Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Abstract

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

“…Majority of the joined products observed by us were consistent with those shown in earlier reports. 15,39,50,53,54 In contrast to some of the earlier reports, we also observed intramolecular circularization. 42,[53][54][55] Comparison of NHEJ activities showed that the joining of DSBs was highest in 14.5-day cephalon, irrespective of the nature of DSBs.…”

“…[42][43][44][45][46] Besides the efficiency of repair, the mechanism of regulation of NHEJ, the regulation of their proteins, and various protein-protein interactions were also studied using such a system. 15,39,[43][44][45][47][48][49][50][51][52][53][54] However, it is not clear whether the end-joining events observed in our studies correspond to in vivo NHEJ events, although the various lines of experimental evidence suggest an in vivo mechanistic correlation. Moreover, we could also correlate the expression of proteins detected by immunoblotting at different developmental stages to the immunofluorescence observed at the intracellular level (see the text below).…”

“…1a). 39 The overall efficiency of end joining varied among extracts prepared from the different day points of fibroblasts, cephalon, and whole embryos ( Fig. 1a and b).…”

Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs

“…Quantification was performed using Multi Gauge V 3.0, as described earlier. 55,56 Electrophoretic mobility shift assay Radiolabeled oligomeric DNA substrates containing the standard 12-RSS and 6-nt bubbles were incubated with RAGs in a buffer containing 25 mM 4-morpholinepropanesulfonic acid (pH 7.0), 30 mM KCl, 30 mM potassium glutamate, and 5 mM MgCl 2 . A 45-bp double-stranded oligomer (0.1 μM) was used as nonspecific DNA.…”

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

“…Bands of interest resulting from independent transfections were cut out from the dried gel, and removal of the bands was confirmed by scanning. The DNA from the bands was eluted in 500 mM NaCl, 10 mM Tris, and 1 mM EDTA and purified (39). The recombinant was then PCR-amplified and ligated to a TA vector (Invitrogen), and positive clones were sequenced (SciGenom).…”