Topoisomerases are essential enzymes discovered by Wang involved in maintaining the correct topology of DNA by breaking and rejoining double stranded DNA. These ‘strand breaks’ formed by Topoisomerase act as ‘transient gates’ for the passage of other DNA strands into this break. These enzymes help to ease knots and tangles and are essential in regulating over winding and underwinding of DNA. During this process covalent attachments are formed between active tyrosyl group in the Topoisomerase and terminal DNA phosphates which help in maintaining the integrity of the genome; a characteristic of all Topoisomerases.
Classification of Topoisomerases:
On the basis of mechanism and structure DNA Topoisomerases are classified as follows in eukaryotic cells:
A) Type I DNATopoisomerases:
These cause a nick in only one strand of DNA duplex through which the unbroken complementary DNA strand passes. This helps in DNA relaxation. This reaction is ATP (adenosine-tri-phosphate) independent.
B) Type II DNA Topoisomerases:
These cause a break in both DNA strands of DNA duplex forming a gate for the passage of another duplex; after the enzyme reseals the break. This reaction eases supercoiled DNA and can decatenate inter-linked DNA circles. ATP is involved in this reaction and the DNA double-strand break (DSB) caused covalently attaches itself to DNA helix via 5’ phosphate end.
Topoisomerase II is involved in DNA recombination, daughter chromosome separation, maintenance of chromosome structure, condensation/ decondensation. Topoisomerase II is an essential enzyme which alters the topology of DNA cleaves and transports one DNA duplex through another via transient double stranded breaks, introduces and removes DNA supercoils and is a target for many anti-cancer drugs. Anti-cancer drugs (Topoisomerase II poisons) interfere with the DNA cleavage cycle increasing the concentration of enzyme DNA cleavage complexes. The action of Topoisomerase II poisons on the enzyme leads to the formation of cleavage complexes consisting of the enzyme, drug and DNA which result in high levels of Topoisomerase II linked breaks in DNA of treated cells. Topoisomerase II is covalently bound to DNA strands at the DSB in drug stabilized cleavage complexes preventing the rejoining of DNA strands leading DNA damage or cell death by apoptosis. Non homologous end-joining (NHEJ) is a major pathway in the repair of damaged DNA which takes place only when the cleavage complex is removed from the DNA DSBs. The repair of these DSBs is necessary for the survival of cell in which DNA proteins play a role.
The mechanism of cleavage complex removal is still under research but DNA repair enzyme Meiotic recombination 11 (Mre11) has been proposed as one of the potential candidates which may influence the repair damaged DNA.
Meiotic Recombination 11 (Mre11):
MRN complex is made up of Mre11; Rad50 and Nbs1(in vertebrates).The reason for MRN complex to be involved in DNA repair is of its characteristics and functions such as telomere maintenance, 3’-5’ exonuclease activity, repair mechanism etc. NHEJ is the repair mechanism used for the repair Topoisomerase II mediated strand breaks for which MRN complex is essential.
Topoisomerase II has a catalytic cycle in which it causes transient DNA double-strand breaks (DSBs). Anti-cancer drugs such as Etoposide interfere with the DNA cleavage cycle and increase the concentration of cleavage complexes. Topoisomerase II with these drugs forms cleavage complexes which bind covalently to DNA to form Topoisomerase II-Drug-DNA cleavage complex which lead to permanent DSB’s causing apoptosis. The removal of cleavage complex is essential for DNA repair. DNA repair protein Mre11 is a potential candidate that might be involved in the repair of DNA.
Thus, the study involves if Mre11 influences the removal of Topoisomerase II from the site of DNA so that less DSBs can occur which means more repair of the cell, less cleavage complexes formation and eventually low cell death.
To study this human colon carcinoma cell lines can be used. For example HT29 and HCT 116 on which various cytotoxicity and immunoblotting assays performed. Cytotoxicity assays like MTT assay performed give the effect of anti-cancer drugs on the cell lines. Resistance of the cell lines towards the drug is observed through this assay.
