Browsing by Author "Salciute, Martyna"

Lynch syndrome is the most common hereditary colorectal cancer (CRC) syndrome caused by inherited mutations in DNA mismatch repair genes. Of those, MLH1 is the most mutated predisposition gene and is best known for its involvement in the DNA mismatch repair (MMR) pathway. In addition to the MMR, MLH1 has proved to have a multifunctional role in assisting in the maintenance of genomic stability. Emerging evidence suggests, that reduced levels of MLH1 directly contribute to an increased number of DNA double-strand breaks (DSBs), leading to chromosomal instability (CIN) through impaired mitochondrial function and homologous recombination directed DSB repair. This study aimed to test this hypothesis by evaluating the DNA damage status and mitochondrial functionality in MLH1 knock-down (KD) fibroblast cell lines with varying expression levels of MLH1. DNA damage levels and repair kinetics were inspected by implementing the Comet assay. Moreover, mitochondrial homeostasis examination was done by utilizing functional mitochondrial staining and analysing mitochondrial DNA copy number. Although there was variability in the results, two KD cell lines exhibiting 30% (line 3A3) and 40% (line 2B7) MLH1 expression levels showed similar outcomes: decreased mitochondrial membrane potential, increased cellular reactive oxygen species (ROS) and stalled DNA damage repair as compared to control cell lines, suggesting the involvement of MLH1 deficiency. It is known, that MLH1 depletion predisposes to DNA damage due to impaired MMR. The findings of this thesis contribute to the growing body of evidence, suggesting that MLH1 deficiency may increase the propensity for DNA DSBs, possibly due to impaired mitochondrial function and subsequent elevation in cellular ROS. Furthermore, this increase in DNA breaks may result in CIN. However, given the limited sample size, the results warrant future studies with larger datasets.