B, viability of transformed and normal thyrocytes after treatment with gml DHMEQ during to hours.Cell growth was estimated by watersoluble tetrazolium saltbased assays in well plates.However, after longer incubation with DHMEQ, IB protein expression was restored to normal levels, which may be explained by both drug degradation andor a recovery of the NFB signaling as has been reported previously. As a result of treatment with DHMEQ, and most XY1 probably because of NFB inhibition, we also observed a decrease in the cellular levels of the IAP family proteins, cIAP, cIAP, and XIAP. Effect of DHMEQ on NFB activation in the FRO cell line.B, inhibition of the nuclear translocation of NFB subunits.Cells were pretreated with various doses of DHMEQ for hour then incubated with ngml TNF for hour.Together with effector caspase and, DHMEQ also activated the cleavage of the upstream procaspase, indicating the initiation of the mitochondrial apoptotic pathway.These findings correlate with the observed release of cytochromecfrom the mitochondria to the cytoplasm. It has also been shown that procaspase can be a substrate for caspase, as part of an apoptosis amplification loop, after initiation of the cytochrome ccaspase pathway. In a time course experiment, both procaspase and PARP cleavage products first appeared after hours of incubation. In contrast to carcinoma cell lines, DHMEQ did not induce cleavage of procaspases or PARP in primary thyrocytes induced cell death after hours incubation, no traces of active forms of caspases were detected.MAPK pathways have been implicated in the modulation of apoptotic cell death in response to chemotherapy pathway is linked to cell proliferation and survival, whereas induction of the stressactivated kinases, JNK and p, is associated with increased apoptosis. In any event, both the extent and duration of MAPK signaling have been shown to be critical for the determination of cell fate. Therefore, we examined the time course effect of DHMEQ on different MAPK pathways, which are possible mediators of apoptosis.We found that DHMEQ enhanced the phosphorylation of ERK, p, JNK, and JNK in transformed thyrocytes within a short time after treatment. ERK activation was transient and peaked at to hours, after which time its phosphorylation levels returned to baseline at hours after treatment.To examine the effects of DHMEQ in model ATC xenografts in vivo, we used nude mice with implanted FRO tumors.After dissecting these tumors, we analyzed their morphology and determined the prevalence of apoptotic cells.During the course of therapy no changes in the behavior of animals were observed.To additionally evaluate the drug toxicity, we also examined the morphologic structure and manifestations of apoptosis in small intestine epithelium of treated animals.The elevation of basal levels of the NFB activity in malignant thyroid tissue plays a critical role in thyroid carcinogenesis, which makes NFB an attractive target for molecular therapy.In a previous report, we showed that inhibition of NFB signaling in anaplastic thyroid cancer cells markedly suppressed the tumor growth in vivo. However, until recently, no effective small molecule compound was available to selectively inhibit the NFB cascade.FRO cell lysates were collected at different time points of incubation with gml DHMEQ.