The development of new blood vessels from pre-existing blood vessels (angiogenesis) is essential for normal tissue repair. However, neovascularization also contributes to the growth and dissemination of solid tumors and thus interfering with tumor angiogenesis presents much promise as a means to limit tumor growth and metastasis. 1,2Tumor-derived stimuli induce quiescent endothelial cells (ECs) to re-enter the cell cycle, express extracellular matrix-degrading proteinases, and up-regulate expression of adhesion molecules to allow migration.3-5 Vascular sprouts then resynthesize basement membranes (BMs), undergo capillary morphogenesis and withdraw from the cell cycle, and form mature quiescent vessels.To understand the complex temporal and spatial regulation of expression of proteinases, adhesion molecules, and extracellular matrix molecules during angiogenesis, we have been investigating the role of Homeobox (Hox) master regulatory genes. In addition to their roles in embryogenesis, Hox genes are also expressed in adult cells, including the vascular endothelium, and regulate expression of genes involved in cell-cell, cell-extracellular matrix interactions, and cell proliferation.6 -11 Previously we showed that HoxD3 induces an angiogenic phenotype and promotes EC migration and invasion via up-regulation of ␣v3 integrin and urokinase plasminogen activator (uPA) and that HoxB3 contributes to angiogenesis by increasing expression of ephrin A1 that facilitates capillary morphogenesis. 9,12The 40 class I vertebrate Hox genes are clustered in four linkage groups (A to D), on four different chromosomes with both the proangiogenic HoxD3 and HoxB3 being located toward the 3Ј end of these Hox gene clusters. During embryogenesis, 3Ј Hox gene expression is followed by the sequential activation of more 5Ј Hox genes, giving rise to nonoverlapping boundaries of expression.13 A similar 3Ј to 5Ј wave of Hox gene expression is also observed when adult hematopoietic progenitor cells are induced to differentiate, 14 while maturing cells attenuate expression of 3Ј Hox genes and begin to express high levels of 5Ј Hox genes such as HoxA10. 15,16 Together these observations suggest that 5Ј and 3Ј Hox genes control different aspects of cell or tissue phenotype.As the majority of adult ECs exist in a quiescent state, we reasoned that after angiogenesis maturing capillaries would begin to express 5Ј Hox genes, which in turn may help to maintain a quiescent, differentiated phenotype. Furthermore, we investigated whether sustaining expression of 5Ј Hox genes in an angiogenic environment could prevent acquisition of an angiogenic phenotype. Materials and Methods Cells, Transfections, and RNA IsolationImmortalized human dermal microvascular ECs HMEC-1 were a gift from T. Lawley, Emory University, Atlanta, GA.17,18 Cells were maintained and cultured on BMs as previously described.9 Primary human dermal microvascular ECs were purchased from BioWhittaker (San Diego, American Journal of Pathology, Vol. 161, No. 6, December 2002 Copyright © Am...
Together our results suggest that restoring Hox A5 expression may provide a novel means to limit breast tumor growth or expansion of hemangiomas.
Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin αvβ3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.
The differentially expressed in adenocarcinoma of the lung (DAL-1) gene, which shares significant homology with members of the 4.1/ezrin/radixin/moesin/neurofibromatosis 2 (ERM/NF2) protein family, has previously been shown to suppress growth in lung cancer cell lines. This gene localizes to chromosome band 18p11.3, which undergoes loss of heterozygosity (LOH) in nonsmall cell lung carcinomas and a significant proportion of ductal carcinomas in situ (DCIS) of the breast. This finding suggests that alteration of gene(s) (possibly DAL-1) within this chromosomal region may be important early in the progression of breast disease. We generated MCF-7 cell lines expressing DAL-1 constitutively or under the control of an inducible promoter and analyzed the effect of DAL-1 expression on growth. These investigations revealed that the DAL-1 protein suppresses the growth of MCF-7 cells and may do so in part through the induction of apoptosis. In addition, expression of DAL-1 increased attachment of these cells to a variety of extracellular matrices. This is the first evidence that the DAL-1 protein functions at the interface between cell adhesion and apoptosis in controlling cell growth.
AbstracthSNF5, the smallest member of the SWI/SNF chromatin remodeling complex, is lost in most malignant rhabdoid tumors (MRT). In MRT cell lines, reexpression of hSNF5 induces G 1 cell cycle arrest, elevated p16INK4a , and activated replicative senescence markers, such as B-galactosidase (B-Gal) and plasminogen activator inhibitor-1. To compare the replicative senescence caused by hSNF5 in A204 cells to normal cellular senescence, we examined the activation of both p16INK4a and p21 CIP/WAF1 . Analogous to normal cellular senescence, both p16INK4a and p21 CIP/WAF1 were up-regulated following hSNF5 restoration. Furthermore, we found that hSNF5 bound the p16INK4a and p21 CIP/WAF1 promoters, suggesting that it directly regulates transcription of these genes. Using p16INK4a RNA interference, we showed its requirement for the replicative senescence caused by hSNF5 but not the growth arrest. Instead, p21 CIP/WAF1 remained activated by hSNF5 in the absence of high p16INK4a expression, apparently causing the growth arrest in A204. Interestingly, we also found that, in the absence of p16 INK4a , reexpression of hSNF5 also increased protein levels of a second cyclin-dependent kinase (CDK) inhibitor, p18 INK4c. However, our data show that lack of hSNF5 does not abrogate cellular responsiveness to DNA damage or growth-inhibitory factors. In summary, our studies suggest that hSNF5 loss may influence the regulation of multiple CDK inhibitors involved in replicative senescence. (Cancer Res 2005; 65(22): 10192-8)
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