Chromosomal location and expression of the genes coding for Ku p70 and p80 in human cell lines and normal tissues

Abstract

Ku protein is a relatively abundant DNA-binding nuclear protein complex composed of two polypeptide subunits, p70 and p80. Ku has been recently identified as the regulatory component of the DNA-dependent protein kinase that phosphorylates RNA polymerase II. To further characterize in vivo regulation of Ku protein, we studied the expression of the transcripts coding for the Ku p70 and p80 subunits in different human cell lines and normal tissues by Northern blot hybridization, using specific cDNA probes. The expression level of both genes was approximately 10-fold higher in established cell lines than in normal tissues. However, mRNA expression levels in permanent cell lines correlated more strongly with their proliferative state than with their level of malignant transformation. In purified T lymphocytes induced to proliferate by the combined action of monoclonal antibodies directed against the CD2 and CD28 adhesion molecules, Ku p70 and p80 mRNA steady-state levels increased as soon as 6 h after activation and lasted at least 72 h. The human genes coding for the Ku p70 and p80 subunits were localized by cytogenetic mapping, using fluorescence in situ hybridization, to 22q13 and 2q33→q35, respectively.

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts

“…The cells were grown to confluence and the gene expression was detected via chemiluminescence by using DIG-labelled specific DNA probes. As demonstrated in Figure 1, each of the tested cell lines possesses a Ku70 signal of about 2.2 kb and two variants of Ku80 transcripts of about 2.4 kb and 3.5 kb, respectively, which is in agreement with literature (Cai et al, 1994). As a control for gel loading, the signal intensity of β-actin was used ( Figure 1C).…”

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein

“…As shown in Figure 7, in the control cell line AHH1, Ku86 probe hybridized to two mRNAs of 2.6 and 3.4 kb as previously described (Cai et al, 1994;Jongmans et al, 1996). The two Ku86 transcripts were also present in PBL and in puri®ed B-lymphocytes and no smaller form of Ku86 mRNA was detected in B-lymphocytes.…”

“…Finally, our results suggest that the mechanism that contributes to the expression of the variant Ku86 protein occurs at the post-transcriptional level. Indeed, the fact that B-lymphocytes express the two Ku86 transcripts also detected in cell lines (Cai et al, 1994; H PBL LB LB 3.4 kb 2.6 kb Figure 7 Northern blot experiments. 5 mg of total RNA obtained from the control cell line AHH1 (H), peripheral blood lymphocytes (PBL) or B-lymphocytes (LB, two independent healthy donors) were separated on a 1.2% agarose ± 7% formaldehyde gel, transferred to nitrocellulose and hybridized with a Ku86 cDNA probe.…”

“…Despite many studies performed with established cell lines, little is known about Ku activity in normal fresh human cells and tissues. Indeed, only a few studies of Ku gene expression have been reported to date (Yaneva and Jhiang, 1991;Cai et al, 1994). Regarding the major role of the Ku protein in DSBs repair, this appears to be an important question.…”

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…The Ku70 and Ku80 genes were found to be distributed widely among eukaryotes. Both genes were mapped to regions of conserved linkage homology among mice, rats and humans (Cai et al, 1994;Koike et al, 1996a). Ku was demonstrated to be a component of the DNAdependent protein kinase (DNA-PK) complex, which is a nuclear serine/threonine protein kinase (Lees-Miller et al, 1990;Gottlieb et al, 1993).…”