s-adenosylhomocysteine and Leukemia--Erythroblastic--Acute

DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in KmDNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Double-reciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dI.dC-dI.dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M. HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.

Topics: Animals; Binding, Competitive; Catalysis; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Isotope Labeling; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Polydeoxyribonucleotides; S-Adenosylhomocysteine; S-Adenosylmethionine; Structure-Activity Relationship; Substrate Specificity; Tumor Cells, Cultured

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

Topics: Actins; Adenosine; Animals; Cell Differentiation; Cell Division; Cycloleucine; Dimethyl Sulfoxide; Erythrocytes; Erythropoiesis; Globins; Hemoglobins; Kinetics; Leukemia, Erythroblastic, Acute; Methylation; Mice; Neoplasm Proteins; Peroxidases; Peroxiredoxin III; Peroxiredoxins; Proto-Oncogene Proteins c-myc; Ribonucleotides; RNA Caps; RNA, Messenger; S-Adenosylhomocysteine; S-Adenosylmethionine; Tumor Cells, Cultured

We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block commitment of murine erythroleukemia (MEL) cells to terminal erythroid maturation. In this study, we further investigated the mechanism of this blockade. Treatment of MEL cells with N6mAdo inhibited cell growth, prevented accumulation of committed cells, suppressed methylation of total cytoplasmic RNA, and erased the expression of "memory" response, an event that precedes initiation of commitment. Furthermore, N6mAdo increased the level of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and altered the SAH/SAM ratio that influences methylation of ribonucleic acid (RNA). Moreover, analysis of the intracellular extracts revealed that N6-mAdo is converted into S-(N6-methyl)-adenosylhomocysteine (N6-SAH) in MEL cells, an active intermediate that affects methylation of RNA. Therefore, we conclude that N6-mAdo prevents induction of MEL cell differentiation by affecting methylation of critical RNA transcripts involved in expression of "memory" and initiation of commitment. It is likely that this inhibition occurs via conversion of N6mAdo into N6-SAH.

Topics: Adenosine; Animals; Cell Differentiation; Cell Division; Kinetics; Leukemia, Erythroblastic, Acute; Methylation; Mice; RNA; S-Adenosylhomocysteine; S-Adenosylmethionine; Tumor Cells, Cultured

The results above show that mammalian cells, as exemplified by MELC, can be selected to be resistant to Adox, and that the resistant cells have greatly decreased nucleoside transport capacity. Since no mutagen was used prior to the selection process and Adox resistance was genetically stable, it appears that within a population of normal cells there is a genetically controlled range of expression of the nucleoside transporter. On the basis of the present data we cannot determine if the low level of nucleoside transporter in AR MELC is due to an altered form of the protein or a decreased amount of the normal protein. However the similarity of the Kd for NBTI in normal and AR MELC suggests that the latter is the case. Considerable indirect evidence is presented that Adox, with its ribose converted to an acyclic dialdehyde, is a substrate for the nucleoside transporter, adding a new type of compound to this list.

Topics: Adenosine; Animals; Carrier Proteins; Cell Division; Drug Resistance; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Membrane Proteins; Mice; Nucleoside Transport Proteins; S-Adenosylhomocysteine; Thioinosine; Tumor Cells, Cultured

Recent studies have demonstrated that 5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase, blocks induction of murine erythroleukemia cell (MEL) differentiation. The nucleoside analogue 3-deazaadenosine (c3Ado) is both an efficient substrate and a potent inhibitor of AdoHcy hydrolase. The present study was undertaken to determine whether c3Ado would similarly inhibit MEL differentiation. The results demonstrate that c3Ado inhibits induction of MEL differentiation by dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, and diazapam. c3Ado blocks the appearance of the differentiated MEL phenotype by inhibiting both MEL heme synthesis and transcription of alpha- and beta-globin RNA. The inhibitory effect of c3Ado on MEL differentiation is concentration dependent, reversible, and potentiated by L-homocysteine thiolactone. Furthermore the AdoHcy/AdoMet ratio increases nearly 3.5-fold after 24 h of treatment with 50 microM c3Ado. In contrast, this c3Ado effect is not associated with polyamine depletion or cytostasis. These findings indicate that c3Ado blocks the induction of MEL differentiation at a transcriptional level and that this effect may be related to inhibition of AdoHcy hydrolase.

Topics: Adenosine; Adenosylmethionine Decarboxylase; Animals; Cell Differentiation; Cell Line; Deoxyadenosines; Dimethyl Sulfoxide; DNA; Leukemia, Erythroblastic, Acute; Methylation; Mice; Ribonucleosides; S-Adenosylhomocysteine; S-Adenosylmethionine; Thionucleosides; Tubercidin