Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Allelotyping for loss of heterozygosity on chromosome 18 in gastric cancer.

AIM: To investigate the association between loss of heterozygosity (LOH) on chromosome 18 and sporadic gastric cancer. METHODS: Multiplex PCR was used to screen 14 highly polymorphic microsatellite markers on chromosome 18 in 45 cases of primary gastric cancer. PCR products were separated on polyacrylamide gels and the electrophoresis maps were analyzed with Genescan and Genotyper. RESULTS: The LOH frequencies in gastric cancer at all 14 markers ranged from 10% to 58%. Eleven markers were found with over 20% LOH frequencies, in which 9 markers located in 18q, and 2 markers in 18p. Two overlapping deleted regions were identified: R1 between D18S61-D18S1161 at 18q22 (9cM) with 24% LOH frequency; R2 between D18S462-D18S70 at 18q22-23(6cM) with 32% LOH frequency. CONCLUSION: LOH of chromosome 18 (18q and 18p) may be involved in gastric tumorigenesis. Two overlapping deleted fragments suggested that there might be unidentified tumor suppressor genes in those two regions.

Simultaneous determination of 7 benzodiazepines in human plasma by UHPLC-MS/MS / 药学学报

The study was designed to develop the method for determination of 7 benzodiazepines concentration in human plasma. UHPLC-MS/MS was adopted to analyze plasma with protein precipitated by acetonitrile. Citalopram was used as an internal standard. Plasma samples were separated on CORTECS UHPLC C18 column with the mobile phase of aqueous solution (0.01% formic acid)-methanol (0.01% formic acid) at a flow rate of 0.3 mL·min-1. Multiple reaction monitoring (MRM) mode was performed in combiation with electrospray ionization source operating in the positive ionization mode. The liner calibration curve of midazolam, nitrazepam, estazolam, clonazepam, lorazepam, triazolam and diazepam were obtained in the concentration range of 1.05-840 (r=0.999 4), 2.06-824 (r=0.998 1), 2.02-1 616 (r=0.994 7), 6.18-2 472 (r=0.997 9), 6.12-2 448 (r=0.997 4), 3.02-2 416 (r=0.990 2), 1.02-816 (r=0.998 8) ng·mL-1, respectively. The lowest detection limit were 0.02, 0.52, 0.51, 1.55, 0.77, 0.76, 0.02 ng·mL-1, respectively. The RSD of inter-day and intra-day were less than 10.81%. The relative recovery was 81.46%-106.53%. The method was successfully applied to clinical analysis of blood samples from patients.