松下 一之

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE). Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and inummohistochemistry. Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 411, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified. Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE). Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and inummohistochemistry. Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 411, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase beta subunit and annexin A2 also were identified. Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Aneuploidy is the hallmark of many human cancers. Recent work has strongly suggested that chromosome missegregation during mitosis is the main cause of aneuploidy and contributes to oncogenesis. Centromere protein (CENP) -A is the centromere-specific histone-H3-like variant essential for centromere structure and function. It plays a central role in the assembly of the protein complex, termed kinetochore, which is indispensable for equal chromosome segregation. In this study, we demonstrate that the kinetochore protein CENP-A was overexpressed in all of 11 primary human colorectal cancer tissues. CENP-A mRNA was also up-regulated, indicating that overexpression of CENP-A occurred at the transcriptional level. Immunostaining with anti-CENP-A antibodies showed increased CENP-A signals in the tumor cells. Moreover, coimmunostaining of CENP-B, a centromere-associated DNA binding protein, with CENP-A showed mistargeting of CENP-A to noncentromeric chromatin in the tumor cells. These results suggest that overexpression of CENP-A could play an important role for aneuploidy in colorectal cancers.

Aneuploidy is the hallmark of many human cancers. Recent work has strongly suggested that chromosome missegregation during mitosis is the main cause of aneuploidy and contributes to oncogenesis. Centromere protein (CENP) -A is the centromere-specific histone-H3-like variant essential for centromere structure and function. It plays a central role in the assembly of the protein complex, termed kinetochore, which is indispensable for equal chromosome segregation. In this study, we demonstrate that the kinetochore protein CENP-A was overexpressed in all of 11 primary human colorectal cancer tissues. CENP-A mRNA was also up-regulated, indicating that overexpression of CENP-A occurred at the transcriptional level. Immunostaining with anti-CENP-A antibodies showed increased CENP-A signals in the tumor cells. Moreover, coimmunostaining of CENP-B, a centromere-associated DNA binding protein, with CENP-A showed mistargeting of CENP-A to noncentromeric chromatin in the tumor cells. These results suggest that overexpression of CENP-A could play an important role for aneuploidy in colorectal cancers.

Background. Increased preoperative serum squamous cell carcinoma antigen (SCC-Ag) concentrations have been found to be associated with advanced stage and poor prognosis in lung and cervical cancers. Because little was known about the significance of SCC-Ag concentration in patients with esophageal cancer, the aim of this study was to analyze the clinicopathologic significance of SCC-Ag in patients with esophageal SCC. Patients and methods. Preoperative SCC-Ak concentration was measured with enzyme-linked immunosorbent assay in 309 patients with primary esophageal SCC. All patients underwent curative radical surgery without any preoperative therapy. In 215 of 309 patients, carcinoembryonic antigen (CEA) was also measured to compare clinical significance of CEA with that of SCC-Ag. The prognostic significance for survival of SCC-Ag concentrations was studied with multivariate analysis with Cox proportional hazards model. Results. The SCC-Ag concentration and the positivity rate of SCC-Ag were significantly elevated in patients associated with tumor progression. Statistically significant differences in SCC-Ag concentrations and SCG-Ag positivity rates were observed depending on tumor size, tumor depth, lymph node status, and distant metastasis. Although CFA was not a prognosticfactor (P = .21), a high SCC-Ag concentration was a significant Prognostic factor (P < . 01). Multivariate analyses indicated that T factor had the best predictive power, but SCC-Ag concentration contained additional, independent prognostic information. Conclusion. Our findings suggest that preoperative serum SCC-Ag concentrations might provide a predictive information for tumor progression and survival in patients with esophageal SCC.

