Membrane vesicles shed by murine melanoma cells selectively inhibit the expression of Ia antigen by macrophages.

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.

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells.

Retinoic acid (RA; beta-all-trans) inhibits the proliferation of both murine 3T3 cells and human HeLa cells. Flow cytometric analyses of exponentially growing cultures show that 3T3 cells are inhibited during the S phase of their cell cycle, while HeLa cells show only a small increase in G1 phase cells. RA (10 microM) causes a 50% increase in total cellular adenosine triphosphate (ATP) pools of 3T3 cells, but not of HeLa cells. We have previously demonstrated that the effects of RA on cellular ATP pools of 3T3 cells are directly related to its inhibition of cellular growth, and now report data which provide a biochemical basis for this process. Established procedures were utilized to investigate the effects of RA on the functional compartmentalization of the nuclear ATP pool which serves as a precursor for RNA synthesis in these cells, and which is shown to be a small pool in comparison with cytoplasmic ATP pools. Expansion of total cellular ATP pools by 1 mM of exogenously supplied unlabeled adenosine is ineffective in reducing the subsequent incorporation of [3H]adenosine into RNA of 3T3 cells. Similar treatment of HeLa cells yields a modest reduction in the incorporation of [3H]adenosine into RNA. RA treatment of HeLa cells does not affect the preferential uptake of exogenous [3H] adenosine into the immediate precursor ATP pool for RNA synthesis. RA treatment of 3T3 cells markedly reduces the incorporation of [3H] adenosine into RNA, indicating a lesser degree of functional compartmentalization of the nuclear ATP pool. Similar conclusions are drawn from correlations of the specific radioactivities of total cellular [3H] ATP pools and the levels of incorporation of radioactive label into cellular RNA. In addition, pulse-chase experiments show that RA-treated 3T3 cells continue to incorporate radioactive label from pools prelabeled with [3H]adenosine despite the presence of a large excess of unlabeled adenosine in the chase medium. Control 3T3 and both control and RA-treated HeLa cells cease to incorporate label immediately upon the start of the chase, suggesting that the functional precursor ATP pool for RNA synthesis is small and readily diluted. These data suggest that RA decreases the degree of functional compartmentalization for 3T3, but not HeLa cell ATP pools, and provides a probable mechanism for expansion of nuclear ATP pools of 3T3 cells. The expanded nuclear ATP pools may provide the biochemical mechanism for the inhibition of DNA synthesis during the S phase of the 3T3 cell cycle.

Correlation between activation of quiescent 3T3 cells by retinoic acid and increases in uridine phosphorylation and cellular RNA synthesis.

Short exposure of cultured quiescent cells to micromolar quantities of beta-all-trans-retinoic acid (RA) has been reported to potentiate the effects of phorbol myristate acetate in promoting the transition from the resting to growing states of these cells. Longer periods of exposure to RA result in substantial inhibition of cellular proliferation. We now show that short-term treatment of quiescent Swiss 3T3 cells with RA yields marked increases in uridine phosphorylation and total cellular RNA synthesis as well as 2-deoxyglucose uptake. Upon subsequent treatment of the cells with phorbol myristate acetate, a direct correlation between the comitogenic activity of RA and its stimulation of uridine phosphorylation and RNA synthesis is apparent. The increases in 2-deoxyglucose uptake persist after long-term exposure of the cells to RA when the growth-inhibitory effects of this agent are observed.

Retinoic acid-promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects.

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.

Growth inhibitory and stimulatory effects of retinoic acid on murine 3T3 cells.

All-trans-beta-retinoic acid (RA) has both comitogenic and antiproliferative effects on murine Swiss 3T3 cells. Treatment of quiescent 3T3 cells for less than 24 hr with micromolar concentrations of RA potentiates subsequent mitogenic response of those cells to phorbol 12-myristate 13-acetate. Longer exposures of 3T3 cells to RA result in inhibition of DNA replication as measured by [3H]thymidine incorporation and decreased growth rates and saturation densities for cells grown in either 2% or 10% serum. Both the comitogenic and antiproliferative activities of RA for 3T3 cells are RA-dose dependent. RA-induced decreases in the 3T3 cell saturation density are reversible only after resuspension of cells by trypsinization and replating. Treatment of 3T3 cells for 48 hr with RA inhibits the rate of [3H]thymidine incorporation by 35--50%, while autoradiographic data show that labeling indices are similar to control values. Equal percentages of control and 48-hr RA-treated quiescent 3T3 cells respond to subsequent stimulation with 10% serum as determined by autoradiographic and flow cytometric analyses. However, the progression of RA-treated cells through the S phase of the cell cycle is slowed. These data suggest that inhibition of 3T3 cell proliferation by RA is established after a minimum 24-hr treatment and that this inhibition is the result of a decreased rate of DNA replication in S-phase cells.

Effects of retinoic acid on plasminogen activator and mitogenic responses of cultured mouse cells.

Treatment of simian virus 40-transformed 3T3 cells with 1 microM beta-all-trans-retinoic acid (RA) resulted in a 5- to 6-fold enhancement of plasminogen activator (PA) release. Intracellular PA levels rose to twice control levels. Confluent 3T3 cells were less responsive to RA. In 6 of 10 experiments, no increase in 3T3 cell PA levels was noted, although up to a 2.5-fold enhancement of PA elaboration has been observed in some experiments at a dose of 10 microM RA. Simultaneous treatments of 3T3 cells with 10 microM RA and 2.1 to 9.3 mM Ca2+, 5 to 40 ng phorbol myristate acetate per ml, or 150 to 600 ng Fraction I from lactalbumin hydrolysate (Fl) protein per ml indicated that RA potentiated the PA stimulatory activities of these agents. Extracellular PA levels of RA-treated cells increased by 4 to 10 times the amount of increase observed for Ca2+, PMA, or Fl alone. A potentiating activity of RA was also evident when quiescent 3T3 cells were pretreated with 10 microM RA and then stimulated to synthesize DNA with Ca2+, PMA, or Fl. For RA-pretreated cells, an increased percentage of nuclei was labeled with [3H]thymidine (24 hr) in response to doses of the three mitogens which were ineffective without RA pretreatment (2.4mM Ca2+, 5 ng PMA per ml, or 150 ng Fl protein per ml). Additional experiments have indicated that, like platelet extracts, Ra renders quiescent 3T3 cells competent to synthesize DNA in response to the progression factors of human plasma as defined by Pledger et al. (Proc. Natl. Acad. Sci. U. S. A., 74: 4481-4485, 1977). Pretreatment of quiescent 3T3 cells for 6 hr with 10 microM RA resulted in a greater than 4-fold increase in the percentage of cells which incorporated [3H]thymidine in response to a 36-hr treatment with 10% human plasma, as compared to cells treated with human plasma alone. Thus, under certain conditions, RA may have cell-activating properties, and caution should be exercised with regard to its suggested use as an antitumor agent.

Alterations in glycosphingolipid patterns in a line of African green monkey kidney cells infected with herpesvirus.

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.