Tel1 and Rad51 are involved in the maintenance of telomeres with capping deficiency.

Vertebrate-like T2AG3 telomeres in tlc1-h yeast consist of short double-stranded regions and long single-stranded overhang (G-tails) and, although based on Tbf1-capping activity, they are capping deficient. Consistent with this idea, we observe Y' amplification because of homologous recombination, even in the presence of an active telomerase. In these cells, Y' amplification occurs by different pathways: in Tel1(+) tlc1h cells, it is Rad51-dependent, whereas in the absence of Tel1, it depends on Rad50. Generation of telomeric G-tail, which is cell cycle regulated, depends on the MRX (Mre11-Rad50-Xrs2) complex in tlc1h cells or is MRX-independent in tlc1h tel1Δ mutants. Unexpectedly, we observe telomere elongation in tlc1h lacking Rad51 that seems to act as a telomerase competitor for binding to telomeric G-tails. Overall, our results show that Tel1 and Rad51 have multiple roles in the maintenance of vertebrate-like telomeres in yeast, supporting the idea that they may participate to evolutionary conserved telomere protection mechanism/s acting at uncapped telomeres.

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome.

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG*CCG repeat tract in the 5' UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of gamma-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.

Tumor cells fail to trans-induce telomerase in human umbilical vein endothelial cell cultures.

The shortening of the telomeres that occurs in most somatic cells and untransformed cell cultures is considered a hallmark of cellular senescence. Re-activation of telomerase, which is usually present in immortal cells, avoids telomere shortening and considerably extends the culture life span. Normal human endothelial cells are characterized by an accelerated rate of telomere shortening and reach replicative senescence after a limited number of cell divisions. It has recently been reported that human telomerase reverse transcriptase expression may be strongly up-regulated in human endothelial cells cocultivated with tumor cells. Due to the important implications of this finding on tumor progression, we have extensively analyzed for the presence of telomerase in primary human endothelial cells either cocultivated with tumor cells or grown with tumor-conditioned medium. We found modest, but readily detectable, amounts of telomerase in all human endothelial cell cultures analyzed that disappeared as the cultures approached senescence. Quantitative reverse transcription-PCR also showed a direct correlation between human telomerase reverse transcriptase expression and the proliferative index of the cultures. Nevertheless, we did not find any evidence of induction of telomerase activity by tumor cells in any of the tested conditions. All data indicate that telomerase in human endothelial cells follows an activation program that is strictly associated to the culture growth rate.

Detection of telomerase activity in prostate massage samples improves differentiating prostate cancer from benign prostatic hyperplasia.

PURPOSE: We performed a case-control study in which we tested the ability of a non-invasive assay to detect telomerase activity and to distinguish between prostatic cancer (Pca) and benign prostatic hyperplasia (BPH) on samples of epithelial cells obtained after prostatic massage. METHODS: Telomerase activity was determined by a telomeric repeat amplification protocol (TRAP) assay. We selected 60 patients with histologically proven Pca (30 cases) or BPH (30 cases). Specimens included in this study were from patients who had no suspicious findings on digital rectal examination for cancer, had clinical evidence of lower urinary tract symptoms, had no sonographic signs of Pca at the transrectal ultrasound evaluation, had total PSA values moderately elevated (2.6-15 ng/ml), and had no evidence of other urological cancers. The whole procedure was conducted in double blind between pathologists and molecular biology operators. RESULTS: Telomerase activity was detected in 90% of Pca cases and in 13% of BPH cases. The sensitivity (90%) and specificity (76%) of this method were calculated. The positive predictive value, negative predictive value, and diagnostic efficiency were 87%, 90%, and 88% respectively. CONCLUSION: Our data indicate that telomerase activity detected by TRAP assay on prostate epithelial cells collected by prostate massage can substantially improve the distinction between Pca and BPH conditions. One of the clinical benefits resulting from the use of this new assay would be to refine the biopsy indication and to avoid for several patients without Pca the unnecessary cost and the complications of prostate biopsy.

Chordoma of the skull base: predictors of tumor recurrence.

OBJECT: Chordomas of the skull base are generally regarded as slow-growing tumors; however, approximately 20% of these lesions have been shown to recur as early as 1 year postsurgery. The classic pathological paradigms are poor predictors of outcome, and additional markers are needed to identify patients at risk for early tumor recurrence. In this study the authors describe such a marker. METHODS: In a series of 26 patients with chordomas of the skull base, the authors investigated the relationship between the biological behavior of the tumor, which was determined according to the interval for its recurrence and volume doubling time, and several pathological and molecular features, which included the histological variant, proliferative activity, mutation of p53 protein, expression of human telomerase reverse transcriptase (hTERT) messenger (m)RNA, loss of heterozygosity (LOH), and microsatellite instability. The major finding in this study was that hTERT mRNA expression in chordoma cells identifies those tumors that exhibit unusually fast rates of growth. The expression of hTERT mRNA was frequently associated with mutation of p53 protein, indicating that telomerase dysfunction combines with abnormal p53 function to initiate the unrestrained clonal expansion of the tumor cells. In cases in which the tumor was partially removed, mutation of p53 protein and expression of hTERT mRNA predicted increased doubling time for residual tumor as well as the probability of tumor recurrence. Cell proliferation, as investigated using the Ki-67 method, was significantly related to the tumor doubling time; however, the authors found that the pattern of cell proliferation was not homogeneous throughout the chordoma tissue, and that the proliferative index might change by a factor as high as 8 among different regions of the same tumor. The LOH and microsatellite instability do not seem to affect the prognosis of skull base chordomas. CONCLUSIONS: Reactivation of telomerase in chordomas is a reliable predictor of outcome. The ability to predict the biological behavior of chordomas might have immediate implications in the management of this disease in patients who undergo surgery.

Erosion of telomeric 3'-overhangs in white blood cells of aged subjects with high frequency of very short telomeres.

After extended proliferation, cells enter a state of replicative quiescence that is probably due to progressive telomere shortening. It is supposed that changes in telomere structure eventually expose the chromosome ends to undesired recombination events and thus promote cell senescence. The telomeric 3'-overhang is crucial for efficient chromosome capping, but its specific role in telomere shortening and in triggering the senescence program is uncertain. We have addressed this issue by measuring the 3'-overhangs of a human tissue cells aging in vivo. The 3'-overhangs were analyzed in blood samples from 41 individuals aged 91-106 years and 89 individuals ranging from 6 months to 85 years. We found that the overall 3'-overhang length did not significantly change with age, but did, however, find extensively eroded 3'-overhangs in 3 subjects of the 91-106 years cohort and one 61 years old subject affected with Down syndrome. These subjects had 3'-overhang length distributions skewed towards shorter tails, the shortest overall telomere lengths and the highest frequencies of very short telomeres. These data raise the possibility that during ageing very short telomeres with very poor 3'-overhangs can reach a critical point for functional telomeres.

Long telomeric C-rich 5'-tails in human replicating cells.

Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3'-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3'-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5'-tail, the incompletely replicated template of the leading strand. We examined the presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3'-overhang, the 5'-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5' C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening.