Seiichi Takenoshita

Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence

Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in approximately 50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b, and mir-34c, which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 (ING2) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b, and mir34c up-regulation and ING2 down-regulation may be involved in the senescence pathway.

Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection test for gefitinib-refractory T790M epidermal growth factor receptor mutation

Mutations in the epidermal growth factor receptor (EGFR) are observed in a fraction of non‐small‐cell lung cancers (NSCLS). EGFR mutation‐positive NSCLS responds to gefitinib. Secondary T790M mutation confers gefitinib resistance to NSCLS. A detection test for the T790M mutation was designed based on the peptide nucleic acid–locked nucleic acid polymerase chain reaction clamp method. The specificity and sensitivity of the test were both greater than 0.99. The test revealed that only a small population of the PC‐13 cells carried the T790M mutation. The test also revealed that the T790M mutation was found in none of 151 NSCLC specimens obtained before gefitinib treatment, whereas it was found in four of four specimens obtained from NSCLS that had become refractory to gefitinib. In one patient in whom the L858R‐positive EGFR allele was amplified to multiple copies, an L858R‐T790M double‐mutant allele emerged during the gefitinib therapy. This allele was expressed highly. The T790M mutation detection test based on the peptide nucleic acid–locked nucleic acid polymerase chain reaction clamp method is sensitive and specific, and is applicable to clinical practice. It detects T790M‐positive cells in the course of gefitinib treatment, and thus will help to devise therapies effective for T790M‐positive NSCLS. (Cancer Sci 2008; 99: 595–600)

Opioid Rotation from Oral Morphine to Oral Oxycodone in Cancer Patients with Intolerable Adverse Effects: An Open-Label Trial

OBJECTIVEnWe prospectively investigated the efficacy of opioid rotation from oral morphine to oral oxycodone in cancer patients who had difficulty in continuing oral morphine treatment because of inadequate analgesia and/or intolerable side effects.nnnMETHODSnTwenty-seven patients were enrolled and 25 were evaluated. The rate of patients who achieved adequate pain control, which provided an indication of treatment success, was evaluated as primary endpoint. The acceptability and pharmacokinetics of oxycodone were evaluated in addition to the assessment of analgesic efficacy and safety during the study period.nnnRESULTSnIn spite of intense pain, the morphine daily dose could not be increased in most patients before the study because of intolerable side effects. However, switching to oral oxycodone allowed approximately 1.7-fold increase as morphine equivalent dose. Consequently, 84.0% (21/25) of patients achieved adequate pain control. By the end of the study, all patients except one had tolerated the morphine-induced intolerable side effects (i.e. nausea, vomiting, constipation, drowsiness). Common side effects (>10%) that occurred during the study were typically known for strong opioid analgesics, and most were mild to moderate in severity. A significant negative correlation between creatinine clearance (CCr) value and the trough concentrations of the morphine metabolites was observed. On the other hand, no significant correlation was found between CCr value and the pharmacokinetic parameters of oxycodone or its metabolites.nnnCONCLUSIONSnFor patients who had difficulty in continuing oral morphine treatment, regardless of renal function, opioid rotation to oral oxycodone may be an effective approach to alleviate intolerable side effects and pain.

Protein and lysate array technologies in cancer research

Capturing quantitative proteomic information provides new insights for enhancing the understanding of cancer biology. There have been several protein microarray formats, and each has an advantage depending on what is being detected. However, in contrast to nucleotide printing, the production of protein arrays generally requires the capability of handling viscous solutions, and the mishandling of various factors, such as temperature and humidity, adversely affect protein status. The requirement for such specifications is critical when increasing the throughput for monitoring a large number of samples for rigorous quantitation. In particular, a new solid pin arrayer has been extremely powerful when highly viscous cell lysates printed for high-density, reverse-phase protein arrays, and acquired data allows for theoretical models of protein signaling networks to be constructed. In this review, applications of currently available protein microarray technology to cancer research are discussed including the advantages of the new solid pin architecture for opening up powerful proteomic applications.

Clinicopathological significance of circadian rhythm-related gene expression levels in patients with epithelial ovarian cancer

Objective. Recent studies implicate circadian genes in the regulation of cell cycle, apoptosis, and cell proliferation at a molecular level. These genesey affect cancer incidence, prognosis, and chemosensitivity. In this study, we measured the expression levels of clock genes and correlated their expression levels with clinicopathological parameters in epithelial ovarian cancer. Methods. The expression levels of core clock genes, per1, per2, per3, cry1, cry2, Bmal1, clock, and CKIɛ were quantified by real‐time quantitative Reverse transcription‐polymerase chain reaction in 83 ovarian cancer tissues and 11 normal ovarian tissues. Results. The expression levels of per1, per2, cry2, clock, CKIɛ in ovarian cancers were significantly lower than those in normal ovaries. In contrast, cry1 expression was highest among the eight examined clock genes, followed by per3 and Bmal1. Cry1 expression was much higher in cancer than that in normal ovaries. Localized circadian gene expression was determined in cancer cells by in situ hybridization analysis. Cry1 expression was significantly reduced in mucinous and grade 3 tumors. Bmal1 expression was also significantly reduced in mucinous adenocarcinomas as compared to other histologies. In a multivariate analysis, the combination of low cry1 expression and low Bmal1 expression was an independent prognostic factor, as well as stage and histological subtype. Conclusions. The antiphase expression of cry1 and Bmal1 may be preserved in ovarian cancers. The combination of cry1 and Bmal1 expression might become a possible prognostic marker in epithelial ovarian cancer.

Primary Adenocarcinoma of the Minor Duodenal Papilla

A 70-year-old man was admitted to our institution due to aggravation of blood-sugar level control and because an abdominal CT showed dilatation of the main pancreatic duct. Upper gastrointestinal endoscopy revealed a flat elevated tumor with central ulceration in the second portion of the duodenum. Subsequent duodenoscopy for a more detailed examination showed that the tumor had originated in the minor duodenal papilla. A biopsy specimen showed moderately differentiated adenocarcinoma. Endoscopic retrograde pancreatography via the major duodenal papilla revealed a slightly dilated main pancreatic duct and obstruction of the accessory pancreatic duct. Endoscopic ultrasonography showed a hypoechoic mass in the minor duodenal papilla with retention of the muscularis propria of the duodenum. These findings suggest that the tumor existed only to a limited extent in the minor duodenal papilla, and that the tumor did not infiltrate into the pancreas. For treatment, pylorus-preserving pancreatoduodenectomy was performed, and histological findings revealed a well- differentiated adenocarcinoma that originated in the minor duodenal papilla. Primary adenocarcinoma of the minor duodenal papilla is extremely rare. Our case is the first report of primary adenocarcinoma of the minor duodenal papilla at an early stage with no infiltration into muscularis propria of the duodenum and pancreas.