Alterations in cellular characteristics associated with transformation were compared among various agents, including simian virus 40, mouse polyoma virus, adenovirus type 12, the ElA region of adenovirus type 12, Rous sarcoma virus, v-Hras oncogene, and N-methyl-N'-nitro-N-nitrosoguanidine. To avoid the difficulty in interpretation due to differences in the original cellular characteristics, all transformed lines (three to four independent lines for each agent) were derived from the same parental line, i.e. rat clonal diploid fibroblasts 3Y1. Doubling time, saturation density, serum requirement, efficiency of colony formation, Ca dependency, anchorage dependency, cell volume, density-dependent inhibition of proliferation, and alteration of morphology were examined. In the lines transformed either by DNA virus, RNA virus, or transforming oncogene, the characteristics were similar among the lines induced by the same transforming agent but distinguishable between the lines induced by different agents. In such cases, a transformed line displayed phenotypes specific to each transforming agent. In contrast, nitrosoguanidine-transformed lines showed different properties. Only one characteristic was shared by all the transformed lines: the ability to attain a higher saturation density than that of the untransformed parent. The fact that (cellular) densitydependent inhibition of DNA synthesis became weak may be responsible for this characteristic.

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5 degrees C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5 degrees C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8 degrees C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, shows a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 compared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synthesis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased markedly depending on the incubation temperature, compared with that in 3Y1, indicating that the lowered levels of D123 protein in 3Y1tsD123 are due to its degradation. Unexpectedly, 2 stably temperature-resistant clones were isolated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein. The D123 protein in both clones degraded extensively at both temperatures, suggesting that the overexpression of the mutant-type D123 protein exceeds its degradation. Both temperature-resistant clones contained higher levels of D123 protein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restrictive temperature induces the temperature-sensitive characteristics of 3Y1tsD123 and SV-3Y1tsD123.

An extensive cytopathic effect occurred in the CD4+ cells expressing gpl60 of human immunodeficiency virus. A protein capable of nuclear location that was microinjected into such cells was not transported into the nuclei at an early stage when little cytopathic effect had yet to occur.

Cytomegalovirus (CMV) causes severe clinical manifestations in immunocompromised hosts; however, it remains unclear whether the virus itself is a cause of immunosuppression or whether it is involved as an opportunistic bystander pathogen. This study was performed to elucidate the effect of CMV infection on the host's immune system. The double-positive thymocytes of BALB/c mice inoculated with a sublethal dose of murine CMV (MCMV) were extensively depleted by a 10-,ug amount of anti-CD3 monoclonal antibody, while such an amount was unable to induce any apparent elimination of thymocytes in noninfected mice. In immature thymocytes of infected hosts, a markedly high level of susceptibility to apoptosis induction was found on treatment with anti-CD3 monoclonal antibody. Analysis of the signal transduction pathway of such double-positive thymocytes demonstrated a profound elevation of the intracellular Ca2+ level after anti-CD3 stimulation, implying that this aberrant mobilization of Ca2+ plays a crucial role in the signaling pathway leading these cells to an extensive apoptosis. Examination of the thymus by PCR was able to detect a low copy number of MCMV DNAs in thymic stromal cells but none at all in thymocytes. Therefore, it is suggested that a mechanism which is not associated with virus replication within the cells exerts a critical effect on rendering the thymocytes highly apoptosis sensitive in hosts infected with MCMV.