Note: Mutant cells defective in DNA repair pathways provide a sensitive high-throughput assay for genotoxicity
Note: Mutant cells defective in DNA repair pathways provide a sensitive high-throughput assay for genotoxicity
DNA repair during replication (S-phase)
1. TLS
2. HR
3. FA
DNA repair throughout cell cycle;
1. base excision
2. nucleotide excision
3. NHEJ
In this paper, the panel that they used;
1. DNA damage checkpoint - ATM
2. HR - Rad54, XRCC3, and UBC13
3. NHEJ - Ku70
4. ICL - FANCC
5. TLS - Rev3
6. NER - XPA and XPG
7. BER - DNA pol-beta
My understanding in this paper - they relied on one particular lesion will be majorly used by one particular pw (how much fold should be different then bc DNA repair has the redundancy?).
Good points for DT40 again (in year 2010) -
1.High targeted integration
2.Stable karyotype/phenotype even for the long subculture
3.Short doubling time
Still, it is chicken - some genes could not be conducted though DNA repair pws have been said to be conserved throughout evolution.
1. Genetic manipulation in mammalian cells - difficult
2. Knockdown - never complete/total
There is the evidents showing that knockdown could reduce to 13% of WT level of mRNA in human - the author raised issue that DNA repair porteins are normally abundant - so it is a bit hard to see the phenotype/epistatic analysis from knockdown.
DT40 has
1. the long S-phase (70% in population) - therefore when applying the DNA damaging agents to the cells, it reflects the DNA synthesis stage.
2. lack of p53 -damage of DNA can entering the cell cycle without getting stuck in the checkpoint - therefore we can see the repair activity.
the author mentioned that false negative and positive numbers are quite high - requiring the new improved method.
Most commonly used assays for genotoxicity;
• the Ames test (a bacterial reverse mutation assay);
• the mouse lymphoma assay (a negative selection for loss of the functional thymidine kinase gene); and
• either the in vitro micronucleus assay (detection of additional small nuclei) or chromosomal aberration assay.
Ames test - very easy and quick but relied on bacterial reverse mutation (points mutations/frame shift mutation) -- evidences these days showed that the mutations are not solely related to carcinogenesis, therefore this model is yet not good enough.
They try to increase the realistic situation by using liver homogenate to biotransform the compound first and observe the phenotype from Ames test.
Micronucleus assay -- prone to false positive and negative.
For the micronuclei assay - using DT40 could improve the specificity (DNA damage agent or agent that inhibits microtubule polymerization).
an efficient G1/S checkpoint may significantly reduce the sensitivity of the micronucleus assay to detect environmental mutagens.
The most promising mutant cells may be cells deficient in translesion DNA synthesis (TLS), because the vast majority of DNA lesions inhibit DNA synthesis by replicative DNA polymerases, and the efficient release from this inhibition is dependent on TLS DNA polymerases.
Firstly, if a large number of chemicals could be screened for toxicity before initiating drug development, those lead molecules that are likely to fail toxicology screening could be eliminated at the outset.
Conventional cancer treatment, basically damaging the DNA content directly which also can harmful to the normal cells (though cancer has the ability to recover from damaging in lesser extent but it can be applied to only some cancer.
Therapeutic index;
Dose required for toxic effect/Dose required for therapeutic effect
If two repair pathways are functionally redundant, loss of one pathway in tumours renders them entirely dependent on the remaining repair pathway.
combination therapy of DNA damaging agent and repair inhibitor is already proving successful.
Since as many as 40 different proteins are involved in HR, many cancers exhibit HR defects. Strikingly, it has been shown that inhibition of PARP-1 in a BRCA2- null background is lethal, even without exogenous DNA damage.
Unfortunately, there are as yet no chemical inhibitors of these polymerases that are both potent and specific.
Whilst the DT40 assay is able to detect all types of genotoxicity, a single genotoxicity assay is unlikely to provide a complete genotoxic assessment. In particular, in vivo assays will remain important.
