Abstract
Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were established in culture by progressive dose escalation. The biochemical basis of resistance was studied. The line with the lowest resistance (R1) had a normal dihydrofolate reductase (DHFR) content but showed decreased MTX transport and polyglutamation. Lines of intermediate resistance (R2 and R3) showed an increased DHFR content and DHFR gene copy number and a defect in MTX transport. The line with the greatest resistance (R4) showed increased DHFR content and gene copy number but nearly normal MTX transport. These results demonstrate that multiple mechanisms of MTX resistance occur in human epithelial cells in culture. We also find evidence of alterations in DHFR gene expression. The MTX-resistant cells were either not cross-resistant or only partly cross-resistant to two lipophilic MTX ester derivatives. These compounds are of potential therapeutic interest for the treatment of MTX-resistant tumors.
Full text
PDF![2873](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f01e/345174/c0d336110698/pnas00610-0280.png)
![2874](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f01e/345174/1f035151a570/pnas00610-0281.png)
![2875](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f01e/345174/e4c5b0964596/pnas00610-0282.png)
![2876](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f01e/345174/34b28dd7d287/pnas00610-0283.png)
![2877](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f01e/345174/e85ba3115bc0/pnas00610-0284.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alt F. W., Kellems R. E., Schimke R. T. Synthesis and degradation of folate reductase in sensitive and methotrexate-resistant lines of S-180 cells. J Biol Chem. 1976 May 25;251(10):3063–3074. [PubMed] [Google Scholar]
- Chen M. J., Shimada T., Moulton A. D., Harrison M., Nienhuis A. W. Intronless human dihydrofolate reductase genes are derived from processed RNA molecules. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7435–7439. doi: 10.1073/pnas.79.23.7435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crouse G. F., Simonsen C. C., McEwan R. N., Schimke R. T. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed] [Google Scholar]
- Diddens H., Niethammer D., Jackson R. C. Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer Res. 1983 Nov;43(11):5286–5292. [PubMed] [Google Scholar]
- Dolnick B. J., Berenson R. J., Bertino J. R., Kaufman R. J., Nunberg J. H., Schimke R. T. Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells. J Cell Biol. 1979 Nov;83(2 Pt 1):394–402. doi: 10.1083/jcb.83.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Domin B. A., Grill S. P., Bastow K. F., Cheng Y. C. Effect of methotrexate on dihydrofolate reductase activity in methotrexate-resistant human KB cells. Mol Pharmacol. 1982 Mar;21(2):478–482. [PubMed] [Google Scholar]
- Domin B. A., Grill S. P., Cheng Y. Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy. Cancer Res. 1983 May;43(5):2155–2158. [PubMed] [Google Scholar]
- Fry D. W., Anderson L. A., Borst M., Goldman I. D. Analysis of the role of membrane transport and polyglutamation of methotrexate in gut and the Ehrlich tumor in vivo as factors in drug sensitivity and selectivity. Cancer Res. 1983 Mar;43(3):1087–1092. [PubMed] [Google Scholar]
- Galivan J. Transport and metabolism of methotrexate in normal and resistant cultured rat hepatoma cells. Cancer Res. 1979 Mar;39(3):735–743. [PubMed] [Google Scholar]
- Goldie J. H., Dedhar S., Krystal G. Properties of a methotrexate-insensitive variant of dihydrofolate reductase derived from methotrexate-resistant L5178Y cells. J Biol Chem. 1981 Nov 25;256(22):11629–11635. [PubMed] [Google Scholar]
- Hill B. T., Bailey B. D., White J. C., Goldman I. D. Characteristics of transport of 4-amino antifolates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate. Cancer Res. 1979 Jul;39(7 Pt 1):2440–2446. [PubMed] [Google Scholar]
- Jackson R. C., Niethammer D. Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofoltate reductase. Eur J Cancer. 1977 Jun;13(6):567–575. doi: 10.1016/0014-2964(77)90118-9. [DOI] [PubMed] [Google Scholar]
- Jolivet J., Schilsky R. L., Bailey B. D., Drake J. C., Chabner B. A. Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. J Clin Invest. 1982 Aug;70(2):351–360. doi: 10.1172/JCI110624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamen B. A., Takach P. L., Vatev R., Caston J. D. A rapid, radiochemical-ligand binding assay for methotrexate. Anal Biochem. 1976 Jan;70(1):54–63. doi: 10.1016/s0003-2697(76)80047-4. [DOI] [PubMed] [Google Scholar]
