compared with their counterparts growing under normal conditions and that the increased metastases only occurred when the cells were exposed to oxygen levels < 1% (8 , 9) . Similar results have been obtained recently in human melanoma cells by Rofstad et al. (10 , 11) , who reported increased metastases in hypoxia-treated cells. They also implicated the involvement of angiogenic growth factors such as vascular endothelial growth factor and interleukin-8 in the hypoxia-enhanced metastasis. Consistent with this finding, Shi et al. (12) have shown that interleukin-8 can be induced by hypoxia in human pancreatic cancer cell lines and that interleukin-8 transfection increases the metastatic potential of these cells.

As a main cause of death in cancer patients, metastasis has been extensively studied. Although the mechanisms controlling tumor cell metastatic efficiency are not completely understood and may vary in different cell types, both experimental and clinical studies indicate that metastatic efficiency of tumor cells can be associated with their resistance to apoptosis (13, 14, 15) . Apoptosis is a genetically controlled, morphologically unique process that plays a central role in regulating tissue homeostasis by eliminating cells that are harmful or no longer required. Deregulation of apoptosis results in abnormal cell growth and death, therefore, promoting tumor expansion (16 , 17) . In studies on metastatic inefficiency, Wong et al. (14) reported that tumor cells injected i.v. underwent apoptosis after being arrested in lungs and that highly metastatic cell lines were found to be more resistant to apoptosis than the control cell lines. They suggested that apoptosis in vivo corresponds to decreased metastases and that sensitivity to apoptosis in vivo is an important component of metastatic inefficiency (14) . In support of these experimental results, clinical evidence also suggests a correlation between apoptosis and metastasis (18 , 19) .

Apoptosis can be induced by environmental stimuli that cause cell damage such as hypoxia. Hypoxia induces apoptosis partly through p53 and may function as a selective pressure for cells in solid tumors, leading to progression to a more malignant phenotype by selection for an apoptosis-resistant cell population (20 , 21) . The level of p53 in cells has been reported to be increased under hypoxic conditions in a number of cell lines (22, 23, 24, 25) , and increased levels of p53 can induce apoptosis by a pathway involving Apaf-1 and caspase-9 (26) . Hypoxia is also able to induce apoptosis in a p53-independent manner, e.g., through Myc, which has been reported to mediate hypoxia-induced apoptosis (27) . In addition to acting as a selective pressure on tumor cells, hypoxia has been reported to regulate the expression of a number of genes that are involved in different cellular biological functions and are important to tumor cell survival such as vascular endothelial growth factor (VEGF), heme oxygenase-1, Adrenomedullin, GLUT1, nuclear factor-{kappa}B (NF-{kappa}B), interleukin-8, and carbonic anhydrase-9 (1 , 28) .

We have been investigating whether the increased metastatic ability of tumor cells exposed to hypoxia may be caused by the altered apoptotic response of the cells, and if so, what mechanisms are involved. For these studies, we chose to use two murine cell lines KHT and SCC VII, which we have shown previously demonstrate a significant increase in metastatic ability after hypoxic exposure and reoxygenation; after 24 h hypoxic exposure, this increase reached a maximum with 18–24 h of reoxygenation and then declined (8 , 9 , 29, 30, 31) . Initially, we performed cDNA microarray analysis to identify hypoxia-regulated genes in KHT cells and found that Mdm2was one of the genes up-regulated by hypoxic exposure. On the basis of the known roles of Mdm2 and its interaction with p53 (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) , we hypothesized that hypoxia may promote KHT cell experimental metastatic efficiency by increasing the survival of the tumor cells in the lung environment. We demonstrated that transient transfection of Mdm2or antisense p53cDNA into KHT cells increased their metastatic potential under aerobic conditions. Our results indicate that increased Mdm2 expression contributes to the increased metastatic efficiency of KHT-C cells after hypoxic treatment, at least partly by increasing the cell resistance to stress-induced apoptosis through the inhibition of p53 activity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Hypoxic Treatment.
KHT-C cells were cultured in {alpha}-MEM (Invitrogen, Carlsbad, CA) supplemented with penicillin/streptomycin and 10% FCS. For all experiments, 2–5 x 105 cells were plated in 10-cm dishes and incubated in 5% CO2 and air at 37°C. To render cells hypoxic, dishes were placed in a modular incubator chamber (Billups-Rothenberg, Del Mar, CA), flushed with 95% N2 and 5% CO2 and incubated at 37°C for designated times. It took between 3 and 6 h to achieve severe hypoxic condition (≤5 mmHg) in the culture medium. For reoxygenation after hypoxic incubation, cells were transferred back to 5% CO2 and air.

Experimental Metastasis Assay and Viability Assay of Lung-Arrested Tumor Cells.
Cells were harvested by trypsin treatment, pelleted, and resuspended in growth medium. They were then passed through a 70 µm cell strainer, counted, and diluted to 1–5 x 105 cells/ml before i.v. injection. Male C3H/HeJ mice (syngeneic to KHT sarcoma), obtained from The Jackson Laboratory (Bar Harbor, ME) were housed in the specific pathogen-free colony of the Ontario Cancer Institute under conditions approved by the Canadian Council on Animal Care.

For the lung metastasis assay, each mouse received 2–10 x 104 cells via the tail vein. Groups of mice were sacrificed 20 days later. The lungs were removed and fixed in Bouin’s solution. The number of pulmonary tumors that had developed on the lung surface was determined with the aid of a dissecting microscope.

