Analysis of the inhibition of commitment of murine erythroleukemia (MEL) cells to terminal maturation by N6-methyladenosine

Abstract

Treatment of cultured murine erythroleukemia (MEL or Friend) cells with N 6-methylated derivatives of adenosine inhibited erythroid cell differentiation induced by various agents. N 6-Methyladenosine (N 6mAdo) inhibited initiation of commitment to terminal maturation and prevented accumulation of hemoglobin in a concentration-dependent manner. Treatment with N 6mAdo slowed cell growth without causing substantial inhibition in the rate of DNA synthesis and a marked decrease in viability and clonogenic potential of MEL cells. Furthermore, N 6mAdo decreased the cytoplasmic accumulation of β major globin mRNA and affected its structural integrity in MEL cells. Cells preexposed to N 6mAdo failed to initiate commitment as early as control cells upon challenge with the inducer dimethyl sulfoxide. N 6mAdo-induced inhibition of commitment was not reversed but rather was potentiated by the presence of adenine, l-homocysteine and/or l-methionine, agents involved in the active methylation cycle. To this respect, N 6mAdo-induced inhibition of commitment was found to be different from that caused by cordycepin (3'-deoxyadenosine, an inhibitor of RNA methylation and mRNA polyadenylation). The latter inhibition was fully reversed by the addition of l-methionine. These findings indicate that N 6-methyladenosine: (a) blocks a central process that is required for initiation of commitment; and (b) decreases accumulation of β major globin mRNA, causes mRNA degradation and prevents hemoglobin synthesis. Due to the differential sensitivity of N 6mAdo- and cordycepin-induced blockade of commitment to l-methionine, these agents inhibit commitment by acting via two different mechanisms impinging on the final pathway of MEL erythroid cell maturation.