Inhibitors of p38 MAP kinase increase the survival of transplanted dopamine neurons.

Fetal cell transplantation therapies are being developed for the treatment of a number of neurodegenerative disorders including Parkinson's disease [10-12,21,22,24,36,43]. Massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. We have explored a novel protein kinase pathway for its role in apoptosis of dopamine neurons. We have discovered that inhibitors of p38 MAP kinase (the pyridinyl imidazole compounds: PD169316, SB203580, and SB202190) improve survival of rat dopamine neurons in vitro and after transplantation into hemiparkinsonian rats. In embryonic rat ventral mesencephalic cultures, serum withdrawal led to 80% loss of dopamine neurons due to increased apoptosis. Incubation of the cultures with p38 MAP kinase inhibitors at the time of serum withdrawal prevented dopaminergic cell death by inhibiting apoptosis. In the hemiparkinsonian rat, preincubation of ventral mesencephalic tissue with PD169316 prior to transplantation accelerated behavioral recovery and doubled the survival of transplanted dopamine neurons. We conclude that inhibitors of stress-activated protein kinases improve the outcome of cell transplantation by preventing apoptosis of neurons after grafting.

IGF-I and bFGF improve dopamine neuron survival and behavioral outcome in parkinsonian rats receiving cultured human fetal tissue strands.

To promote dopamine cell survival in human fetal tissue strands transplanted into immunosuppressed 6-OHDA-lesioned rats, we have preincubated tissue in insulin-like growth factor-I (IGF-I, 150 ng/ml) and basic fibroblast growth factor (bFGF, 15 ng/ml) in vitro for 2 weeks. Growth factor treatment did not affect the rate of homovanillic acid production in vitro but increased overall dopamine neuron survival in animals after transplant from 1240 +/- 250 to 2380 +/- 440 neurons (P < 0.05). Animals in the growth factor-treated group had a significantly greater reduction in methamphetamine-induced rotation (66%) compared to control transplants (30%, P < 0.05). We conclude that in vitro preincubation of human fetal tissue strands with IGF-I and bFGF improves dopamine cell survival and the behavioral outcome of transplants.

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis.

The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.

Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.

The use of human hepatocyte cultures to study the induction of cytochrome P-450.

We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively.