Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdDD mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdDD with gemcitabine, irinotecan or cisplatin resulted in two-to fourfold decreases in EC 50 (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the dl1520 (ONYX-015) and dl922-947 mutants. AdDD replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdDD. Suboptimal doses of AdDD and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdDD has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer.

No abstract

Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of ␣3␤1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin ␣3␤1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-␣3 and anti-␤1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with ␣3␤1, which involved multiple additional contact sites.Adenovirus (Ad) host cell entry requires an initial attachment to cells which is mediated by the fiber interaction with the coxsackievirus-Ad receptor (CAR) (2). The subsequent association of the capsid protein penton base with integrin molecules promotes Ad entry (31). Integrins are a family of structurally and functionally related cell surface heterodimeric receptors that mediate cell migration and adhesion. The major extracellular ligands for integrins are collagens, laminins, fibronectin, tenascin, vitronectin, von Willebrand factor, and fibrinogen, reflecting the primary function of integrins in cell adhesion to the extracellular matrix. The ␣v␤1, -3, -5, -6, and -8 integrins, the ␣5␤1 and ␣8␤1 integrins, and the ␣IIb␤3 integrins form a subgroup that primarily recognizes ligands containing Arg-Gly-Asp (RGD) motifs (see reference 13 and references therein). Many microorganisms utilize integrins to gain entry into cells: the SA11 rotavirus binds to ␣2␤1 and ␣4␤1 (9), ␣v␤3 and ␣v␤1 integrins are receptors of the human parechovirus 1 (30), and ␣v␤5 has been proposed, although not conclusively, as a coreceptor in adeno-associated virus type 2 infection (27, 29). The foot-and-mouth disease virus uses different integrins for cell infection (14,15,16). Integrin ␣3␤1 is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (1). Yersinia pseudotuberculosis binds to members of the ␤1 integrin family in order to enter eukaryotic cells (22).Several Ad serotypes contain an RGD motif in the penton base protein. This feature, and the Ad cell-detaching property, suggested an interaction of the virus with the integrin receptors. Indeed, ␣v␤3 and ␣v␤5 are receptors for human Ad2 and Ad5, and direct binding to isolated ␣v␤5 was shown for human Ad2, Ad3, Ad4, Ad5, and Ad37 (24, 31). In hematopoietic and melanoma cells, respectively, the ␣M␤2 and b1 integrins were found to be implicated in human Ad5 infection (3, 12). More recent evidence indicates ␣v␤1 as an Ad2 and Ad5 coreceptor in the human embryonic kidney (HEK293) cell line (23). Ad interaction with the ␣v␤1, -3, and -5 integrin subtypes is efficiently competed by RGD-containing peptides (23, 31). A second integrin binding motif is...

Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered lambda by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286-393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286-393) includes the principal receptor-binding motif ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda Pb (286-393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.

Given the biological complexity of viral infections, the variability of the host response, and the safety concerns related to viral-mediated gene transfer, recent studies have made use of DNA mircoarrays to integrate multi-layered experimental approaches aimed at completely clarifying virus-host interactions. Particular attention has been given to those viruses that are implicated in clinical use and/or in life-threatening diseases. Examples of such use can be divided into three main categories, including: (i) the use of microarrays to study viral expression; (ii) the use of microarrays to analyze the host response to viral infection; and (iii) the use of microarrays to characterize the host response to viral vector-mediated transduction. Significant information on virus- and viral vector-host interactions can be obtained with the microarray approach, including the recognition of master pathways of virally-induced responses, the identification of new target genes for specific viruses, and indications on the molecular toxicity of specific gene transfer vectors currently used for gene therapy trials (in particular, adeno-associated viruses and adenovirus-derived vectors). We predict that the development of accessible repositories containing most of the DNA microarray data on viral infections will certainly help to elucidate the puzzling pictures of different viral infections. This will be crucially important for the correct handling of viral diseases and the intelligent amelioration of viral vectors for gene therapy.

The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs.