Characterization of the growth inhibited substate induced in murine hepatic tumor cells during in vitro exposure to dimethylsulfoxide

Kinetic events associated with the dimethylsulfoxide (DMSO)-induced inhibition of hepatic tumor cell proliferation were studied using established lines of murine liver tumor cells (BW77-2 and Hepa-1/A1) and conditions of polar solvent treatment (1–3% final concentration in the culture medium for a period of 4 days) previously shown to increase the expression of differentiated functions in BW77-2 cells. Cell-cycle substates of exponentially growing and DMSO-treated liver tumor cell populations were compared by flow cytometric techniques employing recently developed cytochemical criteria to identify hepatocyte cell cycle compartments based on individual cellular RNA and DNA contents (Higgins, 1985). Suppression of hepatic tumor cell proliferation by DMSO (in non-cytotoxic concentrations) persisted only for the duration of the exposure period. Resumption of cell division was readily observed following removal of the polar solvent from the culture medium. During DMSO treatment, BW77-2 and Hepa-1/A1 cells accumulated in the G, phase of the cell division cycle (low-population-density 3% DMSO-treated cultures were composed of 88% G1cells compared to only 48% G1 DNA content cells in control cultures of similar population density) and exhibited a substantial shift to lower mean cellular RNA content. The relatively few S-and G2+M-phase cells in DMSO cultures also possessed lower RNA contents compared to the corresponding cell cycle compartments in exponentially growing cultures. The mean RNA contents for the G1, S, and G2+M compartments of DMSO-treated cells approximated 63.8,78.6, and 74.4%, respectively, of the amounts observed in control cultures. Low-RNA G1 cells in DMSO cultures expressed a continuum of RNA distributions similar in range variation to (but at lower mean cellular RNA content levels than) cycling G1 cells in log-phase growth. Thus, G1 cells in 1% DMSO-treated populations had a mean cellular RNA content of just 25 (arbitrary RNA) units compared to over 40 units for G1 cells in exponential phase growth. Low RNA content, non-replicating, hepatic tumor cells in polar solvent-treated cultures were designated as being in the “Qi” substate (DMSO-induced quiescent-type cells). Release of BW77-2 cells from Qi, after replacement of the DMSO-containlng growth medium by medium without the polar solvent, was characterized by an increase in mean G1 RNA content and recruitment into log-phase growth. Since Qi cells predominate in DMSO-treated hepatic tumor cell cultures (approximating 90% of the total population) and are unresolved, under control exponential-phase growth conditions, it is likely that cells of the Qi phenotype participate in the augmentation of liver functions induced as a consequence of DMSO exposure.