Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis最新文献

DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs. To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.

Taxol is an antitumor drug derived from the bark of the Pacific Yew tree that inhibits microtubule disassembly, resulting in cell cycle arrest in late G2 and M phases. Additionally, Taxol increases cellular oxidative stress by generating reactive oxygen species. We hypothesized that the inhibition of specific DNA repair machinery/mechanisms would increase cellular sensitivity to the oxidative stress capacity of Taxol. Initial screening using Chinese hamster ovary (CHO) cell lines demonstrated that base excision repair deficiency, especially PARP deficiency, caused cellular Taxol hypersensitivity. Taxane diterpenes-containing Taxus yunnanensis extract also showed hypertoxicity in PARP deficient cells, which was consistent with other microtubule inhibitors like colcemid, vinblastine, and vincristine. Acute exposure of 50 nM Taxol treatment induced both significant cytotoxicity and M-phase arrest in PARP deficient cells, but caused neither significant cytotoxicity nor late G2-M cell cycle arrest in wild type cells. Acute exposure of 50 nM Taxol treatment induced oxidative stress and DNA damage. The antioxidant Ascorbic acid 2 glucoside partially reduced the cytotoxicity of Taxol in PARP deficient cell lines. Finally, the PARP inhibitor Olaparib increased cytotoxicity of Taxol in wild type CHO cells and two human cancer cell lines. Our study clearly demonstrates that cytotoxicity of Taxol would be enhanced by inhibiting PARP function as an enzyme implicated in DNA repair for oxidative stress.

Searching for differential genes in lung adenocarcinoma (LUAD) is vital for research. Hyaluronan mediated motility receptor (HMMR) promotes malignant progression of cancer patients. However, the molecular regulators of HMMR-mediated LUAD onset are unknown. This work aimed to study the relevance of HMMR to proliferation, migration and invasion of LUAD cells. Let-7c-5p and HMMR levels in LUAD cells and HLF-a cells were assessed, and their correlation was also detected. Their interaction was determined by dual-luciferase experiments and qRT-PCR. Cell proliferation, migration and invasion potentials in vitro were validated through cell counting kit-8 (CCK-8), colony formation, scratch healing, and transwell assays. The expression of HMMR was examined by qRT-PCR and western blot and the expression of let-7c-5p was assayed by qRT-PCR. It was found that HMMR level was increased in LUAD and negatively correlated with let-7c-5p level. Let-7c-5p directly targeted HMMR to repress LUAD cell proliferation, migration and invasion. The above data illustrated that the let-7c-5p/HMMR axis may provide certain therapeutic value for LUAD patients.

Objective

Examine whether DNA repair capacity (DRC) levels are associated with body mass index (BMI) in adult women.

Design and participants

A nested study composed of 539 women without breast cancer (BC) from a case-control BC study in addition to 104 that were recruited later for a total of 643.

Measurements

DRC levels were measured in lymphocytes using a host-cell reactivation assay with a luciferase reporter gene damaged by UVC. This assay measures the efficiency of nucleotide excision repair (NER). Log-binomial regression model was used. The prevalence ratio (PR) was used to evaluate the magnitude of the association between the BMI and DRC levels. An assessment of interaction terms was performed with the likelihood ratio test. The confounding effect was assessed by comparing the point estimates of the crude and adjusted PR.

Results

The 75th percentiles of DRC levels of the women with a BMI between 18 and 25 and > 25 showed statistically significant differences. The prevalence of a DRC ≤ 5 % among women with BMI > 25 is 1.24 (95 % CI: 1.03, 1.48) times the prevalence of having a DRC ≤ 5 % among the women with BMI ≤ 25 after adjustments for different covariates. This excess was statistically significant (p < 0.05). Women with a family history of cancer had an estimated PR of 1.25 (95 % CI, 0.87–1.39; P ≥ 0.05); and women with no family history of cancer, the estimated PR was 1.6 (95 % CI, 1.14–2.22; p ≤ 0.05).

Conclusions

Women with BMI > 25 tend to have lower DRC levels. When having a family history of cancer, the PR of low DRC levels in overweight/obese individuals was not statistically significant. However, the PR of low levels of DRC in overweight/obese individuals with no family history of cancer was statistically significant.

