,5,3Ј-Triiodo-L-thyronine (T3), but not L-thyroxine (T4), activated Src kinase and, downstream, phosphatidylinositol 3-kinase (PI3-kinase) by means of an ␣ v␤3 integrin receptor on human glioblastoma U-87 MG cells. Although both T 3 and T4 stimulated extracellular signal-regulated kinase (ERK) 1/2, activated ERK1/2 did not contribute to T 3-induced Src kinase or PI3-kinase activation, and an inhibitor of PI3-kinase, LY-294002, did not block activation of ERK1/2 by physiological concentrations of T 3 and T4. Thus the PI3-kinase, Src kinase, and ERK1/2 signaling cascades are parallel pathways in T 3-treated U-87 MG cells. T3 and T4 both caused proliferation of U-87 MG cells; these effects were blocked by the ERK1/2 inhibitor PD-98059 but not by LY-294002. Smallinterfering RNA knockdown of PI3-kinase confirmed that PI3-kinase was not involved in the proliferative action of T 3 on U-87 MG cells. PI3-kinase-dependent actions of T 3 in these cells included shuttling of nuclear thyroid hormone receptor-␣ (TR␣) from cytoplasm to nucleus and accumulation of hypoxia-inducible factor (HIF)-1␣ mRNA; LY-294002 inhibited these actions. Results of studies involving ␣v␤3 receptor antagonists tetraiodothyroacetic acid (tetrac) and Arg-Gly-Asp (RGD) peptide, together with mathematical modeling of the kinetics of displacement of radiolabeled T3 from the integrin by unlabeled T3 and by unlabeled T4, are consistent with the presence of two iodothyronine receptor domains on the integrin. A model proposes that one site binds T3 exclusively, activates PI3-kinase via Src kinase, and stimulates TR␣ trafficking and HIF-1␣ gene expression. Tetrac and RGD peptide both inhibit T3 action at this site. The second site binds T4 and T3, and, via this receptor, the iodothyronines stimulate ERK1/2-dependent tumor cell proliferation. T3 action here is inhibited by tetrac alone, but the effect of T4 is blocked by both tetrac and the RGD peptide. thyroid hormone; phosphatidylinositol 3-kinase; extracellular signal-regulated kinase 1/2; integrin ␣v␤3; glioblastoma cells; Src kinase; mitogenactivated protein kinase; intracellular hormone receptor trafficking ACTIONS OF THYROID HORMONE [L-thyroxine (T 4 ); 3,5,3Ј-triiodo-L-thyronine (T 3 )] that are independent of ligand binding to nuclear thyroid hormone receptors are called nongenomic actions. Nongenomic effects of thyroid hormone are initiated outside the cell nucleus but may culminate in complex cellular events that are nucleus mediated (7, 8, 26 -29). Initiation of nongenomic actions includes a plasma membrane receptor for T 4 and T 3 on integrin ␣ v ␤ 3 that is linked to mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK) 1/2] for transduction of the hormone signal (3) and nuclear receptors residing in the cytosol of unstimulated cells, such as thyroid hormone receptor (TR) ␤1 (28).The phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) pathway is an important regulator of cellular growth, metabolism, and survival (13, 19). Studies of Storey et al. (38) indi...

The stilbene resveratrol (RV) initiates p53-dependent apoptosis via plasma membrane integrin aVb3 in human cancer cells. A thyroid hormone (L-thyroxine, T 4 ) membrane receptor also exists on aVb3. Stilbene and T 4 signals are both transduced by extracellular-regulated kinases 1 and 2 (ERK1/2); however, T 4 promotes cell proliferation in cancer cells, whereas RV is proapoptotic. Thyroid hormone has been shown to interfere with RV-induced apoptosis. However, the mechanisms involved are not fully understood. In this study, we examined the mechanism whereby T 4 inhibits RV-induced apoptosis in glioma cells. RV activated conventional protein kinase C and ERK1/2 and caused nuclear localization of cyclooxygenase-2 (COX-2), consequent p53 phosphorylation and apoptosis. RV-induced ERK1/2 activation is involved in not only COX-2 expression but also nuclear COX-2 accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2 activation, but reduced the nuclear abundance of COX-2 protein and the formation of complexes of nuclear COX-2 and activated ERK1/2 that are required for p53-dependent apoptosis in RVtreated cells. T 4 inhibited RV-induced nuclear COX-2 and cytosolic pro-apoptotic protein, BcLx-s, accumulation. Furthermore, T 4 inhibited RV-induced apoptosis by interfering with the interaction of nuclear COX-2 and ERK1/2. This effect of T 4 was prevented by tetraiodothyroacetic acid (tetrac), an inhibitor of the binding of thyroid hormone to its integrin receptor. Tetrac did not, in the absence of T 4 , affect induction of apoptosis by RV. Thus, the receptor sites on aVb3 for RV and thyroid hormone are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive.

