胎盤型アルカリフォスファタ-ゼのmRNAの発現からみた胃腸上皮化生と胃癌との関連

金沢大学がん研究所単クロ-ン抗体による免疫組織染色により胎盤型ALPは癌特異性の高いことが判明したため,遺伝子レベルでの発現について検討を試みた。胎盤型ALPを産生している新鮮胎盤組織と絨毛上皮癌BeWo細胞株を陽性対象として,胃癌KATOーIII細胞ならびに小腸粘膜組織よりRNAを抽出し,RTーPCR法を用いて胎盤型ALPmRNAの発現の有無について検討を行った。すなわち,抽出した各RNAにrandom hexamerと逆転写酵素を用いて,cDNAを作成した。胎盤型ALPと小腸型ALPのcDNA塩基配列の比較により,胎盤型ALPに特異的と考えられる領域(214bp)をはさみ込む25oligonucleotideをDNA合成した。これをprimerとして用いて,RNAより変換されたcDNA中の胎盤型ALPcDNAの特異領域(214bp)を増幅し,胎盤型ALPmRNAの発現の有無について検索した。その結果,胎盤組織,KATOーIII,BeWo細胞株に胎盤型ALPmRNAの発現が認められたが,小腸粘膜組織にも214bpの胎盤型ALPcDNAの出現が認められた。最近胎盤型ALPのgenomic DNAの塩基配列が解明され設定していた214bpは,exon 9,10,11から構成されており,特異的と考えられたこの領域も,他領域と同様にexon,intron共に,小腸型ALPと非常に類似していることが判明した。1にて,胎盤型ALPの蛋白レベルでの発現は,胃腸上皮化生では認められなかったことより,今回のRTーPCR法の結果は,小腸粘膜組織に胎盤型ALPが発現されているというよりも,小腸型ALPmRNAにも交差反応を示した可能性が強いと思われた。また,KATOーIII細胞では胎盤型ALPcDNAのbandが214bpよりも小さなbandとして出現しており,特異領域内の一部の塩基が欠失している可能性が考えられた。現在,これらの点を確認するために,dideoxy法によるDNA sequencingにより塩基配列を決定するなどの実験を継続中である。Intestinal metaplasia of the gastric mucosa has been suggested to represent a precancerous state. However, how it is related to carcinogenesis remains controversial. We developed specific monoclonal antibodies (MAbs) against placental alkaline phosphatase (PLAP). Imnninohistochemical study with the specific MAb showed that expression of PLAP was apt to occur in more highly differentiated gastric carcinoma, and was highly specific for carcinoma. Therefore, we attempted to elucidate the expression of PLAP MRNA with the RT-PCR method in various benign and malignant gastric diseases. Each RNA was extracted from KATO-III cells (gastric cancer), intestinal mucosal tissue, BeWo cells (chorionic carcinoma), and placental tissue. Primer pairs were selected as synthetic 25 oligonucleotides which detected the PLAP cDNA region (2l4 bp) which was widely different from intestinal alkaline phosphatase cDNA (IAP cDNA). The results of RT-PCR showed that 214 bp band appeared in samples from KATO-III cells, BeWo cells, intestinal mucosal tissue as well as placental tissue. On the other hand, PLAP cDNA is very similar to IAP cDNA. These results indicated that primer pairs probably reacted with both PLAP mRNA and IAP mRNA. Futhermore, the amplified PLAPcDNA region in the sample of KATO- III was recognized as a slightly small weight band in electrophoresis and therefore several base pairs of the amplified region might be deleted in KATO- III cells. To clarify these points, further investigations continue.研究課題/領域番号:02670301, 研究期間(年度):1990 – 1991出典：研究課題「胎盤型アルカリフォスファタ-ゼのmRNAの発現からみた胃腸上皮化生と胃癌との関連」課題番号02670301（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-02670301/026703011991kenkyu_seika_hokoku_gaiyo/）を加工して作

