The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O 6 -methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-KB (NF-KB). Temozolomide inhibits basal and tumor necrosis factor A (TNFA)-induced NF-KB transcriptional activity without altering phosphorylation or degradation of inhibitor of KB-A. Inhibition of NF-KB is secondary to attenuation of p65 DNA binding, not nuclear translocation. Inhibition of DNA binding is shown both in vitro, with gel shift studies and DNA binding assays, and in vivo at KB sites. Consistent with inhibition of NF-KB activity, temozolomide reduces basal and TNFA-induced KB-dependent gene expression. Temozolomide also inhibits NF-KB activated by inducers other than TNFA, including lipopolysaccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-KB is observed to be maximal following pretreatment of cells with temozolomide for 16 h and is also seen with the S N 1-type methylating agent methylnitrosourea. The ability of temozolomide to form O 6

Carboxylesterases are a broad class of enzymes important in the detoxification of many ester- or amide-bond containing xenobiotics. They also activate analgesics, anticancer prodrugs, and other biologically active compounds, such as cocaine and heroin. The objective of this work was to identify the CES2 gene structure, complex 5' untranslated regions and three potential promoters for the initiation of transcription in different human tissues. Using bioinformatics and progressive reverse transcriptase-polymerase chain reaction, we found that the 5' untranslated region is more than 1100 bases longer than previously reported. Rapid amplification of cDNA ends showed three distinctive transcription start sites at -74, -629 and -1187. DNA fragments upstream of each of the three transcription start sites were found to be transcriptionally active in HepG2 cells. The distal promoter is active in both orientations, suggesting its potential role in the transcription of another gene, CGI-128, located immediately upstream to the distal promoter in the opposite direction with respect to CES2. Hybridization analyses showed that CES2 is highly expressed in the heart, skeletal muscle, colon, spleen, kidney and liver, but considerably less expressed in fetal tissues (e.g. fetal heart, kidney, spleen, and liver) and cancer cells. It is also evident that the distal promoter is responsible for low level expression of the gene in many tissues, whereas the other two promoters are tissue specific. These findings shed some light on CES2 gene regulation, a gene important in the metabolism of many drugs.

Carboxylesterases are members of the serine esterase super family important in the metabolism of a wide variety of substrates, including xenobiotics and prodrugs. There are two known carboxylesterases expressed in human liver, small intestine and other tissues, carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2). The aim of this study was to identify polymorphisms in the CES2 gene and determine whether these polymorphisms affect expression levels of CES2 or rate of metabolism of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin). Microsome samples prepared from liver tissues of 78 normal individuals were used to determine the rate of hydrolysis of irinotecan and procaine (an anaesthetic hydrolysed by CES2 but not CES1). The rate of hydrolysis of irinotecan is highly variable among individuals, ranging from 2.7-138 pmol/mg protein/h (mean +/- SD 26.0 +/- 22.9). Fifteen single nucleotide polymorphisms (SNPs) were identified, one is in an exon, 9 are in introns, three are in the 3'-untranslated region (UTR), and two are in the 5'-flanking region. Eight of the 15 SNP loci have rare allele frequencies greater than 5%, of which three were greater than 20%. Genotyping of samples from the SNP Consortium demonstrated different distributions among African-Americans, Asian-Americans and European-Americans. We also analysed the haplotype structure and estimated linkage disequilibrium (LD). A SNP located in the 5'-UTR (5'-UTR-363) was found in LD with loci in intron 1 (Intron1 + 947, Intron1 + 1361, Intron1 + 1643). Haplotypes with homozygous rare alleles on these loci exhibit lower mRNA levels as determined by real time polymerase chain reaction (P < 0.01) and the incorporation of rare alleles in haplotypes correlate with reduced mRNA (P = 0.03). The 5'-UTR-363 SNP is located in one of the three promoters of CES2. However, we did not observe significant differences in CES2 activities (irinotecan and procaine hydrolysis) among individuals with different haplotypes.

ABSTRACT