Overcoming drug resistance has become an important issue in cancer chemotherapy. Among all known mechanisms that confer resistance, active efflux of chemotherapeutic agents by proteins from the ATP-binding cassette family has been extensively reported. The aim of the present study was to determine the involvement of ABCG2 in resistance to SN38 (the active metabolite of irinotecan) in colorectal cancer. By progressive exposure to increasing concentrations of SN38, we isolated 2 resistant clones from the human colon carcinoma cell line HCT116. These clones were 6-and 53-fold more resistant to SN38 than the HCT116-derived sensitive clone. Topoisomerase I expression was unchanged in our resistant variants. The highest resistance level correlated with an ABCG2 amplification. This overexpression was associated with a marked decrease in the intracellular accumulation of SN38. The inhibition of ABCG2 function by Ko143 demonstrated that enhanced drug efflux from resistant cells was mediated by the activity of ABCG2 protein and confirmed that ABCG2 is directly involved in acquired resistance to SN38. Furthermore, we show, for the first time in clinical samples, that the ABCG2 mRNA content in hepatic metastases is higher after an irinotecan-based chemotherapy than in irinotecan-naive metastases. In conclusion, this study supports the potential involvement of ABCG2 in the development of irinotecan resistance in vivo. © 2004 Wiley-Liss, Inc. Key words: colorectal cancer; ABCG2; SN38; drug resistanceChemotherapeutic drug resistance is a frequent cause of treatment failure in colorectal cancer patients. Understanding the cellular mechanisms that lead to this resistance should permit an improvement in the treatment of colorectal cancer. Irinotecan (CPT-11), a semisynthetic water-soluble derivative of camptothecin, is widely used for the treatment of metastatic colon cancer. 1 CPT-11 is a prodrug converted by carboxylesterases into its active form, SN38. 2 Like other camptothecin derivatives, SN38 exerts its cytotoxic activity through the inhibition of topoisomerase I. Human topoisomerase I is a 100 kDa nuclear enzyme needed for replication and transcription and causing single strand breaks in DNA, thus permitting relaxation of supercoiled DNA. 3 SN38 interferes with topoisomerase I function by forming stable ternary complexes at the DNA breakage points and stopping the topoisomerase I-mediated religation. 4 Cellular resistance to camptothecin derivatives can result from a decrease in cellular drug accumulation, alterations in the structure or location of topoisomerase I, changes in the cellular response to the drug-DNA-enzyme ternary complex formation 5 or increased glucuronidation of SN38, resulting in an inactivation of the drug. 6 Members of the ATP-binding cassette (ABC) transporters, notably MDR1 (ABCB1) and MRP1 (ABCC1), confer resistance to chemotherapeutic drugs by active drug efflux. 7,8 Recently, a new member of this family, the ABCG2 transporter also called BCRP 9 or ABCP 10 or MXR 11 has been discovered. This gene, ...

DNA microarray technology enables investigators to measure the expression of several thousand mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix® GeneChip® array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and Degradometer) and our in-house RNA Quality Scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and Degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybrizidation experiments, ensured reliable and reproducible microarray data.3

Chromatin remodeling by posttranslational modification of histones plays an important role in brain plasticity, including memory, response to stress and depression. The importance of H3/4 histones acetylation by CREB-binding protein (CBP) or related histone acetyltransferase, including p300, was specifically demonstrated using knockout (KO) mouse models. The physiological role of a related protein that also acts as a transcriptional coactivator with intrinsic histone acetylase activity, the p300/CBP-associated factor (PCAF), is poorly documented. We analyzed the behavioral phenotype of homozygous male and female PCAF KO mice and report a marked impact of PCAF deletion on memory processes and stress response. PCAF KO animals showed short-term memory deficits at 2 months of age, measured using spontaneous alternation, object recognition, or acquisition of a daily changing platform position in the water maze. Acquisition of a fixed platform location was delayed, but preserved, and no passive avoidance deficit was noted. No gender-related difference was observed. These deficits were associated with hippocampal alterations in pyramidal cell layer organization, basal levels of Fos immunoreactivity, and MAP kinase activation. PCAF KO mice also showed an exaggerated response to acute stress, forced swimming, and conditioned fear, associated with increased plasma corticosterone levels. Moreover, learning and memory impairments worsened at 6 and 12 months of age, when animals failed to acquire the fixed platform location in the water maze and showed passive avoidance deficits. These observations demonstrate that PCAF histone acetylase is involved lifelong in the chromatin remodeling necessary for memory formation and response to stress.