Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Abstract

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.

A 60 kDa prolactin variant secreted by cervical cancer cells modulates apoptosis and cytokine production

“…Whereas in SiHa cells, Bcl-xL and survivin do not show this tendence. These results may be explained since it is well known that antiapoptotic proteins are redundant in their functions (57)(58)(59), and this is why it may occur that one or another protein is expressed and in some cases both; however, more experiments must be performed in order to conclude with more certainty.…”

Cell Cycle–Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells

“…Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described [27]. Cells (2,000 per well for KB3 and 30,000 per well for others) were seeded in 96-well plates, and vincristine was added in a fixed final concentration of 0.1% DMSO.…”

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis

“…This is in accordance with earlier findings indicating that the anti-apototic BCL2 family members have functional redundancy in other malignant tissues, such as testicular germ cell tumors, melanoma, and colon cancer. [38][39][40] In this study, we tested whether GATA4 protects HB cells from Dox-induced apoptosis by regulating BCL2 family members. Transcription factor GATA4 is absent from normal postnatal hepatocytes, but it is expressed in the majority of HBs.…”