膵癌早期診断に対する新しい内視鏡的ならびに分子生物学的アプローチ

金沢大学がん研究所超音波内視鏡(EUS)と膵液の遺伝子診断を組み合わせた一連の内視鏡下の検査が、膵癌の効率的な診断法としていかに有用であるかを明らかにしようとした。対象は、腹痛などの自覚症状、検診でのチェック、膵酵素や腫瘍マーカーの上昇、糖尿病の悪化などより膵の異常が疑われる患者であり、EUSにより膵の一次スクリーニングを行った。腫瘤や膵管拡張などの異常エコーが検出された場合、内視鏡下に純膵液を採取し、ASOプローブ法ないしはPCR-RELP法によりK-rasコドン12の点突然変異を検索した。その結果、(1).腫瘍径1〜2cmの小膵癌は、EUSで辺縁不整な類円形の低エコー腫瘤として描出された。内部エコーも約半数で不均一であったが、大きな癌に比べ軽微なため、周囲との境界は比較的明瞭であった。(2).主膵管は、腫瘍部尾側で急激に拡張した。拡張形態は数珠状で、その腫瘍部断端は、凹凸不整を示した。(3).炎症性膵腫瘤は、一般に角ばった低エコー域として描出され、膵管の走行や径に影響を与えないことが多いが、腫瘤径が1cm以下で、円形の場合には、小膵癌と鑑別できないものも存在した。(4).PCR-RELP法による膵液中K-rasコドン12点突然変異の検出率は、膵管癌78%(29/37)、粘液産生膵腫瘍(腺腫)50%(2/4)、慢性膵炎9%(5/53)であった。K-ras変異の陽性率と、腫瘍占拠部位や腫瘍径との間に一定の関連性はなく、小膵癌でも6例中4例が陽性であった。以上の成績より、膵液中のK-rasコドン12点突然変異やp53の変異検索は、EUSにて検出された小腫瘍性病変の質的診断に有用であり、これら内視鏡を応用した一連の検査は、膵癌の効率的な早期診断法として大いに期待される。The combination of endoscopic ultrasonography and DNA diagnosis using pancreatic juice for the detection of small pancreatic cancer was evaluated. The patients with upper abdominal pain. abnormal laboratory data or images on the pancreas, or worsening of diabetic control were enrolled in this study. When the findings of a small pancreatic mass or dilated pancreatic duct were detected by endoscopic ultrasonography, the pure pancreatic juice was collected under a duodenofiberscope for the analyzes of k-ras point mutation at codon 12. The pancreatic cancers less than 2 cm in a diameter were clearly visualized as round hypoechoic tumors with irregular margin. The pancreatic ducts were abruptly dilated caudal to the tumors. Although inflammatory pancreatic masses were generally visualized as square-shaped hypoechoic area without compression or encasement of the neighboring pancreatic ducts, the differentiation from small pancreatic cancer was not always complete, particularly when the tumor was round and the size was less than 1 cm. The incidence of k-ras point mutation analyzed by PCR-RFLP method was 78% (29/37) in patients with pancreatic cancer and 9% (5/53) in those with chronic pancreatitis. The correlation between the incidence of k-ras mutation and tumor size/location were not seen. These results suggest that the combination of endoscopic ultrasonography and DNA diagnosis using pancreatic juice is useful and promising to the early detection of pancreatic cancer.研究課題/領域番号:06670530, 研究期間(年度):1994 – 1995出典：研究課題「膵癌早期診断に対する新しい内視鏡的ならびに分子生物学的アプローチ」課題番号06670530（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-06670530/066705301995kenkyu_seika_hokoku_gaiyo/）を加工して作

実験胃癌モデルにおける癌の発育進展に関する音響力学的検討

金沢大学がん研究所研究課題/領域番号:63770444, 研究期間(年度):1988出典：研究課題「実験胃癌モデルにおける癌の発育進展に関する音響力学的検討」課題番号63770444（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-63770444/）を加工して作

