Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells

doi:10.1016/j.radonc.2005.06.025

Radiotherapy and Oncology

Volume 76, Issue 2, August 2005, Pages 168-176

https://doi.org/10.1016/j.radonc.2005.06.025 Get rights and content

Abstract

Background and purpose

Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions.

Methods and materials

The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1α-inducing agent, CoCl₂ (100 μM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression.

Results

Induction of hypoxia was confirmed using HIF1α and VEGF expression in gas- and CoCl₂-treated cultures. Hypoxia (48–72 h of 0.2% O₂) decreased RNA expression of a number of HR-related genes (e.g. Rad51, Rad52, Rad54, BRCA1, BRCA2) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70, DNA-PKcs, DNA Ligase IV, Xrcc4) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl₂-treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution.

Conclusions

Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.

Section snippets

Prostate cell cultures and hypoxic or CoCl₂ treatments

The human malignant prostate cell lines DU145 and PC3 were purchased from American Type Culture Collection (ATCC; Manassas, VA) and the 22RV-1 cell line was a kind gift of Dr Yoni Pinthus (PMH). These cell lines were supplemented with 10% fetal calf serum and 1% l-Glutamine in α-Modified Eagles Medium (MEM), Ham's F12K and RPMI1640, respectively. The normal diploid fibroblast strain GM05757 was purchased from Coriell Cell Repository (Camden, NJ). The SV40-immortalized, benign prostatic

Results

Initial experiments confirmed the gas hypoxic response (0.2 or 2.0% O₂ concentrations) in logarithmically-growing normal (normal diploid fibroblast strain, GM05757) and malignant (DU145 prostate cancer) cell cultures. Fig. 1 demonstrates up-regulation of VEGF RNA and increased expression of HIF1α protein at 48 and 72 h for both GM05757 and DU145 cells. Repeated experiments showed similar induction at times ranging from 24 to 72 h for all cultures tested at 0.2% and 2% oxygen gassing conditions

Discussion

To our knowledge, this is the first report that documents gene expression relating to members of the HR and NHEJ pathways following hypoxia in normal, immortalized and malignant cells. We observed decreases in HR-related DNA-dsb repair protein expression within several immortalized and malignant prostate cell lines following gas hypoxia, but not CoCl₂-treatment. In our study, the HIF1α-induced gene expression induced by CoCl₂ was insufficient to alter RAD51 protein expression. In a previous

Acknowledgements

The authors would like to thank Drs D. Hedley, R.P. Hill and M. Milosevic for helpful discussions and encouragement. These studies are supported by operating grants to R.G.B. from the Terry Fox Foundation (Hypoxia Project Program Grant), Ontario Cancer Research Network PMH Foundation, US Army DOD Prostate Cancer Program and a grant to P.M.G. from the NIH (ES05775). R.S.B. was supported by NIH Medical Scientist Training Grant (GM07205). R.G.B. is a Canadian Cancer Society Research Scientist.

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Molecular radiobiologyHypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells

Abstract

Background and purpose

Methods and materials

Results

Conclusions

Section snippets

Prostate cell cultures and hypoxic or CoCl2 treatments

Results

Discussion

Acknowledgements

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Mol Cell

Urology

DNA Repair (Amst)

Mol Cell

Biochem Biophys Res Commun

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Int J Radiat Oncol Biol Phys

Mol Cell

Mol Cell

Mol Cell

DNA Repair (Amst)

Drug Resist Updat

Modulation of c-Myc activity and apoptosis in vivo

Cancer Res

DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis

Nature

Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells

Mol Cell Biol

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

Oncogene

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Nat Rev Cancer

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Molecular radiobiology
Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells

Prostate cell cultures and hypoxic or CoCl₂ treatments