A 74-year-old Japanese woman presented with a 3-month history of anal bleeding. Proctoscopy revealed an unusual polypoid lesion with focal pigmentation at the dentate line, which was histologically diagnosed as a malignant melanoma. Whole-body clinical and radiographic. evaluations revealed no alternative primary source. Endoscopic ultrasonography (EUS) showed well-delineated hypoechoic tumors invading the muscularis propria, and magnetic resonance imaging (MRI) revealed regional lymphadenopathy. Following this evaluation, an abdominoperineal resection with regional lymphadenectomy was performed. The excised tumor was histologically confirmed to be malignant melanoma, and its depth and metastatic lymph nodes proved to have been accurately and precisely evaluated by the preoperative examinations. Thus, EUS and MRI are useful preoperative diagnostic for the tumor staging of primary anorectal malignant melanomas, as for other rectal tumors.

ATP-sensitive K+ (KATP) channels comprise the pore-forming subunit (Kir6.1 of Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). KATP channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 of Kir6.2, functional roles of K ATP channels in various organs have been clarified. Kir6.1 -/- mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2-/- mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K ATP channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2-/- mice lacking the sarcolemmal KATP channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal KATP channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial KATP channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K+ channel openers activating mitochondrial KATP channels, decreased the mitochondrial membrane potential, thereby preventing the Ca2+ overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K+ channel openers and the activating effects on sarcolemmal KATP channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of KATP channels, there would be considerable differences in the tissue selectivity of KATP channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that KATP channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysilogical roles of KATP channels may lead to a new therapeutic strategy to improve the quality of life.

BACKGROUND. Thymidine phosphorylase (dThdPase), which also is referred to as platelet-derived endothelial cell growth factor, is a potent inducer of angiogenesis in malignant tumors. Increased dThdPase expression and activity have been found to be associated with poor prognosis in various solid tumor tissues. Because very little was known about the significance of serum dThdPase concentration (S-dThdPase), the objective of this study was to analyze the clinicopathologic significance of S-dThdPase in the patients with esophageal squamous cell carcinoma. METHODS. The S-dThdPase was measured by enzyme-linked immunosorbent assay in 77 healthy controls and 153 patients with primary esophageal squamous cell carcinoma. A total of 80 patients underwent surgery alone; 46 patients received chemo radiotherapy alone; 17 patients received chemoradiotherapy followed by surgery; and 10 patients did not receive any treatment. Thymidine phosphorylase expression in esophageal carcinoma tissues was examined by immunohistochemistry. The clinicopathologic value and prognostic value of S-dThdPase was determined in 80 patients treated by surgery alone. RESULTS. The S-dThdPase is significantly higher in patients with esophageal carcinoma than in healthy controls (30.8 +/- 31.8 ng/mL vs. 13.8 +/- 7.6 ng/mL; P < 0.001). Statistically significant differences in S-dThdPases were observed depending on tumor size (P < 0.01) and tumor depth (P < 0.01). A S-dThdPase of more than 29.0 ng/mL (which represented the mean plus 2 standard deviation of the concentration in healthy controls) was associated with dThdPase expression (P = 0.022), poor response (P = 0.022), and poor survival (P < 0.01). Because S-dThdPase was associated with tumor depth, S-dThdPase was not an independent prognostic factor (P = 0.095). CONCLUSIONS. A high S-dThdPase is associated with depth of tumor invasion and poor response to treatment.

