The Endogenous Sources of DNA Damage - Lab Report Example

?Lab Report DNA in the living cell is to damage by both endogenous and exogenous agents. The endogenous sources of DNA damage include the Reactive Oxygen Species (ROS) formed during oxidative metabolism, aldehydes derived from lipid peroxidation, some oestrogen metabolites, and many metabolites that can act as methylating agents (De Bont and van Larebeke, 2004). Some of the exogenous agents capable of causing DNA damage are ultraviolet rays and ionising radiation, environmental toxins, e.g., polycyclic aromatic hydrocarbons (PAHs) and heavy metals, and chemicals used in chemotherapy of cancers. The types of DNA damage caused include covalent modification of the four bases (A, T, G and C) in DNA, single- and double-stranded breaks in the DNA backbone, mismatch of base pairing occurring during DNA replication, and intrastrand and interstrand crosslink formation between DNA bases. DNA double-strand breaks (DSBs) represent the most dangerous type of DNA damage since a single DSB is capable of causing cell death or disturbing the genomic integrity of the cell (Jackson and Bartek, 2009). DSBs are difficult to repair and extremely harmful (Khanna and Jackson, 2001). DSBs generally form when two single-strand breaks (SSBs) occur in close proximity, or when a SSB or certain other lesions are encountered during DNA-replication (Jackson and Bartek, 2009). Ionising radiation (IR) and chemotherapeutic compounds used in cancer also generate DSBs. Mediated by DSBs, DNA lesions develop as a result of recombination between different loci (Kongruttanachok et al., 2010). Thus, a cell constantly faces the risk of DNA damage caused exogenously as well as from an incorrect incorporation of base pairs occurring during normal replication. The chemical changes occurring in the DNA structure, therefore, must be corrected in order to preserve the encoded genetic information. Mechanisms of DNA damage repair exist which involve systems that detect DNA damage, signal its location and bring about the repair. The DNA-damage responses (DDR) are physiologically very important as each of the nearly 1013 cells in the human body experiences innumerable DNA lesions per day (Lindahl and Barnes, 2000). Nuclear Excision Repair (NER) is a repair mechanism in the cell to deal with DNA damage caused exogenously by UV rays and ionizing radiation, besides other types of chemical alteration to the DNA (Wood, 1997). Mismatch repair (MMR) is an evolutionarily conserved DNA damage fixing process that is employed by the cell when base pairs are added incorrectly during replication (Kunkel and Erie, 2005). At least ten different DNA repair processes are operative in higher organisms (Reed, 2010). Platinum-based compounds e.g., cisplatin, oxaliplatin, satraplatin etc. interfere with the repair mechanisms to effect programmed cell death, or apoptosis. For example, cisplatin when introduced into the nucleus, forms adducts (Fichtinger-Sherpman, et al., 1985). The formation of the bulky adducts causes torsional strain on the DNA strand prompting the cell to invoke the NER and/or the Mismatch repair enzymes. However, the repair mechanisms are generally unable to effectively correct the damage as the platinum adduct is a non-native structure, and this leads to permanent DNA damage causing apoptosis of the potentially defective cell (Agarwal et al., 1998). The inactivation of repair processes could have wide-ranging consequences, both beneficial and detrimental. In the context of the former, DNA damage and repair has been the major target of anticancer therapy. Serious attempts are being made to modulate these processes in order to improve their efficacy in the treatment of cancer (Reed, 2010). While DSBs are processed by a number of DNA repair pathways depending partly on the phases of the cell cycle, NER is the only mechanism known to remove bulky DNA adducts, including those formed by platinum-based anticancer drugs, from human cells (Readon et al., 1999). A majority of DSBs are repaired by non-homologous end-joining repair (NHEJ) mediated by either the Ku-requiring pathway or the more precise ataxia telangiectasia mutated (ATM) -mediated pathway (Kongruttanachok et al., 2010). One of the earliest DSB repair responses is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX (Rogakou et al.,1998). The phosphorylated form of H2AX, referred to as gammaH2AX (?H2AX), is produced by a reaction catalysed by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ATM, DNA-protein kinase catalytic subunit, and ATM and RAD3-related (ATR). The protein kinases, ATM and ATR are key DDR-signaling constituents of mammalian cells (Shiloh, 2003). The formation of the protein ?H2AX within 1-3 minutes of DNA damage is the first step in recruiting and localizing DNA repair proteins. ?H2AX forms discrete foci consisting of an accumulation of repair proteins in large domains of chromatin around the site of the DSB which are easily visualized by immunofluorescence (Ismail and Hendzel, 2008; Vasireddy et al., 2010), providing a measure of the number of DSBs within a cell (Ismail et al., 2007). The extensive phosphorylation of H2AX following the occurrence of DSBs supposedly “functions as a molecular scaffold that binds DSB signaling and repair proteins” (Ismail and Hendzel, 2008). The H2AX-interacting proteins include NBS1, 53BP1 and MDC1 (Stucki et al., 2005). The observation of intense staining of ?H2AX in S phase cells and faint staining in G1 cells in some studies (Marti et al., 2006) suggest that ?H2AX formation possibly occurs to safeguard cell cycle checkpoints that protect the cell from genomic instability (Ismail and Hendzel, 2008). The evaluation of ?H2AX foci is widely used to investigate DSB formation and repair, especially to assess the efficacy of various radiation modifying compounds and cytotoxic compounds used in cancer treatment (Kuo and Yang, 2008). The high specificity and sensitivity of ?H2AX make it a novel biomarker for DNA double-strand breaks. The general practice has been to use alkaline comet assay to monitor patients undergoing chemotherapy. The gamma-H2AX assay has been reported to detect DNA damage as low as 100-fold below the detection limit of the alkaline comet assay (Ismail et al., 2007). Furthermore, Ismail et al. also observed induction of gamma-H2AX in response to DSBs in all nucleated blood cell types, including the short-lived neutrophils. The main aim of the present study was to investigate the induction of DNA double strand breaks in lymphocytes in response to oxaliplatin and satraplatin treatments, using gamma-H2AX phosphorylation, assayed by an immunofluorescence technique, as a biomarker of DNA double strand breaks. Results Lymphocytes from colon cancer patients treated with 0.02?M, 0.2 ?M and 2.0 ?M oxaliplatin were analysed by immunofluorescence for the presence of ?H2AX foci. Lymphocytes from healthy donors and untreated lymphocytes obtained from colon cancer patients formed the two controls for the assay while ethyl methanesulfonate (EMS) treated cells formed the positive control. As seen from the results of the assay shown in Fig. 1, highly significant induction of ?H2AX foci occurred at all the concentrations of oxaliplatin tested, as also with 1.5mM EMS compared to the relevant control (P Read More