Christine Hohenadl

Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma

Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposis sarcoma‐associated herpes virus (KSHV) or human herpes virus‐8 (HHV‐8) and the development of Kaposis sarcoma (KS). To clarify the role of HHV‐8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7‐kb HHV‐8‐encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS‐KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV‐8‐infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL‐1 established from a body cavity‐based lymphoma (BCBL) and latently infected with HHV‐8. In all KS biopsies examined, independent of their epidemiological type, the late‐stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV‐8 infection of these cells. The presence of T0.7‐expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV‐8 infection in KS pathogenesis. Int. J. Cancer 72:68–71, 1997.

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein‐1 (GBP‐1) as the key and specific mediator of the anti‐proliferative effect of IC on endothelial cells. GBP‐1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposis sarcoma. Experimental modulation of GBP‐1 expression demonstrated that GBP‐1 mediates selectively the anti‐proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP‐1 anti‐proliferative activity did not affect ERK‐1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C‐terminal helical domain of the protein. These results define GBP‐1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC‐activated non‐proliferating phenotype of endothelial cells in vascular diseases.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

Angiostatic immune reaction in colorectal carcinoma: Impact on survival and perspectives for antiangiogenic therapy

Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein‐1 (GBP‐1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP‐1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP‐1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as determined by immunohistochemical investigation of 388 sporadic CRC. Cancer‐related 5‐year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP‐1‐positive CRC. Multivariate analysis showed that GBP‐1 is an independent prognostic factor indicating a reduction of the relative risk of cancer‐related death by the half (p = 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP‐1‐positive (n = 12) and ‐negative (n = 12) tumors showed that particularly IFN‐γ‐induced genes including the major antiangiogenic chemokines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP‐1. Altogether our findings indicated that GBP‐1 may be a novel biomarker and an active component of a Th‐1‐like angiostatic immune reaction in CRC. This reaction may affect patients response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induction of a Th‐1‐like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC.

Guanylate binding protein-1 inhibits spreading and migration of endothelial cells through induction of integrin α4 expression

Human guanylate binding protein‐1 (GBP‐1) is a large GTPase that is induced by inflammatory cytokines and acts antiangiogenically through the inhibition of endothelial cell proliferation and migration. In this study, we detected that GBP‐1‐expressing cells show a significantly reduced spreading and migration on fibronectin matrices. Investigating possible mechanisms of these effects, we found that integrin α4 (ITGA4) was consistently up‐regulated at both the RNA and protein level in GBP‐1‐expressing cell cultures. Inhibition of cell spreading and migration by GBP‐1 was dependent on the binding of ITGA4 to fibronectin. The inflammatory cytokines IL‐1β and TNF‐α induced ITGA4 expression in HUVECs and inhibited spreading and migration. Knockdown of GBP‐1 by shRNA abrogated inflammatory cytokine induced ITGA4 expression and restored spreading and migration capabilities of the cells. These results show that inhibition of endothelial cell spreading and migration by inflammatory cytokines is mediated by GBP‐1 through induction of ITGA4 expression. Endothelial cell migration is a key process during angiogenesis. Therefore, ITGA4 may be a novel molecular target to modulate angiogenesis in human disease.— Weinländer, K., Naschberger, E., Lehmann, M. H., Tripal, P., Paster, W., Stockinger, H., Hohenadl, C., Stürzl, M. Guanylate binding protein‐1 inhibits spreading and migration of endothelial cells through induction of integrin α4 expression. FASEB J. 22, 4168–4178 (2008)

GBP-1 acts as a tumor suppressor in colorectal cancer cells.

The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.

Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.

Interferon γ (IFN-γ) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-γ are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-γ-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-γ-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-γ-induced antitumoral defense system.

Expression of human herpesvirus-8 (HHV-8) encoded pathogenic genes in Kaposi's Sarcoma (KS) primary lesions

Transcription of six different HHV-8 specific mRNAs was examined in early- and late-stage KS primary lesions. Expression of the latency-associated T0.7 mRNA and of VP23 mRNA which is a specific marker of lytic/productive infection suggested that HHV-8 is secondarily recruited into the KS lesions by productively infected monocytes, macrophages. From these cells HHV-8 is transmitted to the KS spindle cells, which are latently infected. v-BCL-2, v-MCP-1 and v-IL-6 were not expressed in latently infected KS spindle cells, therefore the impact of these factors in KS pathogenesis appears to be low. By contrast, v-Cyclin D was highly expressed in almost all latently infected spindle cells and may therefore be an important factor triggering progression of late-stage KS lesions.

Magnetic field-controlled gene expression in encapsulated cells

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches.

Specific packaging of spliced retroviral vector transcripts lacking the Ψ-region

The effect of a cryptic splice acceptor (cSA) site located at the end of the extended packaging signal in murine leukemia virus (MLV)-based vectors was investigated. Although this cSA is also present in wild type MLV, it was found to result in a smaller transcript in which the packaging signal (Psi) had been removed by splicing only in MLV-derived vectors. Splicing occurs both in packaging cells producing the MLV-vectors as well as in the infected target cells. Transcripts lacking the Psi-sequence (Psi(-)) are packaged relatively efficiently into virus particles, even in the presence of wild type Psi(+)-vector transcripts. The Psi(-)-viral RNA is reverse transcribed in vector transduced cells as is any other retroviral genome. The titer obtained from the Psi(-)-vector was only 1000-fold reduced in comparison to the same Psi(+)-vector. These results suggest that Psi(-)-transcripts may be packaged more frequently than previously supposed and that splicing patterns should be carefully analysed on an individual basis for retroviral vectors used in gene therapy.