Masumi Ohtsu

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1

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‡C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5‡ C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8‡ 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.

Accumulation of p60lck in HTLV-I-transformed T Cell Lines Detected by an Anti-Lck Monoclonal Antibody, MOL 171

The lck gene encodes a protein tyrosine kinase of nonreceptor type, p56lck, whose expression occurs almost exclusively in T lymphocytes. MOL 171, an anti‐p56lck monoclonal antibody, was produced by using a 25‐amino‐acid synthetic polypeptide as the antigen, its structure corresponding to the N‐terminal region deduced from the lck cDNA sequence. Immunoblot analysis with MOL 171 showed the accumulation of 60 kD form of Lck protein, p60lck, and the decrease of p56lck in human T cell leukemia virus type I (HTLV‐I)‐transformed T cell lines. Another anti‐Lck monoclonal antibody, MOL 294, raised by using a synthetic peptide corresponding to the C‐terminal region deduced from the lck cDNA sequence, also detected the accumulation of p60lck in those HTLV‐1‐transformed T cell lines. The appearance of p60lck with the decrease of p56lck in normal T lymphocytes after stimulation suggested the origin of p60lck in HTLV‐I‐transformed T cells.

Ineffective control of murine cytomegalovirus by IE1-specific cytotoxic T lymphocytes during protracted infection in the lung

Summary. Interstitial pneumonia caused by cytomegalovirus (CMV) is a fatal disease in immunocompromised patients. In order to examine the defense mechanism against the virus in the lung, we employed an intratracheal infection model in susceptible mice. In mice infected intratracheally with murine CMV, a protracted infection was observed where infectious virus was detected up to 21 days of infection. During this prolonged infection, massive accumulation in the lung of CD8+ T cells with activated phenotypes occurred and these CD8+ T cells showed direct ex vivo cytolytic activity against target cells pulsed with the nonamer peptide derived from IE1 protein of the virus, which has been shown to be the dominant epitope recognized by most of virus-specific CTL. Moreover, adoptive transfer of in vitro induced IE1 peptide-specific CTL line showed no anti-virus effect in the lung, although they were effective in the spleen. Hence, there is reason to assume the IE1-specific CTL induced in vivo or in vitro plays limited roles during the prolonged infection in the lung.

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

SummarySodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.

The high sensitivity of cells transformed by E1A gene of adenovirus type 12 to diacylglycerol-mediated cell killing

Viability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was markedly impaired by the administration of dilinoleoylglycerol (DLG) to the culture medium. To identify the gene(s) of Ad12 responsible for the high sensitivity to DLG, we established several transformed sublines of 3Y1 induced by the viral E1A gene or by the mutants of Ad12 which have mutations in the E1A region. All of the transformed sublines of 3Y1 expressing either the 12S or the 13S message from the E1A region were highly sensitive to the cytotoxicity of DLG. We propose that the high sensitivity of Ad12-transformed cells to the DLG mediated cytotoxicity is attributable to the common function of E1A-12S and E1A-13S mRNA products.

Induction of Interleukin 2-Responsiveness in Thymocytes of the Transgenic Mice Carrying lck -Transgene

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H‐2Kb and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell‐surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL‐2, but not the thymocytes from negative littermates. Further analysis revealed that CD4+8– single positive thymocytes proliferated in response to IL‐2. While surface expression levels of IL‐2Rα and IL‐2Rβ of these CD4+8– thymocytes from transgenic and control mice were almost equal before stimulation with IL‐2, the expression of IL‐2Rβ was induced only in transgenic thymocytes after stimulation with IL‐2. Immunoblot analysis demonstrated that the expression of p56lck of transgenic thymocytes was not down‐reguated at 4 hr after stimulaion with IL‐2, whereas p56lck of control ones were not detectable any more at 4 hr after stimulation with IL‐2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56lck in the thymocytes from transgenic mice: the kinase activities of p56lck did not decrease in thymocytes from transgenic mice after stimulation with IL‐2, while kinase activities of control ones were significantly down‐regulated by stimulation of IL‐2. These results suggested that a significant proliferative response found in the thymocytes from lck‐transgenic mice after the stimulation with IL‐2 was caused by a constitutive expression of p56lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56lck may play a role in the IL‐2R‐mediated signaling system in CD4+8– thymocytes.

Frequency of Cell Transformation by the Small DNA Tumor Viruses: Infection of Proliferating Cells and Quiescent Cells

Small DNA‐containing tumor viruses (simian virus 40, mouse polyomavirus, and adenoviruses) malignantly transform fibroblasts of the susceptible rodents. Fibroblasts can exist, in vitro and in vivo, in either of the two states: the proliferating state or the quiescent state. In the present study, we examined whether the state of fibroblasts at the time of exposure to these DNA viruses affects the frequency of transformation. Dense‐focus formation in monolayer culture of rat 3Y1 fibroblasts was used to quantitate transformation. Results show that the frequency of transformation by simian virus 40 and mouse polyomavirus was reduced when cells were in the proliferating state at the time of virus inoculation as compared to cells in the quiescent state, whereas that by adenovirus type 12 was similar in the two cellular states. The reduction of the frequency of transformation in proliferating cells infected with simian virus 40 was also observed in BALB/c 3T3 mouse cells. Mechanisms underlying the difference between the two cellular states and the difference between the papovavirus and adenovirus in this aspect of transformation remain to be investigated.