Radiosensitivity of hypoxic and proliferating clonogen in a human lung cancer grown in nude mice

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we studied their radiosensitivity by focusing attention on the dynamics of tumor clonogens. The movement of clonogens in the regrowing IA tumor after irradiation can be divided into three phases: first: the early and rapid survival recovery (PLD repair) phase, second, the delay phase involving a certain lag in survival change, and third, the repopulation phase consisting of two stages: the anoxic repopulation before angiogenesis and the hypoxic repopulation in the presence of a poorly-developed vascular network. Clonogens in a regrowing tumor after irradiation were found to actively proliferate even in an anoxic environment before angiogenesis and under the hypoxic conditions prevailing after the formation of a tumor with a poorly developed vascular system. This re-grown tumor was found to be more radioresistant than a sham-treated control tumor. It is believed that these clonogens are genetically selected under hypoxic conditions throughout the process of tumor growth and regrowth, and may be primarily involved in tumor recurrence or accelerated repopulation in fractionated irradiation

Inhibition of potential lethal damage repair and related gene expression after carbon-ion beam irradiation to human lung cancer grown in nude mice

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we previously demonstrated their radiosensitivity by focusing attention on the dynamics of tumor clonogens and the early and rapid survival recovery (potential lethal damage repair: PLD repair) occurring after X-ray irradiation. To the authors knowledge, this is the first study demonstrating gene expression in association with PLD repair after carbon-ion beam or X-ray irradiation to cancer cells. In this study we tried to detect the mechanism of DNA damage and repair of the clonogens after X-ray or carbon-ion beam irradiation. At first, colony assay method was performed after irradiation of 12 Gy of X-ray or 5 Gy of carbon-ion beam to compare the time dependent cell survival of the IA cells after each irradiation pass. Second, to search the genes causing PLD repair after irradiation of X-ray or carbon-ion beam, we evaluated gene expressions by using semi-quantitative RT-PCR with the selected 34 genes reportedly related to DNA repair. The intervals from the irradiation were 0, 6, 12 and 24hr for colony assay method, and 0, 3, 18hr for RT-PCR method. From the result of survival assay, PLD repair was not significantly observed in carbon-ion beam as compared to X-ray. The results of RT-PCR were as follows. The gene showing significantly higher expressions after X-ray irradiation than after carbon-ion beam irradiation was PCNA. The genes showing significantly lower expressions after X-ray irradiation rather than after carbon-ion beam irradiation were RAD50, BRCA1, MRE11A, XRCC3, CHEK1, MLH1, CCNB1, CCNB2 and LIG4. We think PCNA among them could be a most hopeful candidate gene for PLD repair

Classifying the variation in response to ionizing radiation in human cells by gene expression profiling

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Correlation between cellular radiosensitivity and DNA damage measured by the alkaline comet assay in human cancer cell lines.

In order to evaluate the usefulness of alkaline comet assay as a predictive assay of radiosensitivity, two parameters of comet assay and three parameters of cell survival curve were compared using 13 human cancer cell lines. Colony forming assay was performed after irradiation from 1 Gy to 6 Gy, and determined clonogenic survival parameters, such as D10, Dq and SF2. In comet assay, initial DNA damage (ID) immediatery after X-ray irradiation at dose of 8 Gy and residual damage (RD) at each 30 or 60 min after irradiation were assessed. Thirteen cell lines showed a broad spectrum in D10 value, ranging from 2.74 to 8.18, and Dq value, ranging from 0.29 to 3.42. ID varied from 1.87 to 9.09 among the cell lines, and RD varied from 0.66% to 56.51% at 30 min, and from 0% to 55.58% at 60 min. When cell lines were classified into two groups (group A and group B) according to each own D10 value, the group A, whose D10 value was more than 6, showed higher ID (5.4 + 2.3) than the group B (2.6 + 0.7), whose D10 values was less than 6 (P=0.008). There was, however, no correlation between ID and SF2. When cell lines were classified into two groups according to each own Dq value (group C and group D), the average of RD in group C, whose Dq value was more than 2, was 2.9% at 30 min. Group D whose Dq value is less than 2 was 15.6% at 30 min. The significant difference was observed between group C and group D (p= 0.049). These results suggest that the comet assay might be potential to classify a radiosensitive group according to ID and RD.International Comet Assay Workshop No.

Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa α-fodrin, by interfering with MHC class II–peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes

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