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We here describe the characteristics of genome mutations found in cancer cells.

口腔癌における遺伝子異常について検索を行った。その結果、がん抑制遺伝子p53 の変異・欠失が70％以上の症例でみられることや、ets がん遺伝子群転写因子E1AF が細胞外基質分解酵素の活性亢進をもたらすこと、テロメラーゼ活性化により口腔癌の発症・悪性化に関与していることを明らかにした。AU-richelement（ARE）をもつmRNA は生理的状態ではエキソゾーム内で素早く分解されるが、がん細胞ではHuRが恒常的にがん遺伝子や細胞増殖因子のARE に結合・安定化させ発現を亢進し、発がんにおけるnon-coding RNA の重要性を示した。これまで腫瘍周囲間質は正常細胞からなっていると考えられてきたが、近年、腫瘍微小環境の異常性が指摘されている。腫瘍微小環境に存在する腫瘍血管の解析を行ったところ、腫瘍血管内皮細胞は細胞学的に異常性をもち正常血管内皮細胞と異なった遺伝子/遺伝子産物を発現しており、腫瘍血管をターゲットにした医薬分子送達システムにより特異的かつ効率的な治療効果を得られる可能性が示された。, Molecular pathological investigation of oral carcinogenesis was described. Mutation and deletion of p53, a potent anti-oncogene, were observed in more than 70% of oral carcinoma, and close relationship was observed between oral carcinomas and ets transcription factor E1AF. E1AF upregulate MMPs that induce cancer cell invasion by degrading extracellular matrices. The significance of non-coding RNA on carcinogenesis, especially concerning ARE (AU-rich element) mRNA was shown. Many oncogenes contain ARE in its untranslated region, and ARE mRNA is stabilized and transported in cancer cells by CRM-1 independent manner. Blood vessels in tumor microenvironment were investigated. Tumor endothelial cells had abnormalities compared to normal endothelial cells, and specific genes/gene products in tumor endothelial cells will be targets of gene therapy.

The causal relationship between chronic inflammation and cancer is widely accepted. Numerous investigations have identified nuclear factor-κB (NF-κB) as an important modulator in driving chronic inflammation to cancer. This study aimed to determine the prevalence of human papillomavirus (HPV) in penile cancer in Japanese patients and whether NF-κB is subsequently overexpressed in penile cancer. Thirty-four specimens of penile tissue (16 malignant and 18 benign cases) were examined to determine the association of HPV infection. An in situ hybridization (ISH) method was used to detect and localize HPV-DNA. A sensitive HPV polymerase chain reaction (PCR) procedure was used for the detection of HPV-DNA, and DNA sequencing was used to identify the HPV genotype. HPV-DNA was detected in 37.5 and 75% of cases of penile cancer, using ISH and PCR, respectively. Our efforts to detect HPV genotypes were unsuccessful as HPV-DNA could not be extracted from these materials. Using ISH, a prevalence of 68.2% of HPV infection was found in penile cancer in Kenyan patients in east Africa. In the present study, all 9 HPV-positive cases, (100%) were NF-κB-positive in the nucleus and/or cytoplasm. In contrast, of the 25 HPV-negative cases, 15 (60%) were NF-κB-positive in the nucleus and/or cytoplasm. Therefore, ISH is a method which is able to prove infection of a large quantity of HPV more effectively when compared with PCR. Thus, a large quantity of HPV infection leads to the activity of NF-κB. The most prevalent genotype was the HPV-22 found in 83.3% of the penile cancer cases. In addition, HPV-11 was found in 81.8% of the non-cancer cases. For cases with a high level of infection, the activity of NF-κB increased compared with those with a low level of HPV infection., Oncology Letters, 1(2), pp.267-272; 2010

Article, 信州医学雑誌 46(4): 239-248(1998)

