In vitro expansion of human glioblastoma cells at non-physiological oxygen tension irreversibly alters subsequent in vivo aggresiveness and AC 133 expression

Among markers of glioblastoma initiating cells, AC133 has been shown to be associated with glioblastoma resistance and malignancy. Recently, it was demonstrated that increasing oxygen tension (pO(2)) down-regulated AC133 expression in glioblastoma cells in vitro. In order to better understand extrinsic factor regulation of AC133, this work aimed to investigate the relationship between cell culture pO(2), AC133 expression, and tumor development and phenotype. Using treatments with CoCl(2) and HIF-1α shRNA knockdowns on non-sorted human primary glioblastoma cells cultured at low (3%) versus high (21%) oxygen tension, we established a responsibility for low pO(2) in the maintenance of high levels of AC133 expression, with a major but non-exclusive role for HIF-1α. We also demonstrated that human glioblastoma cells previously cultured under high oxygen tension can lose part of their aggressiveness when orthotopically engrafted in SCID mice or lead to tumors with distinct phenotypes and no re-expression of AC133. These observations showed that the specific pO(2) microenvironment irreversibly impacts glioblastoma cell phenotypes, highlighting the pertinence of culture conditions when extrapolating data from xenogenic models to human cells in their source environment. They also raised AC133 as a marker of non-exposure to oxygenated areas rather than a marker of aggressiveness or low pO(2) niches

The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133high situation than in the CD133low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken together, these data extend our knowledge of the function of CD133 and underline the interest of further exploring the CD133-Tf-iron network

Hypoxia induces fructose accumulation in human U87 brain tumor cells: A 13C NMR study

International audienc

Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the amyloid precursor protein is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576 there is increased processing of the amyloid precursor protein to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced by hypoxia

Recognition of CD133 and modulation of its expression interfere with Tf uptake and TfR accessibility in Caco-2 cells.

<p>A) AC133 antibody treatment inhibited Tf uptake. Constitutively CD133-expressing undifferentiated Caco-2 cells were exposed to Tf-Alexa 488 for 1 h at 37°C/5%CO₂ in the presence of 5 or 10 µg/ml of AC133 or IgG1κ immunoglobulin control. Tf-Alexa 488 that was effectively internalized within cells was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the untreated control. Data represented mean ± s.e.m. from a triplicate obtained from one representative experiment. Dunnett's test: *p<0.05, ***p<0.001. B) Flow cytometric analysis of the expression of TfR (CD71) at the surface of CD133^high (Control siRNA) and CD133^low (CD133 siRNA) Caco-2 cells. Data represented mean ± s.e.m. obtained from three independent experiments. IgG2aκ immunoglobulins were used as negative control immunostaining. Dunnett's test: ***p<0.001. C) Analysis of the expression of AC133, TfR/CD71 and CHC within Caco-2 cells by immunocytochemistry combined with confocal laser scanning microscopy. Note the increase of CD71 expression while AC133 was depleted from the Control siRNA to the CD133 siRNA situation. Bar: 50 µm.</p

Implication of CD133 in endocytosis mainly involved the clathrin pathway.

<p>A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133^high (control siRNA) and CD133^low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p<0.05, **p<0.01, ***p<0.001; comparison between control siRNA and CD133 siRNA: Dunnett's test: p<0.05. B) Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells led to an increase in LNC intracellular accumulation. Flow cytometric analysis of intracellular uptake of NR-LNC within CD133^high (Control siRNA) and CD133^low (CD133 siRNA) Caco-2 cells after 1 h of incubation at 37°C/5%CO₂. Results are expressed as percentage of control, thus representing the geomean fluorescence intensity levels obtained for cells treated with vehicle alone. Data represented mean ± s.e.m. obtained from three independent experiments. Dunnett's test: **p<0.01.</p

Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells led to an increase in Tf intracellular accumulation but had no impact on Dx and CTB uptake.

<p>A) Immunofluorescence associated flow cytometric analysis of CD133 expression on non-differentiated Caco-2 cells treated with 30 nM of control or CD133-specific siRNA. Results are expressed as a percentage of the control treatment, representing the geomean fluorescence intensity levels obtained after AC133 immunostaining of cells treated with irrelevant siRNA (CD133^high Caco-2 cells); note the effective down regulation of CD133 when CD133-specific siRNA (CD133^low Caco-2 cells) was used. B–D) Flow cytometric analysis of intracellular uptake of Dx-FITC (B), CTB-FITC (C) and Tf-Alexa 488 (D) within CD133^high and CD133^low Caco-2 cells after 1 h of incubation at 37°C/5%CO₂. Results are expressed as percentage of control, thus representing the geomean fluorescence intensity levels obtained for cells treated with vehicle alone. Data represented mean ± s.e.m. obtained from three independent experiments. Dunnett's test: **p<0.01, ***p<0.001</p

Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells did not affect short-term Tf exocytosis.

<p>CD133^high (control siRNA) and CD133^low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to Tf-Alexa 488 for 2 h at 37°C/5% CO₂ before the extracellular medium was removed, washed and replaced by fresh medium free from Tf-Alexa 488. Amounts of Tf-Alexa 488 that were not recycled to the extracellular compartment were measured by flow cytometric analysis after further cell incubation at 37°C/5% CO₂ for 1 to 3 h. Data are expressed as a % of Tf-Alexa 488 initially internalized. They represent mean ± s.e.m. obtained from three independent experiments.</p

Schematic representation of known IREs and nearest related stem loop sequence located in the 3′UTR of the CD133 mRNA (NCBI GenBank accession number NM_001145847.1, nucleotide sequence: from +3544 to +3574).

<p>Note the perfect match between the 5′-A<u>CAGAGU</u>U-3′ loop sequence of the CD133 mRNA and the one present in the TfR1 3′IRE. Note also the high discrepancy between hairpin structures, notably with the presence of secondary loops (dashed line) in the APP 5′IRE. A secondary loop is also present in the CD133 hairpin selected here. CDC14A: dual specificity protein tyrosine phosphatase. DMT1: divalent metal transporter 1. 75 kDa Fe-S: NADH dehydrogenase (ubiquinone) Fe-S protein 1. APP: Alzheimer amyloid precursor protein. Ft-H: ferritin heavy chain.</p