Enhanced oncolytic adenoviral production by downregulation of death-domain associated protein and overexpression of precursor terminal protein.

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.

Atorvastatin inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to inhibit vascular smooth muscle cell growth, a key event in the pathogenesis of proliferative vascular diseases. The mechanism by which HMG-CoA reductase inhibitors exert their antiproliferative activity is not fully understood, especially their effect on DNA replication. We therefore investigated the effects of atorvastatin on minichromosome maintenance (MCM) protein 6 and 7 expression in vascular smooth muscle cells, two proteins essential for initiation of DNA replication. Stimulation of quiescent rat aortic vascular smooth muscle cells with fetal bovine serum induced MCM6 and MCM7 protein and mRNA expression, which was potently attenuated by atorvastatin in a dose-dependent fashion. Mevalonate completely abrogated the inhibitory effect on serum-induced MCM6 and MCM7 expression, demonstrating that biosynthesis of isoprenoids was likely the specific pathway blocked by atorvastatin. Transient transfection experiments revealed that atorvastatin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. The MCM6 and MCM7 promoters contain several E2F sites critical for their transcriptional activation. Activity of a luciferase reporter plasmid containing four E2F elements was also strongly inhibited by atorvastatin. The inhibitory effect of atorvastatin on MCM6 and MCM7 was reversed by adenoviral-mediated overexpression of E2F, indicating that their downregulation by atorvastatin involves an E2F-dependent mechanism. These findings demonstrate that MCM proteins play an essential role during the proliferation of vascular smooth muscle cells and may provide a novel therapeutic target for proliferative vascular diseases. Inhibition of MCM6 and MCM7 expression by blocking E2F function may contribute importantly to the inhibition of vascular smooth muscle cell DNA synthesis by atorvastatin.

Targeting the cell cycle for cancer therapy.

Most if not all neoplasias show a directly or indirectly deregulated cell cycle. Targeting its regulatory molecules, the cyclin-dependent kinases, as a therapeutic mode to develop new anticancer drugs, is being currently explored in both academia and pharmaceutical companies. The development of new compounds is being focused on the many features of the cell cycle with promising preclinical data in most fields. Moreover, a few compounds have entered clinical trials with excellent results maintaining the high hopes. Thus, although too early to provide a cell cycle target based new commercial drug, there is no doubt that it will be an excellent source of new anticancer compounds.

Early region 4 modulates adenovirus DNA replication by two genetically separable mechanisms.

Three viral proteins, all products of early region 2 (E2), participate directly in adenovirus DNA replication. Three products of early region 4 (E4) also affect viral DNA synthesis: the product of E4 ORF4 inhibits viral DNA accumulation, while the products of E4 ORFs 3 and 6 antagonize that effect of ORF4 expression. Because no E4 products are required for DNA synthesis, these proteins probably act indirectly. The E4 ORF3, 4, and 6 proteins all participate in aspects of the regulation of viral gene expression. To determine whether they modulate DNA replication by effects on expression of viral replication proteins, we examined E2 expression in E4 mutant-infected cells. In cells infected by ORF3-, 6- mutants, expression of ORF4 substantially depressed the steady-state levels of replication proteins and E2 mRNAs, reduced E2 transcription rates, and profoundly inhibited viral DNA replication. Thus, in the absence of E4 ORFs 3 and 6, ORF4 acts as a transcriptional regulator of E2 expression, and reduced replication protein levels largely account for the inhibition of DNA replication by ORF4. Cells infected by viruses that express ORFs 3 and 6 in addition to ORF4 accumulated much larger quantities of viral DNA than did cells infected by the ORF3-, 6-, 4+ mutant. Increased DNA accumulation was not accompanied by a comparable increase in E2 expression. Therefore, the ORF3 and 6 products counteract the ORF4-induced reduction of DNA replication by a mechanism other than reversing the inhibitory effect of ORF4 on E2 expression. The effect of ORF4 on E2 expression is consistent with its ability to regulate levels of the transcription factor AP-1 (Müller et al., 1992, J. Virol. 66, 5867-5878); the mechanism by which ORFs 3 and 6 enhance replication is unknown.

E2A gene products are not required for insulin gene expression.

