Isolation of methylcholanthrene-"initiated" C3H/10T1/2 cells by inhibiting neoplastic progression with retinyl acetate.

A clone of 3-methylcholanthrene-treated 10T1/2 cells has been isolated which possesses basic characteristics expected of "initiated" cells. In the presence of retinyl acetate, this clone exhibits contact inhibited growth control and is morphologically indistinguishable from the parental 10T1/2 cell line. Removal of retinyl acetate in vitro results in neoplastic transformation after a latent period of 3 weeks. The classical 10T1/2 transformation system was reconstructed by coculturing normal and "initiated" 10T1/2 cells formed either Type II or Type III foci after a latent period of 3-4 weeks, and an additional 22% formed Type I foci. Treatment of "initiated" 10T/2 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate accelerated the formation of transformed foci in the coculture system by reducing the length of the latent period to less than 3 weeks. Injection of 10(6) "initiated" cells/mouse s.c. into nude mice resulted in the appearance of progressively growing fibrosarcomas after a latent period of 5-7 weeks. Dietary supplementation with 4-hydroxyphenyl-retinamide prevented tumor formation; after drug withdrawal, tumors developed in all surviving mice after 6 weeks. We believe this cell line possesses all characteristics expected of "initiated" cells. With this new cell line, designated INIT/10T1/2, we can now study the early biochemical changes in growth control mechanisms resulting in neoplastic transformation and the mechanism(s) of chemoprevention of cancer by vitamin A.

Effect of rhesus or vervet cytomegalovirus infection of DNA synthesis in untreated and 5-iodo-2'-deoxyuridine-arrested cells.

Infection of permissive cells with either rhesus or vervet cytomegalovirus resulted in suppression of cellular DNA synthesis, only viral DNA was resolved by isopycnic centrifugation after 24 h postinoculation. Infection of 5-iodo-2'-deoxyuridine (IUdR)-arrested cells with either of the simian cytomegaloviruses, however, induced synthesis of cellular DNA of normal density; synthesis of cellular DNA substituted with IUdR as evidenced by resolution of a heavy DNA peak after isopycnic centrifugation was not observed. Stimulation of DNA synthesis in IUdR-arrested cells was not observed with virus inactivated with UV light.

Blastogenic response of human lymphocytes to human cytomegalovirus.

A method was developed for measuring the blastogenic response of human lymphocytes to human cytomegalovirus (CMV). Viral and control antigens were prepared by extracting disrupted infected and uninfected cell cultures with an alkaline buffer. Lymphocytes from ten donors with complement-fixing (CF) antibody exhibited a blastogenic response, whereas cells from ten seronegative donors did not. A relationship between the stimulation index (SI) and the results of neutralization (NT), indirect haemagglutination (IHA) or CF tests was not observed. The maximum blastogenic response occurred after 5 to 7 days of incubation and was usually greater when the cultures were supplemented with homologous plasma instead of sera. The presence of CMV antibody in the supplementary sera did not appear to affect the reactivity of the lymphocytes.

Three-day assay for human cytomegalovirus applicable to serum neutralization tests.

A fluorescing cell assay (FCA) technique utilizing the indirect fluorescent-antibody method to measure human cytomegalovirus (CMV)-infected cells has been applied to the rapid determination of CMV-neutralizing antibody. Human sera with complement fixation titers to CMV of 1/32 or greater and fluorescein-conjugated rabbit anti-human globulin are the primary and secondary reagents in the fluorescent-antibody test. FCA measured in 3 days the same number of infectious units measured by plaque assay in 2 weeks. FCA and plaque assay yielded identical neutralizing antibody titers to CMV in 20 human sera.