Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells.

Abstract Telomeres are the very ends of chromosomes and protect the genome from recombination, end-to-end-fusion, and recognition as damaged DNA. Telomeres are eroded with each cell division, eventually reaching such critically short length as to cause cell cycle arrest, apoptosis, or genomic instability. In most highly proliferative cells, including hematopoietic stem cells and T lymphocytes, telomere attrition is countered by telomere extension by telomerase reverse transcriptase complex. The majority of cancer cells also express telomerase, which maintains telomere length and allows indefinite cell proliferation. However, about 10% of tumors maintain telomere length in the absence of telomerase by mechanisms collectively termed alternative lengthening of telomeres (ALT). ALT mainly acts through asymmetrical exchange of telomeric material between chromosomes or sister chromatids, producing one daughter-cell with short telomeres and a limited life-span and its sister with long telomeres and higher proliferative capacity. To date, ALT has only been reported in cancer cells or through genetic engineering of mammalian cells. Here we investigated whether ALT mechanisms were active in hematopoietic cells using chromosome orientation fluorescent in situ hybridization (CO-FISH). In standard FISH, a telomeric probe produces fours signals per chromosome, one at each end of the two chromatids. Using CO-FISH, the newly synthesized DNA strand is fragmented by BrdU incorporation and UV light exposure and then digested by exonucleases. In CO-FISH, a telomeric probe produces two signals only, one at each end of the chromosome; in the presence of telomeric recombination, the telomeric signal is split, generating more than two signals per chromosome. Peripheral blood lymphocytes from three healthy volunteers, normal human fibroblasts, K562 cells, telomerase-positive HeLa cells (known to be negative for ALT),and telomerase-negative VA13 cells (known to be positive for ALT) were investigated for telomeric sister chromatid exchange (t-SCE); at least 20 metaphases per cell type were examined. Cultured peripheral blood lymphocytes and VA13 cells both showed increased levels of telomeric sister chromatid exchange in comparison to the other cells (P=0.0001): telomeric probe generated 2.62±0.11 telomeric signals/chromosome in lymphocytes; 2.23±0.04 in VA13 cells; 2.09±0.01 in HeLa cells; 2.02±0.01 in K562 cells; and 2.02±0.01 in human skin fibroblasts. Staining incorporated-BrdU over 24 hours and evaluation of “harlequin” chromosomes point to a similar rate of genomic sister chromatid exchange in lymphocytes, VA13 cells, and HeLa cells, suggesting that high chromatid exchange is confined to the telomeric region. A physical association between promyelocytic leukemia protein (PML) and telomeres is characteristic of some ALT-positive cells, but confocal microscopy failed to co-localize the telomeric probe and anti-PML monoclonal antibody in peripheral blood lymphocytes, suggesting that t-SCE in lymphocytes is not mediated by PML. This is the first demonstration of ALT activation in normal mammalian cells. ALT may be activated in peripheral blood lymphocytes as a complementary mechanism to maintain telomere length, and may explain the differences in age-related telomere shortening observed between lymphocytes and granulocytes.