mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Rots, Marianne G.; Willey, James C.; Jansen, Gert; Zantwijk, C. H. Van; Noordhuis, Paul; DeMuth, Jeffrey P.; Kuiper, E.; Veerman, A. J. P.; Pieters, Rob; Peters, Godefridus J.

doi:10.1038/sj.leu.2401943

Cited by 84 publications

(58 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The principles have been described in detail elsewhere (Willey et al, 1998;Rots et al, 2000;Crawford et al, 2001).…”

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

“…RNA was extracted from 5 Â 10 6 cells by the RNAzol TM method, checked for DNA contamination and reverse transcribed by random hexamers as described by the manufacturer with minimal modifications (Rots et al, 2000). Competitive templates were designed for b-actin (Rots et al, 2000) and TP, using the primer sets shown in Table 1.…”

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

“…Competitive templates were designed for b-actin (Rots et al, 2000) and TP, using the primer sets shown in Table 1. Competitive templates were produced from a cell line known to contain a considerable amount of TP activity (Colo320TP1).…”

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

“…The intensity of the NT and CT bands was quantified by digital image analysis using Scion Image (NIH, Bethesda, DC, USA). Concentrations of NT molecules of TP and b-actin in the cDNA samples were calculated by the ratio of NT/CT after amplification and the molarity of the CT mixture used as described previously (Rots et al, 2000). The relative expression of TP mRNA was given as the ratio of the concentration NT of TP vs NT of b-actin.…”

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

“…The bands were scanned and the OD was used to calculate a ratio between the native cDNA and CT. The contribution of the heteroduplex was calculated as described previously (Willey et al, 1998;Rots et al, 2000). Table 2).…”

Section: Fluoropyrimidine Sensitivity In Relation To Tp Levelsmentioning

confidence: 99%

See 4 more Smart Citations

Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity

et al. 2003

View full text Add to dashboard Cite

Platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (TP) catalyses the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate and is involved in the metabolism of fluoropyrimidines. It can also activate 5 0 -deoxyfluorouridine (5 0 DFUR) and possibly 5-fluorouracil (5FU) and Ftorafur (Ft), but inactivates trifluorothymidine (TFT). We studied the contribution of TP activity to the sensitivity for these fluoropyrimidines by modulating its activity and/or expression level in colon and lung cancer cells using a specific inhibitor of TP (TPI) or by overproduction of TP via stable transfection of human TP. Expression was analysed using competitive template-RT -PCR (CT-RT -PCR), Western blot and an activity assay. TP activity ranged from nondetectable to 70678 pmol h À1 10 À6 cells, in Colo320 and a TP overexpressing clone Colo320TP1, respectively. We found a good correlation between TP activity and mRNA expression (r ¼ 0.964, Po0.01) in our cell panel. To determine the role of TP in the sensitivity to 5FU, 5 0 DFUR, Ft and TFT, cells were cultured with the various fluoropyrimidines with or without TPI and differences in IC 50 's were established. TPI modified 5 0 DFUR, increasing the IC 50 's 2.5-to 1396-fold in WiDR and Colo320TP1, respectively. 5-Fluorouracil could be modified by inhibiting TP but to a lesser extent than 5 0 DFUR: IC 50 's increased 1.9-to 14.7-fold for WiDR and Colo320TP1, respectively. There was no effect on TFT or Ft. There appears to be a threshold level of TP activity to influence the 5 0 DFUR and 5FU sensitivity, which is higher for 5FU. Even high levels of TP overexpression only had a moderate effect on 5FU sensitivity.

show abstract

“…The principles have been described in detail elsewhere (Willey et al, 1998;Rots et al, 2000;Crawford et al, 2001).…”

