12.GENETICS DIVISION
Amplification of RNA from Cells Collected by Laser-capture Microdissection
An effective method, termed TALPAT, has been developed for amplification of the 3' portions of mRNAs. Through a T7 RNA polymerase-mediated RNA amplification reaction combined with an adaptor ligation-mediated PCR reaction, more than 10 mg of cRNA can be produced from 1 ng of total RNA, corresponding to as few as 100 cells. The fidelity and power of TALPAT have been validated on the microarray analysis on minute samples derived from laser-capture microdissection (LCM) of various cancers.
Towards Personalized Cancer Care By Expression Profiling
One of the most important prognostic factors of Esophageal Squamous Cell Carcinoma for surgically treated patients is the status of lymph node metastasis, but preoperative accurate assessment of the nodal status is difficult. Oligonucleotide microarray expression analyses of 12,629 genes were applied to surgical specimens of patients who underwent extended lymph node dissection. Genes that are associated with the increased risk of lymph node metastasis were identified and validated in an additional series of samples. Expression profiles were also correlated with the duration of survival following chemoradiotherapy. Among more than 100 genes found to be associated with prognosis, genes involved in the immune response or drug resistance were noted. Machine learning using SVMs networks were employed successfully to predict a response to the therapy.
Identification of a novel tumor suppressor gene, GDM
Through a detailed structural analysis of the gene amplification at 17q12, a novel tumor suppressor gene candidate, GDM, was identified in the region. The gene is expressed preferentially in the normal esophagus, stomach and mammary gland. In situ hybridization using mouse tissues demonstrated the restricted expression of the gene in the superficial, PCNA-negative region of the gastric mucosa. In contrast, the gene was tightly suppressed in most of the esophageal, gastric and breast cancer cell lines. The promoter region of this gene was often methylated heavily in the cancer cells. Adenovirus-mediated GDM transduction into cancer cells showed that overexpression of this gene induced suppression of the cell growth.
WNT signaling pathway implicated in carcinogenesis
WNT signal is transduced to the b-catenin-TCF pathway, the JNK pathway, or the Ca2+-releasing pathway through WNT receptors encoded by Frizzled (FZD) genes. WNT2B/WNT13, FZD1, FZD2, FZD3, FZD4, FZD6, FZD7, and FZD10 have been previously cloned and characterized. Here, the following WNT signaling molecules were cloned and characterized using bioinformatics, cDNA-library screening, cDNA-PCR and RACE: WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, FZD5, FZD8, NKD1, NKD2, FRAT1, and FRAT2.(162-175) MFRP, RNF26, and LZIC were also cloned and characterized during isolation of WNT signaling molecules. (176-178) Expression and regulation of 19 WNT genes, 10 FZD genes and other WNT-related genes in various types of human cancer, especially in gastric cancer, were then systematically investigated.(179-186) WNT2, WNT2B, WNT5A, WNT10A, and WNT16 were relatively frequently up-regulated in primary gastric cancer. WNT10A was up-regulated by TNFa and Helicobacter pylori in MKN45 cells derived from gastric cancer.
Functions of WNT signaling molecules were investigated by using Xenopus axis duplication assay. (173,187,188) WNT2B1 and WNT2B2 mRNAs, generated due to alternative promoter, encode almost identical WNT family proteins with the N-terminal divergence. WNT2B2 mRNA rather than WNT2B1 mRNA was preferentially expressed in various tumor cell lines including NT2 and also in primary tumors. WNT2B2, but not WNT2B1, was a positive regulator of the WNT-b-catenin pathway. Therefore, up-regulation of WNT2B2 in tumors might lead to carcinogenesis.