BACKGROUND. Serum cross-linked carboxyterminal telopeptide of Type I collagen (ICTP) is a metabolite of Type I collagen and has been reported to serve not only as a marker of bone metastasis but also as an indicator of treatment response and prognosis in several malignant tumors. The objective of this work was to evaluate the value of serum. ICTP as a tumor marker in patients with esophageal squamous cell carcinoma (SCC). METHODS. In this study, pretreatment serum ICTP concentrations were measured by double-antibody radioimmunoassay in 50 patients undergoing tumor resection, chemoradiotherapy, or palliation for newly diagnosed esophageal SCC. The cutoff value for positive ICTP levels was defined as 4.50 ng/mL. As a comparison, serum concentrations of SCC antigen, carcinoembryonic antigen, cytokeratin-19 fragment, alkaline phosphatase, and lactate dehydrogenase were simultaneously evaluated. The results were categorized according to several clinicopathologic variables. RESULTS. Among the markers tested, ICTP showed the highest sensitivity (58.0%) in esophageal SCC sera. Positive ICTP levels were significantly correlated with tumor progression variables, such as tumor depth greater than or equal to T2, regional lymph node metastasis (N1), distant metastasis (M1), TNM stage greater than or equal to II and maximal tumor length greater than 50 mm. Survival analyses of 29 patients who underwent curative resection demonstrated that patients who were positive for ICTP had significantly worse outcomes in terms of overall, disease specific, and disease free survival than those who were negative. Multivariate analyses confirmed that serum ICTP levels provided an independent prognostic indicator of overall and disease specific survival. However, serum ICTP values did not correlate with prognosis or treatment response in 17 unresectable cases treated by chemoradiotherapy. CONCLUSIONS. our results indicated that serum ICTP concentrations might be a novel prognostic tumor marker for assessing the progression of esophageal SCC. (C) 2002 American Cancer Society.

Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane. Delivery of electric pulses to established tumors thereby can improve the susceptibility of tumors to an anti-cancer agent administered. We examined whether electroporation-mediated transfer of cytokine genes into solid tumors could produce anti-tumor effects in the tumor-bearing mice. Plasmid DNA containing cytokine genes were injected into human esophageal T.Tn tumors developed in nude mice and electric pulses were then delivered. Administration of murine GM-CSF or human IL-2 gene followed by electroporation significantly suppressed the subsequent growth of T Tn tumors and prolonged the survival of the inoculated mice. In contrast, electroporation-mediated introduction of a control gene, human GM-CSF gene, whose products do not bind to murine GM-CSF receptors, did not achieve any anti-tumor effects. In vivo transfection of cytokine genes with electroporation could be a possible therapeutic strategy for established solid tumors.

BACKGROUND, The primary objective of this study was to clarify the significance of p21(WAF1/CIP1)(p21) gene expression in the tumorgenicity of hepatitis B virus (HBV) and hepatitis C virus (HCV) infected human hepatocelluar carcinoma (HCC). METHODS. The authors performed Northern blot hybridization to compare the p21 messenger (m) RNA expression levels among 16 HCC cases. They detected tissue HBVx mRNA (Northern blot) and plus- and minus-strand HCV RNA (reverse transcription-polymerase chain reaction) in liver tissues. They also measured alanine transaminase (ALT) levels and indocyanine green retention rate at 15 minutes (ICG-R15). RESULTS. The p21 transcripts of tumor (T) tissues could be identified with lower intensity than nontumor (N) tissues in all 4 HBVx mRNA(+) cases, 8 of 10 HCV RNA(+) cases, and 1 of 3 B(-), C(-) cases (1 case was positive for both viruses). p21 mRNA expression levels of N tissues were significantly higher in HCV RNA(+) cases than in HBVx mRNA(+) cases. p21 mRNA expression levels of N tissues were significantly correlated with serum ALT levels. CONCLUSIONS. In HCV hepatitis, p21 mRNA expression is up-regulated to control cell cycle under regeneration stress. Once the liver develops HCC, the p21 mRNA expression decreases to prominently low levels. The up-regulated p21 expression may play a role as a guard to prevent hepatocytes from tumorgenicity in HCV hepatitis. Cancer 2001;91:2096-103. (C) 2001 American Cancer Society.

