Inhibition of morphological transformation induced with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in cultures of hamster embryo cells by 5′‐bromo‐2′‐deoxyuridine‐photolysis

Abstract

The present study was performed in order to determine whether type III transformed foci induced by N-methyl-N'-nitro-N-nitrosoguanidine originate from the small subpopulation of cells stimulated by the carcinogen to enter DNA synthesis. During the last 30 min of variable treatment periods using different doses of N-methyl-N'-nitro-N-nitrosoguanidine, administered alone or in association with the thymidine analogue, 5'-bromo-2'-deoxyuridine (0.98 x 10(-5)M), the density-inhibited monolayers of hamster embryo cells were exposed to fluorescent light and then assayed for abnormal growth patterns by the focus formation method. Mock-irradiated cultures as well as monolayers whose medium lacked N-methyl-N'-nitro-N-nitro-soguanidine, 5'-bromo-2'-deoxyuridine, or both, served as controls. The cytotoxicity of 5'-bromo-2'- deoxyuridine + N-methyl-N'-nitro-N-nitrosoguanidine + photolysis (BMP) protocol on confluent as well as logarithmically growing hamster embryo cells was estimated in single-cell survival experiments. Plating efficiency determinations have demonstrated that, unlike their actively growing counterparts, confluent hamster embryo cell monolayers are extremely resistant to the cytotoxic effects of the BMP protocol. The quantitative transformation assays indicated that: (1) in non-illuminated cultures addition of 5'-bromo-2'-deoxyuridine to carcinogen-containing medium does affect transformation frequency of hamster embryo cells in the sense that the incidence of type III foci did not subside at later intervals during the post-carcinogen administration period as it did in the absence of the analogue; (2) irradiation of N-methyl-N'-nitro-N-nitrosoguanidine and halogenated pyrimidine analogue-treated cultures with fluorescent light practically suppressed transformation; (3) analogue-added and analogue-removed experiments pointed out that the event(s) on which 5'-bromo-2'-deoxyuridine fluorescent light sensitization of morphological transformation largely depends, takes place between 5 and 15 h after N-methyl-N'-nitro-N-nitrosoguanidine administration, i.e., during the period of maximal carcinogen-stimulated DNA synthesis; and (4) neither fluorescent light nor 5'-bromo-2'-deoxyuridine, singly or in combination, were able to transform cultures of hamster embryo cells. These findings are strong indirect arguments for the concept that carcinogen-induced DNA synthesis and the initiation of transformed clones are causally related.

Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.

“…4) nervous system to ENU induction of neuroectodermal tumors is associated with the development and cellular proliferation of the nervous system (4). Similarly, DNA replication is required for induction of transformation by methylating carcinogens in vitro (16,17). When density-inhibited cultures of HEC are treated with MNNG, a small cell population is released from a Go block and proceeds through a full round of S phase synthesis; transformation occurs only in the replicating population (16).…”

“…Similarly, DNA replication is required for induction of transformation by methylating carcinogens in vitro (16,17). When density-inhibited cultures of HEC are treated with MNNG, a small cell population is released from a Go block and proceeds through a full round of S phase synthesis; transformation occurs only in the replicating population (16). Because DNA replication is considered a requirement for carcinogenesis, the effects of methylating carcinogens on DNA replication was studied.…”

Initiation of carcinogenesis dependence upon MNNG-induced release of the G1 block of density-inhibited syrian hamster cells

“…After MNNG (0.5 pg/ml) 30% of the cells were in S as compared to 5% for nontreated control cultures as determined by autoradiography. By 35 h post-MNNG the level of cells in S approached that of the controls [3]. Reseeding the treated DDIR cultures for either colony or focus assay within 8-30 h posttreatment resulted in neoplastical transformation.…”

“…Since the size of the [3H]-thymidine-responsive subpopulation correlated with the transformation frequency in dose-response experiments, it was postulated that the transformants were derived from this subpopulation [2]. This was confirmed in experiments utilizing BrdUrd photolysis, which specifically eliminates this population [3]. BrdUrd was incorporated into the DNA of only the [W]-thymidine-incorporating cells when MNNG-treated DDIR were incubated in its presence.…”

“…The size of the [3H]-thymidine-responsive population correlated with the number of transformants in time of exposure and dose-response experiments [2, 31. Furthermore, the positive relationship between the thymidine-incorporating population and transformation was shown by a 5-bromodeoxyuridine (BrdUrd) photolysis protocol [3], indicating a specific requirement for DNA synthesis. Neither the nature nor the extent of DNA synthesis is clear; no data exist to indicate whether thymidine incorporation is due to repair replication or semiconservative DNA synthesis.…”