Biomedical subjects

John Boyer

Publications and source records attributed to John Boyer.

8 recordsLinked to original sources

PubMed2006

Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer.

DNA microarray analysis was used to analyze the transcriptional profile of HCT116 colorectal cancer cells that were treated with 5-fluorouracil (5-FU) or oxaliplatin and selected for resistance to these agents. Bioinformatic analyses identified sets of genes that were constitutively dysregulated in drug-resistant cells and transiently altered following acute exposure of parental cells to drug. We propose that these genes may represent molecular signatures of sensitivity to 5-FU and oxaliplatin. Using real-time reverse transcription-PCR (RT-PCR), the robustness of our microarray data was shown with a strong overall concordance of expression trends for > or =82% (oxaliplatin) and > or =85% (5-FU) of a representative subset of genes. Furthermore, strong correlations between the microarray and real-time RT-PCR measurements of average fold changes in gene expression were observed for both the 5-FU (R(2) > or = 0.73) and oxaliplatin gene sets (R(2) > or = 0.63). Functional analysis of three genes identified in the microarray study [prostate-derived factor (PDF), calretinin, and spermidine/spermine N(1)-acetyl transferase (SSAT)] revealed their importance as novel regulators of cytotoxic drug response. These data show the power of this novel microarray-based approach to identify genes which may be important markers of response to treatment and/or targets for therapeutic intervention.

Antineoplastic Agents↗

PubMed2004

The roles of thymidylate synthase and p53 in regulating Fas-mediated apoptosis in response to antimetabolites.

Fas (CD95/Apo-1) is a member of the tumor necrosis factor receptor family. Receptor binding results in activation of caspase 8, leading to activation of proapoptotic downstream molecules. We found that expression of Fas was up-regulated >10-fold in MCF-7 breast and HCT116 and RKO colon cancer cell lines after treatment with IC(60) doses of 5-fluorouracil (5-FU) and raltitrexed (RTX). Combined treatment with the agonistic Fas antibody CH-11 and either 5-FU or RTX resulted in a highly synergistic induction of apoptosis in these cell lines. Similar results were obtained for another antifolate, Alimta. Induction of thymidylate synthase expression inhibited Fas induction in response to RTX and Alimta, but not in response to 5-FU. Furthermore, thymidylate synthase induction abrogated the synergy between CH-11 and both antifolates but had no effect on the synergistic interaction between 5-FU and CH-11. Inactivation of p53 in MCF-7 and HCT116 cell lines blocked 5-FU- and antifolate-mediated up-regulation of Fas. Furthermore, Fas was not up-regulated in response to 5-FU or antifolates in the p53-mutant H630 colon cancer cell line. Lack of Fas up-regulation in the p53-null and -mutant lines abolished the synergistic interaction between 5-FU and CH-11. Interestingly, synergy was still observed between the antifolates and CH-11 in the p53-null HCT116 and p53-mutant H630 cell lines, although this was significantly reduced compared with the p53 wild-type cell lines. Our results indicate that Fas is an important mediator of apoptosis in response to both 5-FU and antifolates.

Antibodies↗

PubMed2004

Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan.

To elucidate mechanisms of resistance to chemotherapies currently used in the first-line treatment of advanced colorectal cancer, we have developed a panel of HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic colorectal cancer cell lines resistant to the antimetabolite 5-fluorouracil (5-FU), topoisomerase I inhibitor irinotecan (CPT-11), and DNA-damaging agent oxaliplatin. These cell lines were generated by repeated exposure to stepwise increasing concentrations of each drug over a period of several months. We have demonstrated a significant decrease in sensitivity to 5-FU, CPT-11, and oxaliplatin in each respective resistant cell line relative to the parental line as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, with increases in IC(50 (72 h)) concentrations ranging from 3- to 65-fold. Using flow cytometry, we have also demonstrated compromised apoptosis and cell cycle arrest in 5-FU-, oxaliplatin-, and CPT-11-resistant cell lines compared with the parental lines after exposure to each drug. In addition, we found that resistance to 5-FU and oxaliplatin was higher in parental p53(-/-) cells compared with parental p53(+/+) cells, with an approximately 5-fold increase in IC(50 (72 h)) for each drug. In contrast, the IC(50 (72 h)) doses for CPT-11 were identical in the p53 wild-type and null cell lines. Furthermore, apoptosis after treatment with 5-FU and oxaliplatin, but not CPT-11, was significantly reduced in parental p53(-/-) cells compared with parental p53(+/+) cells. These data suggest that p53 may be an important determinant of sensitivity to 5-FU and oxaliplatin but not CPT-11. Using semiquantitative reverse transcription-PCR, we have demonstrated down-regulation of thymidine phosphorylase mRNA in both p53(+/+) and p53(-/-) 5-FU-resistant cells, suggesting that decreased production of 5-FU active metabolites may be an important resistance mechanism in these lines. In oxaliplatin-resistant cells, we noted increased mRNA levels of the nucleotide excision repair gene ERCC1 and ATP-binding cassette transporter breast cancer resistance protein. In CPT-11-resistant cells, we found reduced mRNA levels of carboxylesterase, the enzyme responsible for converting CPT-11 to its active metabolite SN-38, and topoisomerase I, the SN-38 target enzyme. In addition, we noted overexpression of breast cancer resistance protein in the CPT-11-resistant lines. These cell lines are ideal tools with which to identify novel determinants of drug resistance in both the presence and absence of wild-type p53.

Antineoplastic Agents↗

PubMed2003

Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling.

