Additional file 1: of HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner

Figure S1. HPV-16 E6 did not affect PLD activity. (PDF 127 kb

Additional file 2: of HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner

Figure S2. HPV-16 E6 and E7 expression affect p53 and pRb expression. (PDF 116 kb

Galectin-3 Up-Regulation in Hypoxic and Nutrient Deprived Microenvironments Promotes Cell Survival

<div><p>Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, <i>e.g</i>. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. <i>In vitro</i> experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7–2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. <i>In vivo</i>, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation <i>in vitro</i> and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.</p></div

Gal-3 knockdown sensitizes cells to cell death in oxygen and nutrient deprivation in the NG97ht cell line.

<p>Analysis of NG97ht cells incubated with specific gal-3 siRNA or scramble siRNA for 6 h and then exposed to either normoxia (N), CoCl₂ (C) or hypoxia (H) in complete or serum-deprived medium for additional 42 h. <b><i>A</i></b>. Protein analysis demonstrated gal-3 knockdown in each experimental condition. Gal-3 knockdown was calculated relatively to its β-actin control. <b><i>B</i></b>. In addition, gal-3 mRNA was evaluated and qRT-PCR demonstrated significant knockdown upon treatment with specific siRNA compared to scramble siRNA. <b><i>C</i></b>. Analysis of hypodiploid cells demonstrated no differences in cell death rates in cells exposed to high serum concentrations, regardless the levels of gal-3 expression. <b><i>D</i></b>. Upon serum-deprivation, cells were sensitized to cell death in hypoxic conditions. Sensitization was much more pronounced upon knockdown of gal-3, as compared to cells transfected with scramble siRNA. T98G cells transduced with gal-3 shRNA demonstrated decreased gal-3 protein by western blot (<b><i>E</i></b>) and mRNA by qRT-PCR (<b><i>F</i></b>) compared to scramble shRNA. Gal-3 knockdown was calculated relatively to its β-actin control. <b><i>F</i></b>. Analysis of hypodiploid cells demonstrated increased cell death in cells transduced with gal-3 shRNA compared to cells transduced with scramble shRNA when exposed both to hypoxia or hypoxia and serum-deprivation. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. **p<0.01, ***p<0.001.</p

U87MG glioma cells transduced with gal-3 shRNA demonstrate decreased tumor size and growth.

<p>U87MG cells transduced with gal-3 shRNA demonstrated decreased gal-3 protein by western blot (<b><i>A</i></b>) and mRNA by qRT-PCR (<b><i>B</i></b>) compared to scramble shRNA. Gal-3 knockdown was calculated relatively to its β-actin control. <b><i>C</i></b>. The inoculation of U87MG gal-3 shRNA cells in the flank of nuce mice (n = 5) demonstrated a decrease in tumor size and growth compared to the U87MG scramble shRNA (n = 6). <b><i>D</i></b>. The delay presented for the decrease in tumor size and growth in the gal-3 shRNA cells was not due to a decreased tumor cell proliferation <i>in vitro</i> when compared to scramble shRNA cells. <b><i>E</i></b>. Gal-3 knockdown cells demonstrated increased tumor free survival time compared to scramble shRNA cells. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Gal-3 upregulation in hypoxia depends on HIF-1α and NF-κB and is blocked by cicloheximide.

<p><b><i>A</i></b>. qRT-PCR analysis of NG97ht cells treated with cycloheximide (CHX) 5 µM and 20 µM and its control (DMSO) and in cells exposed to either normoxia, CoCl₂ or hypoxia in serum-deprived medium for 6 h demonstrated significant increase in gal-3 expression in cells exposed to CoCl₂ and hypoxia compared to normoxia in the group treated with DMSO, but not when cells were exposed to CHX. <b><i>B</i></b>. The exposure to HIF-1α inhibitor 10 µm (CAS 934593-90-5) demonstrated gal-3 decreased induction by qRT-PCR in cells exposed to hypoxia, but not normoxia and CoCl₂. DMSO used as control. <b><i>C</i></b>. The exposure to DHMEQ 5 µg/mL, an NF-κB inhibitor, decreased gal-3 induction by qRT-PCR both in CoCl₂ and hypoxia compared to normoxia after 24 h in serum-deprived medium. DMSO used as control. <b><i>D</i></b>. Western blot analysis with the HIF-1αinhibitor demonstrated a slight decrease in gal-3 only in cells exposed to hypoxia, but not normoxia and CoCl₂, compared to DMSO control (experiment performed once). <b><i>E</i></b>. Western blot analysis also demonstrated decreased gal-3 induction in cells treated with DHMEQ compared to DMSO in hypoxia and CoCl₂ compared to normoxia after 24 h in serum-deprived medium. <b>F</b>. Cells in normoxia display scattered HIF-1α (green) throughout cytoplasm, while cells exposed to CoCl₂ and hypoxia display HIF-1α mainly in cells nuclei (blue). <b><i>G</i></b>. <i>In</i> vivo, NF-κB demonstrates to be upregulated in pseudopalisading areas, which display increased gal-3 expression. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. **p<0,01; ***p<0.001.</p

NG97ht cells demonstrate increased cell death only when exposed to oxygen and nutrient deprivation, which is accompanied by the increase of reactive oxygen species and caspase-7 activation.

<p><i>A</i>. Experiments performed in complete medium did not demonstrate differences in cell death rates in cells exposed to hypoxia compared to normoxia, however, cells in CoCl₂ demonstrated cell death after 48 h of exposure. <i>B</i>. In serum-deprived medium, longer periods of exposure (>24 h) significant cell death rates were observed in CoCl₂ and hypoxia compared to normoxia. <i>C</i>. ROS detection by DHE also demonstrated the increase of these molecules in cells exposed to hypoxia and CoCl₂ regardless serum concentrations and in normoxia in nutrient deprivation compared to normoxia in complete medium. <i>D</i>. NG97ht cells exposed to DHMEQ in serum-deprived medium presented increased cell death in cells exposed to normoxia and hypoxia, but not CoCl₂. <i>E</i>. Caspase 7 analysis showed increased active caspase-7 in cells exposed do CoCl₂ and hypoxia, both in complete or serum-deprived medium. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; *** p<0.001.</p

<i>In vivo</i> analysis shows hypoxia and gal-3 accumulation in pseudopalisades.

<p>NG97ht cells were transplanted subcutaneously in nude mice and tumors formed were excised and analyzed microscopically. <b><i>A</i></b>. Tumors present necrotic foci (* all figures) and pseudopalisades (arrows). HE staining. <b><i>B</i></b>. Tumors demonstrated hypoxic regions (pimonidazole adducts) in pseudopalisades (brown areas, arrows). <b><i>C</i></b>. Immunohistochemistry for gal-3 demonstrated pseudopalisades intensely stained (brown areas, arrows). Sparse cells in non-necrotic areas around blood vessels (BV) were also positive. Scale bar 100 µm.</p

Viral load in gliomas.

<p>Box plot of the viral load for astrocytomas (AST), oligodendrogliomas (ODG) and non tumoral brain (NTB), shown as natural logarithm. p-value = 0.0102.</p

Human Cytomegalovirus positivity in gliomas.

<p>Total samples (Light gray), positive samples (Dark gray) and frequency of positivity are shown for astrocytomas (AST I, AST II, AST III and GBM), oligodendrogliomas (ODG II and ODG III) and non-tumoral brain (NTB) by qPCR (1A), IHC (1B)and ISH (1C).</p