小膵腫瘤性病変の鑑別診断能の向上に関する内視鏡的ならびに分子生物学的研究

金沢大学がん研究所超音波内視鏡(EUS)と膵液の遺伝子診断を組み合わせた一連の内視鏡下の検査が、小膵腫瘤性病変の検出とその鑑別診断にいかに有用であるかを明らかにしようとした。対象は、腹痛などの自覚症状、検診でのチェック、膵酵素や腫瘍マーカーの上昇、糖尿病の悪化などより膵の異常が疑われた患者176例であり、EUSにより膵の一次スクリーニングを行った。腫瘤や膵管拡張などの異常エコーが検出された場合、内視鏡下に純膵液を採取し、PCR-RFLP法によりK-rasコドン12の点突然変異を検索した。同時に、通常のエコー(US)、コンピューター断層撮影(CT)、内視鏡的膵管造影(ERCP)などの各種画像診断を施行し、腫瘤検出能をEUSと比較した。その結果、1)EUSにより36例(20%)の充実性膵腫瘤が検出された。うち19例は膵癌であり、腫瘍径1〜2cmの小膵癌が7例が含まれていた。また、17例は、炎症性膵腫瘤で、そのうち13例は2cm以下の小腫瘤であった。2)各画像診断の検出感度を比較すると、全体では、USが68%、CTが79%、ERCPが84%、EUSが100%を示した。そして、2cm以下の小さな者に限ると、US29%、CT43%、ERCP86%、EUS95%となり、EUSは小病変の検出に優れていることが示された。3)特異性について比較すると、全体では、USが53%、CTが88%、ERCPが88%、EUSが82%を示した。そして、2cm以下の小さな者では、US69%、CT92%、ERCP100%、EUS92%であり、特異性については、ERCPやCTと同等であった。4)EUSと膵液の遺伝子診断を組み合わせたが、感度、特異性ともEUS単独の成績を凌ぐことはできなかった。しかし、、膵液中のK-ras変異の検索は、EUSにて検出された小腫瘍性病変の診断確定には有用であった。以上の成績より、現時点では、これら遺伝子診断を併用した一連の内視鏡検査の早期診断法としての意義は明らかにできなかったが、今後、1cm以下の小病変がどんどん検出されるようになれば、その真の意義が明らかになるであろう。Background & Aims : The early diagnosis of pancreatic cancer is still difficult. The purpose of this prospective study was to assess the utility of a combination of endosonography and genetic analysis of pure pancreatic juice for the early recognition of pancreatic cancer. Methods : One hundred and seventy-six patients with syspected pancreatic injury were enrolled and underwent endosonography. Pure pancreatic juice was collected endoscopically in patients with solid pancreatic masses. K-ras point mutations at codon 12 in the juice were assayd by polymerase chain reaction-restriction fragment length polymorphism. Results : Thirty-six patients (20%) were found to have solid pancreatic masses. They consisted of 19 patients with pancreatic cancer (7 patients, 2cm) and 17 patients with an inflammatory pancreatic mass (13 patients, 2cm). Although endoscopic retrograde cholangiopancreatography showed high accuracy for cancer diagnosis, ultrasonography and computed tomography were less sensitive, particularly in small pancreatic masses, and 65% of them were not disclosed until endosonography. In contrast, endosonography showed high sensitivity (100%) and specificity (92%) even in small masses. Together with K-ras analysis, assayd safely using small samples, the endosonographic diagnosis became more definitive. Conclusions : Both endosonography and K-ras analysis was safely performed. The combination of endosonography and K-ras analysis of pure pancreatic juice may be useful for the early diagnosis of pancre研究課題/領域番号:08670572, 研究期間(年度):1996 – 1997出典：研究課題「小膵腫瘤性病変の鑑別診断能の向上に関する内視鏡的ならびに分子生物学的研究」課題番号08670572（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-08670572/086705721997kenkyu_seika_hokoku_gaiyo/）を加工して作

Comparison in gene expression of secretory human endometrium using laser microdissection

