EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION

3.EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION

    Analysis of susceptibility to various chemical carcinogens and underlying mechanisms in human c-Ha-ras proto-oncogene transgenic rats, prevention of carcinogenesis and metastasis by bovine lactoferrin (bLF), ferulic acid and its derivatives, isoliquiritigenin from plant licorice, anti-angiogenic effects of diaminotriazines and characterization of a tumor suppressing factor were studied.
Characterization of Carcinogen Susceptibility of Human c-Ha-ras Proto-oncogene Transgenic Rats

    A rat line carrying 3 copies of the human c-Ha-ras proto-oncogene, was established and designated Hras128. The transgene mRNA expression was detected in all organs. Multiple mammary carcinoma developed as short as 8 weeks after treatment with N-methy-N-nitrosourea. Most of the tumor tissue contained cells with mutations in codon 12. On the other hand, no mutations were found in codons 12 and 61 of the endogenous rat c-Ha-ras gene. Similarly, mammary carcinomas were induced by other mammary carcinogens within a short period. These results indicated that transgenic rats are extremely susceptible to mammary carcinogenesis and that the roles of mutations of the transgene require further analysis.⁽⁴⁹⁾ The rats were also shown to be highly susceptible to esophagus, bladder ⁽⁵⁰⁾ and skin carcinogenesis. Regardless of the mechanisms involved, the human c-Ha-ras proto-oncogene transgenic rats could be utilized for short-term detection of carcinogens and promoting agents.
Isolation of a Putative Target Gene for the Ras Signaling Cascade in Tumor Cells from the Hras128 Rat

    Primary cell lines were established from spontaneous fibrosarcoma-like subcutaneous tumors in a male Hras128 rat, and genes overexpressed in tumor cells as compared to normal fibroblast cells (3Y1) were isolated by suppression subtractive hybridization. Among 86 highly expressed cDNA clones, rat neuronal leucine rich repeat protein-3 (NLRR-3) was found which was homologous to mouse NLRR-3. Rat NLRR-3 gene expression was found to be regulated by the Ras-MAP kinase signaling cascade. The protein was localized to Golgi and ER membranes suggesting a role on vesicular transportation.
Cancer Chemoprevention by Lactoferrin and Other Plant Factors

    To search for cancer chemopreventive agents, the focus was on food components as candidate compounds because of easier applicability for practical use and known toxicity. bLF, a multi-functional protein in milk, significantly inhibited development of bladder ⁽⁵¹⁾ and tongue ^(52,⁵³⁾ tumors, in addition to the previously observed effects on colon carcinogenesis.⁽⁵⁴⁾ Furthermore, bLF was shown to inhibit expression of CYP1A2, an activating enzyme for heterocyclic amines such as PhIP and MeIQx, resulting in inhibition of liver and colon carcinogenesis. bLF has been found to exhibit anti-HCV activity and clinical trials for patients with chronic hepatitis is underway in the National Cancer Center Hospital. bLF was also found to decrease the colony formation of Helicobacter pyroli in Mongolian gerbils, indicating its potential application to the prevention of Helicobacter pyroli-related stomach carcinogenesis in animals.⁽⁵⁵⁾
    The rice bran component, ferulic acid, its derivative, 3-(4-geranyloxy-3-methoxyphenyl)-2-propionate and isoliquiritigenin from plant licorice (Glycyrrhiza glabla) were shown to suppress colon carcinogenesis.
Analysis of Anti-metastatic and Anti-Carcinogenesis Activity of Bovine Lactoferrin (bLF) in Mice

    Orally administered bLF and its pepsin-digested hydrolysates (bLFH) markedly increased the number of T, B and NK cells in the small intestine and peripheral blood. bLF and bLFH increased production of IL18 and caspase-1 in the epithelial cells of the small intestine, which subsequently induced IFN-g presenting cells.⁽⁵⁶⁾ Enhanced intestinal mucosal immunity may have important roles in prevention of tumor metastasis and carcinogenesis.
Anti-metastatic and Anti-tumor Activities of Diaminotriazine Derivative (4PyDAT) in Mice

    2, 4-Diamino-6-(pyridine-4-yl)-1, 3, 5-triazine (4PyDAT) was found to inhibit the production of urokinase-type plasminogen activator (uPA) in Co26Lu cells. The anti-tumor and anti-metastatic activities of 4PyDAT may be due to its anti-angiogenesis activity.⁽⁵⁷⁾
Analysis of Tumor-regressing Factor (TRF)

    cDNA analysis of the polysaccharide-induced TRF, revealed several cDNAs which have a 5'-terminal sequence identical to that of C3 but have novel sequences at their 3'-end.
Other Studies

    The importance of the tumor suppressing activity of Connexin43 was shown in C6 glioma cells and bladder carcinoma cells by overexpression or dominant-negative inhibition.^(58,⁵⁹⁾ The significance of adducin in control of cytoskeleton-membrane interaction was reviewed.⁽⁶⁰⁾




