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Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs.

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of approximately 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only approximately 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.