Biological indicators of chemoresistance: an ex vivo analysis of γH2AX and p53 expression in feline injection-site sarcomas

Abstract

Abstract Background The response of soft tissue sarcomas to cytotoxic chemotherapy is inconsistent. Biomarkers of chemoresistance or chemosensitivity are needed in order to identify appropriate patients for treatment. Given that many chemotherapeutics kill cells through direct DNA interactions, we hypothesized that upregulation of DNA damage response mechanisms would confer resistance to cytotoxic chemotherapy in sarcomas. To study this, we used spontaneously-occurring feline injection-site sarcomas (FISS). Methods γH2AX and p53 expression were determined in biopsy samples of FISS. γH2AX expression was determined via immunohistochemistry whereas p53 expression was determined via qRT-PCR. Cell lines derived from these sarcoma biopsies were then treated with carboplatin (N = 11) or doxorubicin (N = 5) and allowed to grow as colonies. Colony forming-ability of cells exposed to chemotherapy was compared to matched, untreated cells and expressed as percent survival relative to controls. ImageJ was used for quantification. A mixed model analysis was performed to determine if an association existed between relative survival of the treated cells and γH2AX or p53 expression in the original tumors. Cell lines were validated via vimentin expression or growth as subcutaneous sarcomas in nude mice. Results An association was detected between γH2AX expression and relative survival in cells exposed to carboplatin (P = 0.0250). In the 11 FISS tumors evaluated, γH2AX expression ranged from 2.2 to 18.8% (mean, 13.3%). Cells from tumors with γH2AX expression higher than the sample population mean had fourfold greater relative survival after carboplatin exposure than cells from tumors with γH2AX expression less than the mean. There was no association between relative survival after carboplatin exposure and p53 expression (P = 0.1608), and there was no association between relative survival after doxorubicin exposure and either γH2AX (P = 0.6124) or p53 (P = 0.8645) expression. Four cell lines were validated via growth as sarcomas in nude mice. Vimentin expression was confirmed in the other 7 cell lines. Conclusions γH2AX expression, but not wild type p53, may potentially serve as a biomarker of resistance to platinum therapeutics in soft tissue sarcomas. To further investigate this finding, prospective, in vivo studies are indicated in animal models.

Keywords

• Chemoresistance
• Sarcoma
• Animal model
• GammaH2AX
• DNA damage