Role of Bcl-2 proteins in stress-induced apoptosis in a non-transformed mammary epithelial cell line

Apoptosis is the process of programmed cell death characterized by morphological and physiological changes executed by cells in response to a stimulus. The apoptotic process contributes to the loss of milk secreting cells (i.e. mammary epithelial cells) that occurs in the bovine mammary gland once peak lactation has occurred. However, the specific mechanisms by which this occurs are unknown. The goal of this work was to examine the role of the Bcl-2 family of proteins in regulating apoptosis in the bovine mammary epithelial cell line, MAC-T.;The Bcl-2 family is divided into three categories based on protein function and combinations of four Bcl-2 homology (BH) domains. Anti-apoptotic multidomain proteins, such as Bcl-2 and Mcl-1, prevent apoptosis by binding to pro-apoptotic members of the family. Pro-apoptotic multidomain proteins, such as Bax, help to regulate apoptosis at the mitochondrial outer membrane. Pro-apoptotic BH3-only proteins, such as Bad and Bim, act as messengers between the anti-apoptotic and pro-apoptotic proteins.;To investigate the roles of Bcl-2 proteins in apoptosis, MAC-T cells were treated with anisomycin (ANS), an activator of the intrinsic apoptotic pathway. ANS had little effect on Bcl-2 or Bax mRNA levels while it induced a 40 to 60% decrease in levels of Bad mRNA. Protein expression of Bax did not change during apoptosis, while Bcl-2, Mcl-1, and Bad expression decreased to varying degrees. The greatest change in protein levels was observed for Mcl-1, whose expression was nearly non-detectable after 4 h of treatment with ANS. Bim was phosphorylated in response to ANS but this was not caused by JNK or p38 signaling. Knock-down of Bim using siRNA decreased the ability of ANS to induce apoptosis. Mcl-1 and Bim protein expression changed in a similar time frame, therefore, interactions between the two proteins were evaluated using co-immunoprecipitation experiments. Mcl-1 and Bim interacted both basally and after treatment with ANS. The significance of Bim phosphorylation, the kinase responsible for its phosphorylation, and the functional significance of the interaction between Bim and Mcl-1 in stress-induced apoptosis remain to be determined.