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Many tumor suppressor genes are involved in the multistep process of neoplastic development. However, the signaling mechanism that underlies the development of tumors has not yet been completely elucidated. Therefore, Discovery of a novel tumor suppressor gene plays a crucial role in our understanding of the development and progression of malignant tumors. Chromosome engineering technique that base on Microcell-mediated chromosome transfer （MMCT），which can be generally used to the introduction of a single chromosome to a variety of tumor cells, is one of effective approach for mapping and identification of tumor suppressor genes. We have identified paired-like homeodomain 1（PITX1）gene as a novel telomerase negative regulatory factor that inhibit the expression of telomerase reverse transcriptase （TERT）using chromosome engineering technique. Here, we describe a unique strategy from mapping to identification of tumor suppressor gene by using MMCT approach.

医学部血液内科学教室 泉二登志子教授退任記念特別号

多発尿路上皮癌の経時的な遺伝子解析により, 1)おもに表在性の多発尿路上皮癌症例では, (少なくとも)約80%は同一クローン由来と考えられる.2)表在性尿路上皮癌の多くは再発を繰り返しても遺伝的には安定である.3)多発尿路上皮癌の腫瘍間での遺伝子変化の多様性は第9染色体の異常が生じた後に起る.4)第9染色体の異常が生じた後も臨床的に腫瘍を形成せずにdormantな状態が存在する.5)第9染色体に異常を持つ細胞はそれ自体では腫瘍形成に至らなくとも, 尿路内を播種又は上皮内進展する能力をもっている, One of the most important features of urothelial cancers of the bladder and upper urinary tract is metachronous and/or synchronous multifocal occurrence with high frequency. Since such multifocal recurrent tumors are derived from a common transformed cell, the chronological tracing of genetic alterations in such multifocal tumors may reveal the precise timing and role of genetic alterations in urothelial carcinogenesis. In this study, we tested the presence of microsatellite alterations in synchronous and/or metachronous multifocal urothelial cancers to examine the chronological genetic alterations for the presence of hierarchy of genetic alterations in urothelial cancer development. Genetic alterations at 20 microsatellite loci on 8 chromosomal arms (2q, 4p, 4q, 8p, 9p, 9q, 11p, and 17p) were tested. Judging from the patterns of allelic deletion and microsatellite shifts, multifocal tumors in at least 21 (81%) of the 26 evaluable patients were considered to be derived from a single progenitor cell. In patients with multifocal tumors of an identical clonal origin, discordant microsatellite alterations were observed at significantly lower frequencies on chromosome 9 compared with those on the other chromosomes tested. The heterotopic spread and genetic divergence may occur long before the clinical manifestation of multiplicity from a single transformed cell. The data strengthens the previous view that heterotopic spread of transformed progenitor cells and genetic divergence occur after chromosome 9 alterations in most of urothelial cancers.

Recently, serum antibodies against the oncogene and tumor suppressor gene products have been studied in patients with various types of cancer. Antibody against p53 tumor suppressor gene product among these antibodies was suggested to be useful for early diagnosis and evaluation of prognosis of patients with some types of cancer. In this article,we review clinical significance of antibodies against product of retinoblastoma gene (pRB),one of representative tumor suppressor genes. We also describe methods of detection of antibodies in sera from patients with lung cancer by immunoblotting assays using glutathione-S-transferase (GST)-RB fusion proteins.

Research is carcinogenesis and cancer treatment, commonly uses rats and their usefulness is well known. Alteration of the p53 tumor suppressor gene is the most common genetic defect known to occur in human tumors and it seems to play an important role in carcinogenesis. Recently, a new biological assay for human p53 using a yeast has been developed. This yeast functional aasay is a simple and more sensitive method than previous methods. This study applied this yeast assay to the analysis of the rat p53 gene and estimated its usefulness. The yeast functional assay for rats could be established by a modification of the assay for humans. Thus, cotransformation of a rat p53 PCR products and pLSRP53 plasmid cut with Pst I and Stu I results in repair of the plasmid with the PCR products in vivo and constitutive expression of the full length rat p53 protein in yeast. After cultivation, yeast inserted with wild-type rat p53 forward white colonies and those inserted with mutant type rat p53 forward red colonies effectively. The ratio (white / red colonies) coresponded well with the mutant ratio of at p53 gene (wild / mutant type). The sequence of p53 cDNA obtained from red colonies detects all clonal mutations. This incidence of mutation fonned out in rat p53 was higher than that of previous reports. These results indicate that the yeast functional assay can be applied to for the analysis of rat p53 and that this assay is highly sensitive and specific enough to estimate the genotype of rat p53.