BACKGROUND: The endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique. METHODS: The epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR. RESULTS: In this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR. CONCLUSIONS: Some of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium

Genetics of Endometrial Cancers

Endometrial cancers exhibit a different mechanism of tumorigenesis and progression depending on histopathological and clinical types. The most frequently altered gene in estrogen-dependent endometrioid endometrial carcinoma tumors is PTEN. Microsatellite instability is another important genetic event in this type of tumor. In contrast, p53 mutations or Her2/neu overexpression are more frequent in non-endometrioid tumors. On the other hand, it is possible that the clear cell type may arise from a unique pathway which appears similar to the ovarian clear cell carcinoma. K-ras mutations are detected in approximately 15%–30% of endometrioid carcinomas, are unrelated to the existence of endometrial hyperplasia. A β-catenin mutation was detected in about 20% of endometrioid carcinomas, but is rare in serous carcinoma. Telomere shortening is another important type of genomic instability observed in endometrial cancer. Only non-endometrioid endometrial carcinoma tumors were significantly associated with critical telomere shortening in the adjacent morphologically normal epithelium. Lynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility and is characterized by a MSH2/MSH6 protein complex deficiency, is associated with the development of non-endometrioid carcinomas

Overexpression of DNA Polymerase ζAffects Cisplatin Resistance in Ovarian Cancer: An Immunohistochemical Study

DNA polymerase ζ (Pol ζ) participates in translesional bypass replication. Pol ζ has been shown to be an important contributor to cis-diamminedichloroplatinum (II)(DDP; cisplatin) -induced genomic instability and the subsequent emergence of resistance in vitro. We immunohistochemically examined the expression of Pol ζ in ovarian cancer tissues to determine whether its expression affects the DDP resistance of human ovarian cancers and also to determine whether Pol ζ expression is a prognostic factor for ovarian cancers. We assessed 76 archival, formalin-fixed, paraffin-embedded tissue samples obtained from patients with epithelial ovarian cancers who underwent their first operation between 2003 and 2011. An ovarian cancer tissue array was also used in this study. Immunohistochemical staining of Pol ζ was performed using an anti-human Pol ζ monoclonal rabbit antibody. The strength of expression of Pol ζ was compared with the DDP resistance and clinical features of the study population. The Pol ζ over-expression in ovarian cancer tissue which compared with epithelial cells in normal ovaries was not affected by the histological types, FIGO stage, or patient age, but Pol ζ was significantly more overexpressed in the DDP-resistant group than in the DDP-sensitive group (P = 0.043). Pol ζ over-expression did not significantly affect the survival rate of the ovarian cancer patients; however, the Pol ζ positive group tended to have a poorer long-term prognosis. In conclusion, ovarian carcinoma patients with Pol ζ over-expression are likely to be resistant to DDP, especially in cases of recurrent disease. These results confirm the previous findings in vitro, wherein Pol ζ modulated the cytotoxicity and mutagenicity of DDP

Establishment of Fetal Cranial and Intracranial Structure Volume Measurements using Three-dimensional Ultrasound Imaging in a Japanese Population

The goal of this study was to establish a normogram of the intracranial structure volumes in appropriate-for-gestational age (AGA) Japanese fetuses. This was a cross-sectional and prospective study of 211 AGA fetuses. The total intracranial, cerebrum, ventricle, choroid plexus, cerebellum, cerebellar vermis, and cavum septum pellucidum volumes were measured using three-dimensional (3D) ultrasound. The fetal cranial and intracranial structure volumes significantly increased with gestational age (GA). When using the GA as an independent variable and the fetal cranial or intracranial structure volumes as the dependent variable, the best-fit equation for the fetal brain was a second-order polynomial regression equation. We herein provide the first report of fetal cranial and intracranial structure volumes and their normal growth curves in normal Japanese fetuses. Future 3D ultrasound studies of volume and other intracranial fetal structures could provide valuable information about how such changes may correlate with long-term neurodevelopment and the results may be used for comparisons with fetal growth restriction in the future