ATM and ATR:Sensing DNA damage

Cellular response to genotoxic stress is a very complex process, and it usually starts with the “sensing” or “detection” of the DNA damage,followed by a series of events that include signal transduction and activation of transcription factors.The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair,cell cycle arrest,and cell death. There havebeen extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process,including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood,human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.

Stable expression of human cytochrome P450 2D6*10 in HepG2 cells

AIM:Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes.The most important enzymes are CYPIA2, 3A4,2C9/19,2D6 and 2E1.Although CYP2D6 accounts for<2% of the total CYP liver enzyme content,it mediates metabolism in almost 25% of drugs.In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.METHODS:Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector,cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9.A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells.Expression of mRNA was validated by RT-PCR.Enzyme activity of catalyzing dextromethorphan Odemethylation in postmitochondrial supernant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).RESULTS:The cloned cDNA had 4 base differences, e.g.100 C→T, 336 T→C,408 C→G and 1 457 G→C,which resulted in P34S,and S486T amino acid substitutions, and two samesense mutations were 112F and 136V compared with that reported by Kimura et al (GenBank accession number: M33388).P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele.The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31±0.19nmol·min^-1·mg^-1 S9 protein (n=3),but was undetectable in parental HepG2 cells.CONCLUSION:cDNA of human CYP2D6*IOcan be successfully doned.A cell line,HepG2-CYP2D6*10,expressing CYP2D6*10 mRNA and having metabolic activity,has been established.

Ultrastructural changes in non-specific duodenitis

AIM: To investigate the ultrastructural and morphological changes of non-specific duodenitis (NSD) in an attempt to grade them according to the extent of the lesions.METHODS: Biopsies were taken from the mucosa of duodenal bulb of 44 patients selected from the patients undergoing upper gastrointestinal endoscopy for epigastric discomforts. From each patient, two pinch biopsies on the same area were obtained from duodenal bulb. One was for scanning electron microscopy and the other was stained with hematoxylin-eosin, Warthin-Starry silver and both were then examined under light microscope. A total of 12 specimens (three from each degree of the normal and Ⅰ-Ⅲ of NSD diagnosed and graded by histology) selected from the 44patients were dehydrated, critical point dried, coated with gold palladium and examined under a JEOL JSM-30 scanning electron microscope (SEM) at 20 kV.RESULTS: According to the ultrastructural morphologic changes, non-specific duodenitis was divided into normal (as control group), mild, moderate and severe degrees according to results of SEM. The normal villi of duodenal bulb were less than 0.2 mm. There were inflammation cells,occasionally red blood cells and macrophages on the mucosal epithelial surface. Erosion and desquamation of epithelium could be seen. Three cases (25%, 3/12) had gastric metaplasia and Helicobacter pylori(H pylori) infection could be found in 5 cases (41.67%, 5/12) in duodenal bulb mucosa. The most distinctive feature was the ulcer-like defect on the surface of epithelial cells.CONCLUSION: Non-specific duodenitis is a separate entity disease caused by different factors. SEM is of value as an aid in the diagnosis of mucosal diseases of duodenum.