BACKGROUND, The primary objective of this study was to clarify the significance of p21(WAF1/CIP1)(p21) gene expression in the tumorgenicity of hepatitis B virus (HBV) and hepatitis C virus (HCV) infected human hepatocelluar carcinoma (HCC). METHODS. The authors performed Northern blot hybridization to compare the p21 messenger (m) RNA expression levels among 16 HCC cases. They detected tissue HBVx mRNA (Northern blot) and plus- and minus-strand HCV RNA (reverse transcription-polymerase chain reaction) in liver tissues. They also measured alanine transaminase (ALT) levels and indocyanine green retention rate at 15 minutes (ICG-R15). RESULTS. The p21 transcripts of tumor (T) tissues could be identified with lower intensity than nontumor (N) tissues in all 4 HBVx mRNA(+) cases, 8 of 10 HCV RNA(+) cases, and 1 of 3 B(-), C(-) cases (1 case was positive for both viruses). p21 mRNA expression levels of N tissues were significantly higher in HCV RNA(+) cases than in HBVx mRNA(+) cases. p21 mRNA expression levels of N tissues were significantly correlated with serum ALT levels. CONCLUSIONS. In HCV hepatitis, p21 mRNA expression is up-regulated to control cell cycle under regeneration stress. Once the liver develops HCC, the p21 mRNA expression decreases to prominently low levels. The up-regulated p21 expression may play a role as a guard to prevent hepatocytes from tumorgenicity in HCV hepatitis. Cancer 2001;91:2096-103. (C) 2001 American Cancer Society.

The status of the p53 gene of tumor cells can modify the sensitivity of the tumors to radiation and anticancer agents. Human esophageal cancer cells (T.Tn) bearing mutated p53 gene were retrovirally transduced with wildtype p53 gene. The transduced cells (T.Tn/p53) which stably expressed wild-type p53 proliferated at the same rate as parental cells. However, the sensitivity to radiation was significantly improved by the transduction and T.Tn/p53 cells became markedly susceptible to cisplatin and etoposide compared with parental cells. Administration of cisplatin noticeably suppressed the,growth of T.Tn/p53 tumors but not T.Tn tumors inoculated in nude mice. Forced expression of wild-type p53 gene thereby can increase the sensitivity to DNA damage in esophageal cells.

1974年5月から1995年12月までの当院で施行した腎移植174例に発症した38例45回の腎移植後呼吸器感染症を重症度分類を行い検討した. 死亡例は3例であった.<br>発症は移植後全期間に認められた. シクロスポリン使用と発症頻度, 移植からの期間, 起因病原体との関連はなかった. 間質性肺炎は移植後1年以内の発症が多かった. 治療経過を検討すると, 全例起因病原体の確定前に治療が開始されていた. 発症による移植腎機能への影響は軽微であったが, 感染症治療薬の多剤投与例14例中4例(28.6%)に肝機能障害を認めた. 重症例4例のうち2例は的確な治療が行われず, 1例は多剤併用療法後の肝機能障害により死亡した. 重症のカリニ肺炎の1例は早期の肺生検により治癒した. 腎移植後呼吸器感染症は, 移植後の死因の大きな要素の1つであり, 早期診断が非常に重要であると考えられた.

The immunosuppressive and toxic effects of cyclosporine (Csa) are affected by many factors. We present the first case of a kidney transplant patient who had an onset of hypothyroidism about two months after the transplantation. In this case, trough levels of whole blood CsA and total serum cholesterol levels increased at the same time. But the decrease in renal function was not as severe as expected from the very high trough levels of whole blood CsA. Elevation of the trough level of whole blood CsA may be due to a decrease in CsA clearance resulting from the decreased cytochrome P-450 activity in hypothyroidism. Furthermore, a decrease in thyroid hormone level and increase in plasma lipoprotein level might have affected the distribution of CsA, and this change might have influenced the toxic effect of CsA. In conclusion, our case suggests that thyroid function and plasma lipoprotein level should be considered important factors that affect the pharmacokinetics and action of CsA.

