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A‐type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A‐type lamins and how they contribute to disease are poorly understood. Here, we show that A‐type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A‐type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A‐type lamins are necessary for the processing of dysfunctional telomeres by non‐homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A‐type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin‐related diseases.

Genomic instability due to telomere dysfunction and defective repair of DNA double‐strand breaks (DSBs) is an underlying cause of ageing‐related diseases. 53BP1 is a key factor in DNA DSBs repair and its deficiency is associated with genomic instability and cancer progression. Here, we uncover a novel pathway regulating the stability of 53BP1. We demonstrate an unprecedented role for the cysteine protease Cathepsin L (CTSL) in the degradation of 53BP1. Overexpression of CTSL in wild‐type fibroblasts leads to decreased 53BP1 protein levels and changes in its cellular distribution, resulting in defective repair of DNA DSBs. Importantly, we show that the defects in DNA repair associated with 53BP1 deficiency upon loss of A‐type lamins are due to upregulation of CTSL. Furthermore, we demonstrate that treatment with vitamin D stabilizes 53BP1 and promotes DNA DSBs repair via inhibition of CTSL, providing an as yet unsuspected link between vitamin D action and DNA repair. Given that CTSL upregulation is a hallmark of cancer and progeria, regulation of this pathway could be of great therapeutic significance for these diseases. Earlier work implicated the DNA repair factor 53BP1 in laminopathy‐linked genome stability and progeria phenotypes. Lamin A loss is now shown to cause 53BP1 proteolysis through Cathepsin L, which can be counteracted by vitamin D—thus offering therapeutic potential in these situations.

Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819–E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may influence the CaR response to calcium and R-568. Human embryonic kidney (HEK-293) cells transiently transfected with three different human CaRs (wild-type [A826], variant allele [T826], and artificial mutant [W826]) were used to test the ability of calcium alone or in combination with the calcimimetic R-568 to modulate CaR activity. CaR activation was detected by flow cytometry using a fluorescent probe. Intracellular calcium changes were measured in response to changes in extracellular calcium alone or with different R-568 concentrations. The change of the alanine in the 826 position (A826) for threonine (T826) worsened calcium sensitivity, increasing the EC 50 value from 2.34 ± 0.48 mM (A826, wild-type) to 2.96 ± 0.75 mM (T826) ( P  < 0.05). The T826 receptor reached a similar response with 1 μM R-568 compared with the wild-type receptor. On the contrary, the artificial introduction of a tryptophan in the same position (W826) did not affect calcium sensitivity (EC 50  = 2.64 ± 0.81 mM) but reduced the ability of the receptor to respond to R-568. The results demonstrate the importance of the 826 residue in the CaR response to calcium and calcimimetics. Since the A826T change was described as a natural variant, the differences in the calcium and calcimimetic responses observed between the alleles could have potential clinical impact.

Aluminum posttranscriptional regulation of parathyroid hormone synthesis: A role for the calcium-sensing receptor. Calcium regulates parathyroid hormone (PTH) gene expression by a posttranscriptional mechanism, as well as parathyroid gland growth through the activation of the calcium-sensing receptor. Aluminum decreases both parathyroid cell proliferation and PTH levels by an unknown mechanism. To investigate the possible role of calcium-sensing receptor in the aluminum-induced PTH inhibition we used human embryonic kidney (HEK-293) cells transiently transfected with the human calcium-sensing receptor. We used a parathyroid gland tissue culture model to investigate whether the effect of aluminum in PTH mRNA was a transcriptional mechanism and also its possible role in calcium-sensing receptor expression. We found that Al activated the calcium-sensing receptor with higher efficiency than calcium, its biologic ligand. Aluminum inhibited PTH gene expression by a posttranscriptional mechanism, but only when low calcium is present in the medium. Finally, we found that aluminum is also able to decrease calcium-sensing receptor mRNA levels by a posttranscriptional mechanism; however, no effect was observed on calcium-sensing receptor protein. These findings indicate that aluminum impairs parathyroid function through a calcium-like mechanism due to the lack of specificity of the calcium-sensing receptor. Additionally, aluminum decreases parathyroid calcium-sensing receptor mRNA levels, and the regulatory mechanism was posttranscriptional. These findings demonstrate for the first time a regulatory effect in the calcium-sensing receptor by one of its ligands.

