Buried intramedullary implants for paediatric forearm fractures. Does the refracture rate improve? / Implantes intramedulares enterrados para fracturas de antebrazo pediátricas. ¿Mejora la tasa de refracturas?

INTRODUCTION: Unstable forearm fractures may require surgical management by reduction and osteosynthesis with intramedullary needles. This fixation should be removed early if it has been left exposed, but this could increase the risk of refracture in a bone in the period of remodelling. As an alternative we can keep the needles, buried subcutaneously, for a longer time, to protect the bone callus. OBJECTIVE: To assess whether there are differences between using exposed needles with respect to burying them in paediatric patients with forearm fractures. Our hypothesis is that by burying the needles we keep them longer by reducing forearm refractures. MATERIAL AND METHODS: We present a cohort of 75 paediatric patients with a forearm fracture between 2010 and 2016. Demographic data, surgical technique, complications and patient follow-up were collected. RESULTS: The implants were left exposed in 50 patients and 25 buried. The average time of removal of the exposed implants was 6.8weeks and 17.6weeks in the buried ones. No significant differences were found in terms of consolidation (P=.19) or immobilization time (P=.22). Regarding refractures, a greater number was observed in the exposed osteosynthesis group (4patients) compared to only one case with buried osteosynthesis, but there were no significant differences (P=.49). No postsurgical complications were detected and the functionality was excellent at the end of the follow-up in both groups. CONCLUSION: Leaving implants buried in relation to skin exposed does not cause a decrease in the number of refractures or other complications, with adequate patient functionality in both cases.

Impact of chemotherapy on telomere length in sporadic and familial breast cancer patients.

Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.

Epigenetic regulation of telomeres in human cancer.

Hypomethylation of repeated elements in the genome is a common feature of human cancer, however, the direct consequences of this epigenetic defect for cancer biology are still largely unknown. Telomeres are specialized chromatin structures at the ends of eukaryotic chromosomes formed by tandem repeats of G-rich sequences and associated proteins, which have an essential role in chromosome end protection and genomic stability. Telomeric DNA repeats cannot be methylated, however, the adjacent subtelomeric DNA is heavily methylated in humans. Here, we show that the methylation status of subtelomeric DNA repeats negatively correlates with telomere length and telomere recombination in a large panel of human cancer cell lines. These findings suggest that tumor telomere length and integrity can be influenced by epigenetic factors. Finally, we show that treatment of human cancer cell lines with demethylating drugs results in hypomethylation of subtelomeric repeats and increased telomere recombination, which in turn may facilitate telomere elongation. All together, these findings suggest that tumor telomere length and integrity can be influenced by the epigenetic status of cancer cells.

A possible role for telomerase RNA and telomere length in global mitotic recombination.

Telomeres are specialised structures at the ends of mammalian chromosomes with many unique properties. Recombinational events at telomeres are more frequent than in the remainder of the genome by several orders of magnitude. This study examined the influence of telomerase status and telomere length on genome-wide recombination assessed by genomic sister chromatid exchange (G-SCE). Telomerase deficiency per se appears to increase G-SCE frequencies in splenocytes but as telomeres shorten through subsequent generations of mTerc(-/-) mice this increase is progressively lost. Telomerase status and telomere length also influences the induction of G-SCE by UV light. Even when mitotic recombination is affected by PARP deficiency, mTerc and telomere length interact to further affect G-SCE frequencies. Taken together the data presented here demonstrate that telomerase status and telomere length can affect recombination frequencies genome-wide.

Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling.

Here, we show that ectopic expression of the catalytic subunit of mouse telomerase (mTert) confers a growth advantage to primary murine embryonic fibroblasts (MEFs), which have very long telomeres, as well as facilitates their spontaneous immortalization and increases their colony-forming capacity upon activation of oncogenes. We demonstrate that these telomere length-independent growth-promoting effects of mTert overexpression require catalytically active mTert, as well as the formation of mTert/Terc complexes. The gene expression profile of mTert-overexpressing MEFs indicates that telomerase enhances growth in these cells through the repression of growth-inhibiting genes of the transforming growth factor-beta (TGF-beta) signaling network. We functionally validate this result by showing that mTert abrogates the growth-inhibitory effect of TGF-beta in MEFs, thus demonstrating that telomerase increments the proliferative potential of primary mouse embryonic fibroblasts by targeting the TGF-beta pathway.

Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells.

Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase-deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence-associated beta-galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration.

Putting the stress on senescence.

