EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION

3.EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION

　Our division has been studying the susceptibility to chemical carcinogens and underlying mechanisms in a human c- Ha- ras proto- oncogene transgenic rat, the potential prevention of carcinogenesis by bovine lactoferrin (bLF), ferulic acid and its derivatives, the inhibition of lung metastasis by bLF, the characterization of a tumor regressing factor and the anti- angiogenic effects of diaminotriazines.
Characterization of Susceptibility of Human c-Ha-ras Proto-oncogene Transgenic Rats to Carcinogens

　A rat line carrying human c- Ha- ras proto- oncogene was established. Expression of transgene mRNA was detected in all organs. PCR- RFLP analysis and direct sequencing of codons 12 and 61 of the transgene indicated that most tumor tissue contained cells with mutations in codon 12. However, RFLP analyses and subsequent subcloning and sequencing revealed that almost all tumors contained both cells with mutated and non- mutated transgenes, indicating mixed populations. PCR- SSCP analysis detected no mutations in codons 12 and 61 of the endogenous rat c- Ha- ras gene. However, in non- transgenic rat tumors, mutations were detected in codon 12 of the endogenous rat c- Ha- ras gene. These results indicated that rats carrying the human c- Ha- ras proto- oncogene are highly susceptible to mammary carcinogenesis. The transgenic rats were also found to be highly susceptible to esophagus and bladder carcinogenesis. Furthermore, they exhibit high susceptibility to DMBA- TPA skin carcinogenesis. Irrespective of the mechanisms involved, the human c- Ha- ras proto- oncogene transgenic rat may be a useful model for the short- term detection of carcinogens and promoting agents. P53- deficient mice, in contrast, were found not to be susceptible to heterocyclic amine 2- amino- 3,8- dimethylimidazo[ 4,5- f] quinoxaline (MeIQx)carcinogenesis.⁽⁵¹⁾
Cancer Chemoprevention by Lactoferrin and Other Dietary Factors

　We focused on food components and their derivatives as candidate cancer chemopreventive agents because of easy practical application and low toxicity. Bovine lactoferrin (bLF), an iron- binding glycoprotein known to have antimicrobial and immunomodulating activity, significantly inhibited development of esophagus, lung and tongue tumors, in addition to inhibitory effects on colon carcinogenesis.^(52,⁵³⁾ It also suppressed the growth of intestinal polyps in Apc MIN mice,⁽⁵⁴⁾ indicating its possible application in FAP patients. Furthermore, it was found to exhibit anti- HCV activity and clinical trials in patients with chronic hepatitis have indicated that it is a candidate treatment for this disease.⁽⁵⁵⁾ The rice bran component ferulic acid, and its derivative, 3- (4- geranyloxy- 3- methoxyphenyl)- 2- propionate (EGMP), were shown to suppress colon carcinogenesis.⁽⁵⁶⁾ The plant flavonoid morin was also shown to suppress colon, tongue and forestomach carcinogenesis. ^(57-⁵⁹⁾ Correlations between organ- specific cancer incidences were analyzed using epidemiological data ^(60-⁶⁵⁾ and a review of screening for secondary prevention was undertaken. ⁽⁶⁶⁾
Heterocyclic Amine Environmental Carcinogenesis

　The effects of caffeine on 2- amino- 1- methyl- 6- phenylimidazo[ 4,5- f] quinoline (PhIP)carcinogenicity were assessed. Combined administration of caffeine at 1000ppm and PhIP at 400ppm resulted in an increase in colon aberrant crypt foci (ACFs), the expression of CYP1A2, a PhIP activating enzyme, and DNA- PhIP adduct formation. Thus the enhancing effects of caffeine on PhIP colon carcinogenesis might also occur in humans.⁽⁶⁷⁾ MeIQx was shown to be not carcinogenic in the cynomolgus monkey with the National Cancer Center, Japan and the National Cancer Institute, U.S.A. collaboration study.⁽⁶⁸⁾
Antimetastatic Activity of Lactoferrin and Stimulation of Production of Interleukin-18 (IL-18)

　Oral bLF and its pepsin- digested hydrolysates (bLFH)remarkably inhibited lung metastasis in Co26Lu bearing mice. bLF and bLFH increased the number of CD4⁺ ,CD8⁺ and asialoGM1⁺ cells in the peripheral blood and the small intestine. Cytotoxicity of these white blood cells against Yac- 1 and Co26Lu cells was enhanced by bLF treatment. Increased levels of IL- 18 was found in the epithelium of the small intestine and serum. Thus the results suggest that inhibition of lung metastasis, and possibly colon carcinogenesis, by bLF and bLFH might be due to enhanced cellular immunity mediated by IL- 18 production.⁽⁶⁹⁾
Other Studies

　The polysaccharide- induced tumor- regression factor (TRF)is composed of disulfide- linked monomers whose NH2- terminal amino acid sequence is identical to the b chain of complement C3, but found to be different from complement C3.
　2,4- diamino- 6(pyridine- 4- yl)- 1,3,5- triazine (4PyDAT)was shown to have anti- tumor and anti- metastatic activities against Co26Lu cells in mice, possibly due to its anti- angiogenic activity.