A 49-year-old female who had noticed hard tumors in bilateral breasts visited our hospital. There were depression of nipples and Delle needle biopsy revealed Group Ⅴ for both tumors. Bone scan showed abmormal accumulation in the sternum and bilateral lower ribs. Bone metastasis was suspected. Auchincloss method was performed for bilateral breasts simultaneously because of the advanced stage of cancers and suspected bone metastasis. Histopathologically both lesions were diagnosed as primary bilateral breast carcinoma. We examined hormone recepters, DNA ploidy, amplification of c-myc and c-erbB-2 genes and loss of heterozygosity (LOH) of p 53 and chromosome 16 q, 17 p in bilateral breast tissues. These results showed that the right breast tumor was estrogen recepter-positive, progesteron recepter-negative and of DNA diploidy pattern, while the left breast tumor was estrogen recepter-positive, progesteron recepter-positive and of DNA aneuploidy pattern. Amplification of c-myc and c-erbB-2 and LOH of p 53 and chromosome 16 q, 17 p were not detected in both lesions.

Several oncogenes were introduced into a normal mouse osteoblastic cell line, MC3T3- El, by calcium phosphate-mediated DNA transfection. Transformed foci were formed by cotransfection with adenovirus type 12 E1A （Adl2E1A） and activated c-Ha-ras （EJras） oncogenes, and with c-myc and EJras oncogenes. Transfection with c- myc, Ad12E1A or EJras induced no obvious foci. Transformed cell lines established from each trans- formed focus had high saturation densities in culture and were tumorigenic when injected into athy- mic mice with low-differetiated sarcoma. The transfected oncogene DNA sequences were detected in the cellular DNA of all the transformants. Alkaline phosphatase activity decreased in transformants compared to their parental MC3T3-E1 cells.

Expression of c-erbB-2 mRNA was examined in 33 cultured human cell lines using two synthetic oligonucleotide probes hybridizable to c-erbB-2 mRNA at the portions cording the extracellular and transmembrane domains. The mRNA with molecular size of 5.0 kb was detected in 28 out of 33 cell lines examined, suggesting that cancer cells frequently produce c-erbB-2 protein. Additionally, in three cancer cell lines expressing a large amount of the 5.0 kb mRNA, an aberrant mRNA with molecular size of 2.5 kb was detected, which reacted with the probe corresponding to the extracellular domain, but did not with another probe. The biochemical characteristics of c-erbB-2 protein were examined in a gastric carcinoma cell line, MKN 7 cells, expressing a large amount of c-erbB-2 mRNA. When the cells were radiolabelled with ^Pi, lysed and then labelled proteins were immunoprecipitated with three different antibodies recognizing extracellular, kinase and carboxyl-terminal domains, phosphorylated c-erbB-2 protein with molecular size of 185 kD (p185) was immunoprecipitated by all of these three antibodies. When the cells were radiolabelled with (^S) cysteine, p185 was detected with three antibodies, moreover, a large amount of (^S)-labelled protein with molecular size of 95 kD (p95) was also detected by using the antibody recognizing the extracellular domain. Other two antibodies did not immunoprocipitate this molecule, suggesting that p95 possesses the structure identical to the extracellular domain of p185. Pulse chase experiments suggest that p95 is not likely to be a degenerating protein of p185. Accordingly, it is reasonable to postulate that two types of c-erbB-2-related protein were present in MKN7 cells with molecular size heterogeneity. Further studies are required to clarify the actual biological roles of c-erbB-2 protein produced by cancer cells.

The amplification and rearrangement of five cellular oncogenes (c-myc, c-K-ras, c-fos, c-raf-1, and N-myc) were studied by Southern hybridization in fourteen human bone and soft tissue tumors obtained at surgery. Amplification of c-myc was detected in the two of four osteosarcomas and one of two malignant fibrous histiocytomas. The c-myc genes in these tissues were amplified 4- to 8-fold in comparision with the placenta DNA. One of these osteosarcomas had 16- to 32-fold amplification of c-raf-1 gene without rearrangement. The clinical course of osteosarcoma and malignant fibrous histiocytoma with the amplified c-myc or c-raf-1 gene showed a rapid malignant progress with lung or bone metastasis. There appears to be a correlation between clinical prognosis and oncogene amplification.

