DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.

Insulin-like factor 5 (INSL5), a member of the insulin superfamily, is expressed in the colorectum and hypothalamus. To facilitate studies into the role of INSL5, we generated Insl5(-/-) mice by gene targeting. Insl5(-/-) mice were born in the expected Mendelian ratio, reached normal body weight, but displayed impaired male and female fertility that are due to marked reduction in sperm motility and irregular length of the estrous cycle. Furthermore, Insl5(-/-) mice showed impairment in glucose homeostasis with characteristic elevation of serum glucose levels at an advanced age. Glucose and insulin tolerance tests revealed that the increased blood glucose in Insl5(-/-) mice was due to glucose intolerance resulting from reduced insulin secretion. Morphometric and immunohistological analyses revealed that the Insl5(-/-) mice had markedly reduced average islets area and β-cell numbers. Furthermore, immunohistochemistry showed the expression of INSL5 in enteroendocrine cells in the colorectal epithelium and the presence of its putative receptor relaxin family peptide receptor 4 in pancreatic islet cells. These results suggest the potential role of INSL5 signaling in the regulation of insulin secretion and β-cell homeostasis.

We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.

This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Poly(ADP‐ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage‐induced PARP1 activity. We used human triple‐negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage‐induced PARP1 activity, which was dependent on functional DNA‐binding AT‐hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

In hypoxia, the survival property of cancer cells mediated by the glycolytic enzyme PDK2 is obligatorily linked to alternative splicing and generation of a novel isoform of death gene Bnip3, which suppresses mitochondrial injury and promotes survival.

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