An RNAi screen in human cell lines reveals conserved DNA damage repair pathways that mitigate formaldehyde sensitivity

Abstract

Formaldehyde is a ubiquitous DNA damaging agent, with human exposures occurring from both exogenous and endogenous sources. Formaldehyde exposure can result in multiple types of DNA damage, including DNA-protein crosslinks and thus, is representative of other exposures that induce DNA-protein crosslinks such as cigarette smoke, automobile exhaust, wood smoke, metals, ionizing radiation, and certain chemotherapeutics. Our objective in this study was to identify the genes necessary to mitigate formaldehyde toxicity following chronic exposure in human cells. We used siRNAs that targeted 320 genes representing all major human DNA repair and damage response pathways, in order to assess cell proliferation following siRNA depletion and subsequent formaldehyde treatment. Three unrelated human cell lines frequently used in genotoxicity studies (SW480, U-2 OS and GM00639) were used to identify common pathways involved in mitigating formaldehyde sensitivity. Although there were gene-specific differences among the cell lines, four inter-related cellular pathways were determined to mitigate formaldehyde toxicity: homologous recombination, DNA double-strand break repair, ionizing radiation response and DNA replication. Additional insight into cell line-specific response patterns was obtained by using a combination of exome sequencing and Cancer Cell Line Encyclopedia genomic data. The results of this DNA damage repair pathway-focused siRNA screen for formaldehyde toxicity in human cells provide a foundation for detailed mechanistic analyses of pathway-specific involvement in the response to environmentally-induced DNA-protein crosslinks and, more broadly, genotoxicity studies using human and other mammalian cell lines.

Enhanced cytarabine-induced killing in OGG1-deficient acute myeloid leukemia cells

“…Thus, using the TCGA database of AML samples, we queried an additional 404 genes for correlation between expression levels and AML cytogenetic subtype. These genes included the 318 DNA damage response genes previously reported in our siRNA screen for formaldehyde response (27) and 87 additional genes related to DNA replication, cell cycle, apoptosis, and Ara-C import and metabolism (SI Appendix, Table S1). Strikingly, no other gene in our query demonstrated any significant differences in expression as a function of AML subtype, suggesting a potentially unique role for OGG1 in modulating responsiveness of AML cells to chemotherapy.…”

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells

“…One possible scenario is that formaldehyde-induced DNA lesions might cause the neurological symptoms. NER, Fanconi anemia (FA) pathway, homologous DNA recombination (HR), and the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR) prevent endogenous accumulation of formaldehyde-induced DNA damage (65)(66)(67). Unlike FA or HR, NER plays a crucial role in postmitotic cells including the cells in the central nervous system.…”

Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

“…Prior capillary transfer to the Hybond-Nϩ membrane (GVS), the DNA was denatured by incubating the gel in 0.25 N NaCl for 10 min and solution A (0.5 M NaOH, 1.5 M NaCl) for 1 h, with subsequent neutralization in solution B (1 M Tris [pH 7.4], 1.5 M NaCl) for 30 min at room temperature. Transfer, DNA crosslinking, and hybridization with the linearized HPV18 genome labeled with [␣- 32 P]dCTP were performed as previously described at hybridization temperature of 68°C.…”