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Transport of Methotrexate into Normal Haemopoietic Cells and into Leukaemic Cells and its Effects on DNA Synthesis
Author(s) -
Hoffbrand A. V.,
Tripp Edith,
Catovsky D.,
Das K. C.
Publication year - 1973
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1973.tb01762.x
Subject(s) - methotrexate , dna synthesis , aminopterin , thymidine , puromycin , dna , biology , theophylline , biochemistry , medicine , microbiology and biotechnology , endocrinology , chemistry , immunology , protein biosynthesis
S ummary . Methotrexate uptake by, and action on normal and leukaemic haemopoietic cells has been studied. Peak uptake occurred just prior to peak DNA synthesis, at 48 hr for normal, and at 6–7 days for chronic lymphocytic leukaemic lymphocytes stimulated by phytohaemagglutinin (PHA). Uptake of methotrexate into these cells and into normal bone marrow cells was consistently inhibited by dibutyryl cyclic 3′,5’adenosine monophosphate (dibutyryl cyclic AMP) at concentrations of 10 −3 to 10 −5 m and by inhibitors of phosphodiesterase (theophylline, puromycin aminonucleoside and puromycin). Lower external concentrations of dibutyryl cyclic AMP (10 −5 to 10 −6 m ), however, slightly increased the uptake of methotrexate. Dibutyryl cyclic AMP and the phosphodiesterase inhibitors at high concentrations also inhibited cellular uptake of 5‐methyltetrahydrofolate and of thymidine into DNA. Conversely, none of these compounds inhibited uptake of pteroylglutamic acid (folic acid). These results suggest that the rate of entry of methotrexate and reduced folates (but not of folic acid) into haemopoietic cells may be governed by the intracellular concentration of cyclic AMP and that uptake of methotrexate by cells is related to uptake of natural folates but not to that of folic acid. Methotrexate uptake was also rapidly inhibited by actinomycin D suggesting that it is closely coupled to DNA synthesis. Rate of entry of methotrexate into acute leukaemic blast cells was roughly proportional to their rate of proliferation, measured by 3 H‐thymidine ( 3 H‐TdR) incorporated into DNA. Resistant cells were not found to show a defect in methotrexate transport in relation to their degree of proliferation. Methotrexate was found to inhibit mitosis, but not transformation of PHA‐stimulated lymphocytes and this was presumably due to blocking of de novo thymidylate synthesis from deoxyuridylate, as shown by inhibition of incorporation of tritiated deoxyuridine ( 3 H‐dU) into DNA. Studies on human leukaemic blast cells showed differences between sensitive and resistant cases in the effect of methotrexate in vitro on 3 H‐dU and 3 H‐TdR incorporation into DNA and on dU blocking of 3 H‐TdR incorporation into DNA but none of these tests gave reliable prediction of resistance or sensitivity to methotrexate.