Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.

Abstract

5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 ,LM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subelones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumorforming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I-and Hpa lI-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.The pyrimidine analog 5-azacytidine (azaC) is a potent effector of decreased DNA methylation in mammalian cells. azaC has been shown to substitute for cytosine residues in DNA (1) and to interfere with maintenance methylation (2) We report here that azaC is a potent carcinogen for the Chinese hamster embryo fibroblast cell line CHEF/18, which we have previously shown to be stably diploid (9, 10), nontumorigenic (9), and resistant to one-step induction of tumor-forming ability by chemical mutagens and carcinogens (11). We previously found further that CHEF/18 cells could be induced by a single short treatment with azaC to differentiate into myoblasts, adipocytes, or chondrocytes (6) as in other fibroblastic cell lines of embryonic origin (4,5). In this paper we demonstrate that the cloned and recloned populations of azaC-induced pre-adipocytes that can be driven to differentiate into mature adipocytes by growth with insulin (12) can also give rise to spindle-cell sarcomas when injected into nude mice. Specific changes in DNA methylation patterns as well as a particular chromosomal change have been found in the tumorigenic and tumor-derived cells. The results suggest that decreased DNA methylation is a molecular mechanism involved both in terminal differentiation and in tumorigenesis and that azaC may induce chromosomal changes as well. These complex effects of the drug pose a serious dilemma for the clinician who must balance therapeutic effectiveness against the carcinogenic threat. MATERR...

5-Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis

“…However, several observations support the notion that the hypomethylating effect of 5-AC is responsible for the induction of cell transformation: the results of Walker and Nettesheim (1986) show that the analog 6-azacytidine, which does not cause DNA hypomethylation, did not transform rat tracheal epithelial cells. Hsiao et al (1985) and Harrison et al (1983) have shown that 5-AC can neoplastically transform established cell Iines, suggesting that changes in methylation occurring during later stages of transformation can result in the conversion of immortal cells to neoplastic cells. Further characterization of the process of malignant progression induced by 5-AC and related compounds, and molecular analysis of immortalized and neoplastic cells seem necessary.…”

“…A block in the G 2 phase of the cell cycle was also reported by Zatsepina et al (1989) in pig kidney cells. 5-AC transformed cultures of 10T1 /2 and M2 mouse fibroblasts (Benedict et al, 1977;Harrison et al, 1983), primary rat tracheal epithelial cells (Walker and Nettesheim, 1986) and Balbjc-3T3 cells (Lubet et al, 1990).…”

Carcinogenicity and haemoglobin synthesis induction by cytidine analogues

“…The results of 5-azacytidine action are expected to be complex. On the one hand, 5-azacytidine is capable of enhancing or inducing the metastatic capacity of various tumour cell lines (Olsson & Forchammer, 1984;Trainer et al, 1985;Ormerod et al, 1986), activating silent retroviral genomes (Jaenisch et al, 1985), enhancing the induction by various carcinogens of gamma-glutamyltransferase positive liver foci (Denda et al, 1985), of altering cellular DNA which is incapable of inducing transformation (Venolia et al, 1982), and of inducing tumorigenesis in various cells in culture (Venolia et al, 1982;Harrison et al, 1983;Benedict et al, 1977;Marquardt & Marquardt, 1977), even though it does not appear to be a significant mutagen in mammalian cells (Landolph & Jones, 1982;Frost et al, 1984;Momparler et al, 1984;Delers et al, 1984;Bouck et al, 1984;Jones, 1984). On the other hand, 5-azacytidine has been shown to induce differentiation in both non-transformed as well as in neoplastic cells in culture (Constantinides et al, 1977(Constantinides et al, , 1978Jones & Taylor, 1980;Walker et al, 1984;Creusot et al, 1982;Darmon et al, 1984;Pinto et al, 1984).…”

Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes.

“…De novo methylation has been observed principally with integrated viral genomes (6,26) and has been attributed to site-specific methyltransferase activity (6) or to the appearance of new chromatin conformations that invite methylation by a nonspecific enzyme (2). Methylation losses have been reported during development (26), in terminal differentiation (6), and in tumorigenesis (9,13) and have been attributed to dilution or inactivation of a preexisting enzyme (6) or to the presence of a demethylating enzyme (10). The dynamics of DNA methylation, how changes occur in methylation patterns at specific sites, remains largely unknown.…”