Isolation of possible cancer stem cells from a human esophageal carcinoma cell line

Recently, cancer stem cells (CSCs), which possess the capacity for self-renewal and generate the progenies of different phenotypes from parental cells, were identified in the solid tumors and maintained in vitro (Al-Haij et al, 2003, Singh et al, 2004, Yuan et al, 2004). In this study, we separated the CSCs from a human esophageal squamous cell carcinoma cell line (KYSE70). KYSE70 cells formed morphologically variable colonies in one dish: a densely mounding type (M-type), a flat, diffusive type (F-type), and a type with mixed mounding and flat cells (M/F-type). Classification of colonies into three types made possible to evaluate the extent of morphological heterogeneity. The proportion of M-type colonies decreased with increasing X-ray dose. In contrast, the proportions of M/F- and F-type colonies increased with increasing dose. X-ray irradiation seemed to stimulate the cells to transform from M-type to F-type. Two clones (EC13M and EC13F) were separated from one clone (clone 13) of the KYSE70 cells. The EC13M cells produced three types of colonies. The EC13F cells mainly produced the F-type colonies, but not M-type colonies. Clustering analysis for gene expression level by oligonucleotide microarray demonstrated that the EC13F cells were less closely related to the parental KYSE70 cells than were the EC13M cells. Gene Ontology analysis demonstrated that ARHE, ARHGDIB, COL5A1, CYR61, DLC1, ESDN, LAMC2, NELL2, OLR1, PCDH11Y, PTPNS1, TGFB1, CDC42EP3 and LCP1 were up-regulated in the EC13F cells. These genes were involved in the regulation of cell adhesion, cell motility and/or cellular morphogenesis. The diffusive EC13F cells may be more active in migrating into other tissues than the mounding cells. The tumor recurred after radiation therapy is usually more malignant than the primary tumor. Our data provide a clue as to why metastatic cells appear in the tissues of tumors that recur after radiotherapy.HUGO\u27s 10th Human Genome Meeting (HGM2005

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines

The purpose of this study was to determine the radiosensitivities of 31 human esophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. ATM gene in these cells had five missense mutations and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy

A deficiency in the phosphorylation of DNA-PKcs in a radiosensitive human ESCC cell line

DNA double strand breaks (DSBs) are the most deleterious and lethal form of DNA damage. DSBs are rejoined or repaired by two major pathways in mammalian cells: i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA-PK is a nuclear, serine/threonine protein kinase consisting of three subunits of DNA-PKcs, Ku70 and Ku80, and involved in the NHEJ, V(D)J recombination and modulation of transcription [1]. Cells lacking DNA-PKcs activity are highly sensitive to DSBs-inducing agents such as ionizing radiations [2]. Douglas et al [3] identified 4 phosphorylation sites in the DNA-PKcs protein: i.e. Thr-2609, Ser-2612, Thr-2638 and Thr-2647. These sites were autophosphorylated with own DNA-PKcs. Chan et al [4] demonstrated that autophosphorylation of DNA-PKcs was required for the rejoining of DSBs. Radiation sensitivities of 31 human esophageal squamous cell carcinoma (ESCC) cell lines were investigated with a colony-formation assay. There was a large variation in radiosensitivity among 31 cell lines. In particular, the radiation sensitivity of one cell line (KYSE190) was distinct from a cluster of radiation sensitivities of other 30 cell lines. In order to understand the mechanism behind this hypersensitivity, we investigated the expression of ATM and DNA-PKcs proteins with a western-blotting method and the phosphorylation of DNA-PKcs with a immunohistochemical staining methd. The phosphorylation of DNA-PKcs was not observed in KYSE190 cells. No mutation was detected in the four phosphorylation sites, but one base change in FAT domain of DNA-PKcs gene was observed. These data seem to indicate that the high radiosensitivity of KYSE 190 cells results from the defect in the autophosphorylation of the DNA-PKcs protein.The 5th Japan-France Workshop on Radiobiology and Medical Imagin