Abstract

Abstract 3239

Poster Board III-176

Background

The Ku autoantigen is a heterodimer composed of a 70kDa subunit (Ku70) and an 80kDa subunit (Ku86), which is critical for repairing DNA double-strand breaks (DSB) by non-homologous end joining and for telomere maintenance. Knockdown of either one or both Ku components results in rapid shortening of telomere ends and genomic instability (Fattah et al., DNA Repair 2008; Faure et al., DNA Repair 2008). We and others have demonstrated significantly shorter telomeres in patients with chronic lymphocytic leukemia (CLL) (Röth A et al, Brit. J. Haematol. 2008). In addition, in over 80% of patients with CLL cytogenetic aberrations are found (Döhner H, NEJM, 2000). Based on these observations we hypothesized that differences in Ku protein expression or function could be involved in the pathophysiology of CLL cells. Our aim was to assess the localization and expression levels of the Ku components in leukemic cells of patients with CLL

Patients and Methods

So far, we examined the localization and expression of the Ku-proteins in 20 patients with CLL (14 with mutated IgVH genes, 6 with unmutated IgVH genes), and in peripheral blood lymphocytes of 4 healthy individuals. The localization of the Ku proteins was assessed by immunological staining and fluorescence microscopy. Expression levels of the Ku components in whole cells as well as in the cytoplasmic and nuclear fractions were assessed by Western blotting. DNA sequencing was done by cycle sequencing and capillary electrophoresis. Telomere length measurements were performed by automated multicolor Flow-FISH.

Results

Lymphocytes of healthy individuals with a normal telomere length showed an expression of Ku70 and Ku86 in the nucleus without any cytoplasmic expression. Interestingly, in leukemic cells from CLL patient samples, Ku86 was not detectable in the nucleus, but was abundantly found in the cytoplasm, while Ku70 was found in approximately equal amounts in the nucleus and cytoplasm. The expression levels of both Ku proteins were comparable in lymphocytes from CLL patients and from healthy controls. Sequence analysis of the Ku70 and Ku86 DNA revealed no mutations. All leukemic cells of CLL patients had short telomeres below the 50th percentile of healthy individuals with significantly shorter telomeres in patient samples with unmutated IgVH (mean: 2.8kb ± 0.03kb) than with mutated IgVH (mean: 6.15kb ± 2.92kb). In contrast, no difference in the expression pattern of Ku70 and Ku86 was found between these two prognostic subgroups of CLL patients.

Conclusion

For the first time we can demonstrate clear differences in the expression and localization of the Ku proteins in leukemic cells of patients with CLL with short telomeres compared to lymphocytes of healthy individuals with telomeres in the normal range. The high cytoplasmic expression of both Ku proteins in the leukemic cells of CLL patients and the lack of nuclear Ku86 protein suggested the presence of a genetic alteration. Although we could not find any mutations in the DNA sequence of the Ku proteins, the delocalization could point to changes in the Ku proteins (i.e. by post-translational modifications) and/or in the transport system of the Ku proteins between the nucleus and the cytoplasm. Deficient nuclear expression of Ku86 and/or Ku70 in CLL cells might lead to shortening of telomeres and genomic instability as has been seen in heterozygous Ku86 deficient human cell lines. Since there was no difference in the expression and localization of the Ku proteins in CLL cells with mutated and unmutated IgVH it is unlikely that the absolute telomere length, which differs highly between the two subgroups of CLL patients, is directly related to the level of the nuclear Ku protein expression. Further molecular and functional studies of the Ku proteins are ongoing.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.