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Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. Animal PDA models which reflect the human condition are clearly necessary to develop early diagnostic tools and explore new therapeutic approaches. We have established transgenic rats carrying a mutated H- or K-ras gene (Hras250 and Kras327) controlled by Cre/loxP activation. These animals develop PDA which are histopathologically similar to that in humans. We utilized this model to identify biomarkers to detect early PDA. We report here that serum levels of Erc/Mesothelin are significantly higher in rats bearing PDA than in controls. Importantly, the levels are significantly elevated in rats before grossly visible carcinomas develop. Even in rats with very small microscopic ductal carcinoma lesions, elevated serum Erc/Mesothelin can be detected. We believe this is the first report of a pancreas tumor animal model in which pre-symptomatic lesions can be diagnosed.

The Birt-Hogg-Dubé gene (BHD) encodes the tumor suppressor protein folliculin (FLCN). The function of FLCN has recently been implicated in the regulation of rapamycin-sensitive mTOR complex (mTORC1). Reciprocally, the mTORC1-dependent phosphorylation of FLCN was reported. However, precise mechanism of FLCN phosphorylation and functional interaction of FLCN with tuberin, the product of tuberous sclerosis 2 gene (TSC2) which is a negative regulator of mTORC1, are unclear. Here we report that multiple phosphorylation in FLCN are evoked by downregulation of tuberin as well as by Rheb expression. We found that phosphorylation at Ser62 and Ser302 are differently regulated by mTORC1-dependent pathway. Some unknown kinases downstream of tuberin-mTORC1 are thought to directly phosphorylate FLCN. Interestingly, our results also suggest that the complex formation of FLCN with AMPK is modulated by FLCN phosphorylation. These results suggest that FLCN is involved in a novel mechanism of signal transduction downstream of tuberin.

ERC/mesothelin is expressed on the normal mesothelium and some cancers such as mesothelioma or ovarian carcinoma. A splicing isoform of ERC/mesothelin (known as SMRP), which has an 82-bp insertion and codes for a C-terminus with a hydrophilic, presumably soluble, tail instead of a GPI-anchoring signal, has been reported as a useful marker for the diagnosis of mesothelioma. However, the existence of SMRP has not yet been demonstrated in the serum of mesothelioma patients. To elucidate the existence of SMRP, we have established a new enzyme-linked immunosorbent assay (ELISA) system for SMRP. The ELISA study revealed that N- and C-ERC/mesothelin were detected in sera from mesothelioma patients, but not SMRP, even in these samples. This result showed that the SMRP detected with MESOMARK kit should be lack of soluble C-terminus and indistinguishable from C-ERC/mesothelin. Further study might be necessary to demonstrate the relationship between SMRP and mesothelin.

The Eker rat is an animal model of renal carcinogenesis and carries a transposon insertion in the Tsc2 (tuberous sclerosis-2) gene. We previously generated transgenic Eker rats and identified coding sequences in the Tsc2 gene that are responsible for suppression of renal carcinogenesis in Eker rats. Tsc2-RGH, a transgene that expresses the carboxy terminal region (amino acids 1425–1755) of the Tsc2 product (tuberin), partially suppressed renal carcinogenesis. However, Tsc2-DRG, which expresses a mutant tuberin lacking the carboxy-terminal region (Δaa 1425–1755), did not suppress renal carcinogenesis. Here, we found that introduction of both Tsc2-RGH and Tsc2-DRG in Eker rats completely suppressed renal carcinogenesis and rescued homozygous (Tsc2Ek/Ek) mutants from embryonic lethality in a complementary manner. Co-introduction of Tsc2-RGH and Tsc2-DRG, but not introduction of either alone, efficiently suppressed phosphorylation of p70 S6K. Thus, the functional domains of N-terminal hamartin binding and C-terminal tumor suppression in tuberin can separate in vivo.

