Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex, Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced ( similar to 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically ( similar to 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5 - 20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity, This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents.