Glioblastoma cells deficient in DNA‐dependent protein kinase are resistant to cell death

Abstract

DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.

Thymoquinone Induces Telomere Shortening, DNA Damage and Apoptosis in Human Glioblastoma Cells

“…The increased cell death shown in DNA-PKcs proficient glioblastoma cells supports the view that TQ targets DNA-PKcs proficient glioblastoma cells and that DNA-PKcs activation may play a role in apoptosis. This finding corroborates an earlier study, which showed a greater resistance of DNA-PKcs deficient glioblastoma cells to cell death as compared to DNA-PKcs proficient glioblastoma [29].…”

The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation

“…A prior investigation has reported decreased Bax levels in the M059J glioblastoma cell line, lacking DNA-PKcs expression, as compared with its isogenic M059K control [12]. We have not observed differences in Bax levels, by Western blot analysis, in either murine cortices or in cultured cortical neurons, regardless of DNA-PKcs status, consistent with others [28].…”

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

“…Our functional proteomic data suggest candidates like AKT1 and 2, MAPK14, and BRAF for future investigation (Figure 3C). Disrupting such kinases could reveal the functional contribution that they (and their cognate substrates) make to apoptosis, as has been shown previously for DNA-PK (Bharti et al, 1998; Chakravarthy et al, 1999; Chen et al, 2005a, b; Wang et al, 2000). Finally, there are other potential forms of crosstalk between phosphorylation and proteolytic pathways that may have eluded detection in our study.…”