We analyzed the transformation-associated cell surface antigens （TAAs） that were expressed on a rat fetus-derived fibroblast, WFB, transformed by the activated oncogenes. By developing monoclonal antibodies （mabs） reacting against these TAAs, it was demonstrated that WFB showed four different TAAs on the transforming process of this clonally-derived cell. （1） These four TAAs recognized by mabs # 109, 061, 081 and 067 showed 86, 62, 101 and 67 kDa of molecular weight, respectively, and each of # 109, 081 and 067 antigen was composed of a. single polypep-tide chain. （2） In order to study the expression of these TAAs on WFB, we utilized a large number of WFB transformants and transfectants obtained by transfection with EJras, polyoma MT （PyMT） and c-myc oncogenes. The data showed that # 109- and 061-defined antigens were expressed on almost all of the WFB transformants in addition to the oncogene-transformants of Fisher rat fetus-derived 3Y1 cells. In contrast, the expression of # 081-defined antigen was relatively restricted to WFB transformants rather than 3Y1 transformants. Furthermore, # 067-defiend antigen could be expressed very selectively on some WFB-ras transformants. （3） It was suggested that these TAAs were not the products of oncogenes as assessed by the localization, as well as the biochemical nature, of molecules. （4） # 109-defined antigen could be expressed on a 16th day fetus, Con A-activated lymphocytes and WFB cells stimulated by PDGF and TGF-beta. In contrast, # 061 and 081 antigens were expressed on the WFB transformants and TGF-beta but not PDGF stimulated WFB cells, suggesting that the expression of # 109 antigen is not associated with the transformation of WFB, but that other TAAs are linked phenotypically with the transformation. （5） We analyzed the immunobiological significance of these TAAs on anti-tumor resistance by the hosts. The data indicated that # 109-and 061-defined antigens may play an important role as the target molecules of natural killer cells.

癌遺伝子の脱メチル化がヒト肝癌発生にどのように関与しているかを明らかにするため， 本研究では， 肝癌症例の肝癌組織と非癌部肝組織， 肝癌を合併しない慢性肝疾患組織， コントロール肝組織を対象として， それらにおける三種の癌遺伝子c-myc， c-Ki-ras， c-Ha-rasのメチル化状態を比較検討した。肝癌組織18例中13例(72.2%）， 非癌部肝組織34例中29例（85.2%）， 肝癌を合併しない慢性肝疾患組織34例中25例（73.5%）においてc-myc遺伝子の第2エクソンのCCGG 部位の完全脱メチル化を認めた。一方， コントロール肝組識31例中3例においてのみ同CCGG 部位の完全脱メチル化を認め， 残りの28例に部分的脱メチル化を認めた。完全脱メチル化を認めた患者3例は， 0， 14， 23歳の若年者であり， 一方部分的脱メチル化を認めた患者28例は25歳から83 歳であり， これは肝癌患者および慢性肝疾患患者とほぼ同様の年齢分布であった。また完全脱メチル化を有する頻度は， 肝硬変で肝癌を合併するか否かで明らかな差を認めず， 慢性活動性肝炎でも同様であった。肝癌組織18例中7例（38.9%）にc-myc遺伝子第3エクソンの脱メチル化が生じており， 13例（72.2%）にc-K1-ras遺伝子の脱メチル化が生じており， 非癌部肝組織， 慢性肝疾患組織， コントロール肝組織においては脱メチル化が生じていなかった。肝癌組織13例（72.2%）において同じ症例の非癌部肝組織と比較してc-Ha-ras 遺伝子の脱メチル化が生じていた。また肝癌組織18例中16例（88.9%）において， 三つのうちいずれかの癌遺伝子に脱メチル化が生じていた。c-myc遺伝子第2エクソンのCCGG 部位の脱メチル化は， 持続する肝細胞壊死とそれに対する代償的な再生をきたす慢性肝疾患における病態と関連を有すると考えられる。そこに肝癌が発生する過程でc-myc遺伝子の第3エクソンあるいはc-Ki-ras， c-Ha-ras遺伝子の脱メチル化等が生じると考えられるが， これらの癌遺伝子の脱メチル化が肝癌発生の原因であるのか結果であるのかは， 本研究では明らかにすることが出来なかった。