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

Section: Competitive Template Rt -Pcr To Determine Tp Mrna Expressionmentioning

confidence: 99%

Section: Fluoropyrimidine Sensitivity In Relation To Tp Levelsmentioning

confidence: 99%

See 3 more Smart Citations

Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity

et al. 2003

View full text Add to dashboard Cite

show abstract

Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia

Kaufman

Drori

Cole

et al. 2004

Cancer

View full text Add to dashboard Cite

BACKGROUND Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with combination chemotherapy containing methotrexate (MTX), drug resistance contributes to treatment failure for a substantial fraction of patients. The primary transporter for folates and MTX is the reduced folate carrier (RFC). Impaired drug transport is a documented mechanism of MTX resistance in patients with ALL; however, to the authors' knowledge it is not known whether inactivating RFC mutations are a contributing factor. METHODS The authors devised a genomic polymerase chain reaction‐single strand conformational polymorphism assay followed by sequencing and screened the entire RFC coding region for sequence alterations in DNA from 246 leukemia specimens from patients with diverse ethnic variation, 24 at the time of recurrence and the rest at the time of diagnosis. This cohort was comprised of 203 B‐precursor ALL specimens (82.5%), 32 T‐lineage ALL specimens (13%), and 11 acute myeloblastic leukemia specimens (4.5%). RESULTS Of 246 DNA samples, only 3 diagnosis B‐precursor ALL specimens (1.2%) were found to harbor alterations in the RFC gene, including heterozygous single nucleotide changes resulting in D56H and D522N substitutions in the first extracellular loop and the C‐terminus of this transporter, respectively. The third sample had a sequence alteration in exon 3 that could not be identified because of the lack of availability of DNA. CONCLUSIONS Whereas inactivating RFC mutations are a frequent mechanism of MTX resistance in human leukemia cell lines and in patients with osteosarcoma, they are not common and do not appear to play any significant role in intrinsic or acquired resistance to MTX in childhood leukemia. This is the first study of RFC mutations in multiple pediatric leukemia specimens. Cancer 2004;100:773–82. © 2003 American Cancer Society.

show abstract

Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression after administration of 5‐fluorouracil to patients with colorectal cancer

Mauritz

Groeningen

Smid

et al. 2007

Intl Journal of Cancer

View full text Add to dashboard Cite

This study explores the effect of 5-fluorouracil (5FU) exposure on mRNA levels of its target enzyme thymidylate synthase (TS) and the rate-limiting catabolic enzyme dihydropyrimidine dehydrogenase (DPD) in tumors of colorectal cancer patients. TS and DPD mRNA levels were determined in primary tumor and liver metastasis samples from patients who were either not pretreated (n 5 29) or given one presurgery bolus of 5FU (n 5 67). In both groups a wide variation in TS mRNA levels was observed. Median TS mRNA expression in 17 primary tumors of exposed patients was 3.0-fold higher than in 19 primary tumors of unexposed patients (p 5 0.015). TS mRNA expression in liver metastasis samples of exposed patients (n 5 16) was also higher (5.2-fold) than that of unexposed patients (n 5 48; p < 0.001). Also DPD mRNA expression displayed a large degree of interpatient variation. No difference in DPD expression in liver metastasis samples was observed between exposed and unexposed patients. However, median DPD mRNA expression in 15 primary tumors of exposed patients was 3.2-fold lower than in 18 primary tumors of unexposed patients (p 5 0.027). In conclusion, administration of 5FU in vivo influences the gene expression of TS and DPD. ' 2007 Wiley-Liss, Inc.Key words: 5-fluorouracil; colorectal carcinoma; thymidylate synthase; dihydropyrimidine dehydrogenase; RT-PCR The antimetabolite 5-fluorouracil (5FU) has been included in the standard treatment of patients with advanced colorectal cancer for over 40 years.1,2 Several studies have indicated that 2 important enzymes in 5FU metabolism, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD), are potential markers for response to 5FU-based therapy.3-8 TS is the main target for 5FU, while DPD plays a major role in 5FU degradation.TS is a key enzyme in the de novo synthesis of dTMP and dTTP, a precursor for DNA. TS catalyses the methylation of dUMP to dTMP for which 5,10-methylene-tetrahydrofolate (CH 2 -THF) is the methyl donor. The main mechanism for the action of 5FU is considered to be an inhibitor of TS. 5-Fluoro-dUMP (FdUMP) is an active metabolite of 5FU and it inhibits TS by the formation of a covalent ternary complex between FdUMP, TS and CH 2 -THF, resulting in depletion of dTTP, which can lead to thymine-less death because of inhibition of DNA synthesis. 9,10 Several groups have noted in preclinical models, both cell lines and animal models, an induction of TS activity or TS-FdUMPbinding 4-24 hr after administration of 5FU.11,12 Interestingly, this increase in TS protein after 5FU exposure in vitro is not accompanied by an increase in TS mRNA expression. This observation has led to the hypothesis that this TS induction in vitro by 5FU (or other TS inhibitors) occurs because of disruption of the translational repression caused by the binding of TS to its own mRNA. 11,13 Another explanation that has been proposed is that 5FU exposure results in stabilization of the TS protein because of decreased degradation of the ternary complex.

show abstract

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Cited by 84 publications

References 48 publications

Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity

Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity

Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia

Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression after administration of 5‐fluorouracil to patients with colorectal cancer

Contact Info

Product

Resources

About