上咽頭癌に対するインターフェロン療法とビタミンAの影響(基礎と臨床)

金沢大学医学部研究課題/領域番号60570810, 研究期間(年度):1985出典：研究課題「上咽頭癌に対するインターフェロン療法とビタミンAの影響(基礎と臨床)」課題番号60570810（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-60570810/）を加工して作

上咽頭癌の頸部転移機構に関する分子生物学的解析

金沢大学医学系研究科上咽頭癌の頸部転移機序を分子生物学的に解明し、それを予防する治療法を開発する目的でEBウイルスの唯一の発癌遺伝子といわれるLMP1発現と相関する転移関連遺伝子群のスクリーニングをおこなった。その結果、細胞外基質破壊、細胞接着、血管新生、細胞運動能亢進に関与する転移関連遺伝子群が明らかになった。次いでLMP1によるMMP9の発現誘導をI-κBが阻害することを明らかにした。つまり、NF-κBシグナルを抑制することでLMP1によるMMP9の転写が抑制されることを証明した。しかしLMP1遺伝子発現の直接制御は難しく、LMP1下流のシグナル伝達経路を遮断することで癌の浸潤・転移を抑制する分子標的治療の可能性について検討を加えることにした。最初に、I-κBキナーゼ(IKK)を不活化し、NF-κBの活性を抑制することが知られているアスピリン及びその関連物質であるサリチル酸ナトリウムについて検討した。その結果、アスピリンはLMP1導入後のMMP9の発現を抑制することをゼラチンザイモグラフィーで観察した。ゲルシフトアッセイの実験から、アスピリンはLMP1によるNF-κB, AP-1の活性を抑制したことから、MMP9の発現抑制はLMP1下流シグナル伝達経路の抑制によるものであると考えられた。また、MMP9の転写に関するプロモーターの活性を調べたCATアッセイでも、アスピリンによる抑制が証明されたので、ヌードマウスを用いて、臨床応用への可能性を検討した。形質転換した子宮頚痛由来C33A上皮系細胞とLMP1発現C33A細胞を作成し、それぞれの細胞をヌードマウス背部へ皮下注射により移植した。次いで腫瘍形成ヌードマウスヘアスピリンを3日間、皮下注射した。この移植腫瘍からウェスタンブロット用及びゼラチンザイモグラフィー用細胞抽出液を採取し、それぞれの材料においてLMP1及びMMP9の誘導を確認した後、アスピリン投与有無での両者の発現の差を比較検討した。結果は期待どおりアスピリンはそれらの発現を有意に抑制し、上咽頭癌の頸部転移抑制のための分子標的療法の可能性が証明できた。Nasopharyngeal carcinoma(NPC), an epithelial tumor which is characterized by marked geographic and population differences in incidence, is found to be associated with Epstein-Barr virus(EBV) by serologic evidence, and the relationship was confirmed by the detection of EBVDNA and EB-encoded RNAs in NPC cells. While, NPC is highly metastatic carcinoma whose consistent associated with EBV has been established. Latent membrane protein 1(LMP1), an EBV membrane protein expressed in latent infection is considered to be the EBV oncoprotein. Matrix metalloproteinase 9(MMP9), one of the MMP families, degrades Type IV collagen, a major 4 component of extracellural matrix and is believed to be crucial for cancer invasion and metastasis. Although MMP9 is reported to be expressed in a variety of cancers, no reports concerning NPC have been published. We have shown that LMP1 induces MMP9 in vitro cell line, which suggests the possibility of mechanism in which LMP1 of EBV contributes to the metastasis and tumorgenesis of NPC by the induction of MMP9. Then the expression of LMP1 and MMP9 were immunohistochemically examined, and the relation of these proteins was statistically analyzed. We also analyzed the association of these proteins with clinical features. As results, both LMP1 and MMP9 proteins were predominantly immunolocalized in cancer nests. The expression of MMP9 showed a significant positive correlation with the expression of LMP1. Also, the expression of MMP9 correlated with lymphnode metastasis.We also demonstrated that LMP1 enhances MMP9 expression by activation of nuclear factor(NF)κB and activator protein(AP)-1. We therefore tested whether up-regulation of MMP9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP9 expression in tumors grown in nude mice were also tested. CS3A cells stably expressing LMP1 had increased expression of MMP9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells. Treatment with aspirin and sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells and suppressed both the LMP1-induced MMP9 expewssion in zymographic analyses and LMP1-induced MMP9 promoter activity in CAT reporter assays. The inhibitory effect of aspirin on NF-κB activity was attributable to the inhibition of I-κB kinase activity. Finally, tumors derived from vector-transfected C3SA cells stably expressing LMP1 grown in nude mice showed enhanced MMP9 levels compared with tumors derived from vector-trancfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.研究課題/領域番号:13470358, 研究期間(年度):2001 – 2003出典：「上咽頭癌の頸部転移機構に関する分子生物学的解析」研究成果報告書　課題番号13470358（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-13470358/134703582003kenkyu_seika_hokoku_gaiyo/)を加工して作

