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The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly low metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between intracellular nm23-immunoreactivity and lymph nodal status of human breast cancer. We analyzed 82 surgically removed breast tumors including 67 invasive carcinomas (ductal, lobular and mucinous carcinomas). The nm23 expression was diffusely positive in the benign tumors and non-invasive carcinomas. Of the invasive ductal carcinomas, lymph node metastasis was found in 67.7% (21/31) of the focally positive/negative cases and in 18.2% (4/22) of the diffusely positive cases (p

To study the appropriate period for formalin fixation in order to detect p53 abnormalities in formalin-fixed tissue, we used seven surgically resected human colorectal cancer specimens. The immunohistochemical reactivity of p53 immunostaining and amplification of DNA by polymerase chain reaction (PCR) of the p53 gene were compared after various periods of 10% formalin fixation (1 day, and 1, 2, 4, and 8 weeks). For comparative immunostaining, we used the monoclonal antibody Ki-67 (MIB-1), and for comparative polymerase chain reaction (PCR), K-ras at codon 12 was amplified. Immunostaining was performed by the streptavidin-biotin method with microwave retrieval, and PCR amplifications were performed by the nested PCR method. p53 and Ki-67 immunoreactivity did not change essentially for up to 2 weeks and 1 week, respectively, of formalin fixation. PCR amplification for p53 at exon 8 and K-ras at codon 12 was successful until 1 day and 2 weeks, respectively, of formalin-fixation for the specimens of all seven cases. Thereafter, the amplification tended to worsen as the fixation time lengthened. Further, the DNA was more successfully amplified in the second PCR than in the first. These results suggest that to detect p53 abnormality in specimens that have been formalin-fixed for long periods, immunohistochemical staining may have advantages over DNA analysis with PCR amplification.

Brain-specific angiogenesis inhibitor (BAI) 1 was recently isolated as a novel p53 inducible gene. BAI1 has been suggested to play a significant role in angiostasis. We studied the expression of BAI1 in 49 colorectal cancer specimens by RT-PCR. BAI1 expression was significantly reduced in colorectal cancers as compared to the extraneoplastic tissues (X(2) test, p=0.041). BAI1 expression was inversely correlated with vascular invasion and metastasis (Fisher's exact test, p 0.045). Moreover, vascularity in the colorectal cancer was inversely correlated with BAI1 gene expression (Mann-Whitney U-test, p=0.0003). These observations suggested that BAI1 expression might inhibit angiogenesis and metastasis of colorectal cancer.

Xenografted neoplastic tissues are often utilized to study biology of human cancer. We studied isoform pattern (VEGF206, VEGF189, VEGF165, VEGF121) of an angiogenic factor VEGF in both esophageal primary cancers and cancer xenografts by RT-PCR analysis. Eighteen of 19 (95%) xenografts showed the cell associated aberrant isoform of VEGF189 at a significantly higher incidence than 19 of 31 (61%) primary cancers (P=0.009, Fisher's exact test). These observations suggested that the aberrant isoform of VEGF189 mRNA affects the xenotransplantability of human esophageal cancer.

Human histocompatibility leukocyte antigen (HLA)-G is a nonclassical major histocompatibility complex class I molecule. HLA-G is known to provide tolerance from recognition by natural killer cells. We studied HLA-G expression in 39 human colorectal cancers and 23 extra-neoplastic colon tissue samples by RT-PCR. The expression of HLA-G mRNA was significantly more frequent in colorectal cancer (34 of 39 cases) than in the extraneoplastic tissue (10 of 23 specimens; chi2 test, p = 0.0003). HLA-G expression was also confirmed on the cancer cells immunohistochemically. These results suggested that HLA-G on colorectal cancer cells may be correlated with escape from immunological surveillance during colon cancer development.