Mutagenesis can be thought of as random, in the sense that the occurrence of each mutational event cannot be predicted with precision in space or time. However, when sufficiently large numbers of mutations are analyzed, recurrent patterns of base changes called mutational signatures can be identified. To date, some 60 single base substitution or SBS signatures have been derived from analysis of cancer genomics data. We recently reported that the ubiquitous signature SBS5 matches the pattern of single nucleotide polymorphisms (SNPs) in humans and has analogs in many species. Using a temperature-sensitive single-stranded DNA (ssDNA) mutation reporter system, we also showed that a similar mutational pattern in yeast is dependent on error-prone translesion DNA synthesis (TLS) and glycolytic sugar metabolism. Here, we further investigated mechanisms that are responsible for this form of mutagenesis in yeast. We first confirmed that excess sugar metabolism leads to increased mutation rate, which was detectable by fluctuation assay. Since glycolysis is known to produce excess protons, we then investigated the effects of experimental manipulations on pH and mutagenesis. We hypothesized that yeast metabolizing 8% glucose would produce more excess protons than cells metabolizing 2% glucose. Consistent with this, cells metabolizing 8% glucose had lower intracellular and extracellular pH values. Similarly, deletion of vma3 (encoding a vacuolar H⁺-ATPase subunit) increased mutagenesis. We also found that treating cells with edelfosine (which renders membranes more permeable, including to protons) or culturing in low pH media increased mutagenesis. Analysis of the mutational pattern attributable to 20 µM edelfosine treatment revealed similarity to the SBS5-like TLS- and glycolysis-dependant mutational patterns previously observed in ssDNA. Altogether, our results agree with multiple biochemical studies showing that protonation of nitrogenous bases can alter base pairing so as to stabilize some mispairs, and shed new light on a common form of intrinsic mutagenesis.

The fluctuation assay remains an important tool for analyzing the levels of mutagenesis in microbial populations. The mutant counts originating from some average number of mutations are usually assumed to obey the Luria–Delbrück distribution. While several tools for estimating mutation rates are available, they sometimes lack accuracy or versatility under non-standard conditions. In this work, extensions to the Luria–Delbrück protocol to account for phenotypic lag and cellular death with either perfect or partial plating were developed. Hence, the novel MLE MUtation Rate calculator, or mlemur, is the first tool that provides a user-friendly graphical interface allowing the researchers to model their data with consideration for partial plating, differential growth of mutants and non-mutants, phenotypic lag, cellular death, variability of the final number of cells, post-exponential-phase mutations, and the size of the inoculum. Additionally, mlemur allows the users to incorporate most of these special conditions at the same time to obtain highly accurate estimates of mutation rates and P values, confidence intervals for an arbitrary function of data (such as fold), and perform power analysis and sample size determination for the likelihood ratio test. The accuracy of point and interval estimates produced by mlemur against historical and simulated fluctuation experiments are assessed. Both mlemur and the analyses in this work might be of great help when evaluating fluctuation experiments and increase the awareness of the limitations of the widely-used Lea–Coulson formulation of the Luria–Delbrück distribution in the more realistic biological contexts.

PARP inhibitors inflict severe toxicity to homologous recombination (HR) repair deficient cells because DNA damages induced by PARP inhibition result in lethal DNA double strand breaks in the absence of HR repair during DNA replication. PARP inhibitors are the first clinically approved drugs designed for synthetic lethality. The synthetic lethal interaction of PARP inhibitors is not limited to HR repair deficient cells. We investigated radiosensitive mutants isolated from Chinese hamster lung origin V79 cells to identify novel synthetic lethal targets in the context of PARP inhibition. HR repair deficient BRCA2 mutant cells were used for positive control. Among tested cells, XRCC8 mutants presented hypersensitivity to PARP inhibitor, Olaparib. XRCC8 mutants showed elevated sensitivity to bleomycin and camptothecin similar to BRCA2 mutants. XRCC8 mutants presented an elevation of γ-H2AX foci formation frequency and S-phase dependent chromosome aberrations with Olaparib treatment. Enumerated damage foci following Olaparib treatment were observed to be elevated in XRCC8 as in BRCA2 mutants. Although this may suggest that XRCC8 plays a role in a similar DNA repair pathway as BRCA2 in HR repair, XRCC8 mutants presented functional HR repair including proper Rad51 foci formation and even elevated sister chromatid exchange frequencies with PARP inhibitor treatment. For comparison, RAD51 foci formation was suppressed in HR repair deficient BRCA2 mutants. Additionally, XRCC8 mutants did not display delayed mitotic entry with PARP inhibitors whereas BRCA2 mutants did. XRCC8 mutant cell line has previously been reported as possessing a mutation in the ATM gene. XRCC8 mutants displayed maximum cytotoxicity to ATM inhibitor among tested mutants and wild type cells. Furthermore, the ATM inhibitor sensitized XRCC8 mutant to ionzing radiation, however, XRCC8 mutant V-G8 expressed reduced levels of ATM protein. The gene responsible for XRCC8 phenotype may not be ATM but highly associated with ATM functions. These results suggest that XRCC8 mutation is a target for PARP inhibitor-induced synthetic lethality in HR repair independent manner via the disruption of cell cycle regulation. Our findings expand the potential application of PARP inhibitors in tumors lacking DNA damage responding genes other than HR repair, and further investigation of XRCC8 may contribute to this research.

Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the TAZ gene in two affected patients with congenital cardiomyopathy. Furthermore, women in this family had no mutations in their blood cells, whereas they only had mutations in the oral epithelial cells. The objective of the project was to evaluate the effect of intertissue mosaicisms on the Barth syndrome phenotypes, searching for another disease-related loci on chromosome X and finally to assess the consequences of the mutation. We conducted the advanced genetic study including cytogenetic research (constitutional karyotyping in blood and fibroblasts), NGS sequencing (with custom chromosome X sequencing together with the evaluation of loss of heterozygosity (LOH) and aberrations (CNV) in the whole genome) in four different tissues and sequencing of tafazzin and deoxyribonuclease 1 like 1 transcripts. The presence of deletions within the 5′untranslated region of the TAZ gene and/or the noncoding regions of the DNASE1L1 gene were detected in several tissues. Whereas, there is no intertissue mosaicism regarding point mutation in TAZ gene in all investigated tissues in female carriers. Only the male patient presented biochemical markers and neurological symptoms of Barth syndrome. All the female carriers are healthy and have normal tafazzin and deoxyribonuclease 1 like 1 transcripts in 2 analyzed tissues. The conclusion of this study is that we cannot rule out or confirm mosaicism in the noncoding regions of TAZ or DNASE1L1 genes, but this is not clinically relevant in female carriers because they are healthy. Finally, it has been proven that mutation (c.83T>A, p.Val28Glu) is responsible for disease in males in this family.

Results of previous studies suggested that renal fibrosis and epithelial-mesenchymal transition (EMT) plays an important role in the process of renal fibrosis, but the underlying mechanism remains unclear. Long coding RNA (lncRNA) CRNDE has emerged as potent regulators of EMT programs, therefore, in present work, we examined the roles of LncRNA CRNDE/miR-29a-3p axis in renal fibrosis and the underlying mechanism. We found that in both renal fibrosis animal and cell models, lncRNA CRNDE was dynamically upregulated in animal models or cells by the treatment of TGF-β. Furthermore, knockdown of CRNDE to rat significantly inhibited EMT, prevented renal fibrosis. Finally, CRNDE regulates renal fibrosis through suppression of miR-29a-3p expression. Together, our results demonstrated that CRNDE acted as a regulator of renal fibrosis via targeting miR-29a-3p. Our findings may provide a potential therapeutic target for the treatment of renal fibrosis.

Background

This investigation studied the impacts of the miR-30a-5p/CBX2 axis on esophageal cancer (EC).

Methods

Research objects were ascertained using The Cancer Genome Atlas database. Followed by qRT-PCR, western blot, dual-luciferase reporter, MTT, Transwell, and wound healing approaches, we tested gene expression and varying cell behaviors

Results

Conspicuously miR-30 family members (miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5p, miR-30e-5p) downregulation and CBX2 upregulation were discovered in EC cells. miR-30 family members target CBX2 and inhibited CBX2 expression. EC cell behaviors were inhibited by miR-30a-5p/CBX2 axis.

Conclusion

MiR-30a-5p draws a new inspiration for EC treatment.