前立腺癌の進行や転移と第8, 10, 16染色体の欠失の相関について転移巣を含めた, 様々な臨床病期の前立腺癌検体を用いて検討した。各症例の正常および癌組織よりゲノムDNAを抽出し, PCR-LOH法を用いて各染色体につき2-4個のマイクロサテライト・マーカー部位について解析した。組織学的分化度は特に染色体欠失との相関を見なかったが, それぞれの染色体の欠失と臨床病期との間に有意な相関を認めた。染色体の欠失はその領域における癌抑制遺伝子の存在を意味しており, 前立腺癌の進行および転移に第8, 10, 16染色体上に存在が予想される癌抑制遺伝子は重要な役割を演じているものと考えられた。またこれらの染色体欠失は前立腺癌患者の予後や進展の可能性を把握するのに有効な指標となりうるものと考えられた。, To evaluate the relationship between the progression and/or metastasis of human prostate cancer and allelic losses of chromosomes 8, 10, and 16, this study screened a unique set of prostate cancer tissues representing the specimens of clinical prostate cancer from organ confined to metastatic lethal disease. Genomic DNAs extracted from cancer tissues and corresponding normal tisssues were analyzed by PCR-LOH method with each 2-4 microsatellite markers on chromosomes 8, 10, and 16. The significant association between each chromosomal loss and clinical stage of the specimens was observed, while no relationship was found between histological differentiation and chromosomal losses. Since frequent allelic losses at specific chromosomal loci in several types of human cancer have implied the presence of putative tumor suppressor genes in the regions where deletions were detected, this study suggests that putative tumor suppressor genes on the chromosomes examined here play an important role of the progression and/or metastasis of prostate cancer. Therefore, allelic losses on these chromosomes can be useful markers to predict and grasp clinical course of prostate cancer patients.

Article, 信州医学雑誌 46(4): 239-248(1998)

