[Measurement of anti-acetylcholine receptor antibody using human rhabdomyosarcoma cell line].

We developed a radioimmunoassay for AChR Ab using AChR solubilized from membrane extracts of human rhabdomyosarcoma cell line (RD cell). The binding affinity of this AChR to the alpha-Bungarotoxin (alpha-BTX) was similar to that of AChR from cell surface. Lyophilized AChR was stable within two months at -20 degrees C. In our assay system, sera from patients with Myasthenia Gravis (MG) showed markedly high titers and frequency (94%) of anti-AChR Ab. On the other hand, almost all sera from normal and non-myasthenic patients such as rheumatoid arthritis and thyroid disease were negative (< 0.2nmol/l). Our assay using AChR from RD cells shows sufficient results in the intra- and inter-CV values, and recovery and dilution tests. AChR Ab titers of our assay showed good correlations with those measured by AChRs from both human ischemic muscles and TE671 cells (commercial kit). The radioimmunoassay for AChR Ab using the membrane extracts of RD cells has high sensitivity, specificity and stability. Therefore, the assay is valuable as the routine assay for the diagnosis of MG.

The distinct roles of alpha- and beta-subunits of human thyrotropin in the receptor-binding and postreceptor events.

The roles of alpha- and beta-subunits of human TSH (hTSH) were studied by investigating the inhibitory effects of monoclonal antibodies on the biological activity of hTSH. Four monoclonal antibodies directed toward different epitopes on the beta-subunit (hTSH beta) inhibited completely the receptor binding of hTSH to FRTL-5 rat thyroid cells. In contrast, five monoclonal antibodies directed toward different epitopes on the alpha-subunit had little or no effect on the receptor binding. Furthermore, four of five alpha-subunit-specific antibodies and three of four hTSH-specific antibodies inhibited both hTSH-induced cAMP accumulation and thymidine uptake in FRTL-5 cells, in a dose-response manner. One hTSH (alpha-beta heterodimer)-specific antibody which did not recognize the free subunits also had the inhibitory effect on both the receptor binding and hTSH-induced cAMP accumulation. These results strongly suggest that hTSH beta is indispensable for recognizing the receptors on FRTL-5 cells and that the alpha-subunit is required for signal transduction in postreceptor step(s). In addition, it is also suggested that the highly conformational structure of hTSH is absolutely essential for the biological activity.

Regulation of proliferation by the cholera toxin B subunit in FRTL-5 cells may involve a mechanism independent from the modulation of membrane receptor function.

In quiescent rat thyroid (FRTL-5) cells, the B subunit of cholera toxin, which binds to cell surface ganglioside GM1 specifically, alone induced DNA synthesis and markedly enhanced that induced by insulin in serum-free medium. On the other hand, the B subunit inhibited DNA synthesis induced by thyrotropin (TSH). The B subunit did not activate adenylate cyclase and had no effect on the TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Moreover, the B subunit inhibited DNA synthesis induced by dibutyryl cAMP (Bt2cAMP) or phorbol-12-myristate-13-acetate (PMA). These data demonstrate that the B subunit has both stimulatory and inhibitory effects on DNA synthesis in FRTL-5 cells depending on the presence of other growth factors and that these effects on cell proliferation by the interaction of the B subunit, possibly with cell surface ganglioside GM1, may involve a mechanism independent from the modulation of membrane receptor function through interaction with growth factor receptor.

Phorbol esters have a dual action through protein kinase C in regulation of proliferation of FRTL-5 cells.

In quiescent rat thyroid (FRTL-5) cells, phorbol-12-myristate-13-acetate (PMA) inhibited DNA synthesis induced by a combination of insulin and thyrotropin (TSH) or dibutyryl cyclic AMP (Bt2cAMP). This inhibitory effect of PMA was observed even when PMA was added after addition of these growth factors, and was maximal when PMA was added 2-4 h after the growth factors (late in the G1-phase of the cell cycle). On the other hand, PMA alone induced DNA synthesis and also enhanced that induced by Bt2cAMP or insulin in these quiescent cells. 1-Oleoyl-2-acetylglycerol mimicked these effects of PMA, but 4 alpha-phorbol-12,13-didecanoate had no effect. These data demonstrate that in FRTL-5 cells protein kinase C has a stimulatory effect on the G0 to G1 transition and an inhibitory effect on the G1 to S transition in the cell cycle.