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The effects of culture medium and serum components on immunoglobulin (Ig) production by mouse splenocytes were examined. In this study, we showed that culture medium containing charcoal-dextran-treated fetal bovine serum (CDFBS) supported Ig production more efficiently than culture medium containing FBS. In addition, RPMI 1640 medium supported Ig production more efficiently than Dulbecco modified Eagle medium. In addition, an increase in medium IgA production was observed with the increase in both CDFBS and FBS concentrations, whereas an increase in IgM level was observed in the presence of 5% and 10% FBS and 10% CDFBS. Dose-dependent effects of estrogenic compounds on Ig production were also examined. We found that 17beta-estradiol induced increases in IgM and IgE levels, but diethylstilbestrol enhanced only the IgE level. On the other hand, tamoxifen and bisphenol A enhanced medium IgM level at physiological concentrations. Among flavonoids, daidzein enhanced IgM and IgE levels at concentrations above 10 microM, and genistein induced a decrease in IgM level and an increase in IgE level at concentrations above 10 microm. In addition, quercetin and luteolin enhanced medium IgE level at all concentrations tested, whereas IgA, IgG, and IgM levels were not affected. These results suggest that environmental estrogens affect Ig production of mouse Splenocytes in a complex and class-specific manner.

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR-/- and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27(KIP1). Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.