Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

Division of Molecular Genetic

Tumor endothelial cells with high aldehyde dehydrogenase activity show drug resistance

Tumor blood vessels play an important role in tumor progression and metastasis. We previously reported that tumor endothelial cells (TEC) exhibit several altered phenotypes compared with normal endothelial cells (NEC). For example, TEC have chromosomal abnormalities and are resistant to several anticancer drugs. Furthermore, TEC contain stem cell-like populations with high aldehyde dehydrogenase (ALDH) activity (ALDHhigh TEC). ALDHhigh TEC have proangiogenic properties compared with ALDHlow TEC. However, the association between ALDHhigh TEC and drug resistance remains unclear. In the present study, we found that ALDH mRNA expression and activity were higher in both human and mouse TEC than in NEC. Human NEC:human microvascular endothelial cells (HMVEC) were treated with tumor-conditioned medium (tumor CM). The ALDHhigh population increased along with upregulation of stem-related genes such as multidrug resistance 1, CD90, ALP, and Oct-4. Tumor CM also induced sphere-forming ability in HMVEC. Platelet-derived growth factor (PDGF)-A in tumor CM was shown to induce ALDH expression in HMVEC. Finally, ALDHhigh TEC were resistant to fluorouracil (5-FU) in vitro and in vivo. ALDHhigh TEC showed a higher grade of aneuploidy compared with that in ALDHlow TEC. These results suggested that tumor-secreting factor increases ALDHhigh TEC populations that are resistant to 5-FU. Therefore, ALDHhigh TEC in tumor blood vessels might be an important target to overcome or prevent drug resistance

Energy-efficient refurbishment of public houses Case studies of the application of an energy-efficient design approach to public house refurbishment

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ALDH is expressed in mouse tumor blood vessels in vivo.

<p>Double immunofluorescence staining for endothelial markers CD31 and ALDH in normal mouse tissue (dermis) (a–c), A375SM melanoma xenografts () (d–i), and oral carcinoma xenografts (j–l). Merged images (white arrow) show co-localization of ALDH (green) and CD31 (red) in situ. Nuclei were counterstained with DAPI. Scale bar: 40 µm.</p

Isolation of ALDH^high cells by FACS.

<p>a. TECs with high or low ALDH activity (ALDH^high and ALDH^low TECs, respectively) were isolated by FACS. b. Analysis of ALDH mRNA expression by real-time RT-PCR confirmed that the sorted ALDH^high/low TECs were highly pure (*p<0.01). c. Cell proliferation was measured every day for 3 days by MTS assays. The proliferation of ALDH^high TECs was significantly slower than that of ALDH^low TECs (*p<0.01). d. Relative expression of Sca-1, CD90, MDR1, and IL-6 to GAPDH in ALDH^high/low TECs was measured using real-time RT-PCR (*p<0.01). e. ALDH^high TECs showed spheroid morphological features with a smooth surface and high circularity at 5 days after seeding into microwells in the stemness spheroid assay. ALDH^high TECs formed spheres at a higher frequency than that of ALDH^low TECs. Scale bar: 30 µm.</p

TECs exhibit a stem like phenotype.

<p>a. Relative expression of Sca-1, CD90, and MDR1 to GAPDH in TECs and NECs was measured using real-time RT-PCR (*p<0.01). b. ALDH mRNA expression was upregulated in TECs as determined by real-time RT-PCR (*p<0.01). ALDH activity was higher in TECs than that in NECs based on analysis of ALDH activity by flow cytometry.</p

ALDH^high TECs show a highly angiogenic phenotype.

<p>a. The number of tube junctions (arrows) was counted at 10 and 24 h after seeding onto Matrigel in a tube formation assay in vitro (*p<0.05). Scale bar: 50 µm. b. Relative expression of VEGF-A, FGF-2, and VEGFR2 to GAPDH in ALDH^high/low TECs was measured using real-time RT-PCR (*p<0.05; **p<0.01). c. Levels of p-Akt were determined by western blotting using an anti-p-Akt antibody. The membrane was stripped and re-incubated with anti-total Akt (t-Akt) and -β-actin antibodies. After treatment with VEGF for 20 min, Akt was more activated in ALDH^high TECs than that in ALDH^low TECs.</p

ALDH is expressed in human tumor blood vessels.

<p>Double immunofluorescence staining for endothelial markers CD31 and ALDH in normal human kidney tissue (a–c) and RCC tumor tissue (d–i). Merged images show co-localization of ALDH (green) and CD31 (red) in situ. Scale bar: 40 µm.</p