Endocrine Abstracts (2004) 7 P118

Expression and localisation of BAD and Akt in human breast cancer cell lines pre- and post-irradiation

Y Al-Bazz1, KP Corke2, PRM Dobson1, JCE Underwood2 & BL Brown1

1Academic Unit of Endocrinology, Sheffield University Medical School, Sheffield, UK; 2Academic Unit of Pathology, Sheffield University Medical School, Sheffield, UK.

The effects of radiotherapy, one of the main options currently available for cancer therapy, depend on various factors, including the dose and the radiosensitivity of tumour cells. Radiotherapy is known to induce apoptosis in breast cancer cells, but the effects of irradiation on components of the intracellular pathways involved in apoptosis and survival are largely unknown. BAD, which is a pro-apoptotic member of the Bcl-2 family plays a central role in the interaction between signal transduction and the cell death machinery. In addition, it is involved downstream of the PI3-K/Akt pathway. Akt is also involved in cell survival pathways.

The aim of this study was to examine the expression and localisation of Akt and BAD by immunocytochemistry and to evaluate the effects of gamma irradiation on their expression in human breast cancer cells (MDA-MB231, MCF-7 and T47D cells).

The cells were irradiated, using a Caesium 137 source, with either 50 or 80Gy (at a dose rate of 0.047Gy/sec). Post-irradiation, the cells were trypsinized, and fixed in 1% paraformaldehyde. Immunohistochemical analysis of fixed cytospin cells was performed on both irradiated samples and unirradiated controls using specific antisera for BAD and Akt.

In these three cell types, BAD was expressed in both the cytoplasm and nucleus. Some of the cells also demonstrated a marked nucleolar staining pattern. The nucleolar staining of BAD disappeared following exposure to irradiation in both MCF-7 and T47D cells, but not in MDA-MB231 cells. Both MCF7 and MDA-MB231 cells showed cytoplasmic and nuclear localization of Akt, which disappeared post-irradiation. Akt expression in T47D cells was unaffected by irradiation.

The breast cancer cell lines showed differential expression of BAD & Akt in response to irradiation. MDA-MB231 and T47D cells showed opposite responses. These results might explain the difference in the apoptotic effect caused by irradiation among various breast tumours.

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