MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

Representative images of “Comets” and the corresponding intensity profiles, showing (i) ~ 5% Tail DNA damage, typical of the NSCLC cells treated with no siRNA or scramble siRNA, and analysed by regular Fpg-modified alkaline comet assay (0.8 U Fpg/gel); and (ii) comets showing ~ 10% tail DNA, typical of the NSCLC cells treated with MTH1 siRNA. Superimposed on the Comet images are the image analysis software (Komet 5.5, Andor Technology) determined boundaries demarcating the ‘Comet head’ (pink circle) and ‘tail extent’ (vertical orange line) (Barber RC, Hickenbotham P, Hatch T, Kelly D, Topchiy N, Almeida GM, et al. Radiation-induced transgenerational alterations in genome stability and DNA damage. Oncogene. 2006;25(56):7336–7342). % tail DNA = 100 - % head DNA; % head DNA = (integrated optical head intensity / (integrated optical head intensity + integrated optical tail intensity)) × 100. (PDF 1431 kb

Studying the effects of DNA replication stress on genome integrity and cell viability

Accurate replication of the genome during cell division is essential for maintaining genome integrity and suppressing diseases such as cancer. DNA replication forks can stall, collapse and/or break when they encounter obstacles present in DNA. This causes DNA replication stress, which is a major source of genome instability and mutations during carcinogenesis and cancer evolution. A number of studies have investigated how cells respond to replication stress by using various genotoxic agents such as radiation or therapeutic agents. However, these agents cause genome-wide damage; thereby, preventing control over where replication forks collapse or break, and preventing replication stress-associated molecular events from being studied directly. Here, we aimed to overcome these current methodological limitations by developing an innovative FLP-FRT system. The FLP-FRT system is designed to induce the generation of broken DNA replication forks at specific loci in mammalian cells. The system utilises a mutant FLP recombinase that binds to an integrated FLP recognition target site (FRT) marked with a florescent locus labelling system and generates an irreversible protein adduct and DNA single-strand break. Upon DNA replication, the fork encounters the gap leading to fork breakage and the formation of a single-ended DNA double-strand break. Using the ANCHOR labelling tool, the ANCH3-ANCH4 sites flanking the FRT sequence were simultaneously visualized. Our findings suggest that cells with a broken fork display a notable reduction in cell viability when compared to a direct DSB, suggesting they are more cytotoxic, as hypothesized. In conclusion, our study shows that the features of FRT-FLP system offers a widely applicable and powerful tool to directly study replication stress at a very high resolution at the damage site within single cells in vivo. This novel tool permits genetic, biological and microscopic analyses of broken replication fork resolution in mammalian cells, which can shed light on important aspects of cancer cell biology and the development of new therapeutic clinical strategies

Pancytopenia, Recurrent Infection, Poor Wound Healing, Heterotopia of the Brain Probably Associated with A Candidate Novel de Novo CDC42 Gene Defect: Expanding the Molecular and Phenotypic Spectrum

CDC42 (cell division cycle protein 42) belongs to the Rho GTPase family that is known to control the signaling axis that regulates several cellular functions, including cell cycle progression, migration, and proliferation. However, the functional characterization of the CDC42 gene in mammalian physiology remains largely unclear. Here, we report the genetic and functional characterization of a non-consanguineous Saudi family with a single affected individual. Clinical examinations revealed poor wound healing, heterotopia of the brain, pancytopenia, and recurrent infections. Whole exome sequencing revealed a de novo missense variant (c.101C > A, p.Pro34Gln) in the CDC42 gene. The functional assays revealed a substantial reduction in the growth and motility of the patient cells as compared to the normal cells control. Homology three-dimensional (3-D) modeling of CDC42 revealed that the Pro34 is important for the proper protein secondary structure. In conclusion, we report a candidate disease-causing variant, which requires further confirmation for the etiology of CDC42 pathogenesis. This represents the first case from the Saudi population. The current study adds to the spectrum of mutations in the CDC42 gene that might help in genetic counseling and contributes to the CDC42-related genetic and functional characterization. However, further studies into the molecular mechanisms that are involved are needed in order to determine the role of the CDC42 gene associated with aberrant cell migration and immune response