For the viability assay of the arrested tumor cells, 105 cells were i.v. injected into each mouse. Lungs were removed at designated times. To make a lung cell suspension, minced lungs were incubated in PBS containing 0.15% Bacto-Trypsin (BD Biosciences, Sparks, MD) and 150 Kunitz units/ml DNase I (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C with agitation. The suspension was passed through a 70-µm cell strainer, the supernatant was discarded, and the tissue incubated in {alpha}-MEM containing 0.25% collagenase type IV (Sigma-Aldrich) and 300 Kunitz units/ml DNase I for 1 h at 37°C with agitation. The suspension was shaken to disperse the cells and passed through a 40-µm cell strainer to collect a single cell suspension. A total of 2–10 x 104 cells/dish was plated into 10-cm tissue culture dishes. Colonies of KHT cells were counted 7–10 days later.

Microarray Analysis.
Mouse 15K microarray slides were purchased from the University Health Network microarray center (Toronto, Ontario, Canada). Each slide contained duplicated 15,000 expressed sequence tag unique mouse cDNAs from the National Institute on Aging/NIH Mouse cDNA project. RNA was extracted from KHT-C cells grown under hypoxic conditions (5% CO2 balanced with N2) for 24 h or aerobic (5% CO2 balanced with air) conditions, respectively, and 2 µg of total RNA from each sample were used to synthesize probes for one labeling. Probes labeled with Cy3 or Cy5 (Amersham Biosciences, Piscataway, NJ) by amino allyl labeling from aerobic and hypoxic RNAs were hybridized to one mouse 15K chip. Reverse dye experiments were performed to correct for any dye bias caused by the different binding affinity of Cy3 or Cy5 to various cDNA sequences. Hybridized slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union City, CA). The microarray hybridization was repeated once giving a total of eight separate hybridizations (including dye reverse experiments and cDNA duplicates on each slide) for each gene. The data were analyzed with GeneSight software from BioDiscovery (Marina del Rey, CA) to filter the data and remove the unreliable information.

Cell Transfection and Fluorescence-Activated Cell Sorting.
The day before transfection, 2 x 106 cells were seeded in 10 ml supplemented {alpha}-MEM culture medium/10-cm tissue culture dish. On the day of transfection, the cells were 60–80% confluent. The cells were suspended in 250 µl of serum-free, antibiotics-free {alpha}-MEM at a concentration of 107 cells/ml. The cell suspension was then transferred into an electroporation cuvette (Bio-Rad, Hercules, CA) and mixed with 20 µg of plasmid DNA at the molar ratio of target gene constructs to enhanced green fluorescent protein (EGFP) at 3:1 diluted in 250 µl of serum-free, antibiotics-free {alpha}-MEM. Electroporation was performed at 210 V, 975 µF. Electroporated cells were transferred to a 10-cm tissue culture dish containing 10 ml of prewarmed complete {alpha}-MEM and recovered for 20–24 h. Transfected cells were treated with trypsin and pelleted by centrifugation at 1000 rpm for 10 min at 4°C. Cells were then washed once and resuspended in complete medium at ~107 cells/ml. The sorting for EGFP- positive cells was performed at DF 530/40 on a Moflo cell sorter (DAKO Cytomation, Fort Collins, CO). The number of sorted viable cells was determined by a colony-forming assay.

Plasmid Constructs.
The mammalian expression vector phCMV1 was purchased from Genetherapy Systems, Inc. (San Diego, CA). The murine Mdm2, p53, and Bcl-2 genes were reverse transcription-PCR (RT-PCR) amplified with full-length coding sequence from mouse lung mRNA. The amplified Mdm2 and Bcl-2 cDNAs then were cloned into the vector (phCMV1) in a sense orientation, whereas p53or Mdm2was cloned into phCMV1 in an antisense orientation to construct plasmids phCMV1-MDM2, phCMV1-Bcl-2, phCMV1-p53as, and phCMV1-mdm2as. All of the constructs were sequenced to confirm that no mutation was introduced into the gene sequences during the RT-PCR or gene cloning.

Western Blotting.
After treatment, cells were washed with ice-cold PBS and harvested in 100 µl of radioimmunoprecipitation assay lysis buffer. Lysates were resolved on 10–12% SDS-polyacrylamide gels. Protein was transferred to a nitrocellulose membrane (Bio-Rad) using an electroblotting procedure. Nitrocellulose membranes were blocked with 5% nonfat dry milk in TBS [50 mM Tris (pH 7.6) and 150 mM NaCl] overnight at 4°C or in TBS-T (TBS and 0.1% Tween 20) for 1 h at room temperature. The antibodies used were as follows: anti-Mdm2 Ab-2 for mouse and anti-Mdm2 Ab-1 for human (Oncogene/Calbiochem, San Diego, CA); anti-p53 CM5 for mouse (Novocastra, Newcastle upon Tyne, United Kingdom); and anti-p53 M-19 for mouse, anti-p53 Bp53-12 for human, anti-p21 M-19 for mouse, and anti-ß-actin C-11 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The blots were incubated with the indicated antibodies, respectively, for 1 h at room temperature and thoroughly washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunolabeling was detected by enhanced chemiluminescence according to the manufacturer’s instructions.

Real-Time Quantitative PCR and Northern Blotting Analysis.
Total RNA was isolated using the Qiagen RNeasy Miniprep kit (Qiagen, Hilden, Germany). For real-time PCR, 2 µg of total RNA were reverse transcribed using OmniScript (Qiagen), and 1 µl from 20 µl of the reverse transcription product was mixed with primers (5 pmol each), double-distilled water, and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The PCR primer sequences were as follows: Mdm2, forward, 5'-AAGGAGGAAACGCAGGACAA-3' and reverse, 5'-TCTTGCCGTGAACAATGCA-3'; and L32, forward, 5'-AACCCAGAGGCATTGACAACA-3' and reverse, 5'-TGTTGCTCCCATAACCGATGT-3'. The real-time PCR protocol was a total of 40 cycles at 94°C for 20 s, at 55°C for 30 s, and at 72°C for 30 s. The reactions were run and analyzed with an ABI PRISM 7700 Sequence Detector (Applied Biosystems).