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Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.

Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. Tissue determined LPCs may reside in or near the terminal biliary tree, but it has been difficult to identify authentic LPCs in the normal liver. 2,3 One of the principle lines of evidence for existence of LPCs is appearance of oval cells after different kinds of liver injury, which differentiate into either hepatocytes or cholangiocytes. For example, using carcinogenic regimens where N-2-acetylaminofluorene (AAF) inhibits proliferation of mature hepatocytes, 4,5 oval cells appear in large numbers in duct-like structures. 6,7 Similarly, marked expansions of ductular oval cells occur when centrolobular injury is induced by CCl 4 and bile ductular hyperplasia is stimulated by bile duct ligation 8 or hepatocyte proliferation is inhibited by AAF. 9,10 Although these observations suggest that LPCs exist within biliary ducts, other models implicate nonductal cells in the portal triad. 11 Thus, nondescript periductular oval cells proliferate within 2 days after allyl alcohol (AA)-induced periportal injury, and 2 to 4 days later the periportal necrotic zone is replaced by an expanding population of oval cells and transitional hepatocytes, with little ductular reaction. 12-14 Based on these and earlier observations, 2 we postulated that nontransformed LPC lines, derived from oval cells induced after AA injury, might exhibit different stages of liver-cell differentiation and bipotential capacities of differentiation. We now describe the isolation and characterization of 11 LPC strains and lines from adult livers after periportal injury induced by AA. Materials and Methods Materials.The following materials were obtained commercially: dexamethasone and bFGF (Sigma); Oncostatin M (R&D Systems, Minneapolis, MN); Matrigel (Collaborative Research, Bedford, MA); human epidermoid carcinoma cell line A431 lysates (Santa Cruz Biotechnology, Santa Cruz, CA).Derivation, Culture, and Storage of LPC Strains. Nineweek-old Fisher/344 female rats weighing 140-150 g were obtained from Harlan, India...

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The potent carcinogen aflatoxin B 1 is a weak mutagen but a strong recombinagen in Saccharomyces cerevisiae. Aflatoxin B 1 exposure greatly increases frequencies of both heteroallelic recombination and chromosomal translocations. We analyzed the gene expression pattern of diploid cells exposed to aflatoxin B 1 using high-density oligonucleotide arrays comprising specific probes for all 6218 open reading frames. Among 183 responsive genes, 46 are involved in either DNA repair or in control of cell growth and division. Inducible growth control genes include those in the TOR signaling pathway and SPO12, whereas PKC1 is downregulated. Eleven of the 15 inducible DNA repair genes, including RAD51, participate in recombination. Survival and translocation frequencies are reduced in the rad51 diploid after aflatoxin B 1 exposure. In mec1 checkpoint mutants, aflatoxin B 1 exposure does not induce RAD51 expression or increase translocation frequencies; however, when RAD51 is constitutively overexpressed in the mec1 mutant, aflatoxin B 1 exposure increased translocation frequencies. Thus the transcriptional profile after aflatoxin B 1 exposure may elucidate the genotoxic properties of aflatoxin B 1 .

The hepatocarcinogen aflatoxin B1 (AFB1) is a potent recombinagen but weak mutagen in the yeast Saccharomyces cerevisiae. AFB1 exposure induces DNA damage-inducible genes, such as RAD51 and those encoding ribonucleotide reductase (RNR), through a MEC1 (ATR homolog)-dependent pathway. Previous studies have indicated that MEC1 is required for both AFB1-associated recombination and mutation, and suggested that AFB1-DNA adducts are common substrates for recombination and mutagenesis. However, little is known about the downstream effectors of MEC1 required for genotoxic events associated with AFB1 exposure. Here we show that AFB1 exposure increases frequencies of RAD51-dependent unequal sister chromatid exchange (SCE) and activates Rad53 (CHK2). We found that MEC1, RAD53, and DUN1 are required for both AFB1-associated mutation and SCE. Deletion of SML1, which encodes an inhibitor of RNR, did not suppress the DUN1-dependent requirement for AFB1-associated genetic events, indicating that higher dNTP levels could not suppress the dun1 phenotype. We identified AFB1-DNA adducts and show that approximately the same number of adducts are obtained in both wild type and rad53 mutants. Since DUN1 is not required for UV-associated mutation and recombination, these studies define a distinct role for DUN1 in AFB1-associated mutagenesis and recombination. We speculate that AFB1-associated DNA adducts stall DNA replication, a consequence of which can either be mutation or recombination.