Transcripts for E2A gene products, ubiquitous basic helix-loop-helix transactivating proteins, are expressed at high levels in the pancreatic epithelium. E2A proteins have been shown to bind the cognate E box sequence (CANNTG) of the insulin promoter/enhancer. E2A gene products dimerize with cell-specific basic helix-loop-helix proteins and synergize with the homeodomain transcription factor, PDX-1, in insulin gene transactivation. PDX-1 is also required for normal pancreatic development in mice. We investigated whether pancreatic development and insulin production could occur in the absence of E2A gene products by studying mice with a null mutation for the gene. E2A(-/-) mice demonstrated normal formation of pancreatic endocrine and exocrine tissue in histochemical sections as well as positive and distinct immunostaining for insulin and glucagon in islet tissue, signifying development of mature beta- and alpha-cells. Moreover, E2A(-/-) mice displayed no significant difference in blood glucose levels or pancreatic insulin content compared with wild-type littermates. These data show that although E2A gene products probably play an important role in insulin gene expression, pancreatic development and insulin production can proceed in their absence.

DNA-binding by oncoprotein E2a-Pbx1 is important for blocking differentiation but dispensable for fibroblast transformation.

The t(1;19) chromosomal translocation of pediatric pre-B cell lymphoblastic leukemia produces the E2A-PBX1 oncogene, which can transform fibroblasts, induce acute myeloid leukemia and T cell lymphomas in mice, and immortalize factor-dependent myeloid progenitors in cultured marrow. The homeodomain of Pbx1 binds ATCAATCAA, and while Pbx1 does not activate transcription through this motif, E2A-Pbx1 induces constitutive transactivation. Here, we investigate whether DNA-binding by Pbx1 or transcriptional activation by E2A are essential for the transforming abilities of E2A-Pbx1. Elimination of DNA-binding in E2A-Pbx1 by point mutations in the Pbx1 homeodomain or by large deletions that removed the Pbx1 homeodomain and carboxyl terminus did not alter ability of E2A-Pbx1 to induce focus-formation in fibroblast, even though these mutations completely eliminated its ability to activate transcription through the PRS. These same DNA-binding mutations, however, severely impaired or eliminated the ability of E2A-Pbx1 to immortalize factor-dependent myeloid progenitors in marrow cultures. Elimination of the first transcriptional activation domain of E2A abolished both fibroblast and myeloid transforming activities while elimination of the second altered neither of these activities. We conclude that DNA-binding is important for the ability of E2A-Pbx1 to disrupt differentiation, as evidenced in myeloblast immortalization, but dispensable for its ability to induce focus-formation, and that the aminoterminal domain of E2A, which strongly activates transcription, is essential for both transforming activities.

Control of HPV 18 DNA replication by cellular and viral transcription factors.

Papillomavirus replication in vivo requires the interaction of the virally encoded proteins E1 and E2 with the origin of replication which is localised in the regulatory region (long control region or LCR) of the viral genome. In genital human papillomaviruses (HPVs), the origin overlaps promoter elements of early transcription. In this study, we analysed the replication of HPV18 DNA using the complete LCR containing mutations in transcription regulatory elements. We found that each of the three E2 binding sites proximal to the AT-rich sequence of the origin contributes to the replication rate of DNA, although not identically. In addition, two sequences important for early transcription, an Sp1 binding site and the TATA box, were also found to play a role in replication. In contrast, two AP1 binding sites required for the enhancer-mediated activation of early transcription did not affect the replication, while other upstream sequences in the LCR did contribute to the replication efficiency. Our results indicate that besides a core origin of replication containing an AT-rich sequence and three E2 binding sites, auxiliary elements affect HPV18 DNA replication in the context of the full length LCR, some of which are important for transcription.

Cyclic AMP signaling is required for function of the N-terminal and CR1 domains of adenovirus E1A in Saccharomyces cerevisiae.

We have constructed yeast vectors in which derivatives of the adenovirus E1A gene are expressed from the GAL1 promoter. Cells expressing E1A289 grow poorly and accumulate cells with a 1C DNA content. Using a series of E1A deletion mutants, we have identified three regions within the E1A protein that are necessary for the G1 growth phenotype; each deletion partially relieves the growth defect. These deletions span residues 4-25, 38-60 and 140-186, which fall within the N-terminal, CR1 and CR3 domains of E1A respectively. Expression of the first 82 residues of E1A, spanning just the N-terminal and CR1 domains, strongly inhibits yeast cell growth in G1 showing that these domains can function independently of other domains of E1A. Using this strong growth inhibition, we isolated a yeast mutant in the net1 gene that conferred resistance to the expression of E1A1-82. The mutant was insensitive to expression of both E1A1-82 and full length E1A, but remained sensitive to the toxicity caused by over-expression of a Gal4p-VP16 fusion. Finally, we found that the function of E1A in yeast depends on the cyclic AMP signaling pathway, providing a striking parallel with the action of E1A at the c-fos promoter in mammalian cells. These results suggest that a genetic analysis of the yeast model system will provide relevant new insights into mechanisms of gene regulation by E1A proteins.

Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.