For Northern hybridization, total RNA (10 µg/lane) was electrophoresed on a formaldehyde-containing agarose gel. Resolved RNA was transferred to positively charged nylon membranes (Bio-Rad) by downward capillary transfer and cross-linked to the membranes using a UV cross-linker. Hybridization was carried out at 42°C, using an Mdm2-or L32-specific probe. The probes were generated from cloned Mdm2or L32genes. The probes were 32P-labeled using the Ready-To-Go random labeling beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The levels of gene-specific mRNA were revealed by autoradiography.

Immunohistochemistry.
For the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, 2 x 105 cells prestained with CM-Dil (Molecular Probes, Eugene, OR) were i.v. injected into C3H mice. The lungs were taken out 24 h later and frozen in liquid nitrogen. The frozen lungs or cut lung sections were stored at –80°C before the staining. An In Situ Cell Death Detection kit (Roche, Indianapolis, IN) was used for TUNEL staining on the lung sections.

Statistics.
The Kruskal-Wallis statistic was used to test for the significance in each transfection experiments and the combined data, followed by Dunn’s test for multiple comparisons against a single control group.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxic Exposure Increases KHT-C Cell Viability in Mouse Lungs.
We first investigated the impact of in vitro hypoxic exposure on KHT cell viability in lungs. Previous studies in our lab have suggested that i.v. injected KHT-C cells die extensively in lungs after the injection (43) . One of the mechanisms responsible for this KHT-C cell death seems to be apoptosis because apoptotic KHT-C cells were observed in mouse lungs at 24 h after the i.v. injection (Fig. 1, A–C)Citation . These results are consistent with previously published studies suggesting that apoptosis is an early event responsible for tumor metastatic inefficiency (14) . When we investigated the viability of i.v. injected KHT-C cells, including hypoxia-pretreated (24 h of 0% O2 followed by 18 h of reoxygenation) and control cells, recovered from mouse lungs at various times after injection using a colony-forming assay, we found that hypoxia-pretreated cells had higher viability than control, as shown in Fig. 1DCitation . Our in vitro studies have also indicated that hypoxia-pretreated KHT-C cells are more resistant to hyperthermia-induced apoptosis compared with normoxic controls (data not shown). These results suggest that hypoxia-pretreated KHT-C cells have increased survival under stress conditions, which may be imposed by the lung environment, and one of the responsible mechanisms may be increased resistance to apoptosis.



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  		Fig. 1. KHT-C cells are more resistant to cell death in vivo after hypoxia-pretreatment. A–C, KHT-C cells were prestained with CM-Dil fluorescent dye and i.v. injected into mice. The staining of cells with CM-Dil does not affect KHT-C cell viability as tested with plating efficiency in our pre-experiments. Lungs were taken out at 24 h after the injection to make histological sections. The bars represent a 20-µm indicator. A, the red fluorescence distinguishes the injected KHT-C cell from host lung cells. B, the green fluorescence indicates terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive staining (fluorescein) of the KHT-C cell in A. C, TUNEL assay (horseradish peroxidase-conjugated antibody) shows the apoptotic morphology of a KHT-C cell in the lung section. D, hypoxia-pretreated and reoxygenated (0% O2 for 24 h then normal condition for 18 h) KHT-C cells (H24/O18) or normal KHT-C cells (CON) were i.v. injected and then recovered from mouse lungs at different times after the injection. KHT-C cell viability was determined by colony-forming assay. The proportion (mean ± SE of five mice) of KHT colony-forming cells recovered at different times relative to the number of KHT colony-forming cells recovered at 5–10 min (time 0) after the injection is shown.

 
Hypoxia Regulates the Expression of the Genes Important to Cell Death and Growth in KHT-C Cells.
To understand the mechanisms involved in the hypoxia-induced increase in cell survival and enhanced experimental metastatic efficiency, cDNA microarray analysis was performed using RNAs extracted from hypoxia-pretreated (0% O2 for 24 h) or control KHT-C cells. A cutoff value of 2.0 was determined by self-hybridizations (data not shown) in which >95% of all of the signals had the ratio of green to red fluorescence between 2 and 0.5. Results are shown in Fig. 2Citation . The function annotations of the genes in Fig. 2Citation are either from National Institute on Aging/NIH expressed sequence tag mouse clone list or Gene Ontology annotation. Among all of the KHT-C genes regulated by hypoxia, almost all of them are involved in signal transduction, stress response, cell growth and/or maintenance, energy/metabolism, and gene transcriptional regulation. These data support our hypothesis that hypoxia may affect tumor cell viability by regulating gene transcription.



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  		Fig. 2. Hypoxia-regulated genes in KHT-C cells. Total RNA was extracted from KHT-C cells grown under normal condition or under hypoxic condition (5% CO2 and 95% N2 for 24 h). Hypoxia up-regulated genes are shown in red and down-regulated genes in green. Only genes with expression changes greater than a factor of 2 are listed. The uncharacterized clone sequences or genes with unknown functions are not shown. The color bar on the top right indicates the expression level changes on a log10 scale with up-regulation (positive value) in red and down-regulation (negative value) in green.

