ABSTRACT
BACKGROUND: Notch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood. METHODS: We examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment. RESULTS: Here, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts. CONCLUSION: Our results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.
ABSTRACT
A recent Molecular Cell paper by Randell et al. (2006) sheds light on the role of ATP hydrolysis by Cdc6 in promoting the stable loading of the Mcm2-7 complex onto origins of DNA replication.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphate , Chromosomal Proteins, Non-Histone , DNA Replication , Minichromosome Maintenance Complex Component 7 , Models, Biological , Origin Recognition Complex/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
Initiation of eukaryotic DNA replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in eukaryotic genomes.
Subject(s)
DNA Replication , Eukaryotic Cells/metabolism , Replication Origin , Acetylation , Animals , Cell Cycle , DNA Methylation , DNA Replication/genetics , DNA Replication/physiology , Drosophila melanogaster/genetics , Replication Origin/genetics , Replication Origin/physiology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Transcription, Genetic , Xenopus/geneticsABSTRACT
We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.
Subject(s)
DNA Replication , DNA-Binding Proteins , DNA/metabolism , Animals , Binding, Competitive , Cell Line , Cell-Free System , Dose-Response Relationship, Drug , Humans , Insecta , Origin Recognition Complex , Plasmids/metabolism , Protein Binding , Recombinant Proteins/metabolism , Time Factors , XenopusABSTRACT
The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.