|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
I have repoted that detection of K-ras mutations in pure pancreatic juice (PPJ) was useful for the diagnosis of pancreatic carcinoma (PCa). However, combination assay of K-rag and p53 mutations was not completely fulfiled for the diagnosis of PCa. Therefore, a new specific gene for PCa is needed for the supplemental diagnosis of PCa. p16 is a suppressor gene with the function of cyclin dependent kinase (CDK) inhibitor, and high incidence of p16 inactivation in PCa cell lines was recently reported.
Expression of p1 6 gene product in human PCa was investigated in paraffin-embedded tissue using a monclonal antibody against p16 protein, clone G175-405, by means of immunohistochemical staining. All six cases of normal pancreas and all but 1 of 20 cases of chronic pancreatitis (CP) expressed p16 protein, whereas 41.9% (26 of 62) of PCas lost p16 expression. There was a significant difference between CP and PCa for frequency of the loss of p16 expression. (p< 0.01). Moreover loss of p16 protei
n in pancreatic malignancy was significantly associated with histological grade (G1 versus G2 and G3, p< 0.01) but not with sex, age, clinical stage, tumor location, or resectability. DNA was extracted using microdissection method from the 26 PCa tissues which show the loss of p16 expression by immunohistochemical staining. Incidence of homozygous deletion was found in 19.2% (5/26) in exon 1, 23.1% (6/26) in exon 2, 0% (0/26) in exon 3 ofpl6, and 34.6% (9126) in all by PCR with the corresponding primers. On the other hand, there was no mutaion of p1 6 gene about the remnant 17 PCa cases expressing PCR products by PCR-SSCP.Recently, methylation of CpG island is regarded as a cause of the loss of p16 expression and p16 methylation for PCa is now under investigation. It is very difficult to detect homozygous deletions of p16 in PPJ because an amount of exfoliated normal pancreatic epitheliums are present in PPJ.Therefore, mutation of p16 in PPJ obtained from patients with PCa is under investigation. At present, there has been no detectable mutations of p16 in PPJ.
一方、膵癌組織のp16免疫染色性すなわち蛋白発現の低下した領域をmicrodissection法にて区分し、DNAを抽出した。p16遺伝子の3つのエクソンに対応するプライマーを用いてPCRを行ない、homozygous deletionの有無を判定した。膵癌26例中エクソン1では5例(19.2%)に、エクソン2では6例(23.1%)にPCR産物の欠損が認められたが、エクソン3には認められず、合計すると26例中9例(34.6%)にhomozygous deletionが認められた。さらに、PCR産物が得られた17例については、PCR-SSCPによりp16の突然変異の有無を検索したが、明らかなmutationを認めたものはなかった。最近、p16蛋白発現低下の原因のひとつにCpG island領域のmethylationの関与が示唆されており、現在、p16のmethylationについても検索中である。また、ヒト膵液中のp16変異の検討については、正常膵管上皮が多量に含まれている膵液中では、homozygous deletionの検討は困難であることから、p16のmutationの有無につき検討を行っているが、現在のところ、明らかなp16mutation陽性症例はみられていない。 Less