 
Mdm2 Is Up-Regulated by Hypoxia, Independent of p53.
To examine further the molecular mechanisms for the hypoxia-enhanced tumor cell viability in mouse lungs and to verify if this enhanced viability can increase tumor cell-metastatic efficiency, we initiated a study of the gene Mdm2, which is an important negative regulator of the tumor suppressor gene p53, and was identified to be one of the hypoxia up-regulated genes (Fig. 2)Citation . The up-regulation of Mdm2 mRNA was confirmed by Northern blotting analysis in KHT-C and SCC VII cell lines (Fig. 3A)Citation . Real-time quantitative PCR revealed a ~4-fold induction of Mdm2 by hypoxia in KHT-C and SCC VII cells (Fig. 3B)Citation . The up-regulation of Mdm2 by hypoxia was additionally confirmed at the protein level with Western analysis (Fig. 3C)Citation . The up-regulation of Mdm2 in SCC VII cells, which, similar to KHT-C cells, exhibit enhanced metastatic efficiency upon hypoxic exposure (8 , 9) , indicates that the up-regulation of Mdm2 by hypoxia is not KHT cell specific. Similar results were also observed in B16F10 melanoma cells (data not shown).



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  		Fig. 3. Mdm2 is up-regulated by hypoxia. KHT-C and SCC VII cells were exposed to hypoxia (5% CO2 and 95% N2) for 24 h (H24), or cultured under normal condition for same time (CON). A, the up-regulation of Mdm2 mRNA in KHT-C and SCC VII cells was confirmed with Northern blotting analysis. L32 was used as loading control. The ratio of Mdm2 mRNA level to L32 loading control was determined by PhosphoImager quantification. B, the extent of Mdm2 up-regulation was determined with real-time quantitative PCR. The mRNA levels are shown relative to L32. The relative expression level was estimated from the number of PCR cycles required to detect SYBR Green signal at a certain intensity in the linear range of the amplification curve. C, the up-regulation of Mdm2 protein by hypoxia was characterized with an immunoblotting assay. The ratio of Mdm2 to ß-actin was determined by densitometry.

 
Although Mdm2 has been frequently reported to be amplified in many tumor types and hypoxia is able to increase the frequency of gene amplification and gene mutation (44, 45, 46, 47) , it is unlikely that the increased Mdm2 by 24 h of hypoxic exposure without reoxygenation was because of hypoxia-induced gene amplification or gene mutation. This was confirmed by our Southern analysis results, which showed similar gene copy numbers in hypoxia-treated and untreated KHT-C cells (data not shown). Furthermore, when hypoxia pretreated KHT-C cells were reoxygenated for different times, the level of Mdm2 mRNA started to decrease after 6 h of reoxygenation (Fig. 4A)Citation . Mdm2 protein was at high levels during 3–24 h of reoxygenation and started to decrease by 48 h of reoxygenation (Fig. 4B)Citation . These results suggest that the increased expression of Mdm2 by hypoxia is transient and occurs at the mRNA level.



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  		Fig. 4. The up-regulation of MDM2 is transient and occurs at mRNA level. A, KHT-C cells were cultured under normal (CON) or hypoxic (H24) conditions or hypoxic and reoxygenated for 3, 6, 12, and 24 h (H24/O3, H24/O6, H24/O12, and H24/O24). Total RNA was extracted for real-time quantitative PCR analysis. B, cell lysates were extracted from KHT-C cells grown under control (CON), hypoxic (H24) condition, or hypoxic and reoxygenated for 6, 12, 24, and 48 h (H24/O6, H24/O12, H24/O24, and H24/O48). The protein level of Mdm2 was analyzed by Western blotting. Lysates from the cell line Raw264.7 were used as a positive control for Mdm2 expression. The ratio of Mdm2 to ß-actin was determined by densitometry.

 
It has been reported that hypoxia up-regulates p53 in some cell lines by down-regulating Mdm2 (23, 24, 25) or inhibiting the p53-binding ability of Mdm2 (22) . Because Mdm2 is regulated by p53 at the transcriptional level, it is possible that the up-regulation of Mdm2 we observed in KHT-C cells is the consequence of hypoxia-induced p53. To address this issue, we investigated the expression of Mdm2 and p53 at 3, 6, 12, and 24 h of hypoxic exposure. The Mdm2 protein was consistently induced through the 24 h of hypoxic treatment, and a consistent decrease of p53 protein was also observed (Fig. 5A)Citation . Real-time quantitative PCR analysis showed the up-regulation of Mdm2 mRNA in KHT-C cells exposed to hypoxia within the same period (Fig. 5B)Citation , whereas p53 protein was down-regulated. These results suggest a mechanism other than p53 regulation for the hypoxia-induced Mdm2. To confirm that Mdm2 can be up-regulated by hypoxia independently of p53, we investigated the level of MDM2 mRNA in hypoxia-treated or control human prostate cancer cell lines DU145 and PC3 in which p53 is either mutated or null, respectively (48) . The MDM2 mRNA level was increased by hypoxic exposure in both PC3 and DU145 cells (Fig. 5C)Citation , indicating that hypoxia is able to induce MDM2 at the mRNA level in a p53-independent manner. The up-regulation of MDM2 at the protein level was observed in DU145 cells after hypoxic exposure followed by reoxygenation but not immediately after the hypoxia treatment. However, in PC3 cells, MDM2 was down-regulated by hypoxia at protein level, despite the increased mRNA. The protein level of p53 is very high and did not changed after hypoxic exposure in DU145 cells, suggesting a mutated form of p53. As expected, p53 is not detected in p53-null PC3 cells (Fig. 5D)Citation .



