Yuichi Nagakawa

Differential recognition of response elements determines target gene specificity for p53 and p63.

ABSTRACT p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63+/+ mouse, it is undetectable in these tissues in the p63−/− mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.

Deregulation of miR-92a expression is implicated in hepatocellular carcinoma development

MicroRNAs (miRNAs) belong to a class of the endogenously expressed non‐coding small RNAs which primarily function as gene regulators. Growing evidence suggests that miRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. The miR‐17‐92 cluster especially is markedly overexpressed in several cancers, and is associated with the cancer development and progression. In this study, we have demonstrated that miR‐92a is highly expressed in hepatocellular carcinoma (HCC). In addition, the proliferation of HCC‐derived cell lines was enhanced by miR‐92a and inhibited by the anti‐miR‐92a antagomir. On the other hand, we have found that the relative amount of miR‐92a in the plasmas from HCC patients is decreased compared with that from the healthy donors. Interestingly, the amount of miR‐92a was elevated after surgical treatment. Thus, although the physiological significance of the decrease of miR‐92a in plasma is still unknown, deregulation of miR‐92 expression in cells and plasma should be implicated in the development of HCC.

Histologic features of venous invasion, expression of vascular endothelial growth factor and matrix metalloproteinase-2 and matrix metalloproteinase-9, and the relation with liver metastasis in pancreatic cancer.

Introduction Pancreatic cancer frequently is associated with venous invasion and hematogenous metastasis. Aims To determine morphologic features of invaded veins, intratumoral vascular composition, the correlation with liver metastasis, and expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9, and the mechanism of development of hematogenous metastasis. Methodology We examined 32 patients with resected pancreatic cancer: 18 had postoperative liver metastasis, and 14 had no liver metastasis. Specimens were examined to determine the composition of veins and microvessels by staining of victoria-blue and CD34. We also investigated expression of VEGF, MMP-2, and MMP-9 by immunohistochemical staining. Results Venous invasion was detected in 31 of 32 patients. Invaded venous densities of middle- and large-sized veins were significantly higher in patients with liver metastasis than in those with nonliver metastasis, and they were related to MMP-2 and MMP-9 overexpression. Invaded veins with fragmentation of the lumen through cancer cells were considered to be an intravasation of cancer (destroyed type vein), and their numbers were significantly related to liver metastasis, and MMP-2 and MMP-9 overexpression. Conclusion In conclusion, almost all the patients with pancreatic cancer showed venous invasion, indicating that invasion into large veins and destroyed type veins could be a risk factor for liver metastasis and that increased expression MMP-2 and MMP-9 were related to such invasion.

Oxidative mitochondrial DNA damage and deletion in hepatocytes of rejecting liver allografts in rats: Role of TNF‐α

An orthotopic liver transplant model in the rat was used to evaluate the role of tumor necrosis factor alpha (TNF‐α) in liver transplant rejection. There were significantly increased levels of TNF‐α mRNA and parallel increases in 8‐hydroxy‐2′ deoxyguanosine (8‐OHdG) indicative of oxidative DNA damage present 7 to 12 days after transplantation. Cells staining positively for 8‐OHdG were localized to the cytoplasm of hepatocytes adjacent to the TNF‐α expressing inflammatory cells in the portal areas or in patches surrounded by inflammatory cells in the hepatic sinusoids. Significantly more cells staining for 8‐OHdG were found in the allogeneic grafts that were strongly rejected than in the syngeneic controls or in the grafts placed in species that accepted the allograft permanently after a rejection episode. TUNEL reactivity lagged 2 days behind peak reactivity for 8‐OHdG. On day 12 after transplantation, many cells stained for both 8‐OHdG and TUNEL, indicating that the cells suffering oxidative DNA injury were undergoing apoptosis or death. Oxidative injury resulted in mtDNA deletion consisting of 4,834 base‐pairs. Studies of hepatocytes cultured from normal rats displayed dose‐dependent relationships between TNF‐α concentration and 8‐OHdG and mtDNA mutation. Repetitive intraperitoneal injection of Enbrel, a TNF receptor blocker, significantly decreased hepatocyte 8‐OHdG levels and the frequency of deleted mtDNA while greatly extending graft survival time. In conclusion, the data presented implicate TNF‐α as being capable of causing oxidative DNA damage and mtDNA mutation in hepatocytes. (HEPATOLOGY 2005;42:208–215.)

Over-Expression of AIF-1 in Liver Allografts and Peripheral Blood Correlates with Acute Rejection after Transplantation in Rats

Early and accurate detection of acute cellular rejection (ACR) is important in the management of liver allograft recipients. We hypothesized that expression of allograft inflammatory factor (AIF)‐1 would be associated with liver allograft rejection as previous studies have shown that a relationship exists between kidney and heart transplantation. Indeed using rat orthotopic transplant models we found that the expression of allograft inflammatory factor‐1 (AIF‐1) can be detected in both allograft and peripheral blood leukocytes with peak levels detected 7 days following liver transplantation. Interestingly, AIF‐1 expression increased 2‐fold in acutely rejecting liver allografts compared to chronically accepted livers on days 5, 7 and 10 after transplantation. AIF‐1 expression in peripheral blood leukocytes was also significantly greater in the rejection model than in the acceptance model. Flow cytometric analysis of peripheral blood leukocytes demonstrated that AIF‐1 was expressed in ED2‐positive cells, a marker for Kupffer cells. In vitro studies showed that AIF‐1 expression in Kupffer cells was up‐regulated by coculture with Th1 cytokines. However, neither LPS nor Escherichia coli (E. coli) administration had an affect on AIF‐1 expression. These data indicate that high levels of AIF‐1 expression reflect aggressive liver allograft rejection and suggest a role for monitoring AIF‐1 in peripheral blood leukocytes as a monitor for increased immunosuppression.

Bacterial contamination in ascitic fluid is associated with the development of clinically relevant pancreatic fistula after pancreatoduodenectomy.

Objectives Clinically relevant postoperative pancreatic fistula (POPF) after pancreatoduodenectomy is often accompanied by bacterial infection. To elucidate the mechanism of bacterial infection associated with POPF, we investigated the relationship between POPF and bacteria isolated from ascitic fluid and removed drains. Methods Subjects were 101 patients who had undergone pancreatoduodenectomy. Microbial culture was performed using ascitic fluid obtained from drains. We also compared the isolated bacteria from removed drains on postoperative day (POD) 4 and after POD 7. Results In 23 patients (22.8%), microbial cultures were already positive on POD 1, although purulent discharge was not observed. Among patients with grade B/C POPF, bacteria were detected on POD 1 in 53.8%; these isolated bacteria were the same as those isolated after POPF formation. In contrast, only 7.7% of patients with grade A POPF were positive on POD 1. The number of bacteria isolated from drains removed after POD 7 significantly increased compared with those isolated from drains removed on POD 4. Conclusions Bacterial contamination in ascitic fluid may be an initiating event that leads to the development of clinically relevant POPF. Therefore, it is important to perform both the administration of the appropriate antibiotics and early drain removal.

Hypoxia-inducible factor-1α expression and gemcitabine chemotherapy for pancreatic cancer

The normal pancreas has an abundant blood flow, in contrast to pancreatic cancer, which is a hypovascular tumor. During hypoxia under a hypovascular environment, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is activated. High HIF-1α expression reduces sensitivity to gemcitabine (GEM) which is used as a treatment for pancreatic cancer. The objective of this study was to clarify HIF-1α expression in pancreatic cancer and the association of its effects to GEM treatment. We used the human pancreatic ductal carcinoma cell lines AsPC-1 and BxPC-3 to evaluate cell proliferation, HIF-1α protein expression and sensitivity to GEM in a hypoxic environment of 1% O2 in 48 pancreatic cancer patients who received adjuvant GEM treatment after pancreatectomy. We divided the patients according to HIF-1α expression and the presence of single nucleotide polymorphisms, and we based our evaluation on the adverse events associated with GEM chemotherapy and patient outcome. The hypoxic environment promoted cell proliferation, induced HIF-1α expression and increased GEM resistance, especially in AsPC-1 cells, which included a mutant homozygote for HIF-1α(C1772T). There were no significant differences between the HIF-1α(-) and HIF-1α(+) groups in either adverse events or patient outcomes. HIF-1α enhanced neo-microvascularity in a hypoxic environment and increased drug resistance. The period until recurrence was shorter in the patients with a strong HIF-1α expression, than that in those with a weak HIF-1α expression.

p53 gene mutation and p53 protein overexpression in a patient with simultaneous double cancer of the gallbladder and bile duct associated with pancreaticobiliary maljunction

