Investigation of DNA Damage Induced by Alkylating Agents and Repair Pathways by Cooperating Mechanisms Driving the Formation of Colorectal Adenomas and Adenocarcinomas Using DNA Alkylation and DNA Methylation

In this recent study, DNA data from 900 patients with colorectal cancer were reviewed. Analysis of the data showed a distinct mutation signature, a pattern that had never been identified before but indicated a type of DNA damage called "alkylation." Red meat contains chemicals that can cause alkylation. High levels of tumor alkylation damage are seen only in patients who consume an average of more than 150 grams of meat per day, roughly equivalent to two or more meals. On the other hand, a group of researchers in 2019 in a controversial conclusion stated that they do not have much confidence in reducing deaths from colon cancer by avoiding red meat. Keywords: Cancer; Cells; Tissues, Tumors; Prevention, Prognosis; Diagnosis; Imaging; Screening; Treatment; Management

Pyrvinium pamoate regulates MGMT expression through suppressing the Wnt/β-catenin signaling pathway to enhance the glioblastoma sensitivity to temozolomide

Temozolomide (TMZ) is the mainstream chemotherapeutic drug for treating glioblastoma multiforme (GBM), but the intrinsic or acquired chemoresistance to TMZ has become the leading clinical concern, which is related to the repair of DNA alkylation sites by O6-methylguanine-DNA methyltransferase (MGMT). Pyrvinium pamoate (PP), the FDA-approved anthelminthic drug, has been reported to inhibit the Wnt/β-catenin pathway within numerous cancer types, and Wnt/β-catenin signaling pathway can modulate the expression of MGMT gene. However, whether PP affects the expression of MGMT and enhances TMZ sensitivity in GBM cells remains unclear. In the present study, we found that PP and TMZ had synergistic effect on inhibiting the viability of GBM cells, and PP induced inhibition of MGMT and enhanced the TMZ chemosensitivity of GBM cells through down-regulating Wnt/β-catenin pathway. Moreover, the overexpression of MGMT or β-catenin weakened the synergy between PP and TMZ. The mechanism of PP in inhibiting the Wnt pathway was indicated that PP resulted in the degradation of β-catenin via the AKT/GSK3β/β-catenin signaling axis. Moreover, Ser552 phosphorylation in β-catenin, which promotes its nuclear accumulation and transcriptional activity, is blocked by PP that also inhibits the Wnt pathway to some extent. The intracranial GBM mouse model also demonstrated that the synergy between PP and TMZ could be achieved through down-regulating β-catenin and MGMT, which prolonged the survival time of tumor-bearing mice. Taken together, our data suggest that PP may serve as the prospect medicine to improve the chemotherapeutic effect on GBM, especially for chemoresistant to TMZ induced by MGMT overexpression.

Versatile cell-based assay for measuring DNA alkylation damage and its repair

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Cellular heterogeneity in DNA alkylation repair as a trade-off between cell survival and genetic plasticity

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the Escherichia coli adaptive response that increases resistance to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are absent. Although lack of these proteins causes extreme sensitivity to alkylation stress, cellular heterogeneity in DNA alkylation repair provides a functional benefit by increasing the evolvability of the whole population. We demonstrated this by monitoring the dynamics of nascent mutations during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. This highlighted that evolvability is a trade-off between mutability and cell survival. Stochastic modulation of DNA repair capacity by the adaptive response solves this trade-off through the generation of a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.

Versatile Cell-Based Assay for Measuring Base Excision Repair of DNA Alkylation Damage

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.