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  		Fig. 5. Hypoxia-induced Mdm2 is not regulated by p53. A and B, KHT-C cells were treated with hypoxia for 3, 6, 12, and 24 h (H3, H6, H12, and H24) or grown under normal conditions (CON). Protein or total RNA was extracted to detect the expression of p53 and Mdm2 by Western analysis (A) or the expression of Mdm2 mRNA by real-time quantitative PCR (B). The bottom panel of A shows the densitometry quantification normalized to the ß-actin loading control and to the control lane. C, total RNA was extracted from hypoxia-treated (H24) or normoxic control (CON) human prostate cancer cell lines PC3 and DU145. The level of MDM2 mRNA was determined by real-time PCR shown as relative level to L32 mRNA. D, cell lysates were extracted from human prostate cancer cell lines PC3 and DU145 grown under normoxic condition (CON), treated with hypoxia (0% O2) for 24 h (H24), or reoxygenated for 24 h after 24 h of hypoxic exposure (H/O). The level of MDM2 and p53 was determined by Western blotting analysis and densitometry.

 
p53 Is Functional and Suppressed by Hypoxia in KHT-C Cells.
It is well known that overexpression of Mdm2 prevents p53-mediated apoptosis and cell cycle arrest (49 , 50) . The effect of Mdm2 on p53 and apoptosis, together with the fact that hypoxia up-regulates Mdm2 in KHT-C cells, may explain the increased apoptotic resistance of hypoxia-pretreated KHT-C cells. In addition, the down-regulation of p53 shown in Fig. 5ACitation did not occur at the mRNA level because no change was detected by real-time quantitative PCR (data not shown), suggesting a role of Mdm2 in the degradation of p53 in response to hypoxia. However, whether the degradation of p53 by hypoxia can render tumor cells more resistant to p53-mediated apoptosis depends on the functionality of p53 in KHT-C cells, because if p53 is not functional and cannot mediate apoptosis in KHT-C cells, we would not expect hypoxia to desensitize tumor cells to apoptosis in a p53-dependent manner. Consequently, ionizing irradiation was used to determine p53 functionality. KHT-C cells were irradiated with 5 Gy, and the p53 protein level was found to be increased at 1 h after irradiation and then decreased at later times (Fig. 6A)Citation . A temporary increase of the p53 target gene p21 was also detected at both protein (Fig. 6A)Citation and mRNA (Fig. 6B)Citation levels after the irradiation. The up-regulation of p53 and p21 after irradiation suggests that p53 is functional in KHT-C cells. The presence of wild-type p53 in KHT-C cells was also confirmed by cDNA sequencing (data not shown).



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  		Fig. 6. p53 is functional in KHT-C cells and suppressed by hypoxia. KHT-C cells were irradiated with 5 Gy, and total RNA or protein was harvested at 0–5 h after irradiation. A, the protein levels of p53 and p21 were examined with immunoblotting analysis. ß-Actin was probed as a loading control. B, the mRNA level of p21 was analyzed with real-time quantitative PCR. The expression level shown was relative to L32 mRNA. C, Mdm2 antisense constructs (Mdm2as) or empty vectors (phCMV1) were cotransfected with pEGFP-F into KHT-C cells. The positively transfected cells were enriched by FACS for EGFP-positive cells. Cell lysates were extracted either immediately after the cell sorting (CON) or after 24 h of hypoxic exposure (H24). The hypoxic treatment was initiated after the sorted cells had been cultured under normoxic condition for 6 h, which allowed sorted cells to attach to tissue culture dishes but no cell division to occur. The protein level of Mdm2 and p53 was analyzed with Western blotting. D, KHT-C cells were grown under normal conditions (CON) or pretreated with hypoxia for 24 h and reoxygenated for 18 h (H24/O18) and then irradiated (5 Gy) under aerobic conditions. Proteins were extracted from nonirradiated KHT-C cells or irradiated cells at 1, 2, and 3 h after 5 Gy and analyzed by immunoblotting for p53 protein levels. The ratio of p53 to ß-actin was determined by densitometry. E, the mRNA level of Mdm2 in KHT-C cells was tested with real-time quantitative PCR. The cells were cultured under either normoxic condition (CON), hypoxia (0% O2) for 24 h (H24), or 24 h of hypoxic exposure, then 18 h of reoxygenation (H/O) before irradiation. Irradiation was performed under normoxic conditions with the cells in fully oxygenated complete medium. The top panel shows the expression of KHT-C Mdm2 mRNA relative to L32 before and at 1, 2, and 3 h after 5 Gy of ionizing irradiation. The bottom panel shows the fold increase of Mdm2 mRNA induced by radiation. F, cell lysates were harvested from KHT-C cells grown either under normoxic condition (CON) or hypoxic condition followed by reoxygenation (H24/O18) before and at 1, 3, and 5 h after 5 Gy of ionizing radiation. The protein level of KHT-C Mdm2 was analyzed with Western blotting. G, the mRNA level of p53 downstream proapoptotic genes was tested with real-time quantitative PCR. The RNAs were extracted from KHT-C cells grown under normal conditions. H, real-time quantitative PCR was performed to determine the mRNA level of target genes in control or hypoxia-treated and reoxygenated KHT-C cells before and at 1, 2, and 3 h after 5 Gy of ionizing radiation. The RNAs were obtained from the cells treated the same way as indicated in D.

 
To investigate the role of Mdm2 in the degradation of p53 after hypoxic exposure in KHT-C cells, Mdm2antisense cDNA was used to suppress the expression of Mdm2. In KHT-C cells transfected with Mdm2antisense cDNA, hypoxia failed to induce Mdm2. As expected, the down-regulation of p53 by hypoxia was only observed in empty vector-transfected KHT-C cells but not in antisense Mdm2-transfected cells (Fig. 6C)Citation , suggesting that the down-regulation of p53 by hypoxia is dependent on Mdm2, which can act as an E3 ligase for p53 and target it to the proteosome for degradation. If hypoxia up-regulates Mdm2, which mediates the degradation of p53 and consequently confers KHT-C cells with resistance to apoptosis under stress conditions, we would expect to observe a reduction in the accumulation of p53 and reduced p53-transcriptional activity in hypoxia-pretreated cells when these cells are exposed to stress conditions or DNA-damaging agents such as ionizing radiation.