上咽頭癌発生初期過程におけるEBウイルス遺伝子と細胞側遺伝子との関連性

金沢大学医学部EBVが密接に関与しているNPCにおいて、EBV遺伝子と宿主細胞側の遺伝子発現の相互関係から上咽頭癌発生機序を考察することにした。つまりNPC組織を用いて、EBV遺伝子発現のうち、EBERs (EBV-encorded small nuclear RNA),BHLF1 (diffuse early antigens)は、in situ hybridization法にて,LMP1,EBNA1,EBNA2,BZLF1は免疫組織化学的手法を用いて調べた。細胞側遺伝子発現としては癌抑制遺伝子p53,アポートシス抑制遺伝子bclの発現を免疫組織化学的手法で調べた。このことから上咽頭癌発癌における初期過程を考察することを試みた。その結果、EBERs-1は非角化型癌(WHO-2)、未分化型癌(WHO-3)で高率(90%以上)に確認され、EBV-抗体価陽性率と明らかな相関関係が証明できた。しかし、LMP1は約35%、EBNA2は陰性でNPCの表現型はLMP1(+/-),EBNA2(-)で、いわゆるLatencyクラス2に分類された。一方アポトーシスに対して抑制的であるbcl-2と逆に促進的である野生型p53の発現はそれぞれ約90%、60%と高率であったが、両者間には発現率、染色強度において統計的に有為な相関関係は認められなかった。しかし、同一標本における両者の二重染色で同一領域で陽性細胞が混在することが特徴的であった。このことはp53蛋白による癌抑制作用がbcl-2により抑制される可能性が示唆された。ところで、EBウイルス転写活性化に必要なBZLF1遺伝子により発現されるZ蛋白やウイルスDNA複製に必要な早期蛋白をコードするBHLF1蛋白の発現率はそれぞれ約40%、10%であり、NPCでは低率でもウイルス産生が生じていることが明らかになった。しかし、EBV抗体価陽性NPC患者の100%がEBERs陽性者であることと大きく異なり、NPCにおける高いEBV抗体価陽性率とは相反するものであった。そこでEBウイルス産生抑制機序の存在を考え、Z蛋白とp53との発現関係を統計的に検討したところ、両者は有意に相関していた。つまり、両者蛋白の結合がそれぞれの機能を抑制しあい発癌に結びつくものと考えられた。Nasopharyngeal carcinoma (NPC), an epithelial tumor which is characterized by marked geographic and population differences in incidence, is consistently associated with the Epstein-Barr virus (EBV). Within the tumor, the EBV DNA is homogeneous and clonal with regard to repeat sequences suggesting that the tumor is also clonal.In this work we examined to detect EBV in formalin-fixed paraffin-embedded NPC specimens by using both polymerase chain reaction (PCR) for EBV-DNA and in situ hybridization for EBV-encoded small RNAs (EBERs). EBV-DNA was detected in none of 3 keratinizing squamous cell carcinomas (SCC), 22 of 24 non-keratinizing carcinomas (NKC), all 13 undifferentiated carcinoma (UNPC) and none of 2 adenocarcinomas (AC). EBERs was detected in none of 5 SCC,30 of 32 NKC,16 of 17 UNPC and none of 2 AC.As an additional study in situ hybridization using BHLF oligonucleotide probes and immunohistochemistry using monoclonal antibodies against LMP1, EBNA2, BZLF1 protein, p53 protein and bcl-2 protein were performed in 56 primary NPC.LMP1 was detected in 17 cases (30%) whereas EBNA2 was not detectable. Bcl-2 protein was positive in 50 cases (89%), but its expression did not depend on expression of LMP1, which did not demonstrate induction of bcl-2 by LMP1 as seen in vitro. Cytoplasmic BZLF1 expression was detectable in 18 cases (32%) whereas BHLF was positive only in 6 cases (11%). This finding suggests BZLF1 which disrupts viral latency dysfunctions despite of its expression. p53 protein was positive in 31 cases (55%), and there was a distinct correlation between expression of BZLF1 and p53 protein (p<0.001). This finding suggests the interaction between BZLF1 and p53 which inactivates each other is one of tumorigenic factor in NPC研究課題/領域番号:06454485, 研究期間(年度):1994 – 1995出典：研究課題「上咽頭癌発生初期過程におけるEBウイルス遺伝子と細胞側遺伝子との関連性」課題番号06454485（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-06454485/064544851995kenkyu_seika_hokoku_gaiyo/）を加工して作

