Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.

NF-kappa B is a transcription factor that can protect from or contribute to apoptosis. Here we report identification of HSCO that binds to NF-kappa B and inhibits apoptosis. HSCO mRNA was overexpressed in 20 of 30 hepatocellular carcinomas analyzed. Overexpression of HSCO inhibited caspase 9 activation and apoptosis induced by DNA damaging agents, while it augmented apoptosis induced by TNFalpha. Like I kappa B alpha, HSCO inhibited NF-kappa B activity and abrogated p53-induced apoptosis. However, the underlying mechanism was different. HSCO is a nuclear-cytoplasmic shuttling protein, bound to RelA NF-kappa B, and HSCO sequestered it in the cytoplasm by accelerating its export from the nucleus. These results suggest that overexpression of HSCO suppresses p53-induced apoptosis by preventing nuclear localization of NF-kappa B during signaling and thus contributes to hepatocarcinogenesis.

Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.Gankyrin (gann ankyrin repeat protein, also known as PSMD10 and p28) is an oncoprotein, the expression of which is increased (1, 2) in hepatocellular carcinomas (HCCs). 1Gankyrin consists of six ankyrin repeats and a 38-amino acid N-terminal extension and binds to the retinoblastoma tumor suppressor protein (Rb), the S6 ATPase subunit of the 26 S proteasome (PSMC4, RPT3, TBP7), and cyclin-dependent kinase 4 (Cdk4) (1, 3, 4). Overexpression of gankyrin increases both the phosphorylation and degradation of Rb in vivo and oncogenically transforms NIH/3T3 cells. Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16INK4A and p18 INK4C (4). In a rodent model of hepatocarcinogenesis, gankyrin is overexpressed from the earliest stage of tumor development (5). These findings suggest that gankyrin is a major player in cell cycle control and tumorigenesis in HCCs.The MAGE (melanoma antigen) genes were initially identified because they encode tumor antigens that can be recognized by cytolytic T lymphocytes derived from the blood lymphocytes of cancer patients (6). The MAGE gene family is composed of more than 25 genes in humans and are classified as type I MAGE genes (including MAGE-A, MAGE-B, and MAGE-C genes) and type II MAGE genes, which include those that reside outside of the MAGE-A, MAGE-B, and MAGE-C genomic clusters (7, 8). The MAGE-A subfamily comprises 12 genes (MAGE-A1 to MAGE-A12), and is expressed in various types of tumors but not in normal adult tissues, except for testis and placenta. The MAGE-A antigens are of particular interest for antitumor immunotherapy because they are stri...

We isolated a novel hsp110-related gene, apg-1, from a testis cDNA library. The apg-1 transcripts were constitutively expressed in the testicular germ cells and, in some degree, most tissues examined. In a mouse TAMA26 Sertoli cell line, apg-1 transcripts were induced in 2 h by a temperature shift from 32 to 39°C, but not by a shift from 37 to 42°C, the traditional heat stress, or a shift from 32 to 42°C. The heat response pattern of hsp110 expression was similar to that of apg-1. Although induction of a hsp70 transcript was observed in 2 h by a shift from 32 to 39°C, the induction was more apparent by a shift from 37 to 42°C or from 32 to 42°C. Essentially similar differential response patterns were observed among these genes in NIH/3T3 fibroblasts as well. The nuclear run-on assay and the native gel mobility shift assay demonstrated that, by the 32 to 39°C temperature shift, the apg-1 gene was transcriptionally activated, and heat shock factor 1 bound to the heat shock elements in the 5 -flanking region of the apg-1 gene. These results demonstrated that expressions of apg-1, hsp110, and hsp70 could be heat-induced at a temperature lower than the traditional elevated temperatures in somatic cells of both testis and nontestis origin and suggest that the mechanisms regulating the transcript levels of apg-1 and hsp110 are different from those of hsp70. Furthermore, the constitutive expression in germ cells suggests that APG-1 plays a specific role in spermatogenesis as well as in stress response.Prokaryotic and eukaryotic organisms respond to elevated temperatures by synthesizing a distinct set of proteins termed heat shock proteins (HSPs) 1 (1). Anoxia, ethanol, radiation, inflammation, and certain heavy metal ions also induce HSPs in the cells. An early and long-standing assumption regarding the heat shock response was that the HSPs protected cells from the toxic effects of heat and other stresses. Subsequently, a series of studies revealed that HSPs are also present in cells at normal temperatures. Now members of the HSP family are established as molecular chaperones, assisting in the folding and unfolding, assembly and disassembly, and transport of various proteins (2-5). HSPs are also shown to interact with mutant p53 and p60 v-src (6 -8), suggesting their involvement in cell cycle regulation.Spermatogenesis begins at puberty and consists of three steps: the mitotic proliferation of the spermatogonia, meiosis at the spermatocyte stage, and the distinct cellular structural changes of the spermatids. Unlike somatic cells, the male germ cells are easily damaged at the body cavity temperature (9), indicating the presence of a differential heat sensitivity between somatic cells and germ cells. Sarge (10) recently reported that the temperature threshold for induction of HSP72 encoded by the hsp70 gene was lower in male germ cells than in somatic cells. To date, several HSPs have been found to be constitutively expressed in germ cells at specific stages of development. Two HSP70-related genes, hsp70.2 and hsc70t, ar...