Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Polymerase iota plays a key role during translesion synthesis of UV-induced lesions in the absence of polymerase eta.

Xeroderma pigmentosum (XP) variant cells are deficient in the translesion synthesis (TLS) DNA polymerase Polη (eta). This protein contributes to DNA damage tolerance, bypassing unrepaired UV photoproducts and allowing S-phase progression with minimal delay. In the absence of Polη, backup polymerases perform TLS of UV lesions. However, which polymerase plays this role in human cells remains an open question. Here, we investigated the potential role of Polι (iota) in bypassing ultraviolet (UV) induced photoproducts in the absence of Polη, using NER-deficient (XP-C) cells knocked down for Polι and/or Polη genes. Our results indicate that cells lacking either Polι or Polη have increased sensitivity to UVC radiation. The lack of both TLS polymerases led to increased cell death and defects in proliferation and migration. Loss of both polymerases induces a significant replication fork arrest and G1/S-phase blockage, compared to the lack of Polη alone. In conclusion, we propose that Polι acts as a bona fide backup for Polη in the TLS of UV-photoproducts.

Effects of isolated and combined exposures of Boana curupi (Anura: Hylidae) tadpoles to environmental doses of trichlorfon and ultraviolet radiation.

The biodiversity collapse strongly affects the amphibian group and many factors have been pointed out as catalytic agents. It is estimated that several events in the amphibian population decline worldwide may have been caused by the interaction of multiple drivers. Thus, this study aimed to evaluate the stressful effects of the exposure to environmental doses of trichlorfon (TCF) pesticide (0.5 µg/L; and an additional 100-fold concentration of 50 µg/L) and ultraviolet radiation (UV) (184.0 kJ/m² of UVA and 3.4 kJ/m² of UVB, which correspond to 5% of the daily dose) in tadpoles of the Boana curupi species (Anura: Hylidae). The isolated and combined exposures to TCF happened within 24 h of acute treatments under laboratory-controlled conditions. In the combined treatments, we adopted three different moments (M) of tadpole irradiation from the beginning of the exposures to TCF (0 h - M1; 12 h - M2; and 24 h - M3). Then, we evaluated tadpole survival, change in morphological characters, induction of apoptotic cells, lipid peroxidation (LPO), protein carbonyl content (PCC), glutathione S-transferase (GST), non-protein thiols (NPSH), and acetylcholinesterase (AChE), as well as the induction of genomic DNA (gDNA) damage. UVB treatment alone resulted in high mortality, along with a high level of apoptosis induction. Both UVA, UVB, and TCF increased LPO, PC, and AChE, while decreased GST activity. Regarding co-exposures, the most striking effect was observed in the interaction between UVB and TCF, which surprisingly decreased UVB-induced tadpole mortality, apoptosis, and gDNA damage. These results reinforce the B. curupi sensitivity to solar UVB radiation and indicate a complex response in face of UVB interaction with TCF, which may be related to activation of DNA repair pathways and/or inhibition of apoptosis, decreasing UVB-induced tadpole mortality.

The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy.

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Transcription blockage by DNA damage in nucleotide excision repair-related neurological dysfunctions.

Human genetic syndromes deficient in nucleotide excision repair (NER), such as xeroderma pigmentosum and Cockayne syndrome, may present neurological abnormalities and premature aging symptoms. Unrepaired endogenously generated DNA damage that hampers transcription is a strong candidate that contributes to the development of these severe effects in neuronal tissue. Endogenous lesions include those generated due to byproducts of cellular metabolisms, such as reactive oxygen species. This review presents much of the evidence on the mechanisms related to neurodegenerative processes associated with DNA damage responses. The primary focus is on the effects of the transcription machinery, including the accumulation of DNA•RNA hybrids (R-loops) that, in turn, influence DNA damage and repair metabolism. Moreover, several neuronal tissues present higher expression of long genes, a genomic subset more affected by DNA lesions, which may explain part of the neurological abnormalities in these patients. Also, neuronal tissues have different DNA repair capabilities that might result in different neurological consequences, as observed in patients and NER deficient animal models. The better understanding of how the accumulation of transcription blocking lesions can lead to neurological abnormalities and premature aging-like phenotypes may assist us in finding potential biomarkers and therapeutic targets that might improve the lives of these patients, as well as other neurological disorders in the general population.