ABSTRACT
The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.
Subject(s)
Adenosine Triphosphate/metabolism , Antibodies/metabolism , Extracellular Space/metabolism , Animals , Humans , Mice , Tumor MicroenvironmentABSTRACT
During mitosis, spatiotemporal regulation of phosphorylation at the kinetochore is essential for accurate chromosome alignment and proper chromosome segregation. Aurