11.Hypoxic ventilatory depressionと思われる一症例について(第551回千葉医学会例会・第9回麻酔科例会・第18回千葉麻酔懇話会)

FISH analysis on metaphase nuclei (top panel) of cultured cells derived from peripheral blood leukocytes of the proband of family 2 by using BAC probes for 11p15.5-15.4 (RP11-11A9, 3,236,552-3,356,012, green) and 11q22.3 (RP11-179B7, 104,298,339-104,459,797, red). The green signal on both homologues is visible only at chr11p, demonstrating the presence of an in cis duplication and excluding an unbalanced translocation. FISH analysis on interphase nuclei (bottom panel) using the BACs RP11-699D10 (2.9–3.0 Mb, red) and RP11-11A9 (green), hybridizing within the duplication. Note that single and duplicated signals can be seen on the two homologues, respectively. The red-green-green-red order of the duplicated signals indicates that the duplication is inverted. (PDF 52 kb

Endogenous hTERT expression was not detected in hMSC and hMSC-telo1 cells

<p><b>Copyright information:</b></p><p>Taken from "Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation"</p><p>http://www.biomedcentral.com/1471-2199/8/49</p><p>BMC Molecular Biology 2007;8():49-49.</p><p>Published online 13 Jun 2007</p><p>PMCID:PMC1906829.</p><p></p> Positive control is a MG63 cell which is known to express hTERT. Ectopic hTERT was detected in hMSC-telo1 cells but not in hMSC's. Positive control is TERT transduced cells. β-actin was found in both hMSC's and hMSC-telo1 cells. Positive control is MG63 cells

Keratin23 (KRT23) Knockdown Decreases Proliferation and Affects the DNA Damage Response of Colon Cancer Cells

<div><p>Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression <i>in vitro.</i> Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. <i>In vitro</i> analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.</p></div

Irradiation of colon cancer cells.

<p>A) SW948-ctrl or SW948-sh1506 with stable KRT23 knockdown were irradiated with 0 GY or 5 GY of γ-rays, respectively and seeded on RTCA16-well plates with 16.000 cells/well (n = 4). Non-irradiated SW948-sh1506 cells showed a reduced proliferation rate compared to non-irradiated SW948-ctrl cells. Irradiated SW948-ctrl cells continued proliferation after a short lag period, while the proliferation of the irradiated KRT23-depleted SW948-sh1506 cells decreased after 72 h post-irradiation. The Cell Index CI of the irradiated SW948-sh1506 cells markedly dropped down at about 96 h post-irradiation suggesting a detaching of the cells, possibly induced by cell death upon irradiation of the KRT23 depleted cells. B) A MTT viability assay co-performed at 120 h post-irradiation together with RTCA showed that the viability of KRT23 depleted SW948-sh1506 cells was reduced by 60% upon irradiation with 5GY (p = 8.1E-08) compared to 30% in the SW948-ctrl cells (p = 6.4E-05). C) Visual inspection at 7 days post-irradiation showed a markedly reduced number of cells in KRT23 depleted SW948-sh1506 cells irradiated with 5GY compared to non-irradiated cells.</p

Knockdown of KRT23.

<p><b>A</b>) Western Blot of freshly made SW948-ctrl and SW948-sh1506 cells extracts with 20 µg extract per lane using a monospecific anti-K23 antibody in a 1∶150 dilution, Marker Biorad All Blue. Stable knockdown of K23 in the SW948-sh1506 cells resulted in >80% reduced K23 protein expression compared to the SW948-ctrl cells. Beta-actin was used as loading control. B) Immunofluorescence analysis confirmed a decreased K23 expression in SW948-sh1506 cells; anti-K23 antibody 1∶500, detection with Alexa 488, nuclear stain Hoechst, magnification 630x C) Visual inspection indicated a lower cell density for SW948-sh1506 cells 48 h post seeding. D) SW948-sh1506 cells showed less nuclear expression of the proliferation marker KI67 (green); anti-KI67 1:100 E) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well plates with 4000 cells per well (n = 12) and proliferation was analyzed post-seeding at five time-points. Proliferation of SW948-sh1506 cells was significantly (Fisher’s exact t-test, p<0.0001) decreased at 96 hours and 120 hours post-seeding. F) The MTT assay was repeated by seeding 16000 cells per well (n = 11) on a 96 well plate and proliferation/viability was analyzed at 48 h post-seeding and percentage viability was measured. The proliferation of KRT23 depleted cells was significantly (p = 2.6E-06) decreased by about 30%. G) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well RTCA-plates with 8000 cells/well (n = 3). Values are shown as medians and standard deviations for each group at selected time points for a representative experiment.</p

Ingenuity pathway analyses and protein expression.

<p>A) KRT23 knockdown affected main regulatory pathways, and decreased the expression of key molecules. Key molecules decreased upon KRT23 knockdown essential for the assembly of replication and repair complexes comprise E2F1, BRCA1, BRCA2, RAD51, MRE11A and RPA. blue = decreased, yellow = increased transcript expression. B) Western blotting. MRE11A, E2F1, BRCA1 and RAD51 proteins were markedly decreased in KRT23 depleted SW948-sh1506 cells compared to control cells. 20µg nuclear (nuc) or cytoplasmic extract (cyt) was loaded. Anti-MRE11A antibody 1∶1000 dilution (nuclear extracts), 1∶500 dilution (nuclear and cytoplasmic extracts). Marker All Blue BioRad; C) Immunofluorescence staining of SW948-ctrl and SW948-sh1506 cells also showed that KRT23 knockdown resulted in a decreased expression of E2F1, MRE11A, RAD51 and BRCA1.</p

Methylation versus expression profiling of KRT23.

<p>Comparison of KRT23 transcription data from Exon 1.0 ST arrays (–•–) to methylation data from 2 probes, cg22392708 and cg06378617 from the Illumina Bead arrays (□) showed a negative correlation between methylation and transcription in the 40 tissue samples analyzed (Spearman rank correlation coefficient of −0.64 and −0.74, respectively).</p

KRT23 knockdown affects canonical pathways involved in DNA damage control.

<p>Data were obtained by microarray expression profiling followed by RMA normalization, comparison of SW948 control cells versus SW948-sh1506 with KRT23 knockdown. All molecules are located in the nucleus.</p

Colon cancer cells depleted from KRT23 compared to control cells.

*<p>number of molecules differentially expressed to number of molecules in given pathway.</p

Additional file 7: Figure S7. of Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes

Note that the haplotype associated with the duplication segregates from I-1 to II-2 and III-1 and that only the D11S4088 marker shows allelic imbalances in II-2 and III-1. (PDF 141 kb