DNA Microarray Study Of The Molecular Mechanisms Of CML

Carcinogenesis is a multi-step process involving abnormalities in genomics as well in the expressed genes and proteins. Abnormal gene mutation, depletion, amplification and expression of oncogenes all may contribute to the synergistic outcome of carcinogenesis, together with the compromise of host immunological functionalities.Chronic myeloid leukemia (CML) is a common malignant tumor in hematological system. It is a cellular disorder derived from clonal amplification of stem cells and in most patients, CML exhibits the t [(9:22) (q34; q11)] chromosomal translocation, resulting in Philadelphia (Ph~1) chromosome. The translocation causes a fusion of two genes, BCR and ABL, which rendering in the production of an aberrant fusion protein P210. P210 has tyrosine kinase activity. The formation of Ph~1 chromosomes leading to increased tyrosine kinase activities in the CML cells. Therefore, tyrosine kinase activity may play important role in the carcinogenesis of the CML.In this study, tyrosine kinase inhibitor was used to curtail the tyrosine kinase activity in K562 cells, in a hope to elucidate the cellular and molecular mechanism behind the K562 phenotype before and after the P210 being inhibited. Furtheremployment of the microarray technology may help the research and development of early diagnostics and therapeutic approaches.Tumor is one of the most devastating human diseases, which defies effective therapeutics. To study the molecular mechanisms from genetic level may provide novel technology for early diagnosis and treatment of the CML and other cancer related diseases, which should have significant implications both theoretically and practically.Gene chips or DNA microarray is a new approach of high-throughput biotechnology, which has began to be applied in human genomics studies. The technological processes of DNA microarray include preparation of the molecuLar probes, selection of the chip substrates and surface treatment, microarray printing, sample labeling and molecular hybridization, microarray scanning as well as statistical and bio-informatics analysis of the scanning results. In this study, lab made K562 microarray were prepared and used in analysis of the gene expression profile before and after the K562 being treated with tyrosine kinase inhibitors. Further studies were conducted using the ABI human whole genomic microarray, to analyze the carcinogenesis mechanisms of CML.This project has been studied in three consecutive processes.1. The construction of the expressed cDNA library of the K562 cells. The most important issue of the preparation of the DNA microarray for gene expression studies is to obtain large amount of cDNA probes. By using restriction display technology (RD), cDNA fragments library of K562 cells could be constructed. After grouping the cDNA fragments, PCR could be conducted and the cDNA probes isolated with two approaches. No.l. is through electrophoresis, after PAGE and silver staining, DNA banding could be demonstrated clearly, which could be isolated by cutting the corresponding bands, after a secondary PCRapproaches, the isolated cDNA fragments could be used for probes making microarray. No.2. Approach is to clone the RD fragments directly, after PCR, the fragments ligated directly with T vectors. Subsequent cloning and validating resulted in 800 probes with the length ranging from 250-750 bp. With DNA sequencing, the isolated cDNA sequences were found to be from genes of cell cycle relevant, cellular metabolite pathway, signal transduction and transcription as well as oncogene related.2. Tyrosine kinase inhibitor (STI571) was used to study the changes of K562 cell growth and proliferation. In our experiment, it was demonstrated that after the treatment of STI571 for 24 hours, the K562 cells underwent major morphological changes, which included nuclear shrinkage, membrane bleb and scattered apoptotic bodies. DNA gel electrophoresis also discovered that the typical "DNA ladder" phenomena in the treatment group. All of these indicated that STI571 could effectively inhibit the K562 cell growth and induce k562 cell into apoptosis. The RNAs purified prior and posterior of the STI571 treatment were stored to be used in the following microarray studies.3. Studies using the lab-made microarray on the apoptosis mechanism of K562 cells induced by STI571. After the hybridization, detection and analysis, genes were found to be significantly down- and up-regulated are categorized separately. It was found that these differentially expressed genes include cell cycle related genes, cell metabolizing pathway related genes, signal transduction and transcript regulation related genes and so on. The down-regulated genes CBL and STAT5 had been also reported in previous literatures. Studies using ABI 1700 Microarray Analysis System were also conducted to correlate the studies using lab-made microarrays on K562, and also on the pathogenesis of CML. The total RNAs were extracted and purified from one CML patient and one control healthy person tosynthesize double-stranded cDNAs by reverse transcription. Differences of gene expression profile of the bone marrow mononuclear cells were examined by ABI 1700 Bioanalyzer. 19 genes mRNA levels were up-reguLated while 56 genes mRNA levels were down-reguLated in the CML patient. It showed that ABI 1700 Bioanalyzer could be applied more extensively for the study of differential gene expression.In summary, this study using K562 cell line as well as human CML samples to apply the DNA microarray technology. The microarray systematically analyzed differentially the gene expression profile prior and posterior of the tyrosine inhibitor STI571 treatment. Apply the ABI 1700 chip system CML expression profile were studied in even larger scale. The data generated from these studies shouLd help further the exploration of early gene diagnosis as well as gene therapy against tumor related diseases.