Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics.

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.

Fibroblast-derived PI16 sustains inflammatory pain via regulation of CD206+ myeloid cells.

Originally identified in fibroblasts, Protease Inhibitor (PI)16 was recently shown to be crucial for the development of neuropathic pain via effects on blood-nerve barrier permeability and leukocyte infiltration, though its impact on inflammatory pain has not been established. Using the complete Freund's Adjuvant inflammatory pain model, we show that Pi16-/- mice are protected against sustained inflammatory pain. Accordingly, intrathecal delivery of a PI16 neutralizing antibody in wild-type mice prevented sustained CFA pain. In contrast to neuropathic pain models, we did not observe any changes in blood-nerve barrier permeability due to PI16 deletion. Instead, Pi16-/- mice display reduced macrophage density in the CFA-injected hindpaw. Furthermore, there was a significant bias toward CD206hi (anti-inflammatory) macrophages in the hindpaw and associated dorsal root ganglia. Following CFA, intrathecal depletion of CD206+ macrophages using mannosylated clodronate liposomes promoted sustained pain in Pi16-/- mice. Similarly, an IL-10 neutralizing antibody also promoted sustained CFA pain in the Pi16-/ when administered intrathecally. Collectively, our results point to fibroblast-derived PI16 mediating substantial differences in macrophage phenotype in the pain neuroaxis under conditions of inflammation. The co-expression of PI16 alongside fibroblast markers in human DRG raise the likelihood that a similar mechanism operates in human inflammatory pain states. Collectively, our findings may have implications for targeting fibroblast-immune cell crosstalk for the treatment of chronic pain.

Exposure to endosulfan can cause long term effects on general biology, including the reproductive system of mice.

Increased infertility in humans is attributed to the increased use of environmental chemicals in the last several decades. Various studies have identified pesticides as one of the causes of reproductive toxicity. In a previous study, infertility was observed in male mice due to testicular atrophy and decreased sperm count when a sublethal dose of endosulfan (3 mg/kg) with a serum concentration of 23 µg/L was used. However, the serum concentration of endosulfan was much higher (up to 500 µg/L) in people living in endosulfan-exposed areas compared to the one used in the investigation. To mimic the situation in an experimental setup, mice were exposed to 5 mg/kg body weight of endosulfan, and reproductive toxicity and long-term impact on the general biology of animals were examined. HPLC analysis revealed a serum concentration of ∼50 µg/L of endosulfan after 24 h endosulfan exposure affected the normal physiology of mice. Histopathological studies suggest a persistent, severe effect on reproductive organs where vacuole degeneration of basal germinal epithelial cells and degradation of the interstitial matrix were observed in testes. Ovaries showed a reduction in the number of mature Graafian follicles. At the same time, mild vacuolation in liver hepatocytes and changes in the architecture of the lungs were observed. Endosulfan exposure induced DNA damage and mutations in germ cells at the molecular level. Interestingly, even after 8 months of endosulfan exposure, we observed increased DNA breaks in reproductive tissues. An increased DNA Ligase III expression was also observed, consistent with reported elevated levels of MMEJ-mediated repair. Further, we observed the generation of tumors in a few of the treated mice with time. Thus, the study not only explores the changes in the general biology of the mice upon exposure to endosulfan but also describes the molecular mechanism of its long-term effects.

Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia.

Ribonuclease H2 (RNase H2) is a member of the ribonuclease H family of enzymes involved in removal of RNA from RNA-DNA hybrids as well as ribonucleotides which get misincorporated into the genomic DNA. Recent studies have shown that RNase H2 function is also needed for successful DNA repair through NHEJ events where DNA pol µ uses ribonucleotides during the gap filling stage. Mammalian RNase H2 is composed of three subunits, RNASEH2A, RNASEH2B and RNASEH2C. There have been studies suggesting changes in expression of these genes in various cancers of breast, prostate, colon, liver, and kidney. In this study, we have investigated the functional role of RNASEH2A and RNASEH2B in leukemic T-cells, MOLT4 and Jurkat. shRNA mediated knockdown of RNASEH2A/ RNASEH2B expression led to reduced cell survival and increase in apoptotic cell population. Importantly, knockdown of RNASEH2A or RNASEH2B, led to cell cycle arrest at S phase and increased number of 53BP1 foci due to abrogation of NHEJ. Interestingly, RNASEH2A or RNASEH2B depleted cells showed significantly retarded DSB repair kinetics compared to scrambled shRNA control, when exposed to ionizing radiation suggesting that NHEJ is abrogated due to loss of RNASEH2 activity in T-ALL cells. Thus, we uncover the importance of RNase H2 function in leukemic cells and suggest that it can be targeted for cancer therapy.

G4 DNA present at human telomeric DNA contributes toward reduced sensitivity to γ-radiation induced oxidative damage, but not bulky adduct formation.