Effect of aluminium on calcium-sensing receptor expression, proliferation, and apoptosis of parathyroid glands from rats with chronic renal failure. To assess the effect of aluminium on the calcium-sensing receptor expression, proliferation, and apoptosis in parathyroid glands from rats with chronic renal failure, 21/2-month-old male Wistar rats were 7/8 nephrectomized. Eight weeks after surgery the rats were divided into two groups, one receiving intraperitoneal AlCl3 for 8 weeks and the other receiving intraperitoneal placebo. Serum Al, Ca, P, creatinine, and PTH were measured. Parathyroid glands were removed, formaldehyde-fixed, and paraffin-embedded. Calcium-sensing receptor and proliferation were detected by immunohistochemistry and apoptosis by TUNEL and propidium iodide uptake. At the end of the study, despite higher levels of serum P in the aluminium group (6.27 ± 0.63 vs. 5.56 ± 0.58 mg/dL; P = 0.045), serum PTH was lower (89.6 ± 57.7 vs. 183.1 ± 123.8 pg/mL; P = 0.059). No significant differences were found in the calcium-sensing receptor expression between groups (aluminium: 27.1 ± 7.6; placebo: 25.4 ± 3.5 RU). Rats receiving aluminium showed a significantly lower cell proliferation rate than the control rats (0.54 ± 0.69 vs. 4.43 ± 3.10 cells/mm2; P = 0.003). No apoptotic events were detected. Aluminium was able to reduce the cell proliferation of the parathyroid glands. Due to the low apoptosis rate, however, it was not possible to find any change. Aluminium had no effect on the calcium-sensing receptor expression.

Background/Aims: This study aimed to test the viability and functionality of fresh and cryopreserved human hyperplastic parathyroid glands cultured in vitro. Methods: Small fragments of 18 parathyroid glands from 18 patients with secondary hyperparathyroidism were cultured in vitro, freshly or after cryopreservation, during 60 h. Cell viability and functionality of the parathyroid fragments exposed to calcium and calcitriol were studied. Results: Human parathyroid glands obtained from renal patients with secondary hyperparathyroidism maintained their viability and functionality for 60 h in culture. Sixty percent of the fresh but only 10% of the cryopreserved parathyroid glands showed the expected response with higher intact parathyroid hormone secretion when cultured with 0.6 mM calcium compared to 1.2 mM calcium. On the contrary, 44 of fresh and 40% of cryopreserved glands behaved in the same manner, showing a similar decrease in intact parathyroid hormone synthesis and secretion when cultured with calcitriol (10 –8 M). Conclusion: These results demonstrate differences in the response to calcium between fresh and cryopreserved glands and no differences in the response to calcitriol. This in vitroculture method may be useful to discriminate between responsive and nonresponsive hyperplastic human parathyroid glands.

Telomerase-negative cells maintain their telomeres through an alternative pathway that involves DNA recombination after replication. In this pathway, the recombination endonuclease MUS81 is found to regulate telomeric recombination and maintains the length of telomeres by interacting with the telomere binding protein TRF2. Telomerase-negative cancer cells maintain their telomeres through the alternative lengthening of telomeres (ALT) pathway 1 , 2 , 3 . Although a growing body of evidence demonstrates that the ALT mechanism is a post-replicative telomere recombination process, molecular details of this pathway are largely unknown. Here we demonstrate that MUS81, a DNA structure specific recombination endonuclease, has a key role in the maintenance of telomeres in human ALT cells. We find that MUS81 specifically localizes to ALT-associated promyelocytic leukaemia (PML) nuclear bodies (APBs) and associates with telomeric DNA in ALT cells, which is enriched during the G2 phase of the cell cycle. Depletion of MUS81 results in the reduction of ALT-specific telomere recombination and leads to proliferation arrest of ALT cells. In addition, the endonuclease activity of MUS81 is required for recombination-based ALT cell survival, and the interaction of MUS81 with the telomeric repeat-binding factor TRF2 regulates this enzymatic activity, thereby maintaining telomere recombination. Thus, our results suggest that MUS81 is involved in the maintenance of ALT cell survival at least in part by homologous recombination of telomeres.

mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

The A/J mouse is highly susceptible to lung tumor induction and has been widely used as a screening model in carcinogenicity testing and chemoprevention studies. However, the A/J mouse model has several disadvantages. Most notably, it develops lung tumors spontaneously. Moreover, there is a considerable gap in our understanding of the underlying mechanisms of pulmonary chemical carcinogenesis in the A/J mouse. Therefore, we examined the differences between spontaneous and cigarette smokerelated lung tumors in the A/J mouse model using mRNA and microRNA (miRNA) profiling. Male A/J mice were exposed whole-body to mainstream cigarette smoke (MS) for 18 months. Gene expression interaction term analysis of lung tumors and surrounding nontumorous parenchyma samples from animals that were exposed to either 300 mg/m3 MS or sham-exposed to fresh air indicated significant differential expression of 296 genes. Ingenuity Pathway Analysis® (IPA®) indicated an overall suppression of the humoral immune response, which was accompanied by a disruption of sphingolipid and glycosaminoglycan metabolism and a deregulation of potentially oncogenic miRNA in tumors of MS-exposed A/J mice. Thus, we propose that MS exposure leads to severe perturbations in pathways essential for tumor recognition by the immune system, thereby potentiating the ability of tumor cells to escape from immune surveillance. Further, exposure to MS appeared to affect expression of miRNA, which have previously been implicated in carcinogenesis and are thought to contribute to tumor progression. Finally, we identified a 50-gene expression signature and show its utility in distinguishing between cigarette smoke-related and spontaneous lung tumors