Comparison Of Three Human Lymphoblastoid Cell Lines And Study On The Mechanism For Two Kinds Of Mutation Colonies In TK Gene Mutation Assay

TK gene mutation assay, which was established in the 1970s, is a system to screen carcinogens and mutagens. With the data from different laboratory were gradually accumulated, this assay has been developed to a rather perfect in vitro genotoxicity test and its mechanism has become rather clear. Comparing with other genotoxicity tests, TK gene mutation assay is faster and more sensitive. It can detect a wide range of genetic damage with different end-points, including point mutation, large scale of chromosomal change, recombination, aneuploidy, etc. It has been extensively used for assessing mutagenic activity of compounds and screening carcinogens in many countries, While in our country, application of this assay has just started in recent years. The TK gene mutation assay has been appointed as an optional method in the new version of Standards for Toxicological Assessment of Health Food issued by the Ministry of Public Health in 2003, also in the national standard Procedures and Methods for Toxicological Assessment of Food (GB15193-2003).The main cell lines used in TK gene mutation assay are mouse lymphoma cell L517SY-3.7.2C/tk+/-, human lymphoblastoid cell TK6, WTK1 and TK6-E6, etc. L5178Y cell line is the classical target cell used in this assay, while the human lymphoblastoid cells were just used in this assay in recent years.Two kinds of colonies can be observed in TK gene mutation assay, namely large colony (or nonnally growing colony, NC) and small colony (orslowly growing colony, SC). It is speculated that the colony type are associated with the mutagenic mechanism, i.e. the large colony or NC is caused by small range of mutation, such as point mutation, frameshift mutation, etc, while the small colony or SC is caused by large range of mutation, such as loss of chromosome fragment, which may caused the damage of some growth related genes. Therefore, there is a hypothesis that the clastogen can be judged by the ratio of small colony induced by the mutagens in TK gene mutation assay, but there are not sufficient research data to support this hypothesis.The object of this study is to find out a best cell line of human lymphoblastoid used in the TK locus mutation assay system, and to explore the mechanism resulting in the formations of normal growing colony and slowly growing colony by cytological and molecular-biological methods.This subject includes three parts.Part Ⅰ. Study on the application of TK6-E6 in TK gene mutation assay. The object of the part is to explore the advantage/disadvantage for using this cell line in the TK gene mutation assay system. The result of the study showed that, for this cell line, the doubling time was 16.03(±0.61)h, the spontaneous mutation frequency (MF) was 29.9(±5.47)×10-6, the plating efficiency was 63.8(±4.6)%, the suitable cell number plated in the 96-well culture plate for the MF assay was 10000/well, and the suitable concentration of MMS as positive control was 1.25 μg/mL The mutagenicity of three conventional mutagen (MMS, MMC, NaN3) and one non-mutagen (KQ) was assessed by TK gene mutation assay using TK6-E6 cell. The results showed that the mutagenicity of the three mutagens were positive and that of the KQ was negative, which means that TK gene mutaton assay using TK6-E6 cell may be promising for the application in this assay system.Part Ⅱ. Comparison of TK gene mutation assay using of three human lymphoblastoids (WTK1, TK6 and TK6-E6). The object of this part is to choose a relative better cell line among the three cell lines as the TK gene mutation assay. The mutagenicity of three mutagens (MMS, MMC, NaN3) and one non-mutagen (KQ) were assessed by TK gene mutation assay using the three cell lines. There was no significant difference of their mutagenicity assessed by the three cell lines. The spontaneous mutationfrequency (SMF) and induced mutation frequency (IMF) as well as the tolerance to chemical toxicity in WTK1 cell were the highest, followed by TK6-E6 cell. The plating efficiency of WTKl cell was also the highest, and the second was TK6 cell. Combined with their different p53 state, it is proposed that the TK gene mutation assay using WTKl cell should be popularized firstly. It was also found that the PE3 colonies increased during the latter12 days of incubation as the slowly growing colony, and the there cell lines showed a good dose-response relationship between the number of increased PE3 colonies during the latter12 days and doses of chemicals. So it is recommended that the mutation frequency should be calculated according to PE3 counted on the 24th day.Part Ⅲ. Comparison between NC and SC. The object of this part is to study the mechanism of the two kinds of mutation colonies. Karyotype analysis, loss of heterozygosity analysis and RT-PCR were applied in this study. The results showed that the doubling time of SC was about two times as that of the NC. The highest ratio of SC in SMF was in TK6-E6 cell, while the lowest one was in TK6 cell. The increased ratios of SC induced by MMS and MMC were more obvious in TK6 cell, and also higher than that induced by NaN3, while the same results were not found in TK6-E6 and WTK1 cells.More than 60 mutation colonies of WTKl cell have been analyzed by karyotype analysis, the result showed that many kinds of changes happened on chromosome 17. However, the correlation between the frequency of abnormal chromosome 17 and the colony types was not visible. It was also found that the frequency of abnormal chromosome 17 in the mutation colonies induced was higher than that in the spontaneous mutation colonies.The result of loss of heterozygosity (LOH) anylasis on exon 7 of TK locus showed that the frequency of LOH had no relation to the colony type, but the frequency of LOH in induced SC is higher than that in spontaneous SC.RT-PCR was used to detect the change of tk gene and two growth relevant genes, Le. grb2 gene and survtvin gene. The results showed that most mutation colonies lost their tk fragment, and some colonies lost their survivin and grb2 fragments. It was also found that loss of survtvin and grb2 had no relation with the colony types, which means that the two genes maynot play important roles in controlling the proliferative speed of the colonies. The ratio of loss of the two genes in the mutation colonies induced was higher than that in the spontaneous mutation colonies, which might suggest that the injury extent of mutation colonies induced was more serious than that of spontaneous mutation.Based on the results of our study, the following summaries can be made:(1) The TK6-E6 cell with defected p53 has rather high plating efficiency, SMF and IMF in TK gene mutation assay. Furthermore, the TK gene mutation assay using TK6-E6 can detect out the mutagenicity of three conventional mutagens and one non-mutagen accurately, so it is valuable for the further study on the use of TK6-E6 cell in TK gene mutation assay.(2) The WTK1 cell with mutated p53 has some advantages in TK gene mutation assay, such as the highest SMF and IMF, the highest plating efficiency, and the strongest tolerance to chemical toxicity among the three human lymphoblastoid cell lines, which is maybe the better cell line for TK gene mutation assay.(3) It is proposed that the PE3 colonies should be counted on the 12th day and the 24th day respectively, and the mutation frequency should be calculated according to the PE3 counted on the 24th day.(4) The relationships between the frequency of abnormal chromosome 17, loss of tk, surviving, grb2 fragments and the colony types are still not so clear, which may indicate that the proliferative speed of mutation colonies is maybe caused mostly by cytotoxicity of chemicals rather than the injury extent of TK gene, but it need to be confirmed by further study.