Cultivation of primary cells over many generations eventually results in a reproducible loss of proliferative potential that has been termed 'replicative senescence'. Recent work has revealed the heterogeneity of senescence. Importantly, the analysis of the various aspects and types of senescence has turned out to be very informative about numerous in vivo processes, and particularly about carcinogenesis.

Telomeric sequences, radiation sensitivity and genomic instability.

PURPOSE: To review the basic features of telomeres with particular emphasis on their potential importance in radiation biology. Recent findings suggest that telomere length can influence radiation sensitivity in mouse and that several human radiosensitive disorders also show abnormalities in telomere dynamics. Numerous studies indicate that telomeric sequences may play a role in determining the stability of certain genomic regions both spontaneously and following irradiation. Furthermore, a number of transmissible genomic instability systems have been described in which it appears that telomere metabolism may be contributing to the delayed effects observed. Features of telomeres and telomere biology relevant to these topics are reviewed. CONCLUSIONS: The evidence that telomeres and the molecular pathways of telomere maintenance can play a role in determining the outcome of radiation exposure is now substantial. Thus, the field of telomere biology deserves continued attention from radiobiologists.

Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres.

Reconstitution of telomerase activity is proposed as a potential gene therapy to prevent, or rescue, age-related diseases produced by critical telomere shortening. However, it is not known whether or not short telomeres are irreversibly damaged. We addressed this by re-introducing telomerase in late generation telomerase-deficient mice, Terc-/-, which have short telomeres and show severe proliferative defects. For this, we have crossed these mice with Terc+/- mice and analyzed telomere length, chromosomal instability and premature aging of the progeny. The Terc-/- progeny had one set of chromosomes with normal telomeres, whereas the other set remained with critically short telomeres; these mice presented chromosomal instability and premature aging. In contrast, Terc+/- progeny showed all chromosomes with detectable telomeres, and did not show chromosomal instability or premature aging. These results prove that critically short telomeres can be rescued by telomerase, and become fully functional, thus rescuing premature aging. This has important implications for the future design of telomerase-based gene therapy of age-related diseases.

Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability.

Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

Telomerase inhibition in RenCa, a murine tumor cell line with short telomeres, by overexpression of a dominant negative mTERT mutant, reveals fundamental differences in telomerase regulation between human and murine cells.

In contrast to human primary fibroblasts, mouse embryonic fibroblasts have telomerase activity, immortalize spontaneously in culture, and can be neoplastically transformed by oncogenic insult. Ectopic expression of the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), in human primary cells allows both spontaneous immortalization and neoplastic transformation by oncogenes. This suggests that telomerase activity, as well as the fact that mouse telomeres are longer than human telomeres, may explain some of the differences in cellular control between human and murine cells. Telomerase inhibition in immortal or transformed human cells using dominant negative hTERT mutants leads to telomere shortening and cell death. Here we study the effect of expression of a dominant negative mutant of the catalytic subunit of mouse telomerase, mTERT-DN, in a murine kidney tumor cell line, RenCa, whose telomeres are similar in length to human telomeres. After showing initial telomerase activity inhibition and telomere shortening, all clones expressing mTERT-DN reactivated telomerase and showed normal viability, in contrast with that described for human cells. This efficient telomerase reactivation coincided with a significant increase in the endogenous TERT mRNA levels in the presence of mTERT-DN expression. The results presented here reveal the existence of fundamental differences in telomerase regulation between mice and man.

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age-associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase-transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild-type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase-transgenic mice also shows an increased wound-healing rate compared with wild-type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age-associated diseases based on telomerase upregulation.

The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang.

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs(-/-) cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.

Telomere length abnormalities in mammalian radiosensitive cells.

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.

Identification of functional domains and dominant negative mutations in vertebrate telomerase RNA using an in vivo reconstitution system.

The telomerase holoenzyme consists of two essential components, a reverse transcriptase, TERT (telomerase reverse transcriptase), and an RNA molecule, TR (telomerase RNA, also known as TERC), that contains the template for the synthesis of new telomeric repeats. Telomerase RNA has been isolated from 32 different vertebrates, and a common secondary structure has been proposed (Chen, J.-L., Blasco, M. A., and Greider, C. W. (2000) Cell 100, 503-514). We have generated 25 mutants in the four conserved structural domains of the mouse telomerase RNA molecule, mTR, and assayed their ability to reconstitute telomerase activity in mTR(-/-) cells in vivo. We found that the pseudoknot and the CR4/CR5 domains are required for telomerase activity but are not essential for mTR stability in the cell, whereas mutations in the BoxH/ACA and the CR7 domains affect mTR accumulation in the cell. We have also identified mTR mutants that are able to inhibit wild type telomerase in vivo.