The level of p53 protein and some of its downstream genes, including Mdm2itself, the antiapoptotic gene Bcl-2, and proapoptotic genes Apaf-1, DR5, EI24, Fas, IGFBP3, NOXA, PIDD, PUMA, and WIG1 (50 , 51) , were investigated in control and hypoxia-pretreated KHT-C cells exposed to ionizing radiation. At the time of the irradiation, the hypoxia-pretreated cells had been reoxygenated for 18 h after being incubated under 0% O2 for 24 h because the maximum metastatic potential (8) and the highest Mdm2 protein level (Fig. 4B)Citation were observed in KHT cells after 18–24 h of reoxygenation. As expected, although the up-regulation of p53 protein was still detected in hypoxia-pretreated KHT-C cells after ionizing radiation, the level was decreased compared with control KHT-C cells (Fig. 6D)Citation .

Because Mdm2 is also a target gene of p53-transcriptional activity, it was of interest to examine how hypoxia would affect the response of Mdm2 to ionizing radiation in KHT-C cells. As shown in Fig. 6ECitation , a higher basal level of Mdm2was observed in both hypoxia-pretreated cells and in cells treated with hypoxia followed by reoxygenation, in comparison with the normoxic cells. Although the induction of Mdm2mRNA by ionizing radiation occurred in KHT-C cells grown under all three different conditions, the fold induction was greatly inhibited in both hypoxic cells and reoxygenated cells. Similar results were observed at the Mdm2 protein level (Fig. 6F)Citation . The attenuated accumulation of p53 and its activation in response to stress condition such as ionizing radiation suggest that hypoxia-exposed KHT-C cells may be less sensitive to p53-mediated apoptosis. This was confirmed by analyzing the response of a number of p53 downstream apoptotic genes to ionizing radiation in KHT-C cells. Of the p53 downstream anti- and proapoptotic genes studied, Apaf-1, DR5, EI24, PIDD, PUMA, and WIG1 had high enough basal (Fig. 6G)Citation and radiation-induced mRNA expression levels to be studied quantitatively with real-time PCR. As shown in Fig. 6HCitation , all six of the proapoptotic genes are induced by 5 Gy of ionizing radiation, but the induction is attenuated in hypoxia-pretreated and reoxygenated cells. These results suggest that hypoxic exposure renders KHT-C cells less sensitive to apoptotic stimuli by suppressing p53 activity.

Mdm2 Overexpression Enhances Metastatic Efficiency in KHT-C Cells.
To further test our hypothesis that hypoxia enhances experimental metastatic efficiency by increasing resistance to apoptosis in KHT-C cells, we investigated the effect of Mdm2 overexpression or p53 suppression on metastatic efficiency by transient transfection. Data from four separate experiments in which the transfections were performed with different batches of KHT-C cells are shown in Fig. 7Citation . Mdm2 cDNA transfection caused a transient increase of Mdm2 and a decrease of p53 (Fig. 7, A and B)Citation . The transient effect of Mdm2 overexpression is shown in Fig. 7BCitation . The protein level of Mdm2 had returned to normal after culturing the enriched Mdm2 positively transfected cells for 48 h. Flow cytometry analysis on the green fluorescent protein (GFP) transiently transfected cells, during the in vitro culture after fluorescence-activated cell sorting (FACS), revealed that the percentage of GFP-positive cells was reduced 50% after each 16 h, which is the approximate KHT-C cell doubling time (data not shown). This suggests that the decreased Mdm2 protein at 24 and 48 h after the FACS is the consequence of the cell proliferation, which results in the dilution of the Mdm2 overexpression. Overexpression of Mdm2 in KHT-C cells caused increased metastases in all four experiments (Fig. 7C)Citation . The suppression of the p53 gene by transfecting p53 antisense cDNA sequence into KHT-C cells also increased lung metastases in all four experiments (Fig. 7C)Citation . Transfection of the antiapoptotic gene Bcl-2 cDNA was performed as a positive control because Bcl-2 has a very low expression level in KHT-C cells as determined by real-time PCR (Fig. 6F)Citation , and numerous studies have suggested that the overexpression of Bcl-2increases the resistance of host cells to apoptosis (52) . As shown in Fig. 7CCitation , the KHT-C cells overexpressing Bcl-2 generated more lung metastases after i.v. injection compared with control KHT-C cells transfected with the empty vector. For comparison, results of an experiment in which KHT cells were exposed to hypoxia are shown. The overall lower metastatic potential of transfected KHT-C cells compared with untransfected cells was probably because of the cell damage caused by cell sorting, which was used to enrich for positively transfected cells.