3.EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION



	Analysis of susceptibility to various chemical carcinogens and underlying mechanisms in human c-Ha-ras proto-oncogene transgenic rats, prevention of carcinogenesis and metastasis by bovine lactoferrin (bLF), ferulic acid and its derivatives, isoliquiritigenin from plant licorice, anti-angiogenic effects of diaminotriazines and characterization of a tumor suppressing factor were studied. Characterization of Carcinogen Susceptibility of Human c-Ha-ras Proto-oncogene Transgenic Rats A rat line carrying 3 copies of the human c-Ha-ras proto-oncogene, was established and designated Hras128. The transgene mRNA expression was detected in all organs. Multiple mammary carcinoma developed as short as 8 weeks after treatment with N-methy-N-nitrosourea. Most of the tumor tissue contained cells with mutations in codon 12. On the other hand, no mutations were found in codons 12 and 61 of the endogenous rat c-Ha-ras gene. Similarly, mammary carcinomas were induced by other mammary carcinogens within a short period. These results indicated that transgenic rats are extremely susceptible to mammary carcinogenesis and that the roles of mutations of the transgene require further analysis.⁽⁴⁹⁾ The rats were also shown to be highly susceptible to esophagus, bladder ⁽⁵⁰⁾ and skin carcinogenesis. Regardless of the mechanisms involved, the human c-Ha-ras proto-oncogene transgenic rats could be utilized for short-term detection of carcinogens and promoting agents. Isolation of a Putative Target Gene for the Ras Signaling Cascade in Tumor Cells from the Hras128 Rat Primary cell lines were established from spontaneous fibrosarcoma-like subcutaneous tumors in a male Hras128 rat, and genes overexpressed in tumor cells as compared to normal fibroblast cells (3Y1) were isolated by suppression subtractive hybridization. Among 86 highly expressed cDNA clones, rat neuronal leucine rich repeat protein-3 (NLRR-3) was found which was homologous to mouse NLRR-3. Rat NLRR-3 gene expression was found to be regulated by the Ras-MAP kinase signaling cascade. The protein was localized to Golgi and ER membranes suggesting a role on vesicular transportation. Cancer Chemoprevention by Lactoferrin and Other Plant Factors To search for cancer chemopreventive agents, the focus was on food components as candidate compounds because of easier applicability for practical use and known toxicity. bLF, a multi-functional protein in milk, significantly inhibited development of bladder ⁽⁵¹⁾ and tongue ^(52,⁵³⁾ tumors, in addition to the previously observed effects on colon carcinogenesis.⁽⁵⁴⁾ Furthermore, bLF was shown to inhibit expression of CYP1A2, an activating enzyme for heterocyclic amines such as PhIP and MeIQx, resulting in inhibition of liver and colon carcinogenesis. bLF has been found to exhibit anti-HCV activity and clinical trials for patients with chronic hepatitis is underway in the National Cancer Center Hospital. bLF was also found to decrease the colony formation of Helicobacter pyroli in Mongolian gerbils, indicating its potential application to the prevention of Helicobacter pyroli-related stomach carcinogenesis in animals.⁽⁵⁵⁾ The rice bran component, ferulic acid, its derivative, 3-(4-geranyloxy-3-methoxyphenyl)-2-propionate and isoliquiritigenin from plant licorice (Glycyrrhiza glabla) were shown to suppress colon carcinogenesis. Analysis of Anti-metastatic and Anti-Carcinogenesis Activity of Bovine Lactoferrin (bLF) in Mice Orally administered bLF and its pepsin-digested hydrolysates (bLFH) markedly increased the number of T, B and NK cells in the small intestine and peripheral blood. bLF and bLFH increased production of IL18 and caspase-1 in the epithelial cells of the small intestine, which subsequently induced IFN-g presenting cells.⁽⁵⁶⁾ Enhanced intestinal mucosal immunity may have important roles in prevention of tumor metastasis and carcinogenesis. Anti-metastatic and Anti-tumor Activities of Diaminotriazine Derivative (4PyDAT) in Mice 2, 4-Diamino-6-(pyridine-4-yl)-1, 3, 5-triazine (4PyDAT) was found to inhibit the production of urokinase-type plasminogen activator (uPA) in Co26Lu cells. The anti-tumor and anti-metastatic activities of 4PyDAT may be due to its anti-angiogenesis activity.⁽⁵⁷⁾ Analysis of Tumor-regressing Factor (TRF) cDNA analysis of the polysaccharide-induced TRF, revealed several cDNAs which have a 5'-terminal sequence identical to that of C3 but have novel sequences at their 3'-end. Other Studies The importance of the tumor suppressing activity of Connexin43 was shown in C6 glioma cells and bladder carcinoma cells by overexpression or dominant-negative inhibition.^(58,⁵⁹⁾ The significance of adducin in control of cytoskeleton-membrane interaction was reviewed.⁽⁶⁰⁾