Daniel Öberg

Specific Inactivation of Inhibitory Sequences in the 5′ End of the Human Papillomavirus Type 16 L1 Open Reading Frame Results in Production of High Levels of L1 Protein in Human Epithelial Cells

ABSTRACT The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5′ end of the L1 gene contains strong inhibitory sequences but that the 3′ end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5′ end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein.

ABSTRACT We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3′ UTR) in HPV-16 gene expression. We found that deletion of the early 3′ UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3′ half of the early 3′ UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3′ splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

A Downstream Polyadenylation Element in Human Papillomavirus Type 16 L2 Encodes Multiple GGG Motifs and Interacts with hnRNP H

ABSTRACT Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.

Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

Purpose: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results: The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.

Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells.

ABSTRACT Here we show that the 5′ end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5′ end of the coding region. Inhibitory regions I and II acted in cis to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5′ end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.

Improved potency and selectivity of an oncolytic E1ACR2 and E1B19K deleted adenoviral mutant in prostate and pancreatic cancers.

Purpose: Replication-selective oncolytic adenoviruses are a promising class of tumor-targeting agents with proven safety in hundreds of patients. However, clinical responses have been limited and viral mutants with higher potency are needed. Here, we report on the generation of a novel set of mutants with improved efficacy in prostate and pancreatic carcinoma models. Currently, no curative treatments are available for late-stage metastatic prostate or rapidly progressing pancreatic cancers. Experimental Design: Adenovirus type 5 mutants were created with deletions in the E1ACR2 region for tumor selectivity and/or the E1B19K gene for attenuated replication in vivo; all constructs retain the E3 genes intact. Cell-killing efficacy, replication, and cytotoxicity in combination with chemotherapeutics were investigated in normal cells (PrEC and NHBE), seven carcinoma cell lines, and human (PC3 and DU145) and murine (TRAMPC, CMT-64, and CMT-93) tumor models in vivo. Results: The double-deleted AdΔΔ (ΔE1ACR2 and ΔE1B19K) mutant had high cell-killing activity in prostate, pancreatic, and lung carcinomas. Replication was similar to wild-type in all tumor cells and was attenuated in normal cells to levels less than the single-deleted AdΔCR2 mutant. AdΔΔ combined with the chemotherapeutics docetaxel and mitoxantrone resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in prostate xenografts in vivo. In murine immunocompetent in vivo models efficacy was greater for mutants with the E3B genes intact even in the absence of viral replication, indicating attenuated macrophage-dependent clearance. Conclusions: These data suggest that the novel oncolytic mutant AdΔΔ is a promising candidate for targeting of solid tumors specifically in combination with chemotherapeutics. Clin Cancer Res; 16(2); 541–53

Low-dose paclitaxel synergizes with oncolytic adenoviruses via mitotic slippage and apoptosis in ovarian cancer

The microtubule-stabilizing drug paclitaxel has activity in relapsed ovarian cancer. dl922-947, an oncolytic adenovirus with a 24-bp deletion in E1A CR2, replicates selectively within and lyses cells with a dysregulated Rb pathway and has efficacy in ovarian cancer. In the aggressive A2780CP xenograft, combination treatment with weekly dl922-947 and paclitaxel has significantly greater efficacy than either treatment alone and can produce complete tumor eradication in some animals. We investigated the mechanisms of paclitaxels synergy with dl922-947 in ovarian cancer. The host-cell microtubule network is grossly rearranged and stabilized following adenovirus infection, but paclitaxel does not increase this significantly. Paclitaxel does not synergize by increasing infectivity, viral protein expression or virus release. However, destabilizing the microtubule network with nocodazole reduces viral exit, revealing a novel microtubule-dependent pathway for non-lytic adenoviral exit. dl922-947 can override multiple cell cycle checkpoints but induces cell death by a non-apoptotic mechanism. In combination, dl922-947 and low-dose paclitaxel induces aberrant, multipolar mitoses, mitotic slippage and multinucleation, triggering an apoptotic cell death.

Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.

Tissue-derived mesenchymal stromal cells used as vehicles for anti-tumor therapy exert different in vivo effects on migration capacity and tumor growth

BackgroundMesenchymal stem cells (MSCs) have been promoted as an attractive option to use as cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines. Multiple isolated populations have been described as MSCs, but despite extensive in vitro characterization, little is known about their in vivo behavior.The aim of this study was to investigate the efficacy and efficiency of different MSC lineages derived from five different sources (bone marrow, adipose tissue, epithelial endometrium, stroma endometrium, and amniotic membrane), in order to assess their adequacy for cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurate targeted therapeutic applications.MethodsWe first measured the in vivo migration capacity and effect on tumor growth of the different MSCs using two imaging techniques: (i) single-photon emission computed tomography combined with computed tomography (SPECT-CT), using the human sodium iodine symporter gene (hNIS) and (ii) magnetic resonance imaging using superparamagnetic iron oxide. We then sought correlations between these parameters and expression of pluripotency-related or migration-related genes.ResultsOur results show that migration of human bone marrow-derived MSCs was significantly reduced and slower than that obtained with the other MSCs assayed and also with human induced pluripotent stem cells (hiPSCs). The qPCR data clearly show that MSCs and hiPSCs exert a very different pluripotency pattern, which correlates with the differences observed in their engraftment capacity and with their effects on tumor growth.ConclusionThis study reveals differences in MSC recruitment/migration toward the tumor site and the corresponding effects on tumor growth. Three observations stand out: 1) tracking of the stem cell is essential to check the safety and efficacy of cell therapies; 2) the MSC lineage to be used in the cell therapy needs to be carefully chosen to balance efficacy and safety for a particular tumor type; and 3) different pluripotency and mobility patterns can be linked to the engraftment capacity of the MSCs, and should be checked as part of the clinical characterization of the lineage.

Single molecule analysis of combinatorial splicing

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.