Human hepatocellular carcinoma is one of the most common cancers in the world. We previously showed that dbpA, a member of the Y box family of proteins, could accelerate the process of inflammation-induced hepatocarcinogenesis, and that dbpA is more abundantly expressed in hepatocellular carcinoma than in non-tumorous tissue. In this study, to clarify the mechanism by which expression of dbpA is enhanced in the proliferative state, we examined the transcriptional activity of the dbpA promoter region. We focused on the sequence 5'-TTTGGGGC-3' (-8 to -1 in the promoter region) resembling the E2F binding site (one base mismatch, TFSEARCH score 86.2). By overexpressing E2F1 in Huh-7 cells, transcriptional activity of dbpA was significantly increased, and this increase was abolished by mutating or deleting this sequence. Thus, expression of dbpA was positively regulated by E2F1, suggesting that one of the effects of E2F1 on cell proliferation might be mediated by dbpA at the carcinogenesis step.

The Eker rat develops hereditary renal carcinomas (RCs) due to two hit mutations of the tumor suppressor gene, Tsc2. We previously identified using representational difference analysis (RDA), four genes that were expressed more abundantly in an Eker rat RC cell line than in normal kidney tissue. One gene, Erc (expressed in renal carcinoma) showed sequence homology to the mouse and human megakaryocyte potentiating factor (MPF)/mesothelin gene. The present study determines the full sequence of the cDNA and the exon-intron structure of the rat Erc gene and maps its locus in the chromosome by fluorescence in situ hybridization. Rat Erc and its human homologue were localized in chromosomes 10q12-21 and 16p13.3, respectively, both of which coincided with the locus of the Tsc2/TSC gene. We also found that Erc was expressed at higher levels in primary RCs compared with the normal kidney of the Eker rat. Erc may be related to carcinogenesis in the Tsc2 gene mutant (Eker) rat model.

To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effectors of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas.

For evaluation of anti-hepatitis B virus (HBV) drugs, we have employed the HBV transgenic mouse in which virion-like particles can be assayed in the serum. Bispivaloyloxymethyl-9-(2-phosphonylmethoxyethyl)-adenine [bis (POM) PMEA] 100 mg/kg/day, 2',3'-dideoxy-3'-thiacytidine [(+-)-BCH189] 200 mg/kg/day and a placebo were orally administered to mice twice a day for 14 days. Anti-viral effects were monitored by checking the levels of serum HBV DNA by the semiquantitative polymerase chain reaction, HBsAg and HBeAg by enzyme immunoassay, and replicative intermediates in the liver by Southern blotting. As expected, decrease from the 10(0.5) to 10(3) copies of HBV DNA per microl of sera detected before the treatment to the undetectable level was evident for all five animals treated with bis(POM) PMEA 100 mg/kg/day. However (+-)-BCH189 200 mg/kg/day, which is known to act as the inhibitor of reverse transcriptase for HBV or HIV in vivo and in vitro, did not suppress HBV DNA levels in the transgenic mouse. Thus, we were able to detect the effects of anti-HBV drugs semi-quantitatively, and confirm differences in drug efficacy.

The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin, form a heterodimer. Recently we reported that hamartin directly interacted with Hsp70. However, the physiological implications of this interaction have not yet been clearly defined. Here we show that hamartin localized to the outer membrane of the mitochondria in an Hsp70-dependent manner. Moreover, phosphorylation of the T417 residue of hamartin was required for its localization to the mitochondria as well as its interaction with Hsp70. A non-phosphorylatable hamartin mutant at residue T417 was unable to localize to the mitochondria and suppress apoptosis, whereas non-phosphorylatable hamartin mutants T357A and T390A localized to the mitochondria and suppressed apoptosis. Importantly, non-phosphorylatable mutants (T357A, T390A and T417A) promoted apoptosis after treatment with Hsp 70-inhibitor KNK437. We conclude that hamartin inhibited apoptosis by localizing to the mitochondria and that its phosphorylation and binding to Hsp70 was required for facilitation of this process.

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2(Y1571H) cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2(R611Q), which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.