Pancreaticobiliary maljunction (PBM) is associated with the occurrence of biliary cancer due to pancreatobiliary reflux. We present a case of simultaneous double cancer of the gallbladder and bile duct. A 77-year-old woman who had jaundice, intra- and extra-hepatic biliary ductal dilatation and a space-occupying lesion in the gallbladder and lower bile duct underwent pancreatoduodenectomy. The gallbladder cancer showed papillary carcinoma without mutation of the K-ras gene and with p53 non-sense mutation of CCA (Pro) to CA (Stop) on codon 301 in exon 8. The bile duct cancer revealed a well-differentiated adenocarcinoma without mutation of the K-ras gene and with p53 miss-sense mutation of GTG (Val) to GAG (Glu) on codon 272 in exon 8. There were no mutations of either the K-ras or p53 gene in non-cancerous epithelia. In contrast, only the mucosa of the common channel had p53 protein accumulation and high cell proliferation activity. Therefore, the genetic pathway might be the same in both the gallbladder and bile duct cancer, and a high potential for carcinogenesis might be present in the epithelium of the common channel in patients with PBM.

The VIO soft-coagulation system can prevent pancreatic fistula following pancreatectomy

BACKGROUND/PURPOSE The VIO soft-coagulation system (SC) is a new device for tissue coagulation. We hypothesized that this device would be an effective tool for sealing small pancreatic ducts, thus reducing pancreatic fistula following pancreatectomy. METHODS To confirm whether the SC could be used to seal small pancreatic ducts, we measured the burst pressure in sealed ducts in mongrel dogs. Eight dogs underwent distal pancreatectomy, with the remnant stump coagulated by using the SC. The animals were necropsied on postoperative day 10. In a clinical trial, 11 patients who underwent pancreatoduodenectomy with SC treatment (SC group), and 24 patients who underwent pancreatoduodenectomy without SC treatment (non-SC group) were compared. RESULTS In the experimental study, the burst-pressure test revealed that the SC had efficiently sealed the small pancreatic ducts. Histological examination revealed completely obstructed pancreatic ductal structures, ranging from large pancreatic ducts (diameter, 500 microm) to microscopic ducts. No pancreatic leakage was observed following distal pancreatectomy without main pancreatic duct (MPD) suturing in dogs that had an MPD diameter of less than 500 microm. In the clinical trial, pancreatic fistula developed in only one patient (9.1%) in the SC group, but a pancreatic fistula developed in five patients (20.8%) in the non-SC group. CONCLUSIONS This novel technique using the SC is an effective procedure for preventing the development of pancreatic fistula following pancreatectomy.

Proposed Nomogram Predicting the Individual Risk of Malignancy in the Patients With Branch Duct Type Intraductal Papillary Mucinous Neoplasms of the Pancreas.

Objectives: This study evaluated individual risks of malignancy and proposed a nomogram for predicting malignancy of branch duct type intraductal papillary mucinous neoplasms (BD-IPMNs) using the large database for IPMN. Background: Although consensus guidelines list several malignancy predicting factors in patients with BD-IPMN, those variables have different predictability and individual quantitative prediction of malignancy risk is limited. Methods: Clinicopathological factors predictive of malignancy were retrospectively analyzed in 2525 patients with biopsy proven BD-IPMN at 22 tertiary hospitals in Korea and Japan. The patients with main duct dilatation >10 mm and inaccurate information were excluded. Results: The study cohort consisted of 2258 patients. Malignant IPMNs were defined as those with high grade dysplasia and associated invasive carcinoma. Of 2258 patients, 986 (43.7%) had low, 443 (19.6%) had intermediate, 398 (17.6%) had high grade dysplasia, and 431 (19.1%) had invasive carcinoma. To construct and validate the nomogram, patients were randomly allocated into training and validation sets, with fixed ratios of benign and malignant lesions. Multiple logistic regression analysis resulted in five variables (cyst size, duct dilatation, mural nodule, serum CA19-9, and CEA) being selected to construct the nomogram. In the validation set, this nomogram showed excellent discrimination power through a 1000 times bootstrapped calibration test. Conclusion: A nomogram predicting malignancy in patients with BD-IPMN was constructed using a logistic regression model. This nomogram may be useful in identifying patients at risk of malignancy and for selecting optimal treatment methods. The nomogram is freely available at http://statgen.snu.ac.kr/software/nomogramIPMN.