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.

A high frequency of human cytomegalovirus (HCMV) genome and antigens in tumor samples of patients with different malignancies is now well documented, although the causative role for HCMV in the development of the neoplasias remains to be established. HCMV infection can modulate multiple cellular regulatory and signalling pathways in a manner similar to that of oncoproteins of small DNA tumor viruses such as human papilloma virus or adenoviruses. However, in contrast to these DNA tumor viruses, HCMV infection fails to transform susceptible normal human cells. There is now growing evidence that tumor cells with disrupted regulatory and signalling pathways enable HCMV to modulate their properties including stimulation of cell proliferation, survival, invasion, production of angiogenic factors, and immunogenic properties. In contrast to previously suggested "hit and run" transformation we suggest that persistence in tumor cells is essential for HCMV to fully express its oncomodulatory effects. These effects are observed particularly in persistent HCMV infection and are mediated mainly by activity of HCMV regulatory proteins. In persistently HCMV-infected tumor cell lines - a selection of novel, slowly growing virus variants with changes in coding sequences for virus regulatory proteins takes place. As a result, oncomodulatory effects of HCMV infection may lead to a shift to more malignant phenotype of tumor cells contributing to tumor progression.

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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-β-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, protooncogenes 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 of tumor cells.

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 r DOX 100 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. © 2002 Wiley-Liss, Inc. Key words: neuroblastoma; drug resistance; mdr-1; NCAM; GD2; karyotypeIntensive chemotherapy including vincristine (VCR) and doxorubicin (DOX) is often used for patients with advanced neuroblastoma (NB), 1,2 however drug resistance often develops. 3 Drug resistance in NB might be caused by multiple factors, including overexpression of the gene for the multidrug-resistance-associated protein (MRP) and mdr-1 gene-encoded P-170 glycoprotein (Pgp). 4 -6 High levels of MRP gene expression are frequently found in NB resistant to chemotherapeutic agents and are proven to correlate with poor outcome in patients with neuroblastoma, 7 whereas the contribution of mdr1 gene expression to the biology of childhood NB is still a matter of debate. 8 Recent studies have provided a link between the malignant and chemoresistant phenotypes, indicating that in addition to the development of drug resistance, chemotherapy of tumors may cause various changes to their biological characteristics, including increased malignancy and metastatic potential. 9,10 NB cell lines with drug-resistant phenotype are suitable models for the study of biological changes, accompanying the development of drug resistance. There are only a few reports concerning establishment and characterization of human drug-resistant NB cell sublines, particularly against VCR and DOX. 11,12 Therefore, in our present study, we established VCR-(designated UKF-NB-2 r VCR 20 ) and DOX-resistant (UKF-NB-2 r DOX 100 ) NB sublines and investigated the influence of a long-term treatment with VCR and DOX on cell growth and biological behavior of drug-resistant NB cells. Because changes in biological behavior are often associated with cytogenetic changes, we used spectral karyotyping (SKY) to characterize cytogenetic alterations in our established cell sublines. 13 We also hypothesized that in NB cells with acquired drug resistance, other mechanisms related to increased malignant behavior could develop. Hence we investigated the phenotype of established cells in comparison to parental line to identify possible factors nonrelated to drug resistance, which may additionally develop in tumor cells. MATERIAL AND M...

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.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis. It has been shown that promoter sequences of the TSP-1 gene can be transactivated by the wild-type tumor suppressor protein p53. As human cytomegalovirus (HCMV) infection inactivates wildtype p53 of various cell types , we investigated whether HCMV infection is associated with reduced TSP-1 production. We found , in conjunction with accumulated p53 , that TSP-1 mRNA and protein expression was significantly reduced in HCMV-infected cultured human fibroblasts. To determine whether the observed TSP-1 suppression depends on p53 inactivation , the p53-defective astrocytoma cell line U373MG was infected with HCMV. In these cells TSP-1 expression was also significantly reduced by HCMV infection whereas expression of the p53 mutant variant remained unaltered. In both cell lines the decreased expression of TSP-1 mRNA occurred early after infection (4 hours) , indicating that HCMV inhibits TSP-1 transcription during the immediate-early phase of infection before HCMV DNA replication. Inhibition of HCMV DNA synthesis by ganciclovir did not influence TSP-1 reduction whereas the antisense oligonucleotide ISIS 2922 , complementary to HCMV immediateearly mRNA , completely prevented the HCMV-mediated TSP-1 suppression. These findings strongly suggest a novel role for HCMV in the modulation of angiogenesis due to p53-independent down-regulation of TSP-1 expression. (Am J Pathol 1999, 155:285-292) Human cytomegalovirus (HCMV) has been implicated in the etiology of several human malignancies based on seroepidemiological studies and the presence of HCMV DNA, RNA, and/or antigens in tumor tissues. 1 The definitive establishment of a direct causative role for HCMV in the course of the malignancies has been elusive as HCMV components were not detected after long-term subculture of tumor tissues. 1 Moreover, the presence of genetic information in human tumors is difficult to interpret as HCMV can cause latent infection of numerous organs in a high percentage of normal individuals. 1 On the other hand, in vitro infection of cells with HCMV influences the expression of different cellular genes and/or function of cellular proteins that are associated with cell growth, differentiation, and apoptosis. These changes result in disturbance of normal tissue homeostasis, which indirectly may promote tumor growth. [2][3][4][5][6] In particular, in vitro studies have shown that HCMV gene products such as immediate-early (IE) proteins or morphological transforming region II oncoprotein bind wild-type p53 tumor suppressor protein and thereby down-regulate p53-activated transcription. [7][8][9][10][11]