25.当院における内視鏡的乳頭切開術の臨床的検討(第617回千葉医学会例会・第1内科教室同門会例会)

Primer sequences and amplicon sizes. (DOCX 15 kb

Immunohistochemical analysis of the murine lung tumor origin.

<p>A lung tumor section was stained with antibody against surfactant protein C (SP-C), commonly used as a marker for the alveolar type II cell (A). The same tumor was also stained with antibody against the Clara cell 10 protein (CC10), commonly used as a marker for Clara cell (B). The majority of the lung tumor cells were SP-C positive suggesting that these lesions arose from the alveolar type II cells (C). A small percentage of the lung tumor cells were CC-10 positive indicating of Clara cell lineage (D). The arrows pointed the CC-10 positive cells.</p

Lung tumor rate in age-matched SPC-p53(273H) transgenic mice and non-transgenic littermates.

<p>No difference was found in lung tumor rate between the transgenic mice and non-transgenic controls at the age range of 4–12 and 22–24 months. The transgenic mice (T) have a statistically significant (Fisher's exact test p<0.01) higher lung tumor rate than wildtype controls (C) during the age of 13–21 months.</p

p16INK4a promoter methylations in lung tumors of transgenic and non-transgenic control mice.

<p>Bisulphite-treated tumor DNA was amplified with primers specific for either a methylated (M) or an unmethylated (U) p16INK4a promoter region (A). Comparison of p16INK4a promoter methylational rate between 4–12 months and the 13–24 months transgenic lung tumors (B), the transgenic tumors and the non-transgenic tumors in age of 13–24 months (C).</p

K-ras mutations in lung tumors of transgenic and non-transgenic control mice.

<p>Comparison of K-ras mutation in codons 12–13 and codon 61, (A) between 4–12 months and the 13–24 months transgenic lung tumors, and (B) between the transgenic tumors and the non-transgenic tumors in age of 13–24 months. Point mutations in (C) K-ras codon 12, (D) K-ras codon 13, and (E) K-ras codon 61.</p

Additional file 2: Table S2. of Rad51C-ATXN7 fusion gene expression in colorectal tumors

In silico translation of fusion gene Rad51C-ATXN7. (DOCX 17 kb

Schematic diagram of the role of Piwil2 for DNA repair.

<p>Once DNA damage is induced by genotoxic agents, silent <i>PIWIL2</i> gene is activated, modulating chromatin relaxation through histone H3 acetylation to allow DNA damage signaling proteins and DNA repair proteins migrate to the sites of DNA damage. Thus, Piwil2 might control multiple down-stream pathways for DNA repair. It is possible that <i>PIWIL2</i> could be activated by DNA damage sensor proteins to regulate histone H3 acetylation through effecting on HAT and/or HDAC. In addition, the proteins recruited to DNA damage sites might in turn suppress chromatin relaxation and Piwil2 expression after success of DNA repair (negative feedback). Overall, Piwil2 may mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation up-stream of DDR. DDR: DNA damage response; HAT: histone acetyltransferases; HDAC: histone deacetylase.</p

Piwil2 promotes chromatin relaxation through regulation of histone H3 acetylation in responding to DNA damage.

<p><b>A.</b> Piwil2 has no effect on activation of H2AX and p53 in MEFs after treatment with UV light. The γH2AX and pp53 in mili^-/- and WT MEFs were analyzed by Western blotting. <b>B</b>. Piwil2 is required for chromatin relaxation in MEFs irradiated by UV light, as revealed by MNase assay. Top panel: micrograph of DNA ladders; bottom panel: quantitation of DNA fragments in the top panel. **, p<0.01. <b>C.</b> Piwil2 up-regulate histone H3 acetylation in MEFs irradiated by UV light. Expression of phosphorylated histone H3 [pH3 (S10)] and acetylated histone H3 [AcH3 (K9, 14) and AcH3k18] in mili^-/- and WT MEFs were analyzed by Western blotting after UV irradiation. Tubulin expression was monitored as an internal control. Shown are the data from one of two experiments.</p

UV irradiation induces Piwil2 expression in HDFs.

