Disparate Mechanisms of Antifolate Resistance Provoked by Methotrexate and Its Metabolite 7-Hydroxymethotrexate in Leukemia Cells: Implications for Efficacy of Methotrexate Therapy.

Abstract Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke antifolate resistance that may further disrupt MTX efficacy. For this purpose we developed resistance to 7-OHMTX as well as MTX in two human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (>10 fold) and MTX (>75 fold), respectively. The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (>10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was solely due to a marked decreased (> 95%) in folylpolyglutamate synthetase (FPGS) activity which conferred >100-fold MTX resistance upon a short term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance as compared to the parent drug MTX.

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Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Blood ◽

10.1182/blood.v90.3.1241.1241_1241_1245 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1241-1245

Author(s):

Giuseppe S.A. Longo ◽

Richard Gorlick ◽

William P. Tong ◽

Emine Ercikan ◽

Joseph R. Bertino

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemia Cells ◽

Partial Purification ◽

Acute Myelocytic Leukemia ◽

Human Leukemia ◽

Folylpolyglutamate Synthetase ◽

Leukemia Cell Lines ◽

Short Term Exposure ◽

Long Chain

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.

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Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Blood ◽

10.1182/blood.v90.3.1241 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1241-1245 ◽

Cited By ~ 23

Author(s):

Giuseppe S.A. Longo ◽

Richard Gorlick ◽

William P. Tong ◽

Emine Ercikan ◽

Joseph R. Bertino

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemia Cells ◽

Partial Purification ◽

Acute Myelocytic Leukemia ◽

Human Leukemia ◽

Folylpolyglutamate Synthetase ◽

Leukemia Cell Lines ◽

Short Term Exposure ◽

Long Chain

Abstract Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.

Download Full-text