Hexon Modification to Improve the Activity of Oncolytic Adenovirus Vectors against Neoplastic and Stromal Cells in Pancreatic Cancer

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.