Background: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. Methods: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. Results: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERMS-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. Conclusion: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Vascular endothelial growth factor (VEGF) has four isoforms (VEGF121, VEGF165, VEGF189, VEGF206). We examined the expression patterns of VEGF isoforms in non-small cell lung cancer (NSCLC) by RT-PCR. The cell-associated isoform VEGF189 was more frequently expressed in NSCLC (90.5%) than in extraneoplastic lung tissue (57.6%, p=0.00004). Immunohistochemical examination confirmed VEGF protein expression in 33 of 76 NSCLC expressing VEGF189 (V189p+ group). The V189p+ group showed significantly poorer prognosis than those without VEGF189 mRNA and protein (V189p- group, p=0.01722). These results suggest that expression of the cell-associated isoform VEGF189 is closely associated with progression of NSCLC.

A human lung cancer xenograft, LC-GP, producing granulocyte-colony stimulating factor and parathyroid hormone-related protein was established by transplantation into severe combined immunodeficient (SCID) mice. The lung cancer patient and SCID mice bearing LC-GP showed leukocytosis and hypercalcemia. G-CSF and PTHrP gene expression were demonstrated in the primary lung cancer, metastatic lesions and LC-GP xenograft. Immunohistochemical analysis confirmed the presence of G-CSF protein in LC-GP xenograft cells. LC-GP possessed activated c-Ki-ras oncogene (point mutation at codon 12). This LC-GP lacked apparent G-CSF receptor expression. The expression of G-CSF and PTHrP may be coregulated by the activated c-Ki-ras oncogene, and autocrine stimulation of G-CSF is unlikely.

The aim of this study was to clarify whether or not the status of gene alteration is heterogeneous in intramucosal carcinoma and homogeneous within invasive carcinoma. We selected 10 colorectal carcinoma cases (1 mucosal, 5 submucosal and 4 advanced carcinomas including 2 cases with lymph node metastasis) and analyzed the p53 gene sequence. Six colorectal cancers in this study showed heterogeneity in p53 mutations in cells from the intramucosal part. In the invasive part of a carcinoma, p53 mutation status was homogeneous intratumorally in all cases. These data indicate that, in regard to p53 gene alterations, colorectal cancers can be composed of various subclones when limited to the mucosa, but clonal selection occurs when one of these subclones commences invasion to the submucosa, generating a monoclonal invasive carcinoma.

BACKGROUND The authors sought to elucidate the histogenesis of pancreatic ductal carcinoma by correlating K-ras mutation with mucus type in normal epithelium, mucous cell hyperplasia (MCH), and carcinoma. METHODS Seventy-four solid-type carcinomas (SCs), 23 ductectatic-type carcinomas (DCs), and specimens of 24 normal pancreata were studied. By histochemical staining, normal duct epithelia, areas of MCH, and carcinomas were classified as having sulfo-type or sialo-type mucus. Foci from normal, DC, SC, sulfo-type, or sialo-type specimens were assessed for K-ras mutation at codon 12 by nested polymerase chain reaction and restriction fragment length polymorphism. RESULTS Of the SCs, 9 were sulfo-type and 65 were sialo-type; all DC specimens were sialo-type, and all normal epithelia were sulfo-type. All foci of sulfo-type, nonneoplastic epithelia were negative for K-ras mutation. In contrast, 124 of 313 sialo-type MCH foci (40%) had a K-ras mutation. Of 74 SCs, only 3 of 9 sulfo-type tumors (33%) were positive for the mutation. Sixty of 65 sialo-type SCs (92%) had a K-ras mutation, whereas 15 of 23 sialo-type DCs (65%) had a mutation. K-ras mutant carcinomas (including both SCs and DCs) were associated with K-ras mutant MCH in 109 of 198 MCHs (55%), whereas carcinomas without a K-ras mutation had mutations in 6 of 68 MCHs (9%). MCH in normal pancreata revealed K-ras mutations in 9 of 51 foci (18%). In addition, in K-ras mutant carcinomas, frequency of K-ras mutation in MCH increased from 27% (11 of 41 foci) of nonpapillary MCHs to 62% (98 of 157 foci) of papillary MCHs; but in K-ras wild-type carcinoma, the mutation rate in MCH was unchanged from 12% (3 of 26 foci) to 7% (3 of 42 foci) in nonpapillary and papillary foci, respectively. CONCLUSIONS These results suggest a strong relationship between the risk of pancreatic carcinoma and the presence of combinations of K-ras gene mutation, papillary growth, and expression of sialomucin in foci of MCH. Cancer 1998;82:651-60. © 1998 American Cancer Society.

