EXPRESSION OF MULTIPLE APOPTOSIS-REGULATORY GENES IN HUMAN BREAST-CANCER CELL-LINES AND PRIMARY TUMORS

The expression of several apoptosis-regulating genes was evaluated in 9 human breast cancer cell lines, 2 immortalized human mammary epithel ial lines, 1 normal breast tissue biopsy, and 3 primary breast tumors, using a multiple antigen detection (MAD) immunoblotting method. The a nti-apoptotic proteins Bcl-2, Bcl-X-L, Mcl-1, and BAG-1 were present a t immunodetectable levels in 7, 10, 10, and 9 of the 11 lines. Compari ng these 11 cell lines among themselves revealed that steady-state lev els of Bcl-2, Bcl-X-L, Mcl-1, and BAG-1 were present at relatively hig her levels in 4, 6, 5, and 5 of the lines, respectively. In contrast, the pro-apoptotic proteins Bar and Bak were detected in all 11 cell li nes, and were present at relatively higher levels in 10 and 5 of the 1 1 lines, respectively. The Interleukin-1 beta converting enzyme (ICE) homolog CPP32 (Caspase-3) was expressed in 10/11 breast cell lines. Hi gh levels of p53 protein, indicative of mutant p53, were found in 8 of the 11 lines and correlated inversely with Bar expression (p = 0.01). Bcl-2 and BAG-1 protein levels were positively correlated (p = 0.03). Immunoblot analysis of primary adenocarcinomas revealed expression of the antiapoptotic proteins Bcl-2, Bcl-X-L, Mcl-1, and BAG-1, as well as the pro-apoptotic proteins Bar, Bak, and CPP32, in at least 2 of th e 3 tumors examined. Immunohistochemical analysis was also performed f or all of these proteins using 20 paraffin-embedded breast cancer biop sy specimens that all contained residual normal mammary epithelium in combination with both invasive cancer and carcinoma in situ. All of th ese apoptosisregulating proteins were detected in primary breast cance rs, though the percentage of immunopositive tumor cells varied widely in some cases. Comparisons of the intensity of immunostaining in norma l mammary epithelium and invasive carcinoma suggested that Bcl-2 immun ointensity tends to be lower in cancers than normal breast epithelium (p = 0.03), whereas CPP32 immunointensity was generally higher in inva sive cancers (p < 0.0001). Taken together, the results demonstrate exp ression of multiple apoptosis-modulating proteins in breast cancer cel l lines and primary tumors, suggesting complexity in the regulation of apoptosis in these neoplasms of mammary epithelial origin.