Ø MTT Assay:
MTT assay is a cytotoxicity assay performed for cell viability where tetrazolium salt MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is cleaved into formazon; a coloured product by the mitochondrial enzyme succinate dehydrogenase. As this reaction can only be performed by live cells amount of formazon produced is a direct measure of cell viability.
Hsiao et al in 2008 who subjected HT29 cell line to MTT and observed that these cell lines were very resistant to Etoposide (anti-cancer drug). . This study supports the growth inhibition cytotoxicity assay performed during this project were also HT29 cells showed high levels of resistance towards Etoposide. In another experiment by Fan et al in 2008 HCT116 cell line showed high level of sensitivity towards Etoposide with an IC50 of ~1µM. This study supports the growth inhibition assay performed on HCT116 which also indicated the sensitivity of HCT116 towards Etoposide and maximum mortality was observed at a drug concentration of 1µM.
Cells have also been exposed to various other drugs and then subjected to MTT assay. Cen et al in 2009 subjected both HT29 and HCT116 to curcumin; a Topoisomerase II inhibitor in which both cell lines shown sensitivity towards curcumin. This experiment by Cen et al indicates that HCT116 cell line is not only sensitive towards Etoposide but also other drugs.
Many other scientists have performed MTT on various cell lines such as K562 and determined cell viability like Vistica et al, 1991. These cell lines showed sensitivity toward MTT assay indicating that different cell lines behave differently with exposure of drugs. MTT has also been used to determine the viability of tissues which were proved to be successful and an increase in cell viability was observed.
Ø Immunoblotting Assay:
To detect the influence of Mre11 in the repair of Topoisomerase II mediated DNA damage immunoblotting assay is performed in which anti gamma H2A X antibody is used as primary antibody and anti rabbit IgG used as secondary antibody. Anti gamma H2A X antibody causes phosphorylation effect by which DNA DSBs can be detected. Etoposide treated Whole Cell Extracts of both cell lines with their respective IC50’s can be used and immunoblotting assay performed and DSBs compared in each of the whole cell extract.
Similar experiments have been performed by Rao et al in 2007 on Etoposide treated cell lines (HT29 and HCT116) to detect DSBs by phosphorylation effect and successful DSBs were obtained. This study was supported by Fan et al in 2008 and Takemura et al in 2006 who experimented similarly on Etoposide treated cell lines for which they obtained successful results. Other cell lines such as K526 and human B cell lymphoblastoid TK6 cells have also been subjected to similar procedure for the detection of DSBs by using H2A X phosphorylation by Kantidze et al in 2006 and Tanaka et al in 2007 respectively. Experiments can also be performed on yeast like Saccharomyces cerevisiae. Detection of DSBs by phosphorylation effect using gamma H2A X is widely used and it is one of the reliable methods to detect DSBs.
4. OUTCOME and FUTURE WORK
From the above discussions it is evident that Mre11 is influenced in the repair of Topoisomerase II mediated damage but there are still some issues still to be experimented such as the reason for the difference in behaviour of the cell lines when treated with anti-cancer drug. This might be due to the different levels of Mre11 present in each cell line but it is yet to be proved and assays to be performed to prove the same. MTT assay results are in favour of Mre11 being involved in DNA repair but other experiments are needed to be performed to confirm the same. One such assay is XTT (2, 3-bis [2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay where live cells degrade tetrazolium salt XTT to formazon. The absorbance is taken at 450 nm and the amount of formazon produced is the direct measure of viable cells.
Effect of Mre11 can be observed by isolating the Mre11 and then inserting it into a plasmid. More repair mechanism observed in cell lines leads to the authentication that Mre11 is involved in the repair of damaged DNA.
With numerous questions unanswered there is ample scope for research and hope to prove that Mre11 influences repair of DNA damage which might be a triumph on cancer.