大腸においても表面型腺腫・癌が多く発見されるようになり, 分子生物学的にはK-ras遺伝子変異が極めて低率であることが明らかにされ, その形態像の特異性とともに従来のポリープ・癌相関とは異なる発育進展機序が想定されている。大腸癌の発生・進展に関与する, 第18染色体長腕上に存在する癌抑制遺伝子の候補遺伝子とされている DCC (Deleted in Colorectal Carcinoma) 遺伝子について, 私達はコドン201に (Arg/Gly) 多型性が存在し, 進行大腸癌と201 (Gly) 型が相関することを見出した。今回, DCC遺伝子コドン201 (Arg/Gly)多型性について表面型と隆起型腫瘍とを比較検討した。腫瘍高が正常粘膜以下のもの (平坦・陥凹型) を表面型とし, 組織学的には軽度異型群 (以下 low 群), 粘膜内癌を含む高度異型群 (以下 high 群), 粘膜下層浸潤癌 (以下sm癌) に分類した。末梢血あるいは正常大腸粘膜でのDCC遺伝子コドン201 (Gly) 型の頻度は, 対照 (全大腸内視鏡検査および家族歴で大腸腫瘍性病変を認めない例) 17% (5/30例), 隆起型腫瘍を有する例では 1ow 群18% (3/17例), high 群49% (17/35例), sm癌52% (15/29例), および進行癌例では49% (36/74例) であった。表面型腫瘍を有する例では, high 群64% (9/14例), およびsm癌54% (7/13例) であり, 隆起型 high 群, sm癌および進行癌と同様に高率であったが, 表面型 low 群では67% (6/9例) と, 対照 (17%) および隆起型 low 群 (18%) に比して有意に高率であった。片対立遺伝子の欠失 (LOH) を認めた10例を除いて, コドン201の多型性は正常組織と腫瘍組織とで同一結果であった。腫瘍組織でLOHを認めた10例のうち9例はコドン201 (Arg) アレルが欠失し, (Gly) アレルが残存していた。以上より, DCC遺伝子コドン201 (Gly) 型は大腸癌の有用な genetic marker であるとともに, 表面型腫瘍にも関連していることが示唆された。, Recent studies have identified the distinct existence of flat-type colorectal tumors. The low incidence of ras gene mutations in these tumors suggests that their genetic pathways of tumor progression may be different from those of the polypoid type. To elucidate further genetic alterations in flat-type colorectal tumors, codon 201^ polymorphism in the DCC (deleted in colorectal carcinoma) gene was analyzed in normal tissue (normal colonic mucosa or peripheral lymphocytes), and tumor tissue from 191 patients with colorectal tumors (36 patients with flat-type colorectal tumors, 81 patients with polypoid-type colorectal tumors, and 74 patients with advanced carcinomas) . For normal controls, 30 samples obtained from patients who had neither colorectal tumors (confirmed by total colonoscopy) nor a family history of colorectal carcinoma were analyzed. DCC gene codon 201^ polymorphism was investigated by polymerase chain reaction-based restriction fragment length polymorphism analysis, fluorescence-based dideoxy sequencing, or both. For the flat type, the frequency of codon 201^ of the DCC gene was 64% and 54% in the normal tissue of patients with adenoma with high-grade dysplasia and submucosal carcinoma, respectively. It was 49%, 52%, and 49% in the normal tissue of patients with polypoid-type adenoma with high-grade dysplasia, submucosal carcinoma, and advanced carcinoma, respectively. In the normal tissue, codon 201^ of the DCC gene was more frequently observed in patients with flat-type adenoma with low-grade dysplasia (67%) than in those with polypoid-type adenoma with low-grade dysplasia (18%) or in normal controls (17%) (p polymorphism in tumor tissues did not differ from that in the corresponding normal tissues, except for ten cases of carcinoma with loss of heterozygosity (LOH). In carcinomas with LOH, preferential loss of the codon 201^ allele was noted (9/10 cases) . These results suggest that codon 201^ of the DCC gene is not only associated with flat-type colorectal tumors, but that it may serve as a useful genetic marker for identifying groups at higher risk for colorectal cancer.

Prostate cancer;Progression model;Gene therapy;Osteocalcin promoter;PSA promoter, Hormone treatment, radiotherapy and anti-cancer chemotherapy are often used to treat prostate cancer. However, there is no effective method of treating hormone-independent prostate cancer. In this study, we attempted to establish a new treatment method for hormone-independent prostate cancer. We developed a new recombinant adenovirus vector containing a suicide gene and controlled by a tissue specific promoter, and examined the usefulness of gene therapy for hormone-independence and PSA expression in prostate cancer. We have also examined the usefulness of gene therapy involving an adenovirus and various tumor suppressor genes for human prostate cancer cells, which are under trial in the United States.

Ten glioma cell lines were examined for alterations of the p16, p15 and p53 genes, which were tumor suppressor genes with direct or indirect CDK inhibitory functions. All the cell lines showed abnormalities in at least 1 gene, often in 2 or 3 genes coincidentally, suggesting that dysfunction of these genes is closely related to glioma cell growth. On examination of the primary tumor tissues, the same alterations of the p16/p15 and p53 genes as detected in the cell lines were demonstrated in all 6 cases examined, p16/p15 gene deletion in 1, p16 gene mutation in 1 and p53 gene mutations in 5 cases. This suggested that the p16/p15 and p53 gene alterations and their combinations in at least some glioma cell lines reflected those in the primary glioma tissues.

