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...

Chromosome changes accompanying differentiation and tumorigenesis in azacytidine- (azaC) and insulin-induced preadipocytes of the Chinese hamster embryo fibroblast cell line CHEF/18 are described. Karyotype analysis of 47 clones, subclones, and tumor-derived cells has shown that trisomy for chromosome 3q (mar 1) is characteristic of azaC preadipocytes but not of insulin preadipocytes. AzaC preadipocytes were consistently tumorigenic as well as trisomic for chromosome 3q, whereas most insulin preadipocytes were nontumorigenic and diploid. Only the few insulin preadipocytes that were tumorigenic were also trisomic for chromosome 3q. Among the tumor-derived cell lines recovered from azaC preadipocytes injected into nude mice, four had no additional chromosome changes except trisomy for 3q, as detected by karyotype analysis. Thus trisomy for 3q may be a sufficient chromosome change to induce tumor-forming ability in these cells. The rearrangements of chromosome 3 seen in this and other work pinpoint the trisomic region between the centromere and 3q5.

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serumfree medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, >90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.In embryonic development, secondary stem cells of mesenchymal origin give rise to a number of differentiated cell types including adipocytes, myoblasts, myotubes, chondrocytes, osteocytes, and adult fibroblasts. This process has been mimicked in cell culture by treating fibroblastic cells of embryonic origin with 5-azacytidine for as short a time as a single cell cycle (1-3). Indeed, the response of embryonic fibroblastic cell lines to this drug has provided the basis for their identification as secondary mesenchymal stem cells. Since 5-azacytidine decreases the extent of cytosine methylation in DNA, its effect on stem cells has suggested that decreased DNA methylation is involved in the differentiation process. The results reported here are part of an ongoing investigation of molecular mechanisms underlying specific steps in development and the distribution of stem cells into alternative pathways of terminal differentiation.In a previous report, we described the ability of 5-azacytidine to induce Chinese hamster embryonic fibroblastic (CHEF/18) ~ stem cells to become preadipocytes, myoblasts, or chondrocytes, and showed further that the same clonal CHEF/18 cell populations could be directed exclusively into the adipocyte pathway if the cells were grown in the presence J Abbreviations used in this paper: CHEF cells, Chinese hamster embryonic fibroblastic cells; FBS, fetal bovine serum; GPDH, snglycerol-3-phosphate dehydrogenase.

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