Although induction of interferon gamma (IFN-gamma) and activation of antigen presenting dendritic cells (DCs) are two vital events during an immune response, the impact of endogenous IFN-gamma on DC function has yet to be clarified. The phenotype and function of DCs isolated from mice with high (IFN-gamma-transgenic mouse [Tg]) and undetectable levels of circulating IFN-gamma (normal mice [NM]) were therefore compared. The capacity to stimulate allogenic (p0.05) and antigen-specific T lymphocytes (p0.05), as well as the ability to produce IL-12 (p0.05) and to process soluble protein antigens (p0.05) was found to be significantly higher in DCs from the Tg mice compared to the NM case. The presence of activated DCs in a microenvironment of endogenous IFN-gamma suggests that the IFN-gamma-Tg mouse is a suitable animal model to study cancer immunotherapy in vivo.

A novel DNA virus designated TT virus (TTV) was cloned from a patient with posttransfusion hepatitis and is thought to be a new hepatitis virus. At present, hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to induce hepatocellular carcinoma (HCC). But, actually, in Japan approximately 5 to 10% of HCCs are in HBV-negative and HCV-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we investigated the frequency of the TTV genome in liver tissue of 20 HCC patients. As a result, 3 of 8 NBNC HCC patients and 5 of 12 HBV- or HCV-associated HCC patients were TTV positive, and TTV was shown not to be specific for NBNC HCC. For all TTV-positive patients, we also confirmed that the TTV genome was not integrated into host hepatocyte DNA.

Alteration of the rat homologue of the tuberous sclerosis 2 (TSC2) gene is associated with dominantly inherited cancer in the Eker rat model, indicating a tumor suppressor nature. The ability ofTsc2to activate signal transduction and transcription suggests that genes induced byTsc2may mediate its biological roles. Using a subtractive hybridization approach in combination with tetracycline operator systems, we identified a set of downstream genes affected byTsc2.Regulated expression of wild-typeTsc2gene in Eker renal carcinomas (RCs) resulted in marked expression of cell arrest or programmed cell-death-related genes and stress-induced genes. Thus, the data suggest thatTsc2might contribute to regulation of the cell cycle and cell survival.

We previously isolated subtracted cDNA clones for genes having increased expression in Tsc2 gene mutant (Eker) rat renal carcinomas (RCs). Among them, fra-1 encoding a transcriptional factor activator protein 1 (AP-1) was identified. We have therefore investigated whether other members of the AP-1 transcription factor family might also be involved in renal carcinogenesis in the Eker rat model. In the present study, overexpression of fra-1, fra-2, c-jun, junB, and junD mRNAs was demonstrated in RCs by Northern blot analysis. Interestingly, AP-1 proteins were highly expressed even in the earliest preneoplastic lesions (e.g., phenotypically altered tubules) as suggested by immunohistochemistry. Moreover, 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-binding activity of AP-1 proteins was observed in RC cell extracts by electrophoretic mobility shift assay. As a next step, we transfected antisense oligonucleotides targeting AP-1 genes into RC cells and demonstrated that their growth was strongly inhibited. Thus, the data suggest that overexpression of AP-1 genes might play a crucial role in renal carcinogenesis in the Eker rat model.

The hereditary renal carcinoma (RC) in the rat, originally reported by R. Eker in 1954, is an excellent example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. Recently, we have identified a germline mutation of the tuberous sclerosis ( Tsc2 ) gene in the Eker rat ( Nature Genetics 9, 70–74, 1995), suggested to be a novel tumor suppressor gene, fitting Knudson's two-hit hypothesis. In this study, the effect of wild-type Tsc2 gene expression in Eker RC cells was tested using a tetracycline-responsive promoter system. Transfection and expression of an exogenous Tsc2 gene affected both cell morphology and growth rate. This demonstration of suppression of the neoplastic phenotype provides direct evidence for an essential role of the Tsc2 gene in tumorigenesis.