| Aim: Tumorigenesis occurs through multiple-steps of mutation accumulation of oncogenes and antioncogenes and through selection amplification of malignant cell clones. Recently the research works in the field of oncogenes and antioncogenes were expanded into the field about genes involved damaged DNA repair. The large amount of small DNA lesions, such as oxidized and alkylated bases and single strand break (SSB), weremainly repaired by DNA polymerase β in vivo through one single nucleotidebase excision repair (BER) pathway. The DNA polymerase β expressionlevel was very low in the normal tissues and cells; and possessed cell protection against apoptosis. Most of tumors, including glioma, lymphoma, breast cancer, colorectal cancer, prostate cancer, ovarian tumor and hepatic cancer and etc., exhibited overexpression of polbeta. The overexpressed polbeta with low fidelity repair could decrease sensitivity to the mismatched bases and increase tolerance to them, which could result in genomic instability, translesion repair and mutator phenotype. The polbeta with heterozygous of wild type (w) and mutant type (m) in which the mpolfl displayed dominantnegative effect over the wpolfi showed almost no BER activity. In our previous study there was a mutant frequency of 1/40 in the cancer-adjacent tissue, much lower than that of 16/40 in the cancer foci tissue in human esophageal cancer. Whether human esophageal cancer tissue could exhibit overexpression of polbeta and alteration of polbeta expression could occur earlier than that of sequence mutation? Especially the POLB and XRCC1, a heterodimer of POLB, involved in SSB repair of early damaged DNA could show altered expression in the cancer- adjacent tissue as an early event? Whether human esophageal cancer Eca-109 cell line could show overexpression of polbeta which could be down regulated during cell differentiation? How about the alteration of wp53 required in BER activity and anti-cancer drug resistance, when the cisplatin-treated cells were used as apositive control? If the pcDNA3-wpol β plasmid was transfected intoEca-109 cell line, the overexpressed polbeta could result in further mutator phenotype or normal tendency through functional regulation in the cell itself? As to the above-mentioned problems, especially associated with the localization of signals in human esophageal cancer, so far, no reports have been found.Methods: 1. 51 pieces of tissues, including cancer foci, cancer-adjacent and corresponding normal tissues were removed from 17 untreated patients with esophageal cancer. Each tissue was divided into 2 aliquots, one of them was fixed with 4% formaldehyde and paraffin-embedded specimens were prepared. The total RNA was isolated from another aliquot tissues by 'Trizol' reagent, and 51 RNA dot blots were performed. 2. The Eca-109 cell line treated withdifferentiation drugs and stably transfected with pcDNA-wpol β Eca-109 cellline and their corresponding control cell lines were established respectively. 3. The wpol β DNA was isolated from pcDNA3-wpol6 recombinant plasmid after digesting with BamH1 and Hind III enzymes and electrophoresis. TheBiotin or Digoxgenin labeled wpolbeta cDNA probe, Bio-wp53 and Bio-mp53 cDNA probes were prepared by random primer method. The Bio-labeled wpolbeta antisense RNA probe was prepared by transcription in vitro. The recombinant pcDNA3-wpolβ plasmid was digested only by Hind III enzyme as the linerized DNA template. The transcription in vitro was carried out by addition of SP6 RNA polymerase, rNTPs and Bio-11-dUTP. The sensitivity of each labeled probe was detected by DNA dot blot. 4. Observation on the signal localization and relative quantitation of expression level: the signal/IR localization in the specimens prepared by in situ hybridization or by immunohistochemistry (POLB-IR, PCNA-IR and MRP-IR) were observed under oil-emersion microscope and the integrated value for demonstration of signal /IR intensity was combined with the TLC scanning value in the RNA dot blot or...
|
PDF Full Text Request