Yet another protein has been added to the crowd of players found at the ends of chromosomes. Known variously as PTOP, PIP1 or TINT1, this negative regulator of telomere length connects some of the key proteins already known to be present - TRF1, TIN2, POT1, and TRF2 - and adds even more complexity to telomere protein interactions.

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.

MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

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.

Aberrant hypoxanthine phosphoribosyltransferase (HUGO-approved gene symbol HPRT1; MIM# 308000) mRNA splicing, promoted by splice site mutation or loss, is a common mechanism for loss of the purine salvage enzyme HPRT1 from human cells. We report here two in vivo somatic HPRT1 mutations in human kidney tubular epithelial cells that disrupt HPRT1 intron 1 splicing and lead to the inclusion of intron 1 sequence. We propose an explanation for the use of a common, cryptic intron 1 splice donor site by these two mutations, and by 14 additional human HPRT1 mutations that lead to aberrant splicing with the incorporation of intron 1 sequence into mRNA. Hum Mutat 13:504–505, 1999. © 1999 Wiley-Liss, Inc.