We examined the expression levels of P-glycoprotein (P-Gp)/the human multidrug resistance gene (MDR1) and in vivo chemosensitivity in the 7 osteosarcoma xenografts. Three of seven (43%) osteosarcoma xenografts expressed MDR1 by reverse transcriptase-polymerase chain reaction (RT-PCR). The OSS-516R xenograft selected with vincristine (VCR) from the MDR1-negative xenograft OSS-516, which was sensitive to VCR and doxorubicin (DOX), acquired cross-resistance to DOX. In the OSS-516R, RT-PCR assay showed definite MDR1 expression and immunohistochemical analysis demonstrated P-Gp-positive tumor cells. These results suggest that P-Gp/MDR1 overexpression is related to multidrug resistance in human osteosarcoma in vivo.

Claudin-1 is a membrane protein with four transmembrane domains, that is exclusively localized at cellular tight junctions. Recent studies have reported that claudin-1 plays an important role in cancer invasion and metastasis. However, the significance of claudin-1 in pancreatic cancer is still unknown. In the present study, we investigated the role of claudin-1 expression in pancreatic cancer growth using the PANC-1 human pancreatic cancer cell line. Treatment with tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, resulted in increased detection of 89 kDa products of poly-(ADP-ribose) polymerase (PARP), a marker of apoptosis, and decreased PANC-1 cell proliferation by 23%. Expression of claudin-1 was up-regulated by TNF-alpha in a concentration-dependent manner in PANC-1 cells. PANC-1 cells treated with TNF-alpha and siRNA against claudin-1 showed a 15% increase in proliferation; i.e. the cells transfected with siRNA against claudin-1 showed resistance to TNF-alpha-induced apoptosis. These results suggest that claudin-1 expression is responsible for TNF-alpha-dependent growth signals and the proliferation of pancreatic cancer cells.

We established a xenograft line of human teratocarcinoma (TC-1) and characterized the pluripotency of differentiation of the neoplastic cells. A teratocarcinoma specimen obtained from a primary mediastinal lesion (22-year-old male patient) was inoculated subcutaneously into severe combined immunodeficient (SCID) mice. The carcinoma formed tumors in the mice. We established a xenograft line by serial passage of the tumor in vivo. The primary tumor was composed of papillary and pseudoglandular nests of highly atypical epithelial cells with foci of glomeruloid structures. The metastatic cells showed apparent production of mucin and differentiation to striated muscle. The xenograft line TC-1 retained the basic histopathological features seen in the primary and metastatic cells. The xenograft line showed focal differentiation to cartilage through serial passages. Immunohistochemical studies with anti-alpha-fetoprotein (AFP) demonstrated positive immunoreactivity on the TC-1 cells. Serum AFP levels were also elevated in the TC-1-bearing SCID mice. The human teratocarcinoma xenograft line TC-1 will be useful for studying the differentiation mechanism in human totipotent stem cells.

Publisher Summary This chapter focuses on a general overview of ribozymes and their applications for cancer gene therapy. Ribozymes are small RNA molecules that possess specific catalytic activities and are being actively investigated for their therapeutic applications in the field of gene therapy. Ribozymes have the ability to modulate specific gene expression because of their site-specific cleavage activity. Ribozymes can be designed for any disease in which a specific protein has been linked to its etiology, and may offer some advantages over antisense oligonucleotide strategies. Since the discovery of the protooncogene, cancer has been defined as a genetic disease. Therefore, gene therapy could be a rational and promising strategy for the treatment of specific cancers. So, oncogenes are obvious targets for the therapeutic application of anti-oncogene ribozymes. One of the problems for successful gene therapy is to define the role of specific oncogenes in specific tumors. Ribozyme technology can be used to help define and delineate the role of oncogenes in cancer and can be used as a therapeutic agent as well. Extensive studies have investigated the efficacy of antioncogene ribozymes, and have shown successful alteration of the human malignant phenotype in vivo. The appearance of gene therapy as an alternative treatment for cancer and other diseases has led researchers toward the development of efficient delivery systems. Among the first methods developed for gene delivery, viral systems have been potentially the most promising. Although viral transfer shows huge potential for clinical settings, nonviral delivery systems are receiving increasing attention. Thus, effective delivery systems with minimal toxicity may advance ribozymes as important therapeutic modalities in the clinical field. Ribozymes could have an important impact on the field of gene therapy in the near future.

