Telomere stability correlates with longevity of human beings exposed to ionizing radiations.

Normal somatic cells have a finite number of divisions, a limited capacity to proliferate. Human telomeres contain TTAGGG repeats which are considered a molecular clock marker. The gradual and progressive telomere shortening at each replicative cycle is associated, through the activation of pRB and p53 pathways and genomic instability, to the replicative senescence, a non-dividing state and widespread cell death. There is no information available about telomere status in individuals who live long and have been exposed to ionizing radiations (IR). To determine the telomere stability, we examined telomeres at metaphase, G2-type chromosome aberrations after IR treatment and karyotypic analysis of 15 individuals. Three individuals were above the age of 80 years and 1 among the 3 was estimated to have received more than 10 Gy of occupational exposure about 30 years back. The other 12 were cancer patients that had received more than 50 Gy of gamma-radiation for therapeutic purposes. No telomere instability or defective G2 chromosome repair was found in 3 individuals above the age of 80 years. Whereas, 3 out of 7 prostate and 1 out of 5 breast cancer patients showed higher G2-type chromosome damage as well as a high frequency of telomeric association (also known as chromosome end associations) along with frequent loss of telomeres. Present studies demonstrate that telomere stability along with normal G2 chromosome repair correlates with the longevity of human beings, whereas defective G2 chromosome repair and telomere instability correlate with the radiotherapy related late toxicity.

Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation.

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.

Down-regulation of Myc as a potential target for growth arrest induced by human polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells.

Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPaseold-35), a 3',5'-exoribonuclease. We previously demonstrated that hPNPaseold-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-beta-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action.

hTERT associates with human telomeres and enhances genomic stability and DNA repair.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.