p21Cip1 was first isolated as one of the cyclin-dependent kinase (Cdk) interacting proteins induced by wild-type p53 gene product, and it appears to play an essential regulatory role in the control of cell proliferation as a potent, tight-binding inhibitor of cyclin-Cdk complex that blocks the G(1)/S transition of the cell cycle. We have now examined the p21Cip1 mRNA expression levels in 16 surgically excised human colorectal tumor and non-tumor tissues by Northern-blot analysis with reference to the identification of p53 gene mutations. p53 gene mutations were detected in 6 tumor tissues but not in the other 10 tissues by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method and following direct sequencing. The mean p21Cip1 mRNA expression level in tumor tissues was significantly suppressed compared to that of non-tumor tissues, irrespective of p53 gene mutations. In p53 gene mutation-detected cases, the mean expression level of p21Cip1 mRNAs of tumor tissues was about 60% of that of cases without p53 gene mutation. Moreover, the relative mRNA expression levels of p21Cip1 significantly decreased as the pathohistological stages progressed by Dukes' staging system, while in patients with liver metastasis these levels were significantly suppressed compared to those of patients without organ metastasis. These results indicate that reduced expression of p21Cip1 mRNA is critical for growth activity and malignant potential of human colorectal carcinoma, and that the decrease in p21Cip1 mRNA level is due to p53 gene mutation as well as other mechanisms during human colorectal carcinogenesis. (C) 1996 Wiley-Liss, Inc.

大腸癌において,原発性びまん浸潤型癌は稀な型であり,現在までに本邦で報告されているのは100余名に過ぎない.また,このうち組織学的にスキルスでない症例が半数を占めている.最近,組織学的にも著しい線維化を認めた直腸原発性びまん浸潤型大腸癌症例を経験したので若干の文献的考察を加えて報告する.<br>症例は68歳,女性.約2カ月間下血,肛門痛を認め当科入院となる.入院時の腫瘍マーカー値は高値で,注腸バリウム, MRI検査にて子宮,膣に浸潤した直腸原発性びまん浸潤型大腸癌の診断のもと,後方骨盤内臓全摘術を施行した.傍大動脈リンパ節に転移を認め非治癒切除であった.術後,肛門痛などの症状は消失し良好な経過を示したが,術後2カ月目頃から腰痛,腹痛,腹水が発生した.腫瘍マーカー値も著明な上昇を示し,癌性腹膜炎,悪液質にて術後6カ月で癌死した.

We have isolated from B16 mouse melanoma cells a complementary DNA (Mel-18), whose deduced amino acid sequence possesses a characteristic zinc finger structure. Immunostaining with antibodies raised against partial Mel-18 peptide sequences demonstrated nuclear localization of the gene product. We have also demonstrated that this protein has DNA-binding capacity, and the zinc finger is responsible for the DNA binding. At the transcriptional level the Mel-18 mRNA was detected in all tumor cells examined as well as melanoma cells (ontogenically of neural origin) but was scarcely present in normal tissues except neural organs. The transcript is developmentally regulated. These data suggest that Mel-18 may play a role in transcriptional regulation and also in control of cell proliferation and/or neural cell development.

We have analyzed the mouse melanoma antigen with monoclonal antibodies established by syngeneic immunization. To further understand the structure of this antigen at a molecular level, we have cloned the genomic DNA controlling the expression of melanoma antigen by cosmid library transfection and a monoclonal antibody. In the process of analyzing this DNA fragment we found that it contained a gene related with transformation, which was proved by tumor formation in nude mice inoculated with NIH/3T3 transfectants of this DNA fragment. We discuss the structure of the gene product based on the deduced amino acid sequence of cDNA, which maps the genome bearing transformation-related activity. © 1989.

We summarize our recent studies on the analysis of melanoma antigen and the melanoma gene recently cloned. The melanoma antigen analyzed by syngeneic monoclonal antibodies is composed of a complex of GM3 ganglioside and protein molecules. The epitope recognized by antimelanoma cytotoxic T lymphocytes seemed to be the combinatorial determinants of GM3 and proteins, whereas the epitope for the suppressor T cells was found to be solely GM3 (NeuAc). The genomic DNA controlling the melanoma antigen was recently isolated and was found to possess transforming activity. The structure of this transforming gene is discussed based on the sequence data of the corresponding cDNA clone. Copyright © 1988, Wiley Blackwell. All rights reserved