Ectopic expression of H-1 parvovirus NS1 protein induces alterations in actin filaments and cell death in human normal MRC-5 and transformed MRC-5 SV2 cells.

When grown in human cell lines, oncolytic H-1 parvovirus (H-1PV) replication preferentially occurs in transformed cells, which ultimately die upon infection. H-1PV-induced cytotoxicity is mainly due to P4 promoter-driven NS1 protein expression. Infection of untransformed cells generally does not induce deleterious effects because the P4 promoter is not activated. Here, we show that ectopic CMV-driven NS1 protein expression in normal human MRC-5 cells results in alterations of actin filaments and cell death, and both effects are prevented by a serine 473 mutation. The same substitution preserves actin filaments of transfected MRC-5 SV2 cells, that are MRC-5 transformed counterparts, but does not impair NS1-induced cytotoxicity.

Parvovirus H-1 induces cytopathic effects in breast carcinoma-derived cultures.

Parvovirus H-1 (H-1 PV) preferentially replicates in malignant cells resulting in their death by cytolysis. It has often been considered a potential candidate for use in novel anticancer therapy. To evaluate its potential in a model of natural tumors, we assayed in vitro the effect exerted by H-1 PV on short-term cultures derived from breast tumor samples freshly excised from patients. Our results show that H-1 PV effectively kills tumor-derived cells, whereas normal tissue-derived cells showed no H-1 PV-induced cytopathic effects (CPE). We also determined that the H-1 PV sensitivity (up to 67% sensitive cultures) is related with the quantities of virus assayed. We further examined the expression and phosphorylation state of the parvoviral nonstructural protein 1 (NS1), known to be associated with parvoviruses-induced CPE. Both appear to be impaired in normal tissue-derived cells and resistant cultures. Finally, we show that H-1 PV sensitivity in cultures correlates significantly with higher tumor grades (Nottingham combined histologic grade 2 or 3). This report confirms that H-1 PV can efficiently induce CPE in primary breast tumor cells in vitro. It identifies tumor characteristics representing potential criteria for recruiting patients for clinical evaluation of H-1 PV antitumor effects.

A revised and complete map of the chicken c-mil/raf-1 locus.

The chicken c-mil/raf-1 gene (formerly also known as c-mht) was originally identified in the search for the cellular counterpart to the v-mil oncogene of the Mill Hill 2 retrovirus and was among the first cellular proto-oncogenes discovered. Although the c-mil/raf-1 promotor, as well as the exons transduced into v-mil, were characterized in detail, an entire map of this locus has never been published. Here, we now report the location of five previously unmapped exons. In addition, we have noticed inconsistent numbering of the c-mil/raf-1 exons in the literature and the GenBank database. Thus, we provide here a complete map of the c-mil/raf-1 gene and a revision of the exon numbers. Comparison of the chicken c-mil/raf-1 gene with those of other vertebrates suggests that the numbers and lengths of the translated exons of the raf-1 locus were established early in the vertebrate lineage and have been conserved during the divergent evolution of teleosts and tetrapods.

Characterization and functional analysis of the p42Ets-1 variant of the mouse Ets-1 transcription factor.

We have identified the mouse exon VII splice variant of the Ets-1 transcription factor. The variant is expressed in all cell lines which express ets-1, at lower levels, it is also expressed in the mouse embryo in vivo. The corresponding protein, p42Ets-1, is a transcription factor as it is able to bind to specific DNA sequences and to transactivate a bona fide ETS reporter vector. A comparison of optimal DNA-binding sites shows that p42Ets-1 binds to more various DNA sequences than p51Ets-1; p42Ets-1 recognizes the same optimal consensus sequence as p51Ets-1, but also many variations of it, mainly at base -1, which is located just prior to the GGAA/T core sequence. The binding differences were quantified by surface plasmon resonance analyses and the protein region responsible for the differences in DNA sequence recognition located in the Val280-Glu302 fragment, which is encoded by exon VII. The specific DNA-binding properties of each isoform translates into clear differences in activity, p42Ets-1 transactivates the natural VE-cadherin gene promoter through both ETS-binding site (EBS)2 and EBS4 whereas p51Ets-1 is mainly active on EBS4. Altogether, our data suggest that p42Ets-1 acts as a distinct transcription factor from p51Ets-1.

Mutual repression of transcriptional activation between the ETS-related factor ERG and estrogen receptor.

Transcription factors are known to interact with each other to modulate their transcriptional activity. In this study, we found that the transcriptional activity of human Erg (one of the Ets family-transcription factors) was repressed by several nuclear receptors, including human estrogen receptor ERalpha, nonsteroid receptors and orphan receptors. Conversely, Erg inhibited ERalpha-dependent transcription. These reciprocal functional interactions extended to other nuclear receptors such as thyroid hormone and retinoic acid receptors, as well as to Fli1, an ERG-related ETS factor. Although similarly inhibited by overexpression of the orphan nuclear receptors ERR1 and RORalpha, ERG activity was unaffected by either REV-ERBalpha1 or COUP-TFII. The antagonism between ERG and ERalpha did not depend on DNA binding inhibition or direct protein-protein interactions. Repression of ERalpha-dependent transcription required the carboxyterminal and aminoterminal transactivation domains of Erg whereas the carboxyterminal AF-2 domain of ERalpha was necessary for repression of Erg activity. Reciprocal inhibition between Erg and ERalpha was not alleviated by overexpressing CBP, SRC-1 or RIP 140, three nuclear coactivator proteins. A negative cross-talk observed between Erg and ERalpha expands their potential range of regulation and may be relevant in vivo, particularly in endothelial, urogenital and cartilaginous tissues where both factors are expressed.

VE-statin, an endothelial repressor of smooth muscle cell migration.

The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE-statin for vascular endothelial-statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE-statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE-statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)-like modules. VE-statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE-statin inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE-statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.

ETS-1 transcription factor binds cooperatively to the palindromic head to head ETS-binding sites of the stromelysin-1 promoter by counteracting autoinhibition.

Stromelysin-1 (matrix metalloproteinase-3) is a member of the matrix metalloproteinase family. Regulation of its gene expression is critical for tissue homeostasis. Patterns of increased co-expression of stromelysin-1 and ETS-1 genes have been observed in pathological processes. Stromelysin-1 promoter is transactivated by ETS proteins through two palindromic head to head ETS-binding sites, an unusual configuration among metalloproteinase promoters. By using surface plasmon resonance, electrophoretic mobility shift assay, and photo-cross-linking, we showed that full-length human ETS-1 (p51) binds cooperatively to the ETS-binding site palindrome of the human stromelysin-1 promoter, with facilitated binding of the second ETS-1 molecule to form an ETS-1.DNA.ETS-1 ternary complex. The study of N-terminal deletion mutants allowed us to conclude that cooperative binding implied autoinhibition counteraction, requiring the 245-330-residue region of the protein that is encoded by exon VII of the gene. This region was deleted in the natural p42 isoform of ETS-1, which was unable to bind cooperatively to the palindrome. Transient transfection experiments showed a good correlation between DNA binding and promoter transactivation for p51. In contrast, p42 showed a poorer transactivation, reinforcing the significance of cooperative binding for full transactivation. It is the first time that ETS-1 was shown to be able to counteract its own autoinhibition.