EBウイルス転写調節因子Z蛋白に対する抗体価測定法の開発とその意義に関する研究

金沢大学医学部上咽頭癌(Nasopharyngeal carcinoma,NPC)とEpstein Barr virus(EBV)の関連性は、患者血清中の抗EBV抗体価上昇や腫瘍組織内EBV核酸の証明などにより確立されている。特にウイルス複製サイクルに発現する早期抗原(early antigen、EA)や後期蛋白のEBV粒子抗原(viral capsid antigen,VCA)に対するIgA抗体は発症時にすでに上昇しており、疾患特異性も高いことから、病勢のマーカーとして用いられている。しかし腫瘍細胞ではウイルス複製サイクルはしばしば不完全に終わり、いわゆる流産感染となっている。このことを考えると、ウイルス複製サイクルにおいて早期抗原よりもさらに早く発現する前早期抗原(Immediate early antigen)である、Z蛋白やR蛋白に対する生体反応のほうがより早く病状を反映する可能性が考えられる。そこで今回、Z蛋白やR蛋白に対する血清抗体価を調べることにした。Z蛋白、R蛋白はそれぞれBZLF1,BRLF1遺伝子発現プラスミドを構築し、精製した。これらを抗原とし、担癌NPC患者、NPC緩解後患者の他、EBV関連疾患であるIM,ホジキン病患者や他の頭頚部癌腫瘍患者、および健常者血清との反応を、ウエスタンブロット法、ELISA法で検討した。その結果、担癌NPC患者では、全例抗Z蛋白、R蛋白抗体陽性であり、ELISA法での吸光度も他の群と比べ高値を示した。VCA-IgA抗体価と比較しても、抗Z蛋白、抗R蛋白抗体陽性はNPC特異性が高かった。以上よりNPC患者における抗Z蛋白、R蛋白抗体価測定は病勢の有用な指標となり得ると結論された。Nasopharyngeal carcinoma (NPC), an epithelial tumor which is characterized by marked geographic and population differences in incidence, is found to be associated with Epstein-Barr virus (EBV) by serologic evidence, and the relationship was confirmed by the detection of EBVDNA and EB-encoded RNAs in NPC cells. Elevation of lgG and lgA antibodies against EBV viral capsid antigen and early antigen are often found in NPC patients. Serologic tests of these antibodies have been recognized astumor markers for NPC.EBV persists in a latent form in both epithelial cells and B lymphocytes. A variety of stimulation may activate a virus. The Z protein and the R protein, which are immediate early proteins of the EBV lytic cycle, control the switch of the virus from a latent to a lytic cycle by transactivating several early promoters. But a lytic cycle is sometimes incomplete in tumor. Z protein and R protein are expressed earlier than EA or VCA,so anti-Z,-R antibody titers will be more sensitive markers for NPC.Plasmid presenting BZLF1 or BRLF1 gene were produced, and Z protein and R protein were harvested. Sera from patients with NPC,infectious monomucleosis (IM), non-Hodgkin\u27s lyumphoma (NHL), head and neck carcinoma and healthy controls are examined for its reactivity against Z and R protein by Western blotting and ELISA.NPC patients before treatment all indicated high anti-Z,-R antibody titers, whereas in three NPC patients without recurrence, only one (33%) was positive. IM patients all indicated no anti-Z,-R antibodies. Several patients of other diseases have anti-Z,-R antibodies, but their titers were low. In comparison with anti-VCA antibodies, ant-Z,-R antibodies were specific to NPC.A NPC patients whose anti-VCA antibodies were negative indicated high anti -Z,-R antibody titers, and a NHL patient who shows elevation of anti-VCA antibody titer was negative in anti-Z,-R antibodies. These findings suggest that the anti-Z,-R antibody titers are useful markers for NPC.研究課題/領域番号:08457450, 研究期間(年度):1996 – 1997出典：研究課題「EBウイルス転写調節因子Z蛋白に対する抗体価測定法の開発とその意義に関する研究」課題番号08457450（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-08457450/084574501997kenkyu_seika_hokoku_gaiyo/）を加工して作

