AU-trans-fi-retinoic acid (RA) has both comitogenic and antiproliferative effects on Retinoids are derivatives ofvitamin A, many ofwhich have been shown to have potential value as cancer-chemopreventive (1) or -therapeutic (2) agents. It has been suggested that these potentialities could relate, in part, to direct effects of retinoids on tumor cells (1-3). There is evidence that retinoids can modify patterns ofcell proliferation in vitro. A large number ofcell lines have been examined, yielding data indicating that retinoids can be inhibitory (4-9), ineffectual (4, 5, 7), or stimulatory (5,8,(10)(11)(12).The mechanisms by which retinoids modulate cell proliferation of various cell lines in opposite ways are unknown. We, and others, have approached this question by examining the effects of retinoids on the response of untransformed quiescent murine 3T3 cells to mitogens. These experiments have led to reports ofretinoid potentiation ofthe mitogenic response of3T3 cells to various growth factors and the tumor promoter, phorbol 12-myristate 13-acetate (PMA) (12, 13). We have extended these studies and now report data that clarify the relationship between retinoid potentiation of the 3T3-cell mitogenic response and the long-term effects of retinoids on 3T3-cell proliferation. Our results suggest that continuous treatment of3T3 cells with all-trans-,B3retinoic acid (RA) results in inhibition of DNA synthesis in S-phase cells and prevents a proliferative manifestation of the enhanced mitogenic response observed with shorter exposures. The relevance of serum concentration to RA effects on 3T3-cell proliferation is also considered as is altered density inhibition of cellular proliferation and its reversibility. MATERIALS AND METHODSCells and Tissue Culture. The origin and history of the murine Swiss 3T3 cells used in this study have been described (14). Cells were grown at 370C in Dulbecco's modification of Eagle's minimal essential (DME) medium supplemented with various concentrations of fetal calf serum, penicillin at 250 units/ml, and streptomycin at 250 jig/ml under humidified 90% air/ 10% CO2. All medium components were obtained from GIBCO. Cells were passaged every 3 to 4 days by dispersion with trypsin/EDTA (15), replated at 5.5 x 103/cm2, and used between passages 8 and 15 in our laboratory. These cells have been demonstrated to be free of Mycoplasma contamination (15).Studies of Cell Proliferation. Cells were plated at 2 X 104 per 35-mm-diameter tissue culture dish in DME medium/ 10% serum. After an overnight attachment period, cells were changed to various test media. Cell numbers were determined for duplicate cultures at 24-hr intervals by duplicate cell counts with an electronic particle counter (Coulter Electronics, Hialeah, FL). Cell-doubling times were calculated for the period of exponential growth 24-72 hr after addition of test media and represent the 48-hr period divided by the number of cell doublings (log2 cell number at 72 hr minus log2 cell number at 24 hr) (16).Assay of DNA Synthesis. I...
Exposure of Swiss 3T3 cells to micromolar quantities of beta-all-trans-retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5'-triphosphate (ATP) are markedly increased after 48-hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitor effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle-dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [3H]thymidine incorporation and labeling indices in intact cells and [3H]dTTP incorporation and labeling indices in isolated nuclei of RA-treated and control cultures suggest that cellular acid-soluble nucleotide pools mediate the inhibition of DNA replication in the 48-hour-RA-treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G0/G1-arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)-stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18-hour treatment of G0/G1-arrested cells with RA, before their exposure to PMA.
We previously demonstrated that membrane vesicles shed by the F10 variant of the murine B16 melanoma cell line inhibited the induction by interferon-gamma (IFN) of murine macrophage immune response region-associated (Ia) antigen expression. In this paper we present evidence that the inhibition of macrophage Ia antigen expression is a selective effect of vesicles and characterize its temporal requirements. Membrane vesicles shed from F10 cells did not affect the expression of macrophage H-2K or H-2D antigens under conditions shown to profoundly inhibit Ia antigen expression. Similarly, the induction of plasminogen activator and interleukin 1 from macrophages was not inhibited by the vesicles. The vesicles did not measurably decrease total cellular RNA or protein synthesis. Macrophages were sensitive to the inhibitory effects of the vesicles during the induction and maintenance phases of Ia expression. Pretreatment of macrophages with vesicles before culture with IFN did not reduce the induction of Ia. The rate of decline of Ia expression after removal of IFN was unaffected by the presence of vesicles. Removal of vesicles from cultures of IFN-treated macrophages resulted in only a partial recovery of Ia expression, suggesting that the inhibition of Ia expression may be a slowly reversible process. The selective and partially reversible inhibition of Ia expression by vesicles shed from the plasma membrane of tumor cells is a possible mechanism whereby tumor-bearing hosts may become immunocompromised.
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