Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Abstract

Treatment of normally tumorigenic murine tumor cell lines in vitro with chemical mutagens followed by cloning of the surviving cells, results in the selection, at extraordinarily high frequencies (anywhere from <1% to >90%), of clones unable to grow progressively in normal syngeneic mice (1-7). Such clones, which are phenotypically stable in culture over a period of several weeks or months, have been designated by Boon and his colleagues (1-6) as "tum-" (nontumorigenic in normal hosts). ~ They have been derived using such standard mutagens as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethyl methanesulfonate (EMS), from at least eight different murine tumors of viral, chemical, or spontaneous origin (1-7). Because the turn-clones will grow readily in highly immunosuppressed recipients, e.g., X-irradiated or nude mice (1-8), the turn-phenotype appears to have an underlying immunological basis, a view confirmed by detailed in vitro studies of T cell-mediated cytotoxicity (CMC).These studies have also shown that each turn-clone derived from a particular mutagenized parent line possesses an individual tumor-specific antigen distinct from the new antigen found on any other turn-clone, i.e., there is a startling degree of tumor antigen polymorphism (1-7). Of considerable interest is that this holds true even for totally non-immunogenic tumors of spontaneous origin (5-7) and as such, has very broad and important implications for the field of tumor immunology, especially in relation to the controversies surrounding the antigenic and immunologic status of neoplastic cells (9-11). Nevertheless, very little is known about how mutagens can cause such drastic and heritable changes in the behavioral properties of tumors in vivo, and at such high frequencies.

Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5‐azacytidine

“…Similar phenomena (referred to as "xenogenization" of tumor cells) were also produced by in vitro treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Boon and Kellermann, 1977;Van Pel et al, 1979;Uyttenhove et al, 1980;Boon, 1983;Frost et al, 1983Frost et al, , 1984Kerbel et al, 1984;Altevogt et al, 1985;Gorelik et al, 1985), ethyl methanesulfonate (EMS) (Frost et al, 1983Kerbel et al, 1984), the DNA hypomethylating agent, 5-azacytidine (5-aza-CR) Kerbel et al, 1984; Olsson and Forchhammer, 1984), or by in vivo treatment with an antileukemic drug, 5-(3,3-dimethyl-l-triazene) imidazole-4-carboxamide (DTIC) (Bonmassar et al, 1970). On the other hand, 5-aza-CR treatment was also reported to induce metastatic clones from non-metastatic tumor lines Olsson and Forchhammer, 1984).…”

Carcinogenicity and haemoglobin synthesis induction by cytidine analogues

“…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). 5-Azacytidine is a cell toxin and has been used to treat leukaemia (von Hoff et al, 1976) and can induce the expression of new cell surface antigens and thus increase the effectiveness of immunosurveillance in immunocompetent animals (Frost et al, 1984). Thus, there appear to be opposite sets of biological actions with respect to carcinogenicity.…”

“…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).…”

“…There has been speculation that DNA methylation changes may play some role in tumour initiation or progression (Holliday, 1979;Riggs & Jones, 1983;Nyce et al, 1983;Jones, 1986), and some experimental data are now available. Evidence that DNA methylation changes may be associated with tumorigenesis is as follows: (a) The DNA of tumour cells has often been found to be undermethylated compared to the DNA of normal cells (although exceptions have been found) (Lapeyre et al, 1981;Gama-Sosa et al, 1983;Goelz et al, 1985); the methylation patterns of some genes that are expressed in tumour cells have been found to be altered in association with increased gene expression (Feinberg & Vogelstein, 1983a, b;Cheah et al, 1984); (b) the DNA of tumour metastases is less-methylated than the DNA of primary tumours (Gama-Sosa et al, 1983); (c) the DNA of primary malignant tumours is less-methylated than the DNA of benign tumours (Gama-Sosa et al, 1983); transformed cells have been shown to have their metastatic properties altered by the potent inhibitor of DNA methylation, 5-azacytidine (Olsson & Forchammer, 1984;Trainer et al, 1985;Ormerod et al, 1986); (e) several carcinogens have been shown to cause demethylation (Boehm & Drahovsky., 1979Salas et al, 1979;Wilson & Jones, 1983); (f) 5-azacytidine, an inhibitor of DNA methylase, has been shown to be a carcinogen in some experimental animal studies (Denda et al, 1985;Stoner et al, 1973;National Cancer Institute 1978;Carr et al, 1984;Vesely & Cihak, 1973).…”

Modulation and biological effects of Ly-6.2 expression on EL4 tumour cells

“…The increased expression of Ly-6.2 is thus a specific event during the metastatic cascade and is applicable to the tumour population as a whole. The mechanism for this increase may be enhancement or induction of mRNA analogous to the proposed mechanism for interferon induced modulation of major histocompatibility complex (MHC) gene products (Rosa et al, 1985) or other mechanisms leading to activation or expression of genes such as hypomethylation of DNA (Frost et al, 1984). Gradual hypomethylation due to local in vivo environmental conditions may result in the expression of genes which are involved in tumour progression and increased metastatic aggressiveness.…”