Expression Of Telomere Binding Factor 2 (TRF2) On Leukemia Cell Lines And Primary Leukemia Cells

Telomeres are nucleoprotein complexes that cap the ends of linear eukaryotic chromosomes. Telomere length can be maintained by telomerase. In humans, activation of telomerase, that synthesizes new telomeric repeats onto the chromosome ends and compensates for the loss occurring during cell division, appears to be an essential, limiting step in cellular immortalization and tumor progression. Since the selective expression of telomerase in tumor cells and most cancer cells lacking telomerase show sluggish growth and death, telomerase has to be considered as an attractive therapeutic target for anticancer therapies. However, some reports have shown a lack of correlation between drug-induced cell death and loss of telomerase activity.Recent findings indicate that a functional telomere has to be considered as a 'telosome', a structure composed of telomeric DNA and telomere binding proteins. This 'telosome' functionally acts protection on chromosomes from degradation, recombination, and end-to-end fusion. With regard to senescence, telomere structure appears at least as important to telomere function as absolute telomere length. Several telomere-associated proteins, which play an important role in protection and maintenance of telomeric DNA, have been identified. Among them, telomere binding factor 2 (TRF2) proteins not only regulate telomere length but also maintain telomere integrity. The overexpression of a dominant-negative TRF2 induces loss of the 3' tail, telomere end fusions and either apoptosis or senescence even though telomeres are not critically short, suggesting that TRF2 may play an important role in the fate of cancer cells.In this study, the target gene mRNA was amplified with RT-PCR, then was sequentially electrophoresed and purified for standards. The standard curves of gene expression were established. After that, the expression levels of TRF2 mRNA of leukemia cell lines and primary leukemia cells obtained from leukemia patients were detected with quantitive real-time RT-PCR. The results showed that the correlation coefficient was 0.996 between the amount of template cDNA and the intensity of fluorescence signal that generated during amplifying when gene expression standard curves were established. That of template cDNA amount and grey density of bands that derived from gel electrophoresis of real-time RT-PCR final products was 0.78 (P<0.05). Among leukemia cell lines, expression levels of TRF2 mRNA in HL60 and K562 cells showed no significant difference compared with the normal control, while T-cell leukemia cell lines Molt-4 and Jurkat cells had higher expression of TRF2 compared with bone marrow mononuclear cells (MNCs) obtained normal dornor (P <0.001; P <0.01), and myeloid leukemia cell lines (HL60 and K562), suggesting T-cell leukemia cell lines expressed a high level of TRF2. The results come from western blot analysis were consistent with that of real time RT-PCR. Of all leukemia patients, expression levels of TRF2 mRNA of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were 0.01185±0.01728 attomol and 0.0083±0.00104 attomol, reseparately, which had no significant difference compared with bone marrow MNCs obtained normal dornor (0.00194±0.00116 attomol; P>0.05). Compared with normal control, TRF2 expression of AML patients with MO and Ml subtypes was higher (P <0.05), suggesting that the patients with higher expression of TRF2 may have a poor prognostic.In general, TRF2 is involved in telomere maintenance by acting as negative regulator. The expression levels of TRF2 have only been examined in a limited number of tumors and cell culture experiments. We and others previously have described the increased expression of TRF2 in lung cancer, gastric cancer, and lymphoma. In contrast, decreased expression of this gene has been reported in breast cancer, gastric cancer, and malignant hematopoietic cells. Importantly, TRF2 is overexpressed in in vivo patient's samples from adult T-cell leukemia but not asymptomatic carriers or normal donors. Although no defined common conditions or signals for different expression of TRF2 in tumors are known, an excess of TRF2 is demonstrated to be involved in the progression of multistep hepatocarcinogenesis, and the extended proliferation capacity in T-cell leukemia. Taken together TRF2 may be a novel marker for malignant human T lymphocytic disease progression. Our results may provide a rationale for the treatment targeting on TRF2 in leukemia.In summary, we established the target genes assay using a quantitive real-time RT-PCR. Our data suggested for the first time that T-cell leukemia cell lines expresses high levels of TRF2, while primary leukemia cells gained from AML patients with with MO and Ml subtypes have signicant higher levels of TRF2 compared with normal control and other subtypes AML. Taken together, we believe that the patients with higher expression of TRF2 may have a poor prognostic, and TRF2 could be an attractive target for new therapies of leukemia.