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  		Fig. 7. Transient overexpression of Mdm2 or suppression of p53 increases lung metastases. A, the level of Mdm2 in phCMV1-mdm2-transfected KHT-C cells and the level of p53 in phCMV1-p53as-transfected cells comparing to control KHT-C cells transfected with phCMV1 empty vectors were detected by Western blotting. The protein levels of Mdm2 and p53 were determined with densitometry. B, KHT-C cells were cotransfected with phCMV1-Mdm2 and pEGFP-F. Positively transfected cells were enriched by FACS for EGFP-expressing cells. Cell lysates were extracted immediately after the sorting (0) or after 24 h (24) or 48 h (48) of culture under normoxic conditions. The level of Mdm2 and p53 was determined with Western analysis. The quantification was done by densitometry. C and D, overexpression of Mdm2 or Bcl-2 or down-regulation of p53 increases lung metastases in KHT-C cells. Empty vector phCMV1, phCMV1-Bcl-2, phCMV1-Mdm2, or phCMV1-p53as constructs were cotransfected into KHT-C cells with pEGFP. EGFP-positive cells were sorted with flow cytometry. The sorted cells were concentrated into a certain volume, and an estimated 2–10 x 104 sorted cells were i.v. injected into animals. A colony-forming assay (plating efficiency) of cells to be injected was performed to determine the actual number of viable injected cells. For control (CON) or hypoxia/reoxygenated (H24/O18) KHT-C cells, 105 cells were injected. Macrometastases on the lung surface were scored at ~20 days after the i.v. injection. The mean (±SE) of lung metastatic efficiency (metastases/viable cells injected) corrected separately for the plating efficiency from each one of the four experiments is shown in D. *, P < 0.01 (q' = 3.628, k = 3); **, P < 0.01 (q' = 2.742, k = 2); ***, P < 0.01 (q' = 2.970, k = 4).

 
Mean values of the metastatic efficiency from the four experiments are shown in Fig. 7DCitation . These results are consistent with our expectation that up-regulation of Mdm2 by hypoxia contributes to enhanced metastatic efficiency and that p53 may inhibit tumor metastasis by mediating apoptosis in tumor cells. Finally, the level of hypoxia-induced Mdm2 (Fig. 3C)Citation and the consequent increase of metastatic efficiency in KHT cells (Fig. 7C)Citation were comparable with that of Mdm2 transfected KHT-C cells (Fig. 7, A and C)Citation , also suggesting a role of hypoxia-induced Mdm2 in enhanced metastatic efficiency in KHT-C cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown for the first time the p53-independent up-regulation of Mdm2 by hypoxia in a number of tumor cell lines. We have also demonstrated that this hypoxia-induced Mdm2 contributes to hypoxia-enhanced metastatic efficiency in KHT cells, possibly by inhibiting p53-mediated apoptosis. Our studies reveal a new mechanism involved in hypoxia-enhanced metastatic potential in tumor cells and demonstrate that a transient change in gene expression can modify the metastatic potential of cells.

The effect of hypoxia on tumor cells was investigated in this work. Our microarray analysis suggested that hypoxia affects tumor cell viability in two opposing ways, inducing cell death and desensitizing cells to stress condition-induced cell death. In Fig. 1DCitation , we have shown that hypoxia-pretreated KHT-C cells indeed survived better in lung than control KHT-C cells. Furthermore, we demonstrated the existence of apoptotic KHT cells (Fig. 1A–C)Citation in mouse lungs, suggesting that apoptosis is at least one of the mechanisms accounting for the cell death of KHT-C cells in lungs. We could not measure the extent of apoptosis of KHT cells in mouse lungs or how much this apoptosis is affected by hypoxia due to the technical difficulties associated with the noisy background of lung section fluorescent microscopy and the limited KHT-C cell number (maximum 105 cells/mouse) to be injected without changing the dynamics of the tumor cell metastatic potential (53) . However, studies on the expression of p53-regulated apoptotic genes have shown an attenuated response of hypoxia-pretreated KHT cells to ionizing radiation (Fig. 6H)Citation , suggesting that decreased apoptotic sensitivity may explain the increased viability of hypoxia pre-exposed KHT-C cells in mouse lungs.

The increased expression of Mdm2 after hypoxic exposure was associated with decreased expression of its target p53 protein (Fig. 5A)Citation . Mdm2 is a main cellular regulator of p53 protein, which is usually maintained at low levels by rapid degradation through ubiquitin-dependent proteolysis. Degradation of p53 is regulated by interaction with Mdm2 protein (38 , 54) , which binds to p53 and functions both as an ubiquitin ligase (39) and to shuttle p53 from the nucleus to the cytoplasm, where degradation of p53 is thought to take place (40 , 42) . Mdm2 also inhibits p53-mediated transactivation by binding to the p53 transactivation domain (32) . Mdm2 transcription is regulated by p53 (33 , 34) , establishing a negative feedback loop where increased levels of p53 increase expression of Mdm2, which targets p53 for degradation. This property underlies the oncogenic potential of Mdm2, which is overexpressed in various human tumors such as soft tissue tumors, osteosarcomas, esophageal carcinomas, breast cancer, glioblastoma, prostate carcinoma, melanoma and lung cancer (55, 56, 57, 58) . In addition to its p53-dependent functions, accumulated evidence suggests that Mdm2 also has p53-independent activities that may contribute to tumor progression (35, 36, 37 , 41 , 56 , 59) . Because of these findings, it is reasonable to argue that hypoxia-induced Mdm2 may be an important mechanism accounting for the increased malignancy of tumor cells under a hypoxic environment.

In this study, we used transient transfection instead of stable transfection because this is likely to be more similar to the effect of hypoxia on gene transcription regulation and the consequent biological effects such as increased resistance to apoptosis and metastatic efficiency in tumor cells. Two issues have been clarified by using transient transfection. The first is that hypoxia-regulated gene expression may explain the transient effect of hypoxia on tumor cell metastatic efficiency. As shown in Fig. 4Citation , hypoxia transiently induces Mdm2 mRNA and causes a temporary increase in expression of Mdm2 and decreased expression of p53. Induction of these effects on Mdm2 and p53 expression by transient transfection caused increased experimental lung metastases (Fig. 7)Citation . Interestingly, the pattern of the Mdm2 protein levels after different times of reoxygenation (Fig. 4B)Citation is very similar to that of hypoxia-enhanced KHT-C metastatic potential, which increased to a maximum after 18–24 h of reoxygenation and then declined (8) . The second issue addressed by the transient transfection is that for KHT tumor cells to form metastases in lungs, the survival of the arrested cell is important because only a small proportion of the cells within a metastatic colony will express the transfected cDNA, and consequently, it is unlikely that the gene plays a role in the later stages of the metastatic processes when the metastatic nodule is expanding. Because in this system it is experimental metastases that have been studied, possible effects of hypoxia on the early stages of spontaneous metastasis, e.g., intravasation, are not addressed by this study.

