Interference with DNA Methyltransferase Activity and Genome Methylation during F9 Teratocarcinoma Stem Cell Differentiation Induced by Polyamine Depletion

Abstract

When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by ␣-difluoromethylornithine, F9 teratocarcinoma stem cells are depleted of putrescine and spermidine and as a result differentiate into a cell type which phenotypically resembles the parietal endoderm cells of the early mouse embryo. Simultaneously the level of decarboxylated S-adenosylmethionine (dcAdoMet), the aminopropyl group donor in spermidine and spermine synthesis, increases dramatically, as the aminopropyl group acceptor molecules (putrescine and spermidine) become limiting. When this excessive accumulation of dcAdoMet is prevented by specific inhibition of the AdoMet decarboxylase activity, the differentiative effect is counteracted, despite the fact that the extent of polyamine depletion remains almost identical. Therefore, it may be concluded that dcAdoMet plays an important role in the induction of differentiation. Moreover, this key metabolite acts as a competitive inhibitor of DNA methyltransferase and is therefore capable of interfering with the maintenance methylation of newly replicated DNA. During the course of F9 cell differentiation, the highly methylated genome is gradually demethylated, and its pattern of gene expression is changed. Our present findings, that the DNA remains highly methylated and that the differentiative process is counteracted when the build-up of dcAdoMet is prevented, provide strong evidence for a causative relation between the level of dcAdoMet and the state of DNA methylation as well as cell differentiation.Cells require optimal levels of the polyamines, putrescine, spermidine, and spermine, for their growth and differentiation and are therefore equipped with many intricate mechanisms for the control of these levels (1, 2). The polyamine biosynthetic pathway consists of two highly regulated enzymes, ornithine decarboxylase (ODC) 1 and S-adenosylmethionine decarboxylase (AdoMetDC), and two constitutively expressed enzymes, spermidine synthase and spermine synthase (Fig. 1). Any cell that becomes polyamine-deficient is severely limited in its ability to grow and proliferate. There is also evidence suggesting that polyamines play a role in apoptotic cell death (3, 4). More specifically, the polyamines have been shown to regulate ion channel gating (5-7) and polyamine derivatives are constituents of venoms produced by many invertebrates (e.g. funnel-web spiders), where they act as potent neurotoxins (8). Polyamines also regulate programmed translational frameshifting, which is essential for the expression of ODC antizyme, a protein involved in the regulation of polyamine levels both in yeast (9) and in mammalian (10) cells.Of particular interest, with regard to growth regulation, is the finding that when ODC genes are transfected into cells and overexpressed, the cells go through malignant transformation (11). The fact that inhibition of the ODC activity counteracts malignant transformation suggests that the ODC gene is a proto-oncogene. In this co...

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme

“…Despite the overall tendency toward global DNA hypomethylation in cancer, it remains as a paradoxical mystery of coexisting DNA-MTase increases and genomic hypomethylation. Several studies show increased overall DNA methylation in cancer cells (Belinsky et al, 1996;Frostesjo et al, 1997). Global DNA hypomethylation resulting from reduction of DNA-MTase activity suppressed intestinal neoplasia in mice (Laird et al, 1995).…”

“…adi¯uoromethylornitine (DMFO), retinoic acid and prostaglandin synthesis inhibitors reduce skin tumor development in carcinogen-treated mice (Hickok and Uitto, 1992;Mao et al, 1993;Takigawa et al, 1983;Verma et al, 1980;Weeks et al, 1982). Suppression of ODC activity in DMFO-treated mouse F9 teratocarcinoma cells and human Caco-2 colonic cancer cells induces polyamine depletion, terminal dierentiation and genome-wide DNA hypomethylation (Duranton et al, 1998;Frostesjo et al, 1997).…”

Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota

“…The gut firmicute Butyvibrio crossotus possesses CASDH, CASDC, and SpdSyn orthologues and has been shown previously to accumulate spermidine as its only polyamine (18). An active AdoMetDC in the absence of an aminopropyltransferase like SpdSyn would lead to an accumulation of decarboxylated S-adenosylmethionine, a potent inhibitor of methyltransferases (34). The CASDH/CASDC pathway appears to have replaced the AdoMetDC/SpdSyn pathway in many gut Firmicutes species, with the SpdSyn remaining as a remnant, although it is possible that the SpdSyn orthologue has acquired a new function.…”

Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells

“…Inhibition of ODC by DFMO prevents adipocyte differentiation of 3T3-L1 cells [14,15]. However, DFMO stimulates early cardiac commitment of mesenchymal stem cells [16] [ [17][18][19] and F9 teratocarcinoma stem cells [20][21][22]. Besides, ODC is active during hair follicle development and hair growth [23].…”