3.EXPERIMENTAL PATHOLOGY AND CHEMOTHERAPY DIVISION



	Our division has been studying the susceptibility to chemical carcinogens and underlying mechanisms in a human c- Ha- ras proto- oncogene transgenic rat, the potential prevention of carcinogenesis by bovine lactoferrin (bLF), ferulic acid and its derivatives, the inhibition of lung metastasis by bLF, the characterization of a tumor regressing factor and the anti- angiogenic effects of diaminotriazines. Characterization of Susceptibility of Human c-Ha-ras Proto-oncogene Transgenic Rats to Carcinogens 　A rat line carrying human c- Ha- ras proto- oncogene was established. Expression of transgene mRNA was detected in all organs. PCR- RFLP analysis and direct sequencing of codons 12 and 61 of the transgene indicated that most tumor tissue contained cells with mutations in codon 12. However, RFLP analyses and subsequent subcloning and sequencing revealed that almost all tumors contained both cells with mutated and non- mutated transgenes, indicating mixed populations. PCR- SSCP analysis detected no mutations in codons 12 and 61 of the endogenous rat c- Ha- ras gene. However, in non- transgenic rat tumors, mutations were detected in codon 12 of the endogenous rat c- Ha- ras gene. These results indicated that rats carrying the human c- Ha- ras proto- oncogene are highly susceptible to mammary carcinogenesis. The transgenic rats were also found to be highly susceptible to esophagus and bladder carcinogenesis. Furthermore, they exhibit high susceptibility to DMBA- TPA skin carcinogenesis. Irrespective of the mechanisms involved, the human c- Ha- ras proto- oncogene transgenic rat may be a useful model for the short- term detection of carcinogens and promoting agents. P53- deficient mice, in contrast, were found not to be susceptible to heterocyclic amine 2- amino- 3,8- dimethylimidazo[ 4,5- f] quinoxaline (MeIQx)carcinogenesis.⁽⁵¹⁾ Cancer Chemoprevention by Lactoferrin and Other Dietary Factors 　We focused on food components and their derivatives as candidate cancer chemopreventive agents because of easy practical application and low toxicity. Bovine lactoferrin (bLF), an iron- binding glycoprotein known to have antimicrobial and immunomodulating activity, significantly inhibited development of esophagus, lung and tongue tumors, in addition to inhibitory effects on colon carcinogenesis.^(52,⁵³⁾ It also suppressed the growth of intestinal polyps in Apc MIN mice,⁽⁵⁴⁾ indicating its possible application in FAP patients. Furthermore, it was found to exhibit anti- HCV activity and clinical trials in patients with chronic hepatitis have indicated that it is a candidate treatment for this disease.⁽⁵⁵⁾ The rice bran component ferulic acid, and its derivative, 3- (4- geranyloxy- 3- methoxyphenyl)- 2- propionate (EGMP), were shown to suppress colon carcinogenesis.⁽⁵⁶⁾ The plant flavonoid morin was also shown to suppress colon, tongue and forestomach carcinogenesis. ^(57-⁵⁹⁾ Correlations between organ- specific cancer incidences were analyzed using epidemiological data ^(60-⁶⁵⁾ and a review of screening for secondary prevention was undertaken. ⁽⁶⁶⁾ Heterocyclic Amine Environmental Carcinogenesis 　The effects of caffeine on 2- amino- 1- methyl- 6- phenylimidazo[ 4,5- f] quinoline (PhIP)carcinogenicity were assessed. Combined administration of caffeine at 1000ppm and PhIP at 400ppm resulted in an increase in colon aberrant crypt foci (ACFs), the expression of CYP1A2, a PhIP activating enzyme, and DNA- PhIP adduct formation. Thus the enhancing effects of caffeine on PhIP colon carcinogenesis might also occur in humans.⁽⁶⁷⁾ MeIQx was shown to be not carcinogenic in the cynomolgus monkey with the National Cancer Center, Japan and the National Cancer Institute, U.S.A. collaboration study.⁽⁶⁸⁾ Antimetastatic Activity of Lactoferrin and Stimulation of Production of Interleukin-18 (IL-18) 　Oral bLF and its pepsin- digested hydrolysates (bLFH)remarkably inhibited lung metastasis in Co26Lu bearing mice. bLF and bLFH increased the number of CD4⁺ ,CD8⁺ and asialoGM1⁺ cells in the peripheral blood and the small intestine. Cytotoxicity of these white blood cells against Yac- 1 and Co26Lu cells was enhanced by bLF treatment. Increased levels of IL- 18 was found in the epithelium of the small intestine and serum. Thus the results suggest that inhibition of lung metastasis, and possibly colon carcinogenesis, by bLF and bLFH might be due to enhanced cellular immunity mediated by IL- 18 production.⁽⁶⁹⁾ Other Studies 　The polysaccharide- induced tumor- regression factor (TRF)is composed of disulfide- linked monomers whose NH2- terminal amino acid sequence is identical to the b chain of complement C3, but found to be different from complement C3. 　2,4- diamino- 6(pyridine- 4- yl)- 1,3,5- triazine (4PyDAT)was shown to have anti- tumor and anti- metastatic activities against Co26Lu cells in mice, possibly due to its anti- angiogenic activity.