Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells

K. H. Dixon, T. Mulligan, K. N. Chung, P. C. Elwood, K. H. Cowan

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Abstract

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTX(R)) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTX(R) ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTX(R) ZR-75-1 and WT ZR- 75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTX(R) ZR-75-1 cell lines require concentrations ≥ 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTX(R) ZR- 75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTX(R) ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (≤1 μM), MTX(R) ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTX(R) ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTX(R) ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTX(R) ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTX(R) ZR-75-1 cells to methotrexate.

Original languageEnglish (US)
Pages (from-to)24140-24147
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number33
StatePublished - Dec 1 1992

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Folic Acid
Methotrexate
Transfection
Cells
Breast Neoplasms
Reduced Folate Carrier Protein
Folic Acid Antagonists
Cell Line
Leucovorin
Trimetrexate
Growth

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells. / Dixon, K. H.; Mulligan, T.; Chung, K. N.; Elwood, P. C.; Cowan, K. H.

In: Journal of Biological Chemistry, Vol. 267, No. 33, 01.12.1992, p. 24140-24147.

Research output: Contribution to journalArticle

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abstract = "Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTX(R)) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTX(R) ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTX(R) ZR-75-1 and WT ZR- 75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTX(R) ZR-75-1 cell lines require concentrations ≥ 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTX(R) ZR- 75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTX(R) ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (≤1 μM), MTX(R) ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTX(R) ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTX(R) ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTX(R) ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTX(R) ZR-75-1 cells to methotrexate.",
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AU - Cowan, K. H.

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N2 - Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTX(R)) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTX(R) ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTX(R) ZR-75-1 and WT ZR- 75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTX(R) ZR-75-1 cell lines require concentrations ≥ 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTX(R) ZR- 75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTX(R) ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (≤1 μM), MTX(R) ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTX(R) ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTX(R) ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTX(R) ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTX(R) ZR-75-1 cells to methotrexate.

AB - Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTX(R)) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTX(R) ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTX(R) ZR-75-1 and WT ZR- 75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTX(R) ZR-75-1 cell lines require concentrations ≥ 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTX(R) ZR- 75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTX(R) ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (≤1 μM), MTX(R) ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTX(R) ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTX(R) ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTX(R) ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTX(R) ZR-75-1 cells to methotrexate.

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