Hypoxia mediated down regulation of Bid and Bax in tumours occurs via HIF-1 dependent and independent mechanisms and contributes to drug resistance Erler JT, Cawthorne CJ, Williams KJ, Stratford IJ & Dive C. University of Manchester.	Print Abstract Search PubMed for: Earler JT Cawthorne CJ Williams KJ Stratford IJ Dive C

Hypoxic tumour cells are resistant to radiotherapy and some forms of chemotherapy (Teicher 1994). We have examined the effect of hypoxia on the Bcl-2 family that play a central role in determining the cellular threshold for the induction of apoptosis (Adams & Cory 2001), and assessed the impact of this on cellular response to therapy.

Human colon carcinoma cell lines HCT116, HT29 and SW480 were grown in RPMI media supplemented with 10% foetal bovine serum and antibiotics and cultured at 37°C in 95% air/5% CO₂. Cells were oxygen deprived by maintenance in an anaerobic chamber for 16h at 37°C. Cell death was assessed by trypan blue uptake, and apoptosis by staining with Hoechst. Analyses of protein expression levels were performed by immunoblotting. mRNA expression levels were assessed by quantitative RT-PCR using Taqman (DePreter et al., 2002). For drug studies, cells were incubated for 8h under normoxic or anoxic conditions then treated with various concentrations of etoposide (Sigma, dissolved in DMSO) or Oxalipatin (Alexis Biochemicals, dissolved in water) for 8h with continuous normoxic or anoxic incubation. Cells were re-seeded for short term viability assays and clonogenic assays. All in vitro results are based on n3. For in vivo studies, HCT116 were grown as subcutaneous xenografts in 8 week old female CD-1 nude mice (20-30g weight range) following intradermal injection of 5 x 10⁶ cells in 0.1ml of media 1cm from the tail base on the midline, performed under general anaesthetic (halothane). Mice were housed in an individually ventilated caging system on a 12hlight/12hour dark environment maintained at constant temperature and humidity. They were fed a standard irradiated diet and allowed water ad libitum, and were observed daily. A single dose of the hypoxic marker pimonidazole (0.2ml/IP of 10mg/ml stock dissolved in 0.9% w/v saline) was administered at 100mg/kg in PBS to mice bearing tumours of 600-800mm³ 2h before sacrifice. The study lasted two months. All procedures were carried out in accordance with the Scientific Procedures Act 1986 and in line with the UKCCCR guidelines 1999, by approved protocols (Home Office Project License number 40-1770).

Anoxic incubation resulted in decreased protein and mRNA levels of pro-apoptotic Bcl-2 family members Bid, Bax, Bad and Bim occurring within 8 hours of exposure. In contrast, the levels of the anti-apoptotic proteins Bcl-2, Bcl-xl and Bcl-w remained unchanged. Comparison of HIF-1 proficient and deficient cells demonstrated that anoxia-induced down-regulation of Bid is HIF-1 dependent, consistent with the discovery of hypoxia response elements in the Bid promoter sequence. Short-term viability tests and clonogenic assays showed that oxygen deprivation resulted in resistance to clinically relevant chemotherapeutic drugs (2.6±0.1 fold resistance in clonogenics; 15±2% and 22±6% less apoptosis in 3 and 7 day viability assays respectively; all n3, mean±SE). The contribution of Bid and/or Bax down regulation to drug responsiveness was demonstrated by the relative resistance of normoxic cells that had no or reduced expression of Bid and/or Bax, and that forced expression of Bid in hypoxic cells resulted in significantly increased etoposide sensitivity. Statistical analyses using students two tailed t-test showed significance (p0.05) when comparing relevant data sets.

These data couple hypoxia and cell survival with the down-regulation of key pro-apoptotic Bcl-2 family proteins, and for Bid this occurs via a HIF-1 dependent transcriptional repression, adding impetus to the design of chemotherapeutic strategies to target this transcription factor. The data provide a mechanistic explanation for pleiotropic drug resistance in hypoxic tumour cells.

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