Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. II. Changes in the distribution of DNA content during the reversible transition between macrophage and nonmacrophage states in the cultures of tsA 640‐transformed macrophages

Abstract

Cultures of mouse macrophage cell lines transformed by wild-type or the tsA640 mutant of simian virus 40 (SV40) show a reversible phenotypic transition between the nonmacrophage (proliferating phase) and the macrophage (stationary phase) states (Takayama, 1980; Tanigawa et al., 1983). Distribution of DNA content in the cultures of the tsA640-transformed macrophage lines in the process of the phenotypic transition was determined by flow cytometry. Taking the mean DNA content of mouse peritoneal macrophages as 1 unit in the scale of fluorescence intensity in the flow cytogram, the transformed macrophages showed, at 33 degrees C, two peaks, one located around the 1.0-unit position (peak 1.0) and the other around the 1.6-unit position (peak 1.6), and a plateau distribution continuing to 3.2 units. Peak 1.0 was predominant in the stationary-phase culture, whereas peak 1.6 was predominant in the proliferating-phase culture. Almost the entire population of the strictly resting culture, which was obtained by culturing the stationary-phase culture for a further 5 days at nonpermissive temperature (39 degrees C), was phagocytic, and had accumulated at peak 1.0. Cells in peak 1.0 moved to peak 1.6 and to higher positions, after the strictly resting culture was sparsely reseeded and incubated at 33 degrees C. In contrast, the DNA content distribution of the successively proliferating cells, which were obtained by repeated passage of an extensively proliferating culture and none of which were phagocytic, was similar to that of proliferating hypotetraploid BALB/c3T3 fibroblasts with a G1 peak at 1.6 unit followed by a plateau containing S- and G2-phase cells. The peak 1.0 cell population appeared from the recloned population of the successively proliferating cells in company with the restoration of the culture condition-dependent phagocytic ability when cocultured with primary macrophages. Each peak in the flow cytogram reflected fairly well DNA content per cell as determined by other methods.

Induction of fibronectin expression, actin cable formation, and entry into S phase following reexpression of T antigen in mouse macrophages transformed by the tsA640 mutant of SV40

“…When these resting cells are replated at a low cell density and incubated at 33.0°C, the majority of the cells begin to express large T antigen by 24 hr, and the cells are increased in number by 4 days after shift down. In contrast, parallel cultures incubated at 39.0"C remain in the resting state without cellular changes detectable at 33.0"C (Tanigawa et al, 1983(Tanigawa et al, , 1984(Tanigawa et al, ,1985. In the present experiments, much the same results were obtained (Fig.…”

“…Temporal order of responses of tsA640-transformed macrophages to shifting the temperature to 33.0"C When confluent cultures of tsA640-transformed macrophage lines are further incubated at 39.0"C for 5 days, cells enter a resting state with a diploid DNA content, express no significant amounts of SV40 large T and small t antigens, and fully express the macrophage characteristics, such as immune phagocytosis (Tanigawa et al, 1983(Tanigawa et al, , 1984(Tanigawa et al, , 1985. When these resting cells are replated at a low cell density and incubated at 33.0°C, the majority of the cells begin to express large T antigen by 24 hr, and the cells are increased in number by 4 days after shift down.…”

“…To determine the effect of a nonpermissive temperature on maintenance of fibronectin and actin cable expression in proliferating cells, cells of lines SV-BB-4 and ,4640-%B-2 that had continuously been expressing fibronectin and actin cables were prepared by replating them in their proliferating phase repeatedly at 33.0"C. These successively proliferating cultures are composed of nonphagocytic and SV40 T antigen-positive cells (Takayama et al, 1981;Tanigawa et al, 1984). These proliferating cells were replated at a cell density of 5.0 x lo4 cells per 35-mm dish and incubated at 33.0"C or at 39.0"C for 3 days.…”

Alteration in cyclic adenosine 3′:5′‐monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature‐sensitive mutant (tsA640) of SV40

“…To determine the order of cellular responses after shifting to the permissive temperature, tsA640-transformed macrophages, line A640-BB-2, which had been incubated at the nonpermissive temperature of 39.0"C for 5 days, were replated at a low cell density and incubated at the permissive temperature of 33.0"C. As reported previously (Tanigawa et al, 1983(Tanigawa et al, ,1984 of the cultures at 39.0"C enter a resting state with a 2N DNA content and are negative for SV40 T antigen expression and positive for macrophage marker expression.…”

“…The macrophage lines established by transformation of mouse bone marrow cells by the tsA mutant of SV40 enter a resting state with a 2N DNA content, show a 0 1986 ALAN R. LISS. INC reduced amount of SV40 T antigen, and restore macrophage markers such as Fc-and complement-mediated phagocytosis when confluent cultures are shifted to a nonpermissive temperature (Tanigawa et al, 1983(Tanigawa et al, ,1984(Tanigawa et al, , 1985. When these resting cells are shifted from the nonpermissive temperature to a permissive temperature, tsA640 transformants become positive for SV40 T antigen in the majority of the cells by 24 hr, and then the number of cells begins to increase and nonphagocytic cells, like fibroblasts, appear in 4-5 days (Tanigawa et al, 1983).…”