Isabelle Desbaillets

Regulation of interleukin-8 expression by reduced oxygen pressure in human glioblastoma.

Oxygen deprivation is an important biological feature of tumor growth. We previously showed that in glioma, anoxia increases expression of IL-8, a chemokine and angiogenic factor. Here, we analysed for the first time the biochemical mechanisms inducing the IL-8 gene upon anoxia in glioma cells, and showed that they differ from those inducing the VEGF gene. Both genes are induced in biologically and genetically heterogenous glioblastoma cell lines (LN-229, LN-Z308, U87MG, T98G), whereas, in gliosarcoma cells (D247MG), only the VEGF gene is induced. The kinetics of IL-8 and VEGF mRNA inductions differ in these cells and reoxygenation experiments showed that the induction is due to the anoxic stress per se. Furthermore, in LN-229 and LN-Z308 cell lines actinomycin D, DRB and nuclear run-on experiments showed that anoxia stimulates increased transcription of both genes. Electromobility shift assays show increased protein binding to the AP-1 site on the IL-8 promoter following anoxia treatment. Finally, in situ hybridization on glioblastoma sections shows that the in vivo expression patterns of IL-8 and VEGF genes overlap, but are not identical. Since intratumoral augmentation of IL-8 and VEGF secretion, following microenvironmental decreases in oxygen pressure, may promote angiogenesis, further definition of these pathways is essential to appropriately target them for antitumoral therapy.

Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1α activation cascade: implications for HIF-1α gene therapy

The heterodimeric hypoxia‐inducible factor (HIF)‐1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF‐1α protein stabilization is well established, it is less clear whether and how oxygen‐regulated mechanisms contribute to HIF‐1α protein modifications, nuclear translocation, heterodimerization with the β‐subunit, recruitment of cofactors, and gene fraws‐activation. Because the HIF‐1α protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet‐Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF‐1α under normoxic conditions, allowing to distinguish hypoxia‐dependent from hypoxia‐independent activation mechanisms. Using these cells, we found that normoxically induced HIF‐1α is localized to the nucleus, binds DNA, and fraws‐activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF‐1α protein modifications and fraws‐activation ability but not protein stabilization and DNA‐binding activity. Because overexpressed HIF‐1α is fully localized to the nucleus but displays only partial DNA‐binding and fraws‐activation activity, mitogen‐activated protein kinase‐dependent phosphorylation might be required for full HIF‐1 activation. HIF‐1α protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF‐1 α gene transfer.

Necrogenesis and Fas/APO-1 (CD95) expression in primary (de novo) and secondary glioblastomas.

Glioblastomas may develop rapidly without clinical and histopathological evidence of a less malignant precursor lesion (de novo or primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). Primary glioblastomas typically show overexpression of EGFR, but rarely p53 mutations, while secondary glioblastomas frequently carry a p53 mutation, but usually lack overexpression of EGFR, suggesting that these glioblastoma subtypes develop through distinct genetic pathways. In the present study, we assessed the expression of Fas/APO-1 (CD95), an apoptosis-mediating cell membrane protein, and its relation to necrosis phenotype in primary and secondary glioblastomas. Large areas of ischemic necroses were observed in all 18 primary glioblastomas, but were significantly less frequent in secondary glioblastomas (10 of 19, 53%; p=0.0004). Fas expression was predominantly observed in glioma cells surrounding large areas of necrosis and was thus significantly more frequent in primary glioblastomas (18 of 18, 100%) than in secondary glioblastomas (4 of 19, 21%; p< 0.0001), suggesting that these clinically and genetically defined subtypes of glioblastoma differ in the extent and mechanism of necrogenesis. Necrosis and microvascular proliferation are histologic hallmarks of the glioblastoma. Following incubation of glioblastoma cell lines under hypoxic/anoxic conditions for 24-48 hours, Fas mRNA levels remained unchanged, whereas VEGF expression was markedly upregulated. This suggests that in contrast to VEGF, Fas expression is not induced by ischemia/hypoxia. Analysis of Fas mRNA levels in a glioblastoma cell line containing a p53 mutation and an inducible wild-type p53 gene showed little difference under induced and noninduced conditions, suggesting that in glioblastomas, Fas expression is not directly linked to the p53 status.

Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy. Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.

Elevated levels of MIC-1/GDF15 in the cerebrospinal fluid of patients are associated with glioblastoma and worse outcome

For patients with brain tumors identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebrospinal fluid (CSF), would greatly facilitate patient management. MIC‐1/GDF15 (growth differentiation factor 15) is a secreted protein of the TGF‐beta superfamily and emerged as a candidate marker exhibiting increasing mRNA expression during malignant progression of glioma. Determination of MIC‐1/GDF15 protein levels by ELISA in the CSF of a cohort of 94 patients with intracranial tumors including gliomas, meningioma and metastasis revealed significantly increased concentrations in glioblastoma patients (median, 229 pg/ml) when compared with control cohort of patients treated for non‐neoplastic diseases (median below limit of detection of 156 pg/ml, p < 0.0001, Mann–Whitney test). However, plasma MIC‐1/GDF15 levels were not elevated in the matching plasma samples from these patients. Most interestingly, patients with glioblastoma and increased CSF MIC‐1/GDF15 had a shorter survival (p = 0.007, log‐rank test). In conclusion, MIC‐1/GDF15 protein measured in the CSF may have diagnostic and prognostic value in patients with intracranial tumors.

Production of interleukin-1 receptor antagonist by human glioblastoma cells in vitro and in vivo

We investigated the expression and production of the interleukin-1 receptor antagonist (IL-1ra) in three human glioblastoma cell lines (LN443, LN444, LN859). Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the expression of IL-1ra mRNA transcripts in the three cell lines. These three cell lines also expressed mRNA for IL-1 alpha, IL-1 beta, as well as IL-1 receptor type I and type II, suggesting the presence of an IL-1 autocrine loop in these cell lines. Northern blot analysis demonstrated that the IL-1ra mRNA expression increased with IL-1 beta or tumor necrosis factor (TNF)-alpha but not with GM-CSF stimulation in both LN443 and LN444 cell lines. PMA stimulation increased the mRNA expression in LN444 but not in LN443 cells. Immunocytochemical staining showed IL-1ra immunoreactivity in these three cell lines. ELISA on culture supernatants demonstrated that the IL-1ra was secreted from the cell lines in agreement with the mRNA expression. RT-PCR with isoform-specific primers showed that both intracellular and secreted forms of IL-1ra were expressed by the three cell lines, with a predominance of the intracellular form. In vivo study with RT-PCR and Northern blot analysis demonstrated IL-1ra mRNA in six out of 12 human glioblastoma and two out of five anaplastic astrocytoma tissues, although the expression level was not high in some cases. Immunohistochemistry demonstrated the presence of IL-1ra within the cytoplasm of tumor cells in six out of 10 glioblastomas in vivo. These results suggest a potential role of IL-1ra in regulation of the IL-1 autocrine loop in glioblastomas.

Glioblastoma Secrete Mcp-1, A Monocyte Chemotactic Factor

Immunohistological studies on glioblastoma tumor sections have demonstrated the presence of variable amounts of infiltrating macrophages. To understand how these macrophages are recruited from the circulation through the blood brain barrier, we have tested for the presence of macrophage chemotactic factors such as MCP-1. MCP-1 is a human monocyte chemoattractant protein produced by several cell types such as fibroblasts, PMA stimulated PBMC and human glioma cell-line U-105MG. We found various constitutive levels of different specific MCP-1 mRNAs in 10/13 cell lines. The inflammatory cytokines IL-lβ and TNFα could induce or increase the MCP-1 mRNA in all cell lines analyzed. To demonstrate in vivo MCP-1 production we showed that ex vivo extracted tumors contained MCP-1 mRNA, both in low and high grade astrocytomas. In situ hybridization is actually performed on glioblastoma tissue sections to determine the in vivo cellular origin of MCP-1.