In mouse models of lung metastasis, before the appearance of significant metastases, localized changes in vascular permeability have been observed, which appear to set the stage for tumour growth. However, it is unclear whether this is also true in human patients. Here, we show that MD-2, a coreceptor for Toll-like receptor 4 that has a key role in the innate immune response, triggers the formation of regions of hyperpermeability in mice by upregulating C-C chemokine receptor type 2 (CCR2) expression. The CCR2–CCL2 system induces the abundant secretion of permeability factors such as serum amyloid A3 and S100A8. Disruption of MD-2 or CCR2 abrogates the formation of hyperpermeable regions, resulting in reduced tumour cell homing. Furthermore, fibrinogen, which is processed during permeability-mediated coagulation, is also localized in areas of elevated CCR2 expression in tumour-bearing human lungs. Our findings raise the possibility that CCR2 upregulation might represent a marker for regions of increased susceptibility to metastatic homing in lung cancer., Tumours are thought to pave the way for metastases to distant organs by secreting factors create regions of increased vascular permeability. Hiratsuka et al. identify innate immune pathways that underlie this process in the pre-metastatic lungs of tumour-bearing mice and patients.

In this study, we have compared the efficacy of a tissue-specific promoter (tyrosinase promoter) with a viral promoter to express anti-ras ribozyme RNA in human melanoma cells. The retroviral vector containing the tyrosinase promoter was superior in its ability to suppress the human melanoma phenotype in vitro as characterized by changes in growth, melanin synthesis, morphology and H-ras gene expression. These data support the use of tissue-specific expression of anti-oncogene ribozymes as a rational therapeutic strategy in human cancers.

Point mutations in the ras gene have been found in approximately 90% of human pancreatic carcinomas. These alterations can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. We have evaluated the efficacy of a hammerhead ribozyme directed against codon 12 (GUU) of the activated K-ras gene in a Capan-1 human pancreatic carcinoma cell line using different delivery systems. Our results have demonstrated that the anti-Kras ribozyme cloned into the pHs plasmid was able to efficiently suppress K-ras gene expression and to inhibit the proliferation of transfected Capan-1 cells. In contrast, the anti-K-ras ribozyme was less efficient against the Capan-1 cells when cloned into a pLNCX retroviral plasmid. In addition, our results showed that adenoviral-mediated expression of the ribozyme RNA was more effective than the two other plasmid vectors. Our studies have characterized different viral and non-viral delivery systems for the therapeutic application of an anti-K-ras ribozyme against a human pancreatic carcinoma cell line. In the near future, ribozymes could emerge as important therapeutic agents against human malignancies, and optimal delivery systems are necessary to achieve maximal gene therapy benefit.

PURPOSE: Submucosa-invasive colorectal carcinoma is a colorectal carcinoma extending only into the submucosal layer. To clarify the metastatic potential of submucosa-invasive colorectal carcinoma, we studied the relationship between the immunohistochemical staining pattern of carcinoembryonic antigen (CEA) and that of lymphatic invasion/ lymph node metastasis. METHODS: We investigated 49 submucosa-invasive colorectal carcinomas resected surgically or endoscopically. CEA distribution patterns of the neoplastic tissues were divided into three patterns: Pattern 1 = luminal type; Pattern 2 = apical cytoplasmic type; and Pattern 3 = diffuse cytoplasmic type. We also observed the submucosal stromal staining of CEA. RESULTS: Lymphatic invasion and lymph node metastasis were found in 48.8 percent (21/43) and 11.6 percent (5/43) of the Pattern 2/Pattern 3 cases, whereas these were seen in none (0/6) of Pattern 1 cases. Lymphatic invasion and lymph node metastasis were found in 63.3 percent (19/30) (chi-squared =21.94;P

