A. V. Hoffbrand

Transport of Methotrexate into Normal Haemopoietic Cells and into Leukaemic Cells and its Effects on DNA Synthesis

Summary. 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 10−5m and by inhibitors of phosphodiesterase (theophylline, puromycin aminonucleoside and puromycin). Lower external concentrations of dibutyryl cyclic AMP (10−5 to 10−6m), however, slightly increased the uptake of methotrexate.

Uptake of Vitamin B12 by Phytohaemagglutinin-Transformed Lymphocytes

Phytohaemagglutinin (PHA)‐transformed lymphocytes have been used as a model cell system to study the uptake of radioactive vitamin B12 by haemopoietic cells. Both mature granulocytes and PHA‐transformed lymphocytes took up more vitamin B12 than mature, non‐transformed lymphocytes. Uptake of vitamin B12 by PHA‐stimulated lymphocytes was greatest at 48–72 hr of culture, i.e. at about the time or just before the time of peak DNA synthesis.

Unbalanced deoxyribonucleotide synthesis caused by methotrexate.

The effect of methotrexate on the free intracellular pools of thymidylate triphosphate (dTTP) and deoxyadenosine triphosphate (dATP) in normal human phytohaemagglutinin-transformed lymphocytes has been studied. Methotrexate caused a fall in the dTTP pool ranging from 38% to 88% and a rise in the dATP pool ranging from 24% to 185%. A rise in the free intracellular pool of dATP is thought to inhibit both rubonucleotide reduction and polynucleotide ligase, an enzyme concerned in DNA synthesis and repair. The hypothesis is suggested here that folate deficiency per se, as well as a functional folate deficiency induced by methotrexate may cause reduced DNA synthesis, megaloblastic changes, and chromosome abnormalities by producing a rise in the free intracellular pool of dATP as well as by causing a fall in free intracellular dTTP.

Sephadex-gel filtration and heat stability of human jejunal and serum pteroylpolyglutamate hydrolase (folate conjugase). Evidence for two different forms.

Abstract Pteroylpolyglutamate hydrolase (PPH, folate conjugase, γ-glutamyl-carboxypeptidase) is an enzyme intimately concerned with folate absorption and with transport of folate from cells to fluid compartments of the body. The apparent molecular weight of human jejunal PPH determined by Sephadex G-100 and G-200 filtration is 83,000 and of the human serum determined by the same techniques is 110,000. Further evidence which suggests that PPH exists in human tissues at least in two different forms is provided by heat-stability studies, since the intestinal enzyme is stable on heating at 50°C whereas serum PPH is rapidly inactivated to only 30% of its original activity by heating to 50°C. Further studies are now needed of purified human PPH to determine its exact MW and to clarify whether it exists in one or more forms.