In vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas

BACKGROUND: Human breast cancer metastasizes mainly to lymph nodes, lungs, liver, and bone; in the majority of cases, it is the development of metastases which leads to death. In order to suppress mammary cancer metastasis, we applied in vivo electrogene transfer (non-viral method) as a means of interleukin-12 (IL-12) gene therapy on highly metastatic murine mammary cancer model. METHODS: Metastatic mammary tumors induced by inoculation in BALB/c female mice were treated by intratumoral injections of either a plasmid vector containing IL-12 or empty vector and then subjected to in vivo electrogene transfer once a week for 8 weeks. RESULTS: Treatment with IL-12 resulted in elevation of both IL-12 and IFNgamma levels in mammary tumors and in serum and intratumoral levels of CD4 and CD8 proteins were also increased. Tumor volumes and lymphatic and pulmonary metastases were significantly reduced. The histopathological changes induced by IL-12 characteristically included marked inflammation, increased apoptosis, decreased DNA synthesis, peripheral influx of significantly greater numbers of active macrophages, and reduced blood microvessel density, and apoptotic vascular endothelial cells were frequently seen. Western blotting showed decreases in VEGFR-3 of tumors exposed to IL-12 gene therapy. In adjuvant immunofluorescence studies, the CD31-positive endothelial cells of microvessels showed decreased VEGFR-3 expression in IL-12-treated tumors. However, apparent alterations in VEGFR-3 expression of podoplanin-positive lymphatic endothelial cells were not observed in IL-12-treated tumors. Although recombinant IL-12 did not inhibit tubular formation of human umbilical vein endothelial cells in a Matrigel assay, recombinant IFNgamma did completely suppress the tubular formation. CONCLUSIONS: In vivo electrogene transfer of IL-12 exerts strong anti-tumorigenic and anti-metastatic effects likely due to T-cell-mediated immune responses as well as anti-angiogenic action.     

Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas

ABSTRACT: BACKGROUND: Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffinembedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. RESULTS: The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p =0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. CONCLUSIONS: Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.     

Presumptive lymph node organizers are differentially represented in developing mesenteric and peripheral nodes

During murine embryogenesis, the formation of Peyer's patches (PPs) is initiated by CD45(+)CD4(+)CD3(-) lymphoid tissue inducers that trigger adhesion molecule expression and specific chemokine production from an organizing stromal cell population through ligation of the lymphotoxin-beta receptor. However, the steps involved in the development of lymph nodes (LNs) are less clear than those of PPs, and the characteristics of the organizing cells within the LN anlagen have yet to be documented. In this study, we show for the first time that the early anlage is bordered by an endothelial layer that retains a mixed lymphatic and blood vascular phenotype up to embryonic day 16.5. This in turn encompasses CD45(+)CD4(+)CD3(-) cells interspersed with ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1, lymphotoxin-beta receptor-positive, chemokine-producing cells analogous to the organizing population previously observed in PPs. Moreover, these LN organizers also express the TNF family member, TRANCE. Lastly, we show that the ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1 cells present in peripheral and mesenteric LN form two discrete populations expressing either intermediate or high levels of these adhesion molecules but that the former population is specifically reduced in PLN. These findings provide a possible explanation for the well-known differences in developmental requirements for nodes at peripheral or mesenteric locations.     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci.     

Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca²⁺ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca²⁺ pathway and whether it regulates gene expression.     

UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells

Background

Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology

Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal Findings

Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions

UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.     

Multiple catalase genes are differentially regulated in Aspergillus nidulans

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (approximately 84 kDa) and CatB (approximately 79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H(2)O(2)-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H(2)O(2) treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H(2)O(2) detoxification in filamentous fungi.     

Cyclins of phases G1, S and G2/M are overexpressed in aneuploid mammary carcinomas

Expression of cyclins A, B1 and D1 in human breast cancer was analyzed using dual-parameter flow cytometry with simultaneous evaluation of the DNA content. The asynchronous MCF-7 breast adenocarcinoma cells were used to implement flow cytometry analysis and to analyze the cell cycle distribution of cyclins. The patterns of the cyclin expression were also analyzed in vivo in fresh tissue specimens of human breast carcinomas. The combined measurement of DNA and cyclins showed a higher cyclin expression in aneuploid (11.5 +/- 2.0%, 4.3 +/- 1.1%, and 19.5 +/- 3.4% positive cells for cyclins A, B, and D1, respectively) than in diploid carcinomas (3.9 +/- 1.2%, 1.1 +/- 0.4%, and 5.0 +/- 1.2% positive cells for cyclins A, B, and D1, respectively). A positive relationship was also found between cyclin A and D1 expression and H(3)-thymidine labeling index. In the in vitro model, the asynchronous growing MCF-7 cells showed a variable number of cells expressing cyclins in an unscheduled way, unrelated to the phase at which these cyclins are expressed in normal cells. A similar condition was also observed in tumors. In conclusion, the data showed a deregulated expression of cyclins in a transformed adenocarcinoma cell line and in breast tumors. Furthermore, overexpression of these proteins is related to the aneuploid and high proliferative activity of human mammary carcinomas.     

TGF-beta(1) and prepro-ANP mRNAs are differentially regulated in exercise-induced cardiac hypertrophy.

The induction of transforming growth factor (TGF)-beta and prepro-atrial natriuretic peptide (ANP) mRNAs represent hallmark features of pathological cardiac hypertrophy. The present study examined whether this pattern of mRNA expression was conserved in a physiological model of cardiac hypertrophy. To address this thesis, female Sprague-Dawley rats were individually housed and permitted to run freely. Voluntary exercise for 3 and 6 wk resulted in biventricular hypertrophy and increased cytochrome c oxidase activity in the triceps muscle. In the hypertrophied left ventricle, the steady-state mRNA level of the cardiac fetal gene prepro-ANP and the extracellular matrix proteins preprocollagen-alpha(1) and fibronectin were similar in exercise-trained and sedentary rats. By contrast, an increased expression of TGF-beta(1) mRNA was observed, whereas TGF-beta(3) mRNA level was unchanged in the hypertrophied left ventricle of exercise-trained compared with sedentary rats. These data highlight a heterogeneity in the regulation of TGF-beta isoforms, and the increased expression of ventricular TGF-beta(1) mRNA in physiological cardiac hypertrophy may contribute to myocardial remodeling.     

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down- regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine- specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.     

The Notch targets Esr1 and Esr10 are differentially regulated in Xenopus neural precursors

The HES family of bHLH repressors plays a key role in regulating the differentiation of neural precursors in the vertebrate embryo. Members of the HES gene family are expressed in neural precursors as targets of the Notch signaling pathway, but how this occurs in the context of neurogenesis is not known. Here, we address this issue by identifying enhancers driving Notch-dependent gene expression of two Hes5-like genes expressed in Xenopus called Esr1 and Esr10. Using frog transgenesis, we identify enhancer elements driving expression of Esr1 and Esr10 in neural precursors or in response to ectopic expression of the proneural protein, Xngnr1. Using deletion and mutation analysis, we define motifs required for enhancer activity of both genes, namely Notch-responsive elements and, in the case of Esr10, E-box motifs. We find that Esr1 and Esr10 are differentially regulated both in terms of Notch input and its interaction with heterologous factors. These studies reveal inputs required for proneural expression of genes encoding bHLH repressors in the developing vertebrate nervous system.