Despite tremendous strides in understanding the molecular basis of cancer (Weinberg, 1989), treatment of human cancer is still limited by the toxicity of chemotherapeutic agents and the development of intrinsic or acquired resistance to these drugs. cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely-used anticancer agents, active in the treatment of ovarian, testicular, head-and-neck, non-small cell lung and brain tumors, among others (Rosenberg, 1985). However, the rapid development of resistance to cisplatin represents an important challenge to clinicians and laboratory investigators alike. Therefore, understanding the biochemical and molecular basis of cisplatin resistance may potentially result in the development of rational approaches to circumvent this problem. At the core of understanding cisplatin resistance lies the realization of both the similarities and differences between the mechanisms of cisplatin action and resistance and that of other chemotherapeutic agents. Cisplatin-resistant cells display a unique cross-resistance pattern to multiple agents, including anti-metabolites such as 5-fluorouracil and methotrexate, DNA polymerase inhibitors such as azidothymidine (AZT), and topoisomerase inhibitors such as camptothecin and etoposide. This “atypical” multidrug resistance is both phenotypically and molecularly distinct from the “classical” multidrug resistance which may involve overexpression of the MDR-1 gene (Gottesman and Pastan, 1993).

e14566 Background: Esophageal squamous cell carcinoma (ESCC) has been generally considered as one of the most aggressive cancers with poor prognosis. Vimentin has been regarded as a marker of epith...

Five argyrophil, non- argentaffin classical carcinoids of the appendix were found in 19 appendiceal classical carcinoids and were investigated histo-chemically, immunohistochemically and ultrastructurally. All tumors consisted entirely of argyrophil cells. Three of the five carcinoids were composed almost totally of peptide YY cells and were negative for serotonin. One of them consisted of peptide YY cells (60%), somatostatin cells (40%), and a few cells with glucagon-like immunoreactivity (GLI). The remaining one without peptides was homogeneously immunoreactive for serotonin alone. Ultrastructurally, each of the four peptide-positive carcinoids was composed of one kind of endocrine cell type with round secretory granules. Average diameter of granules were 150, 160, 190, and 210 nm, respectively. The non-argentaffln, serotonin-positive carcinoid showed predominant round secretory granules and a few irregular ones, both being 150 nm in largest diameter. It is suggested that the argyrophil, non-argentaffln carcinoids of the appendix are subdivided into two groups; carcinoids composed mainly of peptide (especially, peptide YY)-positive cells with round granules of D, and/or L cell type and those of serotonin-positive cells with pleomorphic granules of ECn cell type.

A case of amputation neuroma which occurred in the common bile duct after surgery for cholelithiasis is reported. The patient, a 53-year-old woman was admitted to our hospital with a chief complaint of abdominal pain. Seventeen years previously, she had undergone cholecystectomy, choledochotomy, T-tube drainage and papillotomy under the diagnosis of cholelithiasis. The endoscopic retrograde cholangiopancreatography and percutaneous transhepatic cholangiography disclosed a stenosis of the common bile duct with a residual cystic duct and an elevated mass was found adjacent to the upper end of the stenotic area with a smooth margin. It was clarified by laparotomy and choledochotomy that the mass was covered with normal mucosa of the bile duct. The mass was excised including the residual cystic duct and the stenotic segment and then anastomosis of the common hepatic duct and jejunum was performed. The excised mass was slightly hard in elasticity and 12×7×8mm in size. Pathological findings showed the mass to be a submucosal tumor appearing as if many nerve fascicles were buried in densely proliferative connective tissue. Immunohistological findings revealed that S-100 protein, the neuron-secific protein, and neuron-specific enolase were both positive in the tumor, and amputation neuroma was diagnosed.

