Human POT1 facilitates telomere elongation by telomerase.

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.

Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.

We directly compared two methods of immortalizing human mammary epithelial cells (HMECs). Cells were transfected with an expression plasmid either for hTERT, the catalytic subunit of telomerase, or for the simian virus 40 (SV40) early region genes. Under standard culture conditions, HMECs were not immortalized by hTERT unless they had spontaneously ceased expression of the p16(INK4a) tumor suppressor gene. Untransfected HMECs had low levels of telomerase expression, and immortalization by both methods was associated with an increase in telomerase activity and prevention of telomere shortening. SV40-induced immortalization was accompanied by aberrant differentiation, loss of DNA damage response, karyotypic instability and, in some cases, tumorigenicity. hTERT-immortalized cells had fewer karyotypic changes, but had intact DNA damage responses, and features of normal differentiation. Although SV40-immortalized cells are useful for studies of carcinogenesis, hTERT-immortalized cells retain more properties of normal cells.

The unexpected landscape of in vivo somatic mutation in a human epithelial cell lineage.

Few data exist on somatic mutation in the epithelial cell lineages that play a central role in human biology and disease. To delineate the "landscape" of somatic mutation in a human epithelial cell lineage, we determined the frequency and molecular nature of somatic mutations occurring in vivo in the X-linked HPRT gene of kidney tubular epithelial cells. Kidney epithelial mutants were frequent (range 0.5 to 4.2 x 10(-4)) and contained a high proportion of unreported HPRT base substitutions, -1-bp deletions and multiple mutations. This spectrum of somatic mutation differed from HPRT mutations identified in human peripheral blood T lymphocytes and from germ-line HPRT mutations identified in Lesch-Nyhan syndrome or hyperuricemia patients. Our results indicate that DNA damage and mutagenesis may have unusual or mechanistically interesting features in kidney tubular epithelium, and that somatic mutation may play a more important role in human kidney disease than has been previously appreciated.

SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype.

In most human primary bone cells, SV40 T-antigen expression was able to expand life span for a few passages before cells undergo growth arrest, described as crisis. In this study, telomerase activity was reconstituted in human osteoblast precursors (hPOB cells) and marrow stromal cells (Saka cells) transformed with the SV40 T antigen. Bone cells with telomerase activity were able to bypass crisis and show unlimited life span. Despite chromosomal aberrations observed in hPOB-tert cells, these immortalized precursors were able to differentiate into osteoblasts like precrisis hPOB cells. Saka-tert cells enhanced the formation of human osteoclast-like cells in a similar manner as Saka cells. These results demonstrate that reconstitution of telomerase activity in transformed SV40 T-antigen human osteoblast precursors or marrow stromal cells leads to the generation of immortalized cells with a preserved phenotype.