Although Mdm2 is well known to target p53 for degradation, the up-regulation of Mdm2 may not be the only mechanism by which hypoxia can reduce p53 protein levels. For example, it has been reported that hypoxia inhibits global protein synthesis via the phosphorylation of the translation initiation factor eIF2{alpha}; however, the phosphorylation of eIF2{alpha} was a modification that was readily reversed upon reoxygenation (60) . In our studies, hypoxia suppressed p53 protein accumulation and the activation of p53 target gene expression after ionizing radiation at 18 h of reoxygenation after the hypoxic exposure, indicating that the effect of hypoxia on p53 activity is not a consequence of hypoxia-inhibited global protein expression. It has also been reported that hypoxia causes transient inhibition of p53 transcription activation at the initiation of hypoxic exposure, which would be expected to decrease the transcription of Mdm2 and p21 and result in the accumulation of p53 and increased p53 activity (61) . However, this again does not explain the hypoxia-reduced p53 activity we observed because the p53 protein did not increase at later time points of hypoxic exposure (for 24 h), and even after 18 h of reoxygenation, the p53 activity still had not recovered to its normal level.

The down-regulation of p53 we observed in KHT-C cells is opposite to some other studies, which reported hypoxia-induced p53 in some human and murine cell lines. This discrepancy may be because of an altered response of Mdm2 to hypoxia. The studies reporting the up-regulation of p53 by hypoxia have described a role of Mdm2 in regulating p53 upon hypoxic exposure. The involved mechanisms include decreased expression of Mdm2 or inhibition of Mdm2 and p53 interaction (22, 23, 24, 25 , 62 , 63) . However, the response of Mdm2 to hypoxia can be cell specific; for example, the up-regulation of Mdm2 by hypoxia was observed in DU145 human prostate cell line but not in PC3 (Fig. 5D)Citation . Interestingly, MDM2 mRNA was up-regulated in both cell lines, suggesting that the MDM2 can be regulated by hypoxia at both mRNA and protein level. Another possibility is that some mechanisms involved in Mdm2 protein degradation by hypoxia may still function in KHT-C cells because the up-regulation of Mdm2 is >4-fold at the mRNA level (Fig. 3B)Citation , but the induction of Mdm2 protein is only slightly >2-fold (Figs. 3CCitation and 4BCitation ). The overall higher Mdm2 level after hypoxia treatment could be the result of excessive Mdm2 mRNA in hypoxic KHT-C cells. The hypoxia-induced Mdm2 mRNA may result from a differently activated stress response pathway in KHT-C cells. Wu et al. (64) have reported p53-independent regulation of Mdm2, suggesting the potential involvement of mechanisms other than p53 in KHT-C cell Mdm2 transcriptional regulation upon hypoxic exposure. Interestingly, Mdm2 is not the only p53 regulator that shows a different response to hypoxia in KHT-C cells. For example, inhibitor of NF-{kappa}B{alpha} has also been reported to inhibit p53 activity by interacting with p53 protein in Mv1Lu epithelial cells, and this interaction is abolished under hypoxic condition (65) . As with Mdm2, inhibitor of NF-{kappa}B{alpha} is up-regulated by hypoxia in KHT-C cells (Fig. 2)Citation , suggesting an alternative transcriptional regulation mechanism for these p53 regulators in response to hypoxia in KHT-C cells. The pathways regulating Mdm2 transcription may be used by KHT-C and other tumor cell lines to adapt to stress conditions such as hypoxia to survive the hostile in vivo microenvironment. The mechanisms of Mdm2 up-regulation by hypoxia and the involvement of mitogen-activated protein kinase pathways are currently being investigated. For example, NF-{kappa}B, a transcriptional regulator activated by mitogen-activated protein kinase pathways, can respond to stress factors, including hypoxia, and can activate Mdm2 at the transcriptional level (66) . Recently, a role of activated NF-{kappa}B in decreased p53 stability and increased resistance to chemotherapy has also been reported (67) .

The studies reported in this article suggest a role for apoptotic resistance induced by hypoxia in enhanced metastatic efficiency. Although we would expect that the mechanisms of hypoxia-increased resistance to apoptosis and hypoxia-enhanced metastases are more complicated than just up-regulation of Mdm2 and down-regulation of p53, the identified involvement of p53 and Mdm2 establishes a new view in tumor cell metastasis under hypoxic condition and provides a new rationale for the cancer therapy of inhibiting the p53-MDM2 interaction in p53-positive cells (50) .


   ACKNOWLEDGMENTS
 
We thank Robert Kuba for his help on animal experiments and Dr. Sam Benchimol for helpful discussion.


   FOOTNOTES
 
Grant support: National Cancer Institute of Canada Grant with funds raised by the Terry Fox Run.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Richard P. Hill, Research Division, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9 Canada. Phone: (416) 946-2979; Fax: (416) 946-2984; E-mail: hill{at}uhnres.utoronto.ca

4 Internet address: http://www.geneontology.org. Back

Received 9/26/03. Revised 2/13/04. Accepted 4/13/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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