Jens-Uwe Vogel

Modulatory Effects of Human Cytomegalovirus Infection on Malignant Properties of Cancer Cells

Although there is no definitive evidence of the association of human cytomegalovirus (HCMV) infection with human cancers, the oncogenic potential of HCMV has been well established by in vitro studies demonstrating the ability of UV-irradiated or infectious virus to transform a variety of cells. After prolonged passaging the transformed cell type was maintained while HCMV DNA sequences were no more detectable. Three morphological transforming regions (mtr) of HCMV have been identified. The effects of HCMV on cellular functions which may be associated with the malignant phenotype include the expression of oncogenes and transcriptional activation of growth factors and interleukin synthesis. In infected cells, HCMV induces cytoskeletal alterations and changes in expression of cell surface receptors for extracellular matrix proteins which could result in increased motility and dissemination of cancer cells. Several human neuroblastoma cell lines undergo maturation in different neural crest derived cell types upon treatment with oncogenic potential agents, i.e. retinoic acid. The persistent HCMV infection of neuroblastoma cells (> 1 year) is accompanied by the increased expression of oncoproteins (i.e. N-myc) and decreased expression of tyrosine hydroxylase and dopamine-beta-hydroxylase. The activation of the cellular metabolism is due to HCMV binding to cellular receptors (prior to virus gene expression) and to the activity of HCMV immediate early (IE) gene products. IE proteins act directly as transcriptional activators or their activity is mediated by a variety of cellular transcription factors. HCMV infection may result in activation of promoters of cellular genes coding for cytokines, replication enzymes, proto-oncogenes and viral promoters. Recently it has been demonstrated that HCMV IE proteins block apoptosis probably by suppressing the ability of the antioncogene p53 to activate a reporter gene. The interactions of HCMV with tumor suppressor proteins such as p53 or retinoblastoma (pRb) susceptibility protein are reminiscent of those mediated by the oncoproteins of DNA tumor viruses. The acquisition of a fully malignant phenotype by normal cells is thought to require several mutations in a number of cellular genes. In this connection, HCMV may play the role of a nonobligate either direct or indirect cofactor for tumor genesis, e.g. by blocking apoptosis, which may be an essential requirement for tumor progression. Due to the stimulation of growth factors and/or inhibition of antioncogenes by its gene products, HCMV may modulate the malignant potential for tumor cells.

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis.

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro α and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.

Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model.

Cytotoxic drug treatment of neuroblastoma often leads to the development of drug resistance and may be associated with increased malignancy. To study the effects of long‐term cytotoxic treatment on malignant properties of tumor cells, we established 2 neuroblastoma cell sublines resistant to vincristine (VCR) and doxorubicin (DOX). Both established cell lines (UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100) were highly resistant to VCR, DOX and vice‐versa but retained their sensitivity to cisplatin. UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 expressed significant amounts of P‐glycoprotein, while parental cells were P‐glycoprotein negative. GD2 expression was upregulated, whereas NCAM expression was decreased in both resistant cells. Spectral karyotype (SKY) analysis revealed complex aberrant karyotypes in all cell lines and additional acquired karyotype changes in both resistant cells. All cell lines harbored high levels of N‐myc amplification. Compared to parental cells, UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 exhibited more than 2‐fold increase in clonal growth in vitro, accelerated adhesion and transendothelial penetration and higher tumorigenicity in vivo. We conclude that development of drug resistance and acquisition of certain karyotypic alterations is associated with an increase of additional malignant properties that may contribute to the poor prognosis in advanced forms of NB. The 2 novel neuroblastoma cell sublines also provide useful models for the study of drug resistance in aggressive forms of neuroblastoma.

Human Cytomegalovirus Circumvents NF-κB Dependence in Retinal Pigment Epithelial Cells

The human CMV (HCMV) is a persistent virus that may cause severe inflammatory responses especially in immunocompromised hosts. In different cell types, HCMV infection leads to the activation of the pleiotropic transcription factor, NF-κB, which triggers virus replication but also propagates cell-mediated inflammatory mechanisms that largely depend on PG synthesis. We investigated the interactions of HCMV and the NF-κB-dependent PG synthesis pathway in cultures of retinal pigment epithelial (RPE) cells that are known to be infected in HCMV retinitis patients. Unlike in other cell types, HCMV increased neither NF-κB activity nor p65 and p105/50 mRNA levels in RPE cells. Both TNF-α and phorbol ester 12,0-tetradecanoylphorbol 13-acetate (TPA) enhanced NF-κB activity but only TPA increased HCMV replication. Cyclooxygenase-2 expression and PGE2 release was increased by TPA and TNF-α but not by HCMV infection. Stimulatory activity of TPA on HCMV replication was suppressed by protein kinase C inhibitors and inhibitors of p42/44 and p38 mitogen-activated protein kinases but not by NF-κB inhibitors. In conclusion, HCMV circumvents the NF-κB route in favor of the protein kinase C-dependent mitogen-activated protein kinase pathway in RPE cells. This virus/host cell interaction might be a mechanism that promotes HCMV persistence in immune-privileged organs such as the eye.

Infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus.

To identify a model for the study of intestinal pathogenesis of severe acute respiratory syndrome (SARS) we tested the sensitivity of six human intestinal epithelial cell lines to infection with SARS coronavirus (SARS-CoV). In permissive cell lines, effects of SARS-CoV on cellular gene expression were analysed using high-density oligonucleotide arrays. Caco-2 and CL-14 cell lines were found to be highly permissive to SARS-CoV, due to the presence of angiotensin-converting enzyme 2 as a functional receptor. In both cell lines, SARS-CoV infection deregulated expression of cellular genes which may be important for the intestinal pathogenesis of SARS.

Antiviral and Immunomodulatory Properties of New Pro-Glutathione (GSH) Molecules

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Cytomegalovirus Infection Decreases Expression of Thrombospondin-1 and -2 in Cultured Human Retinal Glial Cells: Effects of Antiviral Agents

In fibroblasts, infection with human cytomegalovirus (HCMV) inhibits expression of the extracellular matrix proteins thrombospondin-1 and -2 (TSP-1 and TSP-2). These effects may depend on expression of HCMV immediate-early (IE) genes, which are activated by cellular transcription factor NF-kappaB. The influence of HCMV infection on TSP-1 and TSP-2 expression and the ability of different antiviral drugs to prevent these cellular changes in permissive cultures of human retinal glial cells were observed. Ganciclovir inhibited only HCMV late antigen (LA) expression, whereas antisense oligonucleotide ISIS 2922 and peptide SN50, inhibitors of HCMV IE expression and NF-kappaB activity, respectively, inhibited both IE and LA expression. ISIS 2922 and SN50, but not ganciclovir, prevented down-modulation of TSP-1 and TSP-2. The results showed that HCMV-induced down-modulation of TSP-1 and TSP-2 in retinal glial cells is prevented by inhibition of HCMV IE expression. These findings may be relevant to pathogenesis and treatment of HCMV retinitis.

Cytomegalovirus infection blocks apoptosis in cancer cells.

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.

Proinflammatory Potential of Cytomegalovirus Infection

We observed the effects of antiviral therapy on CMV and/or oxidative-stress-induced stimulation of proinflammatory molecules including interleukin-8 (IL-8), melanoma growth stimulatory activity-α (GRO-α) and intercellular adhesion molecule 1 (ICAM-1) using human foreskin fibroblasts. Ganciclovir, foscarnet or cidofovir completely suppressed virus replication, as demonstrated by CMV late (L) antigen production. These drugs did not influence CMV immediate-early (IE) antigen expression and had no effects on CMV-induced cellular changes in IL-8, GRO-α and ICAM-1 levels. Phosphorothioate oligonucleotide (ISIS 2922) suppressed both CMV IE and L antigen by 99%. ISIS 2922 completely suppressed CMV-induced upregulation of both chemokines and ICAM-1. Induction of oxidative stress by H2O2 upregulated IL-8 expression. Oxidative stress and CMV infection showed synergistic effects on IL-8 expression. ISIS 2922 only partially inhibited the upregulation of IL-8 in infected cells treated with H2O2, whereas cotreatment with ISIS 2922 and antioxidants inhibited the upregulation almost completely. The results showed that inhibition of CMV IE expression alone or in combination with antioxidants is promising for the treatment of CMV disease.

Typing and strain differentiation of clinical herpes simplex virus type 1 and 2 isolates by polymerase chain reaction and subsequent restriction fragment length polymorphism analysis.

In the present study, different combinations of primers were investigated for PCR amplification and typing of clinical herpes simplex virus type 1 (HSV-1) (n = 22) and herpes simplex virus type 2 (HSV-2) (n = 12) isolates. Intratypic strain differentiation was performed by restriction fragment length polymorphism (RFLP) analysis of different PCR amplified HSV genome regions. Enzymatic amplification of HSV DNA from all the clinical isolates could be achieved using primer combinations DNAP5/DNAp3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2). With the primer pair corresponding to the thymidine kinase (TK) genome region, amplification of all the HSV-1 isolates was only possible by nested PCR. With primers HSV UP/DOWN, 10 of 12 HSV-2 isolates could be detected. HSV typing by type-specific primers DNAP5/DNAP3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2) or restriction enzyme analysis (Ava II) of amplified DNA with HSV-specific UP/DOWN primers showed results which concorded with serotyping by monoclonal antibodies. RFLP analysis of the PCR products using selected restriction enzymes showed sufficient diversity of profiles among strains to differentiate all the HSV-1 isolates and to distinguish four groups of HSV-2 isolates. Amplification of HSV DNA from clinical isolates with subsequent typing and strain differentiation represents a valuable alternative to conventional methods (virus isolation, serotyping and restriction fragment analysis of the entire genomic DNA) and may be suitable for the study of HSV transmission and pathogenesis, especially in labour-intensive clinical samples with low levels of virus replication (i.e. cerebrospinal fluid, vitreous fluid and corneal transplants).