頚部転移性腫瘍のPCR法によるEBウィルスDNAの検出に関する基礎的研究

金沢大学医学部上咽頭癌とEpstein-Barrウイルスの密接な関係に注目し、上咽頭癌頚部転移リンパ節のパラフィン包埋組織からポリメラーゼ連鎖反応(polymerase chain reaction,PCR)を用いてEBV DNAの増幅を行い、サザンブロットハイブリダイゼーション法にてEBV DNAの検出を試みた。14例中、12例にヒトβグロビンDNAが検出され、うち9例にEBV DNAの検出がみられた。EBV DNAの検出が見られた例は、世界保健機構の上咽頭癌病理組織分類別に見ると非角化型が4例、未分化型が5例であり、全例にEBV粒子抗原対するIgA抗体価の陽性が見られた。一方対照群として他の頭頚部悪性腫瘍からの転移リンパ節19例と反応性リンパ節1例を選んだ。20例中18例にヒトβグロビンDNAが検出されたが、EBV DNAは全例に検出されなかった。以上から、頚部転移組織からのEBV DNAの検出は上咽頭癌に特異的であった。上咽頭癌は、原発巣不明のまま頚部リンパ節転移を起こし、他の頭頚部腫瘍との鑑別が困難である場合がしばしば生ずる。そこでEBVゲノム保有上皮系細胞株NPC-KT細胞をヌードマウスに移植し、形成された腫瘍において穿刺吸引細胞診(fine needle aspiration biopsy,FNA)を施行した。FNABから得られた微小組織からPCR法を用いてEBV DNAの検出を行った。FNABに使用された穿刺針は23,21,18ゲージ針であるがいずれの針でもEBV DNAの検出が認められた。以上から原発巣不明頚部転移癌症例においてFNAB手技とPCR法を用いてEBV DNAの検出を行うことは、上咽頭癌頚部転移の分子生物学的補助診断法となり得ることが証明できた。なお、その感度はNPC-KT細胞の希釈系列を用いた簡易PCR法による実験結果から10^2個以上の癌細胞があればEBV DNAの検出が可能であった。研究課題/領域番号:05671420, 研究期間(年度):1993出典：研究課題「頚部転移性腫瘍のPCR法によるEBウィルスDNAの検出に関する基礎的研究」課題番号05671420（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-05671420/）を加工して作

Malignant phosphaturic mesenchymal tumor, mixed connective tissue variant of the tongue

金沢大学医薬保健研究域医学系The majority of the oncogenic osteomalacia-associated mesenchymal tumors are considered to belong to the category of phosphaturic mesenchymal tumors, mixed connective tissue (PMTMCT) variant, of which malignant cases are very rare. Here we report a case of a recurrent malignant PMTMCT variant which arose in the tongue. The patient was treated with surgery at an initial treatment and the first recurrence. In accordance with the tumor recurrence and resection, the hypophosphatemia progressed and improved. However, hypophosphatemia did not progress after receiving radiation therapy at the second recurrence even though the recurrent tumor gradually increased its size. These results suggest clinical feature of malignant PMTMCT could be changed by radiation therapy. Thus, this report could add an insight to the nature of PMTMCT. © 2008 Elsevier Ireland Ltd. All rights reserved

