Keisuke Tomoda

PIK3CA Mutation Status in Japanese Esophageal Squamous Cell Carcinoma

BACKGROUND A somatic mutation of the PIK3CA (phosphatidylinositol 3-kinase catalytic subunit) gene has been found in human cancer patients. However, this mutation has not yet been extensively studied in esophageal squamous cell carcinomas. MATERIALS AND METHODS We analyzed a mutation of the PIK3CA gene in 88 Japanese cases of esophageal squamous cell carcinomas that had all undergone surgery at the Department of Surgery II, Nagoya City University Medical School, between 1996 and 2003. The TE and KYSE series of cell lines are human esophageal cancer cell lines. Two PIK3CA mutation hot spots (exon 9 and exon 20) were analyzed by a real time polymerase chain reaction (PCR)-based assay and the data were confirmed by direct sequencing. We performed a cell proliferation assay to determine the effects of a PI3K inhibitor LY294002. RESULT In exon 9, a somatic mutation was found in two patients (2.2%) and in two cell lines. The mutations included three E545K (G1633A) mutations and one E545Q (G1633C) mutation. However, in exon 20, no mutation was observed in our esophageal cancer patients. PI3K inhibitor (LY294002) inhibited the growth of an esophageal cancer cell line with a PIK3CA mutation (E545K) in vitro. CONCLUSIONS We found LY294002 to reduce the proliferation of the esophageal cancer cell line in vitro. Importantly, a cell line with a PIK3CA gene mutation was more susceptible to a PI3K inhibition than those without any such mutation. Further functional analyses of the PIK3CA mutations are warranted to determine whether or not they may be potentially useful targets of therapy for esophageal cancer.

Identification of candidate genes involved in the radiosensitivity of esophageal cancer cells by microarray analysis

Radiotherapy plays a key role in the control of tumor growth in esophageal cancer patients. To identify the patients who will benefit most from radiation therapy, it is important to know the genes that are involved in the radiosensitivity of esophageal cancer cells. Hence, we examined the global gene expression in radiosensitive and radioresistant esophageal squamous cell carcinoma cell lines. Radiosensitivities of 13 esophageal cancer cell lines were measured. RNA was extracted from each esophageal cancer cell line and a normal esophageal epithelial cell line, and the global gene expression profiles were analyzed using a 34 594-spot oligonucleotide microarray. In the clonogenic assay, one cell line (TE-11) was identified to be highly sensitive to radiation, while the other cell lines were found to be relatively radioresistant. We identified 71 candidate genes that were differentially expressed in TE-11 by microarray analysis. The up-regulated genes included CABPR, FABP5, DSC2, GPX2, NME, CBR3, DOCK8, and ABCC5, while the down-regulated genes included RPA1, LDOC1, NDN, and SKP1A. Our investigation provided comprehensive information on genes related to radiosensitivity of esophageal cancer cells; this information can serve as a basis for further functional studies.

Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins.

The p53 binding protein 2 (53BP2) has been identified independently as the interacting protein to p53, Bcl‐2, and p65 subunit of nuclear factor κB (NF‐κB). It was demonstrated that over‐expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF‐κB activation elicited by a physiological NF‐κB inducer, interleukin‐1β (IL‐1β), and anti‐apoptotic Bcl‐2 family proteins on the 53BP2S‐mediated apoptosis. We found that both NF‐κB activation and Bcl‐2 family proteins could prevent the 53BP2S‐mediated depression of mitochondrial transmembrane potential, activation of caspase‐9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti‐apoptotic Bcl‐2 family proteins. The regulatory network of the 53BP2S‐mediated apoptosis cascade including its interacting proteins is discussed.

Gene expression profiling of the response of esophageal carcinoma cells to cisplatin

Cisplatin is the most common chemotherapeutic agent used in esophageal cancer. However, sensitivity to cisplatin varies greatly between patients. It is important to identify the gene(s) that are related to the sensitivity to cisplatin in esophageal cancer patients. The IC50 for cisplatin was measured for 15 esophageal cancer cell lines (TE1-5, TE8-15, KYSE140, and KYSE150). RNA was extracted from each of these cell lines and a normal esophageal epithelial cell line, namely, Het1A, and gene expression profiles were analyzed using an oligonucleotide microarray consisting of 34 594 genes. TE4 was highly resistant and TE12, 14, and 15 were sensitive to cisplatin. Thirty-seven genes were differentially expressed in the cisplatin-resistant esophageal cancer cell line. Our investigation provides a list of candidate genes that may be associated with resistance to cisplatin in esophageal cancer cells, which may serve as a basis for additional functional studies.

Adenocarcinoma arising in a colonic interposition following a total gastrectomy: Report of a case

A segment of the transverse colon can be used for gastric reconstruction after a total gastrectomy. This report presents the case of a 68-year-old woman with primary adenocarcinoma of the colon in a segment used for reconstruction after a total gastrectomy. The interposed colon developed colon carcinoma 9 years after the gastric reconstruction. The possibility of a primary carcinoma arising in a gastric colon interposition must be considered when employing the transverse colon as a gastric substitute.

Molecular docking analysis of the protein–protein interaction between RelA-associated inhibitor and tumor suppressor protein p53 and its inhibitory effect on p53 action

RelA‐associated inhibitor (RAI) was initially identified as a protein that interacts with the p65 subunit (RelA) of nuclear factor‐κB. It was recently found to interact with the p53 tumor suppressor protein. RAI is a structural homolog of the p53‐binding protein 2 and IκB family proteins, and is known to inhibit the DNA‐binding activities of p65 and p53. In the present study, we have attempted to predict the 3‐dimensional structure of RAI in complex with p53 using computational chemistry. In order to evaluate the predicted structure model, we created a series of RAI mutants in which the amino acid residues involved in the interaction with p53 were mutated, and examined their activities in blocking p53‐mediated bax gene expression. Our observations support the validity of the predicted 3‐dimensional model of the p53–RAI protein complex. Based on the p53–RAI complex model, we have demonstrated the biological importance of the R248 and R273 residues of p53, and the D775 and E795 residues of RAI, in the protein–protein interaction between p53 and RAI and the biological actions of these proteins. These findings will further clarify the biological actions of RAI in carcinogenesis and can be used for the development of a novel strategy in blocking the actions of RAI. The possible biological implications of RAI are also discussed. (Cancer Sci 2008; 99: 615–622)