<p><b>A &B.</b> Kinetics of Piwil2 expression in responding to DNA damage induced by UV light. HDFs were irradiated with UV (20 J/m²) and harvested at 0, 1, 2, 4, 8 and 24 h later and analyzed by Western blotting for piwil2 expression, using polyclonal rabbit antibody to Piwil2 (1∶1000 dilution). <b>C & D.</b> Dose-dependent expression of Piwil2 in responding to UV-induced DNA damage. HDFs were treated with various dose of UV, harvested 2 hrs after treatment and analyzed by Western blotting for Piwil2 expression. <b>E & F.</b> HDFs were treated as in A, and analyzed by RT-PCR for Piwil2 transcript expression. A, C & E: micrographs of Piwil2 proteins or transcripts; B, D & F: quantitation of the Piwil2 proteins or transcripts in A, C & E by normalization to β-actin. The data shown are a representative of two experiments.</p

Piwil2 is required for repair of DNA damage induced by IR and cisplatin.

<p><b>A, B & C.</b> Piwil2 is required for repair of DNA damage induced by cisplatin. (<b>A</b>) The survival rate of mili^-/- MEFs was significantly reduced in a dose-dependent manner as compared to WT MEFs after cisplatin treatment in various doses. The relative cell survival rate was determined by methylene blue staining (n = 3). **, p<0.01. (<b>B</b>) DNA repair in the cisplatin-treated mili^-/- and WT MEFs. The MEFs were treated with cisplatin for 1 h, cultured and harvested at the indicated time for ISB assay to determine amounts of Pt-GG in the cells (n = 3). (<b>C</b>) Cisplatin induced Piwil2 expression <i>in vivo</i>. Male mice were treated i.p. with cisplatin (20 mg/m²) or vehicle (PBS) for 5 consecutive days and kidney, liver and testis were harvested and whole cell lysates from the tissue were prepared and subjected to Western blotting with monoclonal anti-piwil2 IgM antibody (Kao2 supernatant; 1∶50). The data shown were a representative of two experiments. <b>D & E.</b> Piwil2 is required for repair of DNA damage induced by IR. (<b>E</b>) Mili^-/- and WT MEFs were seeded at 1×10⁵/well in 6-well plates in triplicates. When cells grew to 50–60% confluence (2 days) they were exposed to various doses (0, 0.5, 2, 5, 10 Gy) of X-ray (RS 2000 Biological Irradiator; Rad Source Technologies, Inc. Alpharetta, GA). Four days after irradiation, cells were harvested and counted with trypan blue exclusion of dead cells. Cell survival rate was calculated as percentage of viable cells of each dose normalized to untreated counterparts (n = 3). *, p<0.05; **, p<0.01. (<b>E</b>) DNA repair in IR-treated MEFs. Mili^-/- and WT MEFs were X-rayed at exponential growth phase and comet assay was performed with standard protocol. DNA damage was estimated by measuring the distance of the tail against the edge of far side of the nuclei for 50 random selected cells (n = 50; **, p<0.001). The data shown are representative of two experiments. <b>F & G.</b> Different size of γH2AX foci in Mili^-/- MEFs versus WT MEFs irradiated by X-ray. (<b>F</b>) Representative micrographs of γH2AX foci in MEFs at 3 h after X-ray irradiation (3 Gy). Arrows indicate the MEFs with large γH2AX foci; (<b>G</b>) Quantitation of γH2AX foci in MEFs at 1 and 3 h after X-ray irradiation (n = 3). **, p<0.01 compared between Mili^-/- and WT MEFs. Note that there is no significant difference between Mili^-/- and WT MEFs in the formation of large γH2AX foci at 1 h after irradiation.</p