Counting Points for Hyperelliptic Curves of type y2=x5+axy^2=x^5+ax over Finite Prime Fields

Counting rational points on Jacobian varieties of hyperelliptic curves over finite fields is very important for constructing hyperelliptic curve cryptosystems (HCC), but known algorithms for general curves over given large prime fields need very long running times. In this article, we propose an extremely fast point counting algorithm for hyperelliptic curves of type $y^2=x^5+ax$ over given large prime fields \Fp, e.g. 80-bit fields. For these curves, we also determine the necessary condition to be suitable for HCC, that is, to satisfy that the order of the Jacobian group is of the form $l\cdot c$ where $l$ is a prime number greater than about $2^{160}$ and $c$ is a very small integer. We show some examples of suitable curves for HCC obtained by using our algorithm. We also treat curves of type $y^2=x^5+a$ where $a$ is not square in \Fp

Changes in calbindin-D28k and parvalbumin expression in the superior olivary complex following unilateral cochlear ablation in neonatal rats

金沢大学医薬保健研究域医学系Conclusion. Unilateral congenital deafness with a volume reduction in cochlear nucleus (CN) induced changes in the calcium-binding proteins (CaBPs) in the contralateral superior olivary complex (SOC) in the rat. With the loss of neurons and a volume reduction in the CN, a decrease in the input to the contralateral SOC may occur, which results in the down-regulation of CaBPs in these nuclei. This study may provide some implications regarding the neurochemistry in the auditory brainstem of deaf children. Objective. Hearing loss produced by cochlear damage during early development can result in persistent changes in the organization of the central auditory system in adults. The purpose of the present study was to investigate the neurochemical changes produced in the auditory brainstem of rats with unilateral cochlear ablation conducted before the onset of hearing. Materials and methods. Following unilateral cochlear ablation during early development, we examined the changes in the distribution of two CaBPs, calbindin-D28k (CB) and parvalbumin (PV), in the SOC. Results. Upon reaching adulthood, a marked decrease in CB- and PV-immunoreactive neurons was observed in the contralateral SOC, particularly in the medial nucleus of the trapezoid body (MNTB), although no neuronal cell death was observed. A volume reduction in the ipsilateral CN was also observed. © 2009 Informa UK Ltd

Expression and Localization of the Cell Adhesion Molecule SgIGSF during Regeneration of the Olfactory Epithelium in Mice

Spermatogenic immunoglobulin superfamily (SgIGSF) is a cell adhesion molecule originally discovered in mouse testis. SgIGSF is expressed not only in spermatogenic cells but also in lung and liver epithelial cells and in neurons and glia of the central and peripheral nervous systems. In the present study, we examined the expression and localization of SgIGSF in mouse olfactory epithelium before and after transection of the olfactory nerves, by RT-PCR, Western blotting and immunohistochemistry. In normal olfactory mucosa, SgIGSF showed 100 kDa in molecular weight, which was identical with that in the lung but different from that in the brain. SgIGSF was expressed on the membrane of all olfactory, sustentacular and basal cells, but more abundantly in the apical portions of the olfactory epithelium where the dendrites of olfactory cells are in contact with sustentacular cells. After olfactory nerve transection, mature olfactory cells disappeared in 4 days but were regenerated around 7–15 days by proliferation and differentiation of basal cells into mature olfactory cells through the step of immature olfactory cells. During this period, both the mRNA and protein for SgIGSF showed a transient increase, with peak levels at 7 days and 11 days, respectively, after the transection. Immunohistochemistry showed that the enriched immunoreactivity for SgIGSF at 7–11 days was localized primarily to the membrane of immature olfactory cells. These results suggested that, during regeneration of the olfactory epithelium, the adhesion molecule SgIGSF plays physiological roles in differentiation, migration, and maturation of immature olfactory cells