PURPOSE: DNA, the hereditary material of a human cell generally exists as Watson-Crick base paired double-stranded B-DNA. Studies suggest that DNA can also exist in non-B forms, such as four stranded G-quadruplexes (G4 DNA). Recently, our studies revealed that the regions of DNA that can fold into G-quadruplex structures are less sensitive to ionizing radiation (IR) compared to B-DNA. Importantly, we reported that the planar G-quartet of a G4 structure is shielded from radiation induced DNA breaks, while the single- and double-stranded DNA regions remained susceptible. Thus, in the present study, we investigate whether telomeric repeat DNA present at the end of telomere, known to fold into G4 DNA can protect from radiation induced damages including strand breaks, oxidation of purines and bulky adduct formation on DNA. MATERIALS AND METHODS: For plasmid irradiation assay, plasmids containing human telomeric repeat DNA sequence TTAGGG (0.8 kb or 1.8 kb) were irradiated with increasing doses of IR along with appropriate control plasmids and products were resolved on 1% agarose gel. Radioprotection was evaluated based on extent of conversion of supercoiled to nicked or linear forms of the DNA following irradiation. Formation of G-quadruplex structure on supercoiled DNA was evaluated based on circular dichroism (CD) spectroscopy studies. Cleavage of radiation induced oxidative damage and extent of formation of nicks was further evaluated using base and nucleotide excision repair proteins. RESULTS: Results from CD studies showed that the plasmid DNA harboring human telomeric repeats (TTAGGG) can fold into G-quadruplex DNA structures. Further, results showed that human telomeric repeat sequence when present on a plasmid can protect the plasmid DNA against IR induced DNA strand breaks, unlike control plasmids bearing random DNA sequence. CONCLUSIONS: Human telomeric repeat sequence when present on plasmids can fold into G-quadruplex DNA structures, and can protect the DNA against IR induced DNA strand breaks and oxidative damage. These results in conjunction with our previous studies suggest that telomeric repeat sequence imparts less sensitivity to IR and thus telomeres of chromosomes are protected from radiation.

Acute toxicity analysis of Disarib, an inhibitor of BCL2.

Small molecule inhibitors targeting BCL2 are explored as anticancer therapeutics. Previously, we have reported identification and characterization of a novel BCL2 inhibitor, Disarib. Disarib induced cancer cell death in a BCL2 dependent manner in different cancer cell lines and mouse tumor models when it was administered intraperitoneally. In the present study, using two syngeneic mouse models, breast adenocarcinoma (EAC) and Dalton's lymphoma (DLA), we show that oral administration of Disarib resulted in significant tumor regression in a concentration dependent manner. Importantly, tumor developed in both female and male mice were equally sensitive to Disarib. Further, we have investigated the toxicity of Disarib in normal cells. Single dose toxicity analysis of Disarib in male and female mice after oral administration revealed no significant variations compared to control group for parameters such as body weight, food and water consumption and behavioural changes which were analysed for the entire period of study. Haematological and histopathological analyses also did not show any significant difference from the control groups. Thus, our results reveal safe use of Disarib as a small molecule inhibitor and provide the foundation for investigation of other preclinical studies.

G-quadruplex Structures Contribute to Differential Radiosensitivity of the Human Genome.

DNA, the fundamental unit of human cell, generally exists in Watson-Crick base-paired B-DNA form. Often, DNA folds into non-B forms, such as four-stranded G-quadruplexes. It is generally believed that ionizing radiation (IR) induces DNA strand-breaks in a random manner. Here, we show that regions of DNA enriched in G-quadruplex structures are less sensitive to IR compared with B-DNA in vitro and inside cells. Planar G-quartet of G4-DNA is shielded from IR-induced free radicals, unlike single- and double-stranded DNA. Whole-genome sequence analysis and real-time PCR reveal that genomic regions abundant in G4-DNA are protected from radiation-induced breaks and can be modulated by G4 stabilizers. Thus, our results reveal that formation of G4 structures contribute toward differential radiosensitivity of the human genome.

T Cells as an Emerging Target for Chronic Pain Therapy.

The immune system is critically involved in the development and maintenance of chronic pain. However, T cells, one of the main regulators of the immune response, have only recently become a focus of investigations on chronic pain pathophysiology. Emerging clinical data suggest that patients with chronic pain have a different phenotypic profile of circulating T cells compared to controls. At the preclinical level, findings on the function of T cells are mixed and differ between nerve injury, chemotherapy, and inflammatory models of persistent pain. Depending on the type of injury, the subset of T cells and the sex of the animal, T cells may contribute to the onset and/or the resolution of pain, underlining T cells as a major player in the transition from acute to chronic pain. Specific T cell subsets release mediators such as cytokines and endogenous opioid peptides that can promote, suppress, or even resolve pain. Inhibiting the pain-promoting functions of T cells and/or enhancing the beneficial effects of pro-resolution T cells may offer new disease-modifying strategies for the treatment of chronic pain, a critical need in view of the current opioid crisis.