- Kaufman R. J., Bertino J. R., Schimke R. T. Quantitation of dihydrofolate reductase in individual parental and methotrexate-resistant murine cells. Use of a fluorescence activated cell sorter. J Biol Chem. 1978 Aug 25;253(16):5852–5860. [PubMed] [Google Scholar]
- Kaufman R. J., Brown P. C., Schimke R. T. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines. Mol Cell Biol. 1981 Dec;1(12):1084–1093. doi: 10.1128/mcb.1.12.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirkwood J. M., Canellos G. P., Ervin T. J., Pitman S. W., Weichselbaum R., Miller D. Increased therapeutic index using moderate dose methotrexate and leucovorin twice weekly vs. weekly high dose methotrexate-leucovorin in patients with advanced squamous carcinoma of the head and neck: a safe new effective regimen. Cancer. 1981 May 15;47(10):2414–2421. doi: 10.1002/1097-0142(19810515)47:10<2414::aid-cncr2820471016>3.0.co;2-w. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lippke J. A., Gordon L. K., Brash D. E., Haseltine W. A. Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleoside-cytidine sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3388–3392. doi: 10.1073/pnas.78.6.3388. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masters J. N., Yang J. K., Cellini A., Attardi G. A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA. J Mol Biol. 1983 Jun 15;167(1):23–36. doi: 10.1016/s0022-2836(83)80032-1. [DOI] [PubMed] [Google Scholar]
- Masters J., Keeley B., Gay H., Attardi G. Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines. Mol Cell Biol. 1982 May;2(5):498–507. doi: 10.1128/mcb.2.5.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morandi C., Attardi G. Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines. J Biol Chem. 1981 Oct 10;256(19):10169–10175. [PubMed] [Google Scholar]
- Niethammer D., Jackson R. C. Changes of molecular properties associated with the development of resistance against methotrexate in human lymphoblastoid cells. Eur J Cancer. 1975 Dec;11(12):845–854. doi: 10.1016/0014-2964(75)90083-3. [DOI] [PubMed] [Google Scholar]
- Ohnoshi T., Ohnuma T., Takahashi I., Scanlon K., Kamen B. A., Holland J. F. Establishment of methotrexate-resistant human acute lymphoblastic leukemia cells in culture and effects of folate antagonists. Cancer Res. 1982 May;42(5):1655–1660. [PubMed] [Google Scholar]
- Rheinwald J. G., Beckett M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 1981 May;41(5):1657–1663. [PubMed] [Google Scholar]
- Rosowsky A., Abelson H. T., Beardsley G. P., Ensminger W. D., Kufe D. W., Steele G., Modest E. J. Pharmacologic studies on the dibutyl and gamma-monobutyl esters of methotrexate in the rhesus monkey. Cancer Chemother Pharmacol. 1982 Dec;10(1):55–61. doi: 10.1007/BF00257240. [DOI] [PubMed] [Google Scholar]
- Rosowsky A., Beardsley G. P., Ensminger W. D., Lazarus H., Yu C. S. Methotrexate analogues. 11. Unambiguous chemical synthesis and in vitro biological evaluation of alpha- and gamma-monoesters as potential prodrugs. J Med Chem. 1978 Apr;21(4):380–386. doi: 10.1021/jm00202a013. [DOI] [PubMed] [Google Scholar]
- Rosowsky A., Forsch R., Uren J., Wick M. Methotrexate analogues. 14. Synthesis of new gamma-substituted derivatives as dihydrofolate reductase inhibitors and potential anticancer agents. J Med Chem. 1981 Dec;24(12):1450–1455. doi: 10.1021/jm00144a016. [DOI] [PubMed] [Google Scholar]
- Rosowsky A., Lazarus H., Yuan G. C., Beltz W. R., Mangini L., Abelson H. T., Modest E. J., Frei E., 3rd Effects of methotrexate esters and other lipophilic antifolates on methotrexate-resistant human leukemic lymphoblasts. Biochem Pharmacol. 1980 Feb 15;29(4):648–652. doi: 10.1016/0006-2952(80)90391-3. [DOI] [PubMed] [Google Scholar]
- Rosowsky A. Methotrexate analogs. 2. A facile method of preparation of lipophilic derivatives of methotrexate and 3',5'-dichloromethotrexate by direct esterification. J Med Chem. 1973 Oct;16(10):1190–1193. doi: 10.1021/jm00268a029. [DOI] [PubMed] [Google Scholar]
- Sirotnak F. M., Moccio D. M., Kelleher L. E., Goutas L. J. Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1210 clonal cell lines derived in vivo. Cancer Res. 1981 Nov;41(11 Pt 1):4447–4452. [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Trus M. D., Sodroski J. G., Haseltine W. A. Isolation and characterization of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus. J Biol Chem. 1982 Mar 25;257(6):2730–2733. [PubMed] [Google Scholar]
- Wolman S. R., Craven M. L., Grill S. P., Domin B. A., Cheng Y. C. Quantitative correlation of homogeneously stained regions on chromosome 10 with dihydrofolate reductase enzyme in human cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):807–809. doi: 10.1073/pnas.80.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]