The fluoropyrimidine 5-Fluorouracil (5-FU) is widely used in the treatment of cancer. To identify novel downstream mediators of tumor cell response to 5-FU, we used DNA microarray technology to identify genes that are transcriptionally activated by 5-FU treatment in the MCF-7 breast cancer cell line. Of 2400 genes analyzed, 619 were up-regulated by >3-fold. Highly up-regulated genes (>6-fold) with signal intensities of >3000 were analyzed by Northern blot. Genes that were consistently found to be up-regulated were spermine/spermidine acetyl transferase (SSAT), annexin II, thymosin-beta-10, chaperonin-10, and MAT-8. Treatment of MCF-7 cells with the antifolate tomudex and DNA-damaging agent oxaliplatin also resulted in up-regulation of each of these targets. The 5-FU-induced activation of MAT-8, thymosin-beta-10, and chaperonin-10 was abrogated by inactivation of p53 in MCF-7 cells, whereas induction of SSAT and annexin II was significantly reduced in the absence of p53. Moreover, each of these genes contained more than one potential p53-binding site, suggesting that p53 may play an important regulatory role in 5-FU-induced expression of these genes. In addition, we found that basal expression levels of SSAT, annexin II, thymosin beta-10, and chaperonin-10 were increased (by approximately 2-3-fold), and MAT-8 expression dramatically increased (by approximately 10-fold) in a 5-FU-resistant colorectal cancer cell line (H630-R10) compared with the parental H630 cell line, suggesting these genes may be useful biomarkers of resistance. These results demonstrate the potential of DNA microarrays to identify novel genes involved in mediating the response of tumor cells to chemotherapy.

Antimetabolites, Antineoplastic↗

PubMed2003

The interaction of thymidylate synthase expression with p53-regulated signaling pathways in tumor cells.

Thymidylate synthase (TS) is a chemotherapeutic target for the fluoropyrimidine 5-fluorouracil (5-FU) and antifolate tomudex (TDX). Using the MCF-7 breast cancer line, we have developed a cell line with inducible TS expression termed M7TS90. Inducible TS expression in this line resulted in a moderate (approximately 3-fold) increase in 5-FU 50% inhibitory concentration at 72 hours (IC-50(72 h)) dose and a dramatic (approximately 24-fold) increase in the IC-50(72 h) dose of TDX, but did not affect chemosensitivity to cisplatin, oxaliplatin, irinotecan, and paclitaxel. In the absence of drug treatment, inducible TS expression had no effect on expression of the p53 tumor suppressor gene. However, TS induction abrogated p53, p21, Fas, and Bak induction in response to TDX, but not 5-FU. Similarly, downregulation of Bcl-2 was reversed by inducible TS expression in TDX, but not 5-FU-treated cells. Our results indicate that inducible TS expression in M7TS90 cells modulates p53 and p53 target gene expression in response to TDX, but not 5-FU.

Antimetabolites, Antineoplastic↗

PubMed2002

The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Breast Neoplasms↗

PubMed2002

Group learning improves case analysis in veterinary medicine.

INTRODUCTION: Group learning has become important to professional students in the healing sciences. Groups share factual and procedural resources to enhance their performances. METHODOLOGY: We investigated the extent to which students analyzing case-based evaluations as teams acquired an immediate performance advantage relative to those analyzing them as individuals and the extent to which group work on one problem led to better performance by individual students on related problems. We blinded written evaluations by randomly assigning numbers to groups of students and using removable tracers. Differences between groups and individuals were evaluated using Student's t statistic. Similar comparisons were evaluated by meta-analysis to determine overall trends. RESULTS: Students who analyzed evaluations as a group had an 8.5% performance advantage over those who analyzed them as individuals. When evaluations were divided into those asking questions related to treatment, differential diagnosis, and prognosis, specific performance advantages for groups relative to individuals were 8.9%, 5.9%, and 6.1% respectively. Students who had previously been trained by group evaluations had a 1.5% advantage relative to those who received their training as individuals. CONCLUSIONS: Answers by students analyzing evaluations as groups suggested a deeper understanding, in large part because of their improved ability to explain treatment and to conduct differential diagnosis. These improvements suggested limited abilities to use previous experience to improve present performance.

Animals↗

PubMedDate not supplied

5-Fluorouracil: identification of novel downstream mediators of tumour response.

BACKGROUND: 5-Fluorouracil (5-FU) is routinely used in the treatment of gastrointestinal, breast and head and neck cancers. A major limitation to the use of this drug is acquired or inherent resistance. MATERIALS AND METHODS: To examine the downstream molecular signals activated in response to 5-FU, we used DNA microarray technology to examine global transcriptional changes in 5-FU-treated MCF-7 breast cancer cells. RESULTS: We identified several novel 5-FU-inducible target genes that have not previously been linked to 5-FU response, including spermine/spermidine acetyl transferase (SSAT) and annexin II. Treatment of MCF-7 cells with the antifolate tomudex (TDX) and the DNA damaging agent oxaliplatin also caused up-regulation of each target gene. Inactivation of wild-type p53 abrogated the 5-FU-mediated induction of SSAT and annexin II. Inducible expression of thymidylate synthase completely abrogated TDX-, but not 5-FU-mediated induction of each gene. Furthermore, basal expression of SSAT and annexin II was elevated in cells resistant to 5-FU. CONCLUSION: These data demonstrate the potential of microarray analysis to identify novel genes associated with response or resistance to chemotherapeutic agents.

Acetyltransferases↗