The ultimate goal of detecting toxicity associated with test chemical compounds is to develop a method for in silico prediction of toxicity from their chemical structures -- really depending on the good database which high-throughout can generate this kind of data.
DNA repair during replication (S-phase)
1. TLS
2. HR
3. FA
DNA repair throughout cell cycle;
1. base excision
2. nucleotide excision
3. NHEJ
In this paper, the panel that they used;
1. DNA damage checkpoint - ATM
2. HR - Rad54, XRCC3, and UBC13
3. NHEJ - Ku70
4. ICL - FANCC
5. TLS - Rev3
6. NER - XPA and XPG
7. BER - DNA pol-beta
My understanding in this paper - they relied on one particular lesion will be majorly used by one particular pw (how much fold should be different then bc DNA repair has the redundancy?).
Good points for DT40 again (in year 2010) -
1.High targeted integration
2.Stable karyotype/phenotype even for the long subculture
3.Short doubling time
Still, it is chicken - some genes could not be conducted though DNA repair pws have been said to be conserved throughout evolution.
1. Genetic manipulation in mammalian cells - difficult
2. Knockdown - never complete/total
There is the evidents showing that knockdown could reduce to 13% of WT level of mRNA in human - the author raised issue that DNA repair porteins are normally abundant - so it is a bit hard to see the phenotype/epistatic analysis from knockdown.
DT40 has
1. the long S-phase (70% in population) - therefore when applying the DNA damaging agents to the cells, it reflects the DNA synthesis stage.
2. lack of p53 -damage of DNA can entering the cell cycle without getting stuck in the checkpoint - therefore we can see the repair activity.
the author mentioned that false negative and positive numbers are quite high - requiring the new improved method.
Most commonly used assays for genotoxicity;
• the Ames test (a bacterial reverse mutation assay);
• the mouse lymphoma assay (a negative selection for loss of the functional thymidine kinase gene); and
• either the in vitro micronucleus assay (detection of additional small nuclei) or chromosomal aberration assay.
Ames test - very easy and quick but relied on bacterial reverse mutation (points mutations/frame shift mutation) -- evidences these days showed that the mutations are not solely related to carcinogenesis, therefore this model is yet not good enough.
They try to increase the realistic situation by using liver homogenate to biotransform the compound first and observe the phenotype from Ames test.
Micronucleus assay -- prone to false positive and negative.
For the micronuclei assay - using DT40 could improve the specificity (DNA damage agent or agent that inhibits microtubule polymerization).
an efficient G1/S checkpoint may significantly reduce the sensitivity of the micronucleus assay to detect environmental mutagens.
The most promising mutant cells may be cells deficient in translesion DNA synthesis (TLS), because the vast majority of DNA lesions inhibit DNA synthesis by replicative DNA polymerases, and the efficient release from this inhibition is dependent on TLS DNA polymerases.
Firstly, if a large number of chemicals could be screened for toxicity before initiating drug development, those lead molecules that are likely to fail toxicology screening could be eliminated at the outset.
Conventional cancer treatment, basically damaging the DNA content directly which also can harmful to the normal cells (though cancer has the ability to recover from damaging in lesser extent but it can be applied to only some cancer.
Therapeutic index;
Dose required for toxic effect/Dose required for therapeutic effect
If two repair pathways are functionally redundant, loss of one pathway in tumours renders them entirely dependent on the remaining repair pathway.
combination therapy of DNA damaging agent and repair inhibitor is already proving successful.
Since as many as 40 different proteins are involved in HR, many cancers exhibit HR defects. Strikingly, it has been shown that inhibition of PARP-1 in a BRCA2- null background is lethal, even without exogenous DNA damage.
Unfortunately, there are as yet no chemical inhibitors of these polymerases that are both potent and specific.
Whilst the DT40 assay is able to detect all types of genotoxicity, a single genotoxicity assay is unlikely to provide a complete genotoxic assessment. In particular, in vivo assays will remain important.
The ultimate goal of detecting toxicity associated with test chemical compounds is to develop a method for in silico prediction of toxicity from their chemical structures -- really depending on the good database which high-throughout can generate this kind of data.
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