Multivalent immunity targeting tumor-associated antigens by intra-lymph node DNA-prime, peptide-boost vaccination.

Active immunotherapy of cancer has yet to yield effective therapies in the clinic. To evaluate the translatability of DNA-based vaccines we analyzed the profile of T-cell immunity by plasmid vaccination in a murine model, using transcriptome microarray analysis and flow cytometry. DNA vaccination resulted in specific T cells expressing low levels of co-inhibitory molecules (most notably PD-1), strikingly different from the expression profile elicited by peptide immunization. In addition, the T-cell response primed through this dual-antigen-expressing plasmid (MART-1/Melan-A and tyrosinase) translated into a substantial proliferation capacity and functional conversion to antitumor effector cells after tyrosinase and MART-1/Melan-A peptide analog boost. Furthermore, peptide boost rescued the immune response against the subdominant tyrosinase epitope. This immunization approach could be adapted to elicit potent immunity against multiple tumor antigens, resulting in a broader immune response that was more effective in targeting human tumor cells. Finally, this study sheds light on a novel mechanism of immune homeostasis through synchronous regulation of co-inhibitory molecules on T cells, highly relevant to heterologous prime boost approaches involving DNA vaccines as priming agents.

Osteoprotegerin is an alpha vbeta 3-induced, NF-kappa B-dependent survival factor for endothelial cells.

Osteopontin protects endothelial cells from apoptosis induced by growth factor withdrawal. This interaction is mediated by the alpha(v)beta(3) integrin and is NF-kappaB-dependent (Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093). In the present study we used differential cloning to identify osteopontin-induced, NF-kappaB-dependent genes in endothelial cells. One of the genes identified in this screen was osteoprotegerin, a member of the tumor necrosis factor receptor superfamily. By Northern and Western blot analysis, osteoprotegerin mRNA and protein levels were very low in endothelial cells plated on the non-integrin cell attachment factor, poly-d-lysine. In contrast, osteoprotegerin mRNA and protein levels were induced 5-7-fold following alpha(v)beta(3) ligation by osteopontin. Osteoprotegerin induction by osteopontin was time-dependent and observed as early as 3 h following treatment. NF-kappaB inactivation achieved by over expression of an IkappaB super repressor in endothelial cells completely inhibited osteoprotegerin induction by osteopontin. Finally, purified osteoprotegerin protected endothelial cells with inactive NF-kappaB from apoptosis induced by growth factor deprivation. These data suggest that alpha(v)beta(3)-mediated endothelial survival depends on osteoprotegerin induction by NF-kappaB and indicate a new function for osteoprotegerin in endothelial cells.

Upstream stimulatory factor 1 regulates osteopontin expression in smooth muscle cells.

Vascular smooth muscle cells (SMCs) undergo a dramatic phenotypic transition in response to injury and ex vivo culture that includes enhanced proliferation, migration, matrix deposition, and alterations in gene expression. Osteopontin is a good marker for the injury-induced SMC phenotypic state in vivo and in vitro. To identify transcription factors that might control the regulation of osteopontin expression, we investigated cultured vascular SMCs that express high and low levels of osteopontin. Using nuclear run-on assays, mRNA stability studies, and deletion analysis, we demonstrate that regulation of osteopontin steady-state mRNA levels in SMCs occurs at the transcriptional level. Transient transfection and gel-shift analyses of osteopontin promoter indicated that a region between -123 and +66 was involved in the expression of osteopontin. Supershift EMSAs identified the bHLH-leucine zipper transcription factor upstream stimulatory factor-1 (USF1) as the protein binding to this sequence. Finally, we show that USF1 protein is induced in vivo within 24 h of balloon angioplasty of rat carotids coordinately with osteopontin induction. These data suggest that USF1 governs expression of osteopontin in cultured vascular SMCs and might contribute to initial osteopontin expression observed post carotid injury and in vascular pathologies in vivo.

Osteopontin regulation in cultured rat renal epithelial cells.

