Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum–Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57–66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in β2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8+ T lymphocytes in vivo.

Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UVirradiation, or TNFa. The effects were specific for Livinexpressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology.

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A substantial proportion of the worldwide liver cancer incidence is associated with chronic hepatitis B virus (HBV) infection. The therapeutic management of HBV infections is still problematic and novel antiviral strategies are urgently required. Using the peptide aptamer screening system, we aimed to isolate new molecules, which can block viral replication by interfering with capsid formation. Eight peptide aptamers were isolated from a randomized expression library, which speci®cally bound to the HBV core protein under intracellular conditions. One of them, named C1-1, eciently inhibited viral capsid formation and, consequently, HBV replication and virion production. Hence, C1-1 is a novel model compound for inhibiting HBV replication by blocking capsid formation and provides a new basis for the development of therapeutic molecules with speci®c antiviral potential against HBV infections. Oncogene (2001) 20, 6579 ± 6586.

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In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.

The enhancer of zeste homolog 2 (EZH2) gene has been recently linked to human malignancies where it may serve as a new target for cancer therapy. Here, we analyzed EZH2 expression in primary renal cell carcinoma (RCC) specimens and in nontumorous tissue samples from adult kidney. EZH2 transcripts were detectable in all RCC specimens examined. Expression levels were significantly higher in tumor tissue (p 0.0001) than in samples from normal adult kidney. Moreover, inhibition of endogenous EZH2 expression in RCC cell lines by RNA interference (RNAi) led to reduced proliferation and increased apoptosis in RCC cells. These data show that EZH2 is overexpressed in RCC. Furthermore, they indicate that the EZH2 gene plays a role for both the proliferation and the apoptosis resistance of RCC cells. Targeted inhibition of EZH2 could therefore represent a novel strategy to improve the therapeutic response of RCC. ' 2008 Wiley-Liss, Inc.Key words: renal cell carcinoma; enhancer of zeste homolog 2 (EZH2); RNA interference; tumor therapy Renal cell carcinoma (RCC) is estimated to account for more than 51,000 new cases and almost 13,000 cancer-related deaths in the United States in 2007, making it the second most lethal of the urological cancers. 1 RCCs typically are highly resistant toward chemotherapy with a concomitant poor prognosis in advanced stages. 2 Therefore, the identification of novel therapeutic targets and the development of new strategies for RCC treatment are urgently required.The enhancer of zeste homolog 2 (EZH2) gene encodes a polycomb group (PcG) protein, which acts as a histone methyltransferase 3-5 and also can directly control DNA methylation. 6 EZH2 is involved in several key regulatory mechanisms within eukaryotic cells, such as control of embryonal development or cell proliferation. 7,8 Moreover, there is accumulating evidence indicating that EZH2 may also play a pivotal role in the etiology of several tumor forms, which include prostate cancer 9,10 and breast cancer. 10,11 For both of these cancers, EZH2 expression is often observed in proliferative and more aggressive tumor subgroups and has diagnostic and/or prognostic value. [9][10][11] Notably, however, EZH2 appears to be not only a potential tumor marker but may itself contribute to the deregulation of cell growth as a bona fide oncogene. Overexpression of EZH2 conferred cellular growth advantage in vitro, 11-13 promoted invasion 11 and exhibited oncogenic properties in nude mice. 14 Vice versa, inhibition of EZH2 expression by antisense constructs or RNA interference (RNAi) did result in growth inhibition of some cancer cells. 9,15 Furthermore, RNAi-mediated inhibition of EZH2 expression induced anoikis in circulating prostate carcinoma precursor cells 16 and apoptotic cell death in breast cancer cells. 17 A possible role of EZH2 for RCC has not been studied so far. Here, we analyzed EZH2 expression in primary RCC specimens and in nontumorous tissue. In addition, we investigated the functional role of EZH2 for the proliferation control and apop...

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.