| Even with the introduction of new anti-leukemic agents and approaches, most patients are still not cured. The occurrence of cross-resistance to structurally and functionally unrelated drugs, called multidrug resistsance(MDR), is a main cause of failure in the chemotherapy of leukemia. Several mechanisms of MDR have been identified, such as overexpression of drug transporter proteins, enhancement of detoxification enzymes and DNA repair enzymes, reduction of DNA topoisomerase , and decreased ability to initiate apoptosis. However, all the known alterations are not enough to explain fully the occurrence of MDR. In clinical situations, many patients have been reported to acquire resistance to chemotherapy without undergoing these changes, suggesting that other mechanisms could be involved.Difference in gene expression are likely to explain the phenotypic difference between resistant tumor cells and sensitive tumor cells. Thus, it is an important approach to identify and characterize genes expressed exclusively or preferentially in resistant cancer cells. Suppression subtractive hybridization (SSH) is a new method to identify differentially expressed genes and proved to be a more rapid and reliable method than other techniques such as DDRT-PCR.In this research, SSH was first performed to screen the differentially expressed genes between the MDR cell line K562/DOX and its parent cell line K562. Then the over-expressed genes in resistant leukemia cells were analyzed and further studied. Our goal is to find out novel mechanisms of MDR in leukemia.PartiIdentification of differentially expressed genes between mult id rug resistant leukemic cell line K562/DOX and its parent cell line K562 using suppression subtractive hybridizationThe multidrug resistanct leukemia cell line K562/DOX established at Cancer Institute of Zhejiang University is cross-resistant to quite a lot of antitumor drugs. Previous research has proved that overexpression of mdrlgene and P-glycoprotein is the main mechanism in the occurrence of MDR in this cell line but not the sole one.First of all, we performed SSH to amplify the differentially expressed cDNA fragments between K562/DOX cell and K562 cell. After SSH, the subtracted and amplified products were cloned into pGEM-T Easy vector. Through T/A cloning, a subtracted library of about 200 clones in total was constructed. Thereafter, Dot Blot was carried out to further screen the subtracted library using cDNA probes from K562 cell and K562/DOX cell respectively. From 73 randomly selected clones, 14 clones were identified to be differentially expressed in MDR cell line K562/DOX. Sequence analysis and homology search of these clones indicated that these 14 clones represent 11 known genes:Human mitochondria gene, complete genome,Human NADH dehydrogenase subunit 2(ND2),Human voltage-dependent anion channel 3 (VDAC3)Human S3 ribosomal protein(S3rp),Human ribosomal protein L7a,Human Cu/Zn superoxide dismutase(Cu/Zn SOD),Human l-8u gene from interferon-inducible gene family,Human hemoglobin zeta,Human activator of s phase kinase,Human leukemia-associated phosphoprotein) (LAP 18)(Stathmin),Human My023 protein(My023).Among these gene products, increased expression of SOD has been shown to diminishthe cytotoxoic effects of certain chemotherapeutic agents. Enhancement of mitochondria energy-yielding pathways in drug resistant cancer cell has also been observed. However, so far the relationship between the other genes and the occurrence of multidrug resistance in cancer hasn't been reported. Thus, these genes may indicate novel mechanisms of MDR in leukemia.Part II Expression of S3rp, ND2 and My023 genes in leukemia cell linesIn this research, semi-quantitative reverse transcription -polymerase chain reaction (RT-PCR), nucleic acid in situ hybridization and Northern Blot were used to study differential expression of S3rp, ND2 and My023 genes between sensitive and resistant leukemia cell linesResults from semi-quantitative RT-PCR showed th...
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