Osteopontin is a secreted, arginine-glycine-aspartate (RGD)-containing phosphoprotein that is up-regulated in kidney cortical tubular epithelial cells in many experimental models of tubulointerstitial fibrosis. Its close association with infiltrating macrophage in this disease and its ability to directly stimulate macrophage migration has made it a key target as a molecule likely to be important in mediating renal inflammation. The mechanism responsible for osteopontin up-regulation in kidney disease is unknown, but may involve induction by specific cytokines released by damaged glomeruli or other parts of the kidney, prior to the onset of interstitial disease. We have investigated this hypothesis by testing the effects of angiotensin II, bFGF, TGF beta 1, EGF, and IGF, important renal cytokines, on osteopontin regulation in cultured NRK52E cells, a rat renal epithelial cell line. Using Northern blot, Western blot, and ELISA analyses, we find that NRK52E cells constitutively express low levels of osteopontin mRNA and protein. TGF beta 1 and EGF are potent inducers of osteopontin mRNA and protein in those cells. mRNA stability and nuclear run on assays suggest that induction of osteopontin expression by TGF beta 1 and EGF is via increased transcription of the osteopontin gene. In contrast, IGF-1, angiotensin II, and PDGF BB did not significantly modulate osteopontin expression in NRK52E cells. These studies are consistent with the hypothesis that release of potent cytokines by the injured kidney might be one mechanism whereby elevated levels of osteopontin are synthesized by cortical tubular epithelial cells early in tubulointerstitial disease.

Instability of endogenous MRP/proliferin transcripts in the nucleus of mouse embryo fibroblasts contrasts with their stability when produced during transient transfections.

The mitogen regulated protein/proliferin (MRP/PLF) gene is transcribed in primary mouse embryo fibroblasts (MEFs), but the pre-mRNA is not properly converted into a stable cytoplasmic mRNA and instead is rapidly degraded, apparently in the nucleus [Malyankar et al. (1994): Proc Natl Acad Sci USA 91:335-359]. In 3T3 cells derived from the MEFs by the standard 3T3 immortalization protocol, stable MRP/PLF mRNA is produced. We show here that the processing of intron sequences is similar in the two cell types and that some of the MRP/PLF transcripts are polyadenylated in the MEFs. We also document the production of stable MRP/PLF mRNA generated by transcription of various plasmid constructs containing different portions of the MRP/PLF3 gene after calcium phosphate-mediated transfection into the MEFs. We conclude that the inability of the MRP/PLF mRNA to accumulate in the MEFs is unlikely to result solely from a single localized sequence in the primary transcript (or the mRNA) that causes it to be subject to rapid breakdown; possibly export of the mRNA from the MEF nucleus is defective or some aspect of the transcriptional process marks the transcript for degradation.

The mitogen-regulated protein/proliferin transcript is degraded in primary mouse embryo fibroblast but not 3T3 nuclei: altered RNA processing correlates with immortalization.

An understanding of what changes occur in the control of gene expression when mammalian cells "spontaneously" immortalize is important to our knowledge of how cancer develops. We describe here an alteration in regulation that occurs when primary mouse embryo fibroblasts (MEFs) are immortalized according to a 3T3 regimen. Mitogen-regulated protein/proliferin mRNA is undetectable in northern blots of RNA from (mortal) MEFs, whereas it is readily detected in immortal 3T3 cell lines derived from the MEFs. Incompletely processed nuclear transcripts of the mitogen-regulated protein/proliferin gene can be detected in MEF RNA preparations by northern blotting and reverse transcriptase polymerase chain reaction analyses, although at roughly half the abundance observed in 3T3 cells. We hypothesize that some attribute of the primary unprocessed transcript determines its assignment to this unique degradative pathway. These results reveal that during passage of MEFs according to a 3T3 regimen the ability of the primary cells to suppress the expression of certain genes by degrading the nuclear transcript is lost concomitantly with immortalization.

Tissue inhibitor of metalloproteinases (TIMP, aka EPA): structure, control of expression and biological functions.

The TIMPs play an important role in regulating the activity of the secreted metalloproteinases (collagenases, stromelysins, gelatinases). Two different TIMPS have been well characterized, each capable of inhibiting all tested eukaryotic metalloproteinases but showing specific binding to a particular gelatinase at a site distinct from the active site. They influence the activation of the prometalloproteinase and act to modulate proteolysis of extracellular matrix, notably during tissue remodeling and inflammatory processes. On certain cell types, they can exhibit growth factor-like activity, and they can inhibit the tumorigenic and metastatic phenotype of cancer cells.