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Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 ( Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29pis localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.Chicken pox (varicella) is a manifestation of lytic infection of cutaneous epithelial cells by varicella-zoster virus (VZV), a ubiquitous human alphaherpesvirus. Following lytic infection, VZV establishes latency in the sensory ganglia and can reactivate later during the host's life to cause shingles (zoster), a painful and potentially debilitating disease that may lead to postherpetic neuralgia (1). Recrudescence occurs when cellmediated immunity is decreased, as seen in the elderly and immunocompromised individuals, where it is associated with significant morbidity that may not be relieved by antiviral and analgesic therapy (63). An understanding of the cellular and viral factors that govern VZV latency and reactivation is critical to developing preventive methods and potential therapies for zoster and postherpetic neuralgia.During latency, viral DNA replication, late protein expression, and virion assembly do not occur. It has been reported that transcripts corresponding to the immediate-early and early open reading frames (ORFs) ORF4, -21, -29, -62, -63, and -66 are present in neurons harboring latent VZV (9-13, 15, 25, 36, 50). Proteins expressed by these ORFs are present in the cytoplasm of latently infected neurons (9,12,15,36,49,50). In particular, Lungu et al. demonstrated ...

In the present study we have analyzed the genetic regulation of increased expression of transformationassociated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (Ell-NMT'") and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFCIP). Parental Ell-NMT"e°cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFP cells exhibited phenotypes opposite to those of E11-NMT'e°cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFCaP and Ell-NMTn" cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which stil exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with Ell-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 ElA and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in AdS-transformed cells and that this suppression is not associated with changes in the expression of AdS transforming genes.Although recent advances have led to a better understanding of the potential role of defined genetic elements in the etiology of cancer, the molecular details by which a cell becomes transformed and ultimately evolves into a population of cells possessing tumorigenic and metastatic potential have not been delineated (reviewed in references 9, 24, and 27). Transforming genes (oncogenes) have been isolated and characterized from viral and mammalian tumor cell genomes which when transferred and expressed in appropriate recipient cells display a dominant-acting phenotype (reviewed in references 2 and 4). In addition to these dominant-acting transforming genetic elements in the genomes of certain tumor cells, several lines of experimental evidence indicate that specific genes exist in normal eucaryotic cells which may function as inhibitors of expression of the transformed phenotype and tumor formation, and these have been referred to as suppressor genes, repressor genes, antioncogenes, or emerogenes (1,3,18,23,28,36). Evidence supporting the existence of suppressor genes has come from (i) the a...

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Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with wild-type 5 adenovirus (H5wt) or a temperature-sensitive mutant of Ad5 (H5ts125) results in an MMS dose-dependent enhancement of viral transformation. With both viral isolates, MMS enhanced the transformation frequency when normalized for cell toxicity but did not induce a carcinogen dose-dependent increase in the absolute number of foci above solvent-treated controls. In contrast, pretreatment of CREF cells with MMS prior to infection with a host-range mutant of Ad5 (H5hr1) which contains a single basepair deletion in the E1a-transforming region of Ad5 and displays a cold-sensitive transformation phenotype, results in an MMS dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for cell toxicity. To explore the possible mechanism by which H5hr1 displays its unique carcinogen-enhancement of transformation (CET) phenotype we have examined the effect of MMS pretreatment on the frequency of transformation of CREF cells infected with mutants of Ad5 which were engineered to contain either a deletion (H5dl101) or an insertion (H5in106) mutation in the E1a gene region resulting in a cold-sensitive transformation phenotype similar to H5hr1. MMS-pretreated CREF cells infected with H5dl101 or H5in106 did not demonstrate a dose-dependent increase in the absolute number of transformed foci as was observed with carcinogen-pretreated H5hr1-infected CREF cells. These findings suggest that the unique CET phenotype displayed by H5hr1 may result from a second site mutation in a region of H5hr1 other than the E1a-transforming region and/or a novel interaction between gene products resulting from the specific mutation in E1a and other region(s) of the H5hr1 genome. Our investigations also indicate that the CREF/H5hr1 system should prove useful in analyzing chemical-viral interactions in cell transformation.

E1 region replacement adenoviruses are replication defective and are propagated in cells providing adenovirus E1A and E1B proteins. Although they are being developed for antitumor therapies, the proliferative behaviors of these viruses in normal brain tissues or in brain tumors are unknown. To address this, freshly cultured cells from normal human brain and common brain tumors (astrocytomas and meningiomas) were infected using wild-type species C adenoviruses and adenoviruses missing E1A (H5dl312) or E1A plus E1B (H5dl434). Viral DNA replication, late viral protein expression, and production of infectious progeny were characterized. Wild-type adenoviruses grew efficiently in normal brain and brain tumor cells. In comparison, E1-deleted adenovirus DNA replication was delayed and lower in cells derived from normal brain tissues, meningiomas, and low-grade astrocytomas. However, in contrast, E1-deleted adenovirus DNA replication did not occur or was extremely low in cells derived from malignancy grade III and IV astrocytic tumors. Because wild-type adenoviruses infected and replicated in all cells, the malignancy grade-based differential E1-deleted adenovirus DNA replication was not explained by differential virus uptake. Infectious H5dl312 and H5dl434 production correlated with viral DNA replication. Compared with a 5-day average for wild-type infections, advanced cytopathology was noted f4 weeks after H5dl312 or H5dl434 infection of meningioma, astrocytoma, and normal brain cells. Cytopathology was not observed after H5dl312 or H5dl434 infection of glioblastoma, anaplastic astrocytoma, and gliosarcoma cells. Because of this tumor grade-based differential growth, the E1-deleted adenoviruses may represent novel tools for studies of brain tumor malignancy. (Cancer Res 2005; 65(19): 8936-43)