Extrahepatic bile duct cancer (cholangiocarcinoma) has a poor prognosis. Since surgical resection is the only way to prolong the patient’s life, it is of critical importance to correctly determine the extent of lesions. However, conventional pre-operative assessments have insufficient spatial resolution for determining the surgical margin. A fluorescent contrast agent might provide a more precise measure to identify anomalies in biliary surface, when combined with probe-based confocal laser endomicroscopy (pCLE). We have previously shown that 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-l-glucose (2-NBDLG), a fluorescent derivative of l-glucose (fLG), is specifically taken up into spheroids consisting of cells showing heterogeneous nuclear-cytoplasm ratio, a feature of malignant cells in clinical settings. In addition, a combined use of 2-TRLG, a membrane-impermeable fLG, with 2-NBDLG visualized membrane integrity as well. We therefore explored in the present study the availability of the fLGs in vivo as a contrast agent for pCLE by using a hamster model of cholangiocarcinoma. Extrahepatic cholangiocarcinoma developed in mid common duct in ~20 % of the animals subjected to cholecystoduodenostomy with the ligation at the distal end of the common duct followed by injection of a carcinogen N-nitrosobis(2-oxopropyl)amine. After infusing bile duct with a solution containing 2-NBDLG and 2-TRLG, the lumen was surgically exposed and examined by pCLE. Fluorescence pattern characterized by bright spots and dark clumps was detected in the areas diagnosed with cholangiocarcinoma in later histopathology, whereas no such pattern was detected in control animals. These findings may form a basis for elucidating a potential availability of fLGs in imaging cholangiocarcinoma by pCLE. Electronic supplementary material The online version of this article (doi:10.1007/s13577-015-0131-5) contains supplementary material, which is available to authorized users.

Carcinomas of the gallbladder can be grossly divided into two types: carcinoma in adenoma (CIA) and carcinoma without adenoma (CWA). The histogenesis of both types of gallbladder carcinoma (CIA and CWA) was investigated in association with metaplastic changes in 35 early carcinomas larger than 5 mm in diameter and 16 microcarcinomas up to 5 mm in largest diameter. In five early CIAs and two micro-CIAs, the carcinoma was surrounded by the adenoma, and the areas of both carcinoma and adenoma mainly showed gastric-type metaplasia (GM). On the other hand, 90% of the other 30 early CWAs showed GM and/or intestinal-type metaplasia (IM) in the tumor, and all of them were surrounded by GM and/or IM. Seven of the other 14 micro-CWAs showed GM and/or IM in the tumor as well as in the surrounding mucosa, and had non-neoplastic metaplastic glands underneath the carcinoma tissue. The remaining seven micro-CWAs showed no or only mild metaplasia in the tumor and were surrounded by proper mucosa without metaplasia. From these data, it is concluded that carcinoma of CIA possibly arises from adenoma mainly with GM, and that CWA originates either from the upper part of the metaplastic mucosa or from the proper mucosa of the gallbladder. In addition, some CWA may undergo secondary metaplastic changes during tumor growth.

The basic helix-loop-helix (bHLH) transcription factors, DEC2 and DEC1, play critical roles in the circadian rhythm of the suprachiasmatic nucleus (SCN). It is known that mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative-feedback loop, and CLOCK/BMAL1 and CLOCK/BMAL2 enhance DEC2 transcription via CACGTG E-boxes. To understand the role of arginine 57 ((57)Arg) within the basic region of DEC2, we examined the effect of substituting this residue into DEC2 on CLOCK/BMAL2-mediated transactivation. A luciferase assay showed that substituting (57)Arg for Ala or Lys in DEC2 diminished the suppressive activity of wild-type DEC2 on CLOCK/ BMAL2-mediated transactivation, while substituting (48)Pro for Ala in DEC2 did not alter it, and the same was true for wild-type DEC2. We also showed that proteins which were wild-type and substitution mutants of DEC2 were expressed at nearly equivalent levels by Western blotting. These findings demonstrate that (57)Arg in the basic region of DEC2 is essential for its activity in suppressing CLOCK/BMAL2-mediated transactivation.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression, These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.