The Human Lung Adenocarcinoma Multidrug Resistant Cell Cloning And Preliminary Identification Of Differentially Expressed Genes In

Chemotherapy treatment is expected to prolong the survival of lung cancer patients, but multidrug resistance is considered to be a major cause of failure of such treatment. Multidrug resistance (MDR) to anticancer agents is observed frequently in lung cancer cases, so how to overcome drug resistance is one of the major fields of lung cancer researching. The extensively studied mechanisms of lung cancer multidrug resistance are P-glycoprotein (P-gp), multidrug-resistance protein (MRP), breast cancer resistance protein (BCRP), and lung resistance-related protein (LRP). Alterations in the essential nuclear enzymes, DNA topoisomerases and factors determining cell-killing pathways play an important role. However, the latest studies have shown that some additional mechanisms are believed to contribute to the MDR of lung cancer. As the developing of strategies and methods of identification of novel genes, various methods to compare patterns of gene expression have been described, it is becoming possible to identify some novel genes related with lung cancer multidrug resistance. To isolate and characterize the differentially expressed genes in multidrug resistance cell line from human lung adenocarcinoma are useful to clarify the MDR mechanism and design the reversal reagent. The aim of our studies is to clone and identify multidrug resistance related gene of human lung adenocarcinoma cell. ? III In the studies described here, the human lung adenocarcinoma cell line SPC-A-1 was exposed to stepwise and high concentration of cisplatin (CDDP), the multidrug resistance cell line (SPC-A-1/CDDP) was established after 192 days. The relative resistance was tested by MTT assay, the morphology observed by electronic microscopy and the chromosome analyzed by Giemsa staining. The results indicated that SPC-A- l/CDDP cell had 11 .2 resistance index to cisplatin and had various cross-resistances to 5-Fu, doxorubicin, mitomycin, vincristine, and etoposide, except hydroxycamptothecine. Electron microscopic examination showed anoplastic nuclei with enlarged nucleoli and abundant microvilli. Our data suggest that a multidrug resistance cell line (SPC-A-1/CDDP) from human lung adenocarcinoma is established. It can be used to downstream application. After the SPC-A-11CDDP cell line was established, the suppression subtractive hybridization (SSH) was performed on human lung adenocarcinoma multidrug resistance cell line (SPC-A-1/CDDP, as tester) and human lung adenocarcinoma cell line (SPC-A-1, as driver). With the I/A cloning method the cDNA fragments generated by SSH were cloned into a TA cloning plasmid (pT-Adv), the subtracted cDNA library being constructed, The dot blots was used to screen the subtracted cDNA library with forward-and reverse-subtracted cDNA probes. The differentially expressed cDNA fragments in SPC-A-1/CDDP cell was sequenced and analyzed in GenBank with Blast 2.0 search. The novel cDNA sequences were examined by Northern blots. The results showed that a high quality subtracted cDNA library was constructed. Twenty-one differentially expressed eDNA fragments in SPC-A-l/CDDP cell were identified. Two of them were novel eDNA sequences and the others had ?3%-l00% homology with the known genes respectively. Northern blots indicated the novel cDNA sequences only expressed in SPC-A-1/CDDP cell. In summary, our data suggest that the novel...