手術的に得られた腎癌24例, 前立腺癌18例, 腎孟移行上皮癌11例に制限酵素切断多型性 (RFLP) 分析を施行し, 各種癌遺伝子の増幅, あるいは癌抑制遺伝子の不活化の有無につき検討した. Harvey ras, c-myc, c-fos, c-myb, EGFR, PDGFRいずれの癌遺伝子も各腫瘍型にてDNAコピー数の過剰増加を呈さなかった. 一方, RFLP分析にて癌抑制遺伝子の不活化を示す染色体座位欠失所見が各腫瘍型で検出された. 腎癌では, 第3染色体短腕 (3p) に68%, 第18染色体長腕 (18q) に33%, Y染色体に29%, 17pに27%の順に高頻度に座位欠失が見られた. 前立腺癌では, 16q (67%), 8p (50%), 18q (43%) 10p (40%), 10q (38%) に, 腎孟移行上皮癌では, 17p (73%), 11p (64%), 9p (40%) に高頻度の座位に欠失が認められた. 各腫瘍型において癌細胞の悪性度が高い症例ほど多数の座位欠失を伴う傾向を示した. We applied restriction fragment length polymorphism (RFLP) analysis to 24 cases of renal cell carcinomas (RCC), 18 cases of prostate adenocarcinomas (PC), and 11 cases of transitional cell carcinomas (TCC) in the renal pelvis to study the oncogene amplification and inactivation of tumor suppressor genes. All of the cases showed no amplification nor gross rearrangements of the Harvey ras, c-myc, c-fos, c-myb, EGFR and PDGFR. In contrast, RFLP analyses demonstrated allelic losses interpreted as inactivational events of TSGs among the tumor forms studied. RCC had allelic losses on the short arm of chromosome 3 (3p) (68%), the long arm of chromosome 18 (18q) (33%), Y chromosome (29%), and 17p (27%) at high frequencies. PC showed frequent allelic losses on 16q (67%), 8p (50%), 18q (43%), lop (40%), and 10q (38%). TCC had allelic losses on 17p (73%), 11p (64%), and 9p (40%). It was likely that the cases with the more malignant grade tumor had the more allelic losses., 出版者照会後に全文公開 / 許可を得て公開

BACKGROUND Many researchers have attempted to correlate p53 mutation and spontaneous apoptosis with the effectiveness of radiochemotherapy and with prognosis in several malignancies. METHODS The current study group consisted of 70 Japanese patients with maxillary sinus squamous cell carcinoma (SCC). Fifty seven patients were treated with radiochemotherapy followed by total or partial maxillectomy, and the remaining13 patients were treated with radiotherapy alone. Tumor biopsy specimens at pretreatment status were examined for apoptosis-related proteins such as p53 protein, Fas, bax, bcl-x, and apoptosis using immunohistologic methods. The proportion of apoptotic cells labeled by single stranded DNA antibody was expressed as an apoptotic index (AI). p53 mutations at exons 5 through 8 were analyzed by direct sequence on polymerase chain reaction amplified products obtained from laser microdissected tissues. The effectiveness of radiochemotherapy was investigated histologically on surgically dissected specimens. RESULTS p53 mutations were identified in 20 (29%) of 70 patients. p53 protein was overexpressed in 39 patients (56%), Fas in 20 patients (29%), bax in 40 patients (57%), and bcl-x in 33 patients (47%). Overexpression of bax was associated with negativity of bcl-x (P = 0.015) and with high AI (P = 0.024). Low AI and/or p53 mutation in the pretreatment tissues correlated with low histologic effectiveness of radiochemotherapy (P = 0.048, P = 0.019, respectively). Kaplan-Meier analysis as well as univariate analysis using the Cox proportional hazards model showed that low histologic effectiveness of radiochemotherapy (P = 0.0281, P = 0.0284, respectively), p53 mutations (P = 0.0095, P = 0.0187, respectively), negativity of bax (P = 0.0069, P = 0.0191, respectively), and low AI (P = 0.0134, P = 0.0407, respectively) were significantly related to worse disease-free survival. Multivariate analysis showed AI as an independent factor predicting for disease-free survival (P = 0.0455). CONCLUSIONS The p53 mutations, expression of bax, and levels of spontaneous apoptosis have prognostic value in maxillary sinus SCC; AI especially is an independent factor for disease-free survival. A high level of spontaneous apoptosis induced by overexpression of bax may increase sensitivity of radiochemotherapy resulting in good prognosis, while p53 mutation may lead to resistance against radiochemotherapy. resulting in poor prognosis. Cancer 2002;94:1968–80. © 2002 American Cancer Society. DOI 10.1002/cncr.10388

Objectives Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. Methods Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. Results CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity. Conclusions CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

2016