Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development.

We previously reported that the telomere-targeting drug telomestatin induces apoptosis accompanied by G-tail reduction and dissociation of binding protein TRF2 from telomeres in cancer cell lines but not normal or human telomerase reverse transcriptase (hTERT)-immortalized cells. Because telomere-targeting drugs induce growth arrest in normal cells at higher doses, their development is dependent on the ability to predict toxicity before in vivo use, but no models for this are available. Here, we established two new cell lines, telomerase immortalized human fetal hepatocytes, Hc3716-hTERT, and telomerase immortalized hepatic stellate cells, NPC-hTERT. Examinations showed that Hc3716-hTERT maintained normal mammalian cell morphology, cell growth, albumin expression, and wild-type p53 responsiveness, whereas NPC-hTERT maintained hepatic stellate-like morphology, expression of hepatic stellate markers, alpha-smooth muscle actin, and secretion of type I collagen, an extracellular matrix protein. Given our finding that telomere G-tail length in Hc3716 cells was decreased in senescence and increased by hTERT infection, we next examined the effect of high-dose telomestatin-induced telomere dysfunction and G-tail shortening on cellular functions in Hc3716-hTERT cells. Interestingly, telomestatin decreased expression of cytochrome P450 (CYP) family members CYP3A3/4, CYP3A5, and CYP3A7, mRNA and induced albumin expression at both mRNA and protein levels. These gene expression responses to telomestatin were similar to those of the normal parental cell Hc3716. These established cell lines thus represent the first model for predicting the side-effects of telomere-targeting drugs in normal cells, and should be powerful tools in the development of these drugs.

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation.

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.