Differences in the Mechanisms of Proapoptotic BH3 Proteins Binding to Bcl-XL and Bcl-2 Quantified in Live MCF-7 Cells

Abstract

Overexpression of antiapoptotic proteins including Bcl-XL and/or Bcl-2 contributes to tumor initiation, progression, and resistance to therapy by direct interactions with proapoptotic BH3 proteins. Release of BH3 proteins from antiapoptotic proteins kills some cancer cells and sensitizes others to chemotherapy. Binding of Bcl-XL and Bcl-2 to the BH3 proteins Bad, Bid, and the three major isoforms of Bim was measured for fluorescent protein fusions in live cells using fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer. In cells the binding of the proteins at mitochondria is similar to the results from in vitro measurements. However, mutations in the BH3 region of Bim known to inhibit binding to Bcl-XL and Bcl-2 in vitro had much less effect in MCF-7 cells. Moreover, the BH3 mimetic ABT-737 inhibited Bad and Bid but not Bim binding to Bcl-XL and Bcl-2. Thus, the selectivity of ABT-737 also differs markedly from predictions made from in vitro measurements.

Antiapoptotic potency of Bcl-2 proteins primarily relies on their stability, not binding selectivity

“…20,21 Also, removal of the membrane anchor from the prosurvival Bcl-2 proteins can alter binding properties, [22][23][24] and BH3-peptides may not represent the full-length proteins. 25,26 Most likely for these reasons, BH3-binding profiles of Bak and Bax determined in vitro differ greatly from those determined by coimmunoprecipitation from cells 27 ( Figure 2). However, immunoprecipitation studies are not fully consistent either (Figure 2 lines 2-3), because the detergents required to solubilize the mitochondrial membrane can induce or disrupt Bcl-2 family interactions 28,29 or alter Bcl-2 protein conformation, as shown for Bcl-xL, 30 Bcl-w, 31 and Bax.…”

Effects of Rapamycin on Cell Apoptosis in MCF-7 Human Breast Cancer Cells

“…The MCF-7 cell line is an ideal model to study the pathway of malignant progression (Aranovich et al, 2012). Prior to the MCF-7 discovery, there was no mammary cell line that could live longer than a few months.…”

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737

“…6,7 These differences explain why currently known BH3-mimetics only inhibit subsets of anti-apoptotic proteins. 8 One of these compounds is ABT-737, which occasionally presents in vitro monotherapy toxicity. 5 It potently inhibits the BH3-binding activity of Bcl-2, Bcl-xL and Bcl-w but not that of Mcl-1 and Bfl-1.…”

“…It is notable that, whereas the pro-apoptotic activity of ABT-737 relies on the nearly immediate ability of this compound to disrupt pre-existing complexes, 8,22 its effects on whole cells sometimes take numerous days to be manifest, implying that de novo synthesis of key actors might intervene. ABT-737 treatment was shown to induce the transcription of death receptor 5 23 and to induce a twofold change in the transcription of nearly 430 genes when added to renal carcinoma cells.…”