Translation initiation in eukaryotes is a rate-limiting step in protein synthesis. It is a complicated process that involves many eukaryotic initiation factors (eIFs). Altering the expression level or the function of eIFs may influence the synthesis of some proteins and consequently cause abnormal cell growth and malignant transformation. P170, the largest putative subunit of eIF3, has been found elevated in human breast, cervical, esophageal, and lung cancers, suggesting that p170 may have a potential role in malignant transformation and/or cell growth control. Our recent studies suggested that p170 is likely a translational regulator and it may mediate the effect of mimosine on the translation of a subset mRNAs. Mimosine, a plant nonprotein amino acid, inhibits mammalian DNA synthesis, an essential event of cell growth. The rate-limiting step in DNA synthesis is the conversion of the ribonucleotides to their corresponding deoxyribonucleotides catalysed by ribonucleotide reductase of which the activity is regulated by the level of its M2 subunit. It has been reported that inhibiting the activity of M2 also inhibits cell growth. To understand the relationship between protein and DNA synthesis and between p170 and cell growth control, we investigated in this study whether p170 regulates the synthesis of M2 and, thus, cell growth. We found that altering the expression level of p170 changes the synthesis rate of both M2 and DNA. Decreasing p170 expression in human lung cancer cell line H1299 and breast cancer cell line MCF7 significantly reversed their malignant growth phenotype. However, the overall [ 35 S]methionine incorporation following dramatic decrease in p170 expression was only B25% less than the control cells. These observations, together with our previous findings, suggest that p170 may regulate the translation of a subset mRNAs and its elevated expression level may be important for cancer cell growth and for maintaining their malignant phenotype.

The NF-B family of transcription factors plays a fundamental role in development, maintenance of the immune system, and cell viability (1-3). NF-B is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (p65 (RelA), RelB, or c-Rel). In unstimulated cells, binary complexes of these subunits are restricted to the cytoplasm by interaction with members of a family of inhibitory proteins, inhibitors of B (IBs) 1 (4, 5). In response to extracellular stimuli such as cytokines or UV radiation, IB proteins are phosphorylated, polyubiquitinated, and then degraded by the 26 S proteasome (6 -13). Dissociation from IBs unmasks the nuclear localization sequence of NF-B, permitting it to move into the nucleus, bind the promoters of target genes, and alter gene expression and cell function (10,13,14). The demonstration that phosphorylation of IB proteins initiates events necessary for activation of NF-B led to the discovery of IB kinase (IKK) complexes composed of IKK␣, IKK␤, and IKK␥ (NEMO) (15)(16)(17)(18)(19). IKK␣ and IKK␤ are serine-threonine kinases, and IKK␥ is a scaffolding protein essential for the function of IKK␣ and IKK␤. IKK␣ and IKK␤ share a high degree of amino acid homology and domain organization. The kinases are composed of an N-terminal kinase domain, a leucine zipper that facilitates homo-and heterodimerization, and a helix-loop-helix domain (20). IKK␣ and IKK␤ can be activated by diverse kinases, among which are NF-B-inducing kinase (NIK), MEKK1, Cot, NF-Bactivating kinase (NAK/TBK), protein kinases C␤ and C␦, and MEKK3 (21-28). However, interest remains sustained in identifying other kinases that affect IKK complexes and NF-B activity. One of these is the Akt serine-threonine kinase, a downstream target for activated phosphatidylinositol 3-kinase (PI 3-kinase) (29 -32). Akt is activated by mitogens and cytokines that function as survival factors. Akt mediates its functions by phosphorylating substrates that decrease the activity of pro-apoptotic proteins or increase the activity of anti-apoptotic proteins (32)(33)(34)(35)(36)(37)(38)(39)(40)(41).Akt may affect NF-B through multiple mechanisms. We demonstrated previously that TNF activates Akt, which phosphorylates and activates IKK␣, thus promoting NF-B function (42). TNF and interleukin-1 can also increase the transactivation potential of the RelA/p65 subunit of NF-B through a mechanism in which Akt has been implicated (43-45). PI 3-kinase activated by phorbol esters or lipopolysaccharide and PI 3-kinase/Akt signaling induced by signaling through CD40, interleukin-1, or G protein-coupled receptors activates [46][47][48]. However, PI 3-kinase/Akt signaling induced by TNF in human umbilical vein endothelial cells inhibits apoptosis without playing a significant role in activation of NF-B (49). Furthermore, Akt can activate a member of the mitogenactivated protein kinase kinase kinase (MAP3K) family, Cot, and indirectly affect IKK activity and NF-B (50). Thus, PI 3-kinase/Akt signaling is upstream of diver...

Tissue transglutaminase (TG2) is involved in Ca 2+ -dependent aggregation and polymerization of proteins. We previously reported that TG2 mRNA is up-regulated in epithelial ovarian cancer (EOC) cells compared with normal ovarian epithelium. Here, we show overexpression of the TG2 protein in ovarian cancer cells and tumors and its secretion in ascites fluid and define its role in EOC. By stable knockdown and overexpression, we show that TG2 enhances EOC cell adhesion to fibronectin and directional cell migration. This phenotype is preserved in vivo, where the pattern of tumor dissemination in the peritoneal space is dependent on TG2 expression levels.

Summary Lung cancer is the leading cause for cancer death in both male and female populations. Although many molecular markers for lung cancer have been developed and useful for early detection of lung cancer, their function remains unknown. In this paper, we report our findings that a 170-kDa protein (p170) is over-expressed in all types of human lung cancers compared with normal tissues and it is identified as a subunit of translation initiation factor eIF3 by cDNA cloning. Translation initiation factors are a family of proteins that promote the initiation step of protein synthesis and are regulators of cell growth at the translational level. Further studies showed that p170 mRNA is ubiquitously expressed with higher levels in adult proliferating tissues (e.g. bone marrow) and tissues during development (e.g. fetal tissues). This study suggests that p170 and eIF3 may be important factors for cell growth, development, and tumorigenesis.

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-κB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21WAF1/CIP1, and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1β in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.

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P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.