Evaluation of dimethylaminosulfonates of alkane diols as a novel group of anticancer agents.

A series of title compounds has been synthesized and evaluated by the cytotoxicity assays conducted in vitro in seven human tumor cell lines, initially in MT-4 and H-9, followed by U-937, PM-1, MCF-7, Hep-3B, and K-562. These compounds were simultaneously compared with the existing clinical drug, busulfan and also with an experimental drug, hepsulfam. IC(50) values of these agents in T-cell lymphoma and leukemic cell lines indicate that two of these agents hexsulfamyl and octsulfamyl (compounds 3 and 4) were significantly more potent than busulfan and were comparable in antileukemic activity with hepsulfam. In order to determine the effect of these agents on normal proliferating cells, the toxicity of 3 and 4 was also determined in vitro against human peripheral blood mononuclear cells (PBMC) and against murine bone marrow progenitor cells. PBMC assay data indicate that these agents were generally less toxic than hepsulfam. The results of the colony forming unit-erythroid (CFU-E) and granulocyte-macrophage colony forming unit (CFU-GM) assays, however, indicate that these agents were more toxic than hepsulfam to erythroid progenitor cells than to granulocyte-macrophage progenitors. The toxicity of octsulfamyl was further assessed in vivo in normal Swiss mice by measuring drug-induced changes in hematological parameters, femoral bone marrow cellularity and splenic cellularity as well as hepatotoxicity and nephrotoxicity on day 7 and 14 following drug treatment at the dose of 1.0 mg/kg body weight from days 1 to 5. The results indicate that the compound did not adversely affect hematopoiesis. Marginal bone marrow suppression was observed on day 7, which gradually tends to reach normalcy on day 14. The other parameters were within normal limit.

Modulation of antigen presentation and class II expression by a class II-associated invariant chain peptide.

During the process of MHC class II assembly, the class II-associated invariant chain peptide (CLIP) remains bound within the peptide binding groove until its subsequent removal, which is mediated by H-2 M. We have defined the functional role of CLIP, through saturation of the endosomal compartment with exogenous CLIP-(85-101), resulting in reduced class II MHC on the surface of APCs and an impeded T cell response. Conversely, incubation of the same cells with immunogenic peptides or proteins resulted in an up-regulation of surface class II MHC. T cells from CLIP- plus Ag-immunized mice showed a marked decrease in Ag-specific response over that in mice primed with Ag alone. A B cell hybridoma, TA3 (H-2d,k) incubated with CLIP in vitro showed dramatically reduced MHC class II I-A surface expression. APCs derived from CLIP-immunized mice exhibited down-regulation of surface class II MHC, but not of CD45 (B220). Electrophoretic studies showed that the addition of exogenous CLIP resulted in a relative decrease in SDS-stable MHC class II heterodimers in TA3 cells. Studies with FITC-CLIP and FITC-OVA-(323-339) peptides demonstrated that exogenously added CLIP peptide does not bind to surface class II molecules via the endogenous route, whereas OVA peptide does. This suggests that exogenously added CLIP acts intracellularly, perhaps in the compartment where H-2 M intersects with class II molecules. These findings demonstrate the functional role of CLIP to regulate MHC class II-mediated Ag presentation in CD4+ T cell responses.

Immune responses to self peptides naturally presented by murine class II major histocompatibility complex molecules.

Peptides eluted from murine Major Histocompatibility Complex (MHC) class II molecules are predominantly fragments of self proteins, which include apolipoprotein E, cystatin-c, transferrin receptor, MHC class II and Ii chains. These naturally processed self peptides are expected to be presented during ontogeny. Therefore, immune responses to these peptides in syngeneic hosts may be under physiological control so as to modulate auto-reactivity. As would be expected from our current understanding, T cells reactive to such antigens should be deleted or clonally anergized. To explore this possibility, we investigated the immunogenicity of a number of these self peptides in mice that express MHC class II, from which these peptides were eluted. T cell and antibody responses were measured following immunization of mice with the appropriate peptide. Surprisingly, many of these peptides were highly immunogenic in normal mice. T cells reactive to these self peptides are restricted by syngeneic MHC class II and were blocked by alpha CD4 antibodies. T cells primed with the native protein in vivo could be challenged with the appropriate self peptide in vitro. Some of the self epitopes induce Th1 cells as indicated by IFN-gamma but not IL-4 production and others induce Th2 cells. Antipeptide antibodies were detected only at higher doses of antigen. Our results suggest that T cells specific for many of the naturally processed self peptides are not deleted but tolerance to these peptides is still maintained in vivo. Presumably the high-affinity self-reactive T cells are deleted in the thymus and the low-affinity self peptide reactive T cells remain unresponsive to antigen challenge in vitro. Upon antigen priming in vivo, many of these self-reactive T cells become activated and readily respond to antigen challenge in vitro. These results point to the physiological control of the maintenance of tolerance to naturally processed self peptides.

Regulation of metallothionein mRNA in human hepatoma (HEP3B) cells.

PURPOSE: Metallothionein (MT) has been shown to protect cells from the injurious effects of ionizing radiation. MT is an inducible protein and heavy metals can upregulate transcription of the MT gene. The present study was initiated to investigate regulation of MT mRNA synthesis in a human hepatocellular carcinoma (Hep3B) cell line. METHODS AND MATERIALS: MT levels in Hep3B cells were measured by the cadmium-hemoglobin assay. Zinc acetate was used as an inducing agent. Levels of the MT mRNA were determined by the slot blot hybridization technique. Cycloheximide was used as an inhibitor of protein synthesis and actinomycin D was used to block transcription. RESULTS: Zinc acetate (0.1 mM) treatment increased the intracellular levels of MT in Hep3B cells. MT levels peaked at 10 h and remained stable for up to 48 h. A time-dependent increase in the MT mRNA was also observed peaking at 16 h and then declining. Addition of cycloheximide and zinc acetate simultaneously, resulted in a decrease in the levels of MT, whereas MT mRNA levels were increased. There was no significant change in the decay rate of MT mRNA when the cells were treated with actinomycin D (7.5 micrograms/ml) either in the presence or absence of Zn. CONCLUSION: These results suggest that neither the increased synthesis of a metal regulatory factor (MRF) nor an increase in half-life of MT mRNA is involved in the mechanism of increased MT biosynthesis upon addition of Zn. These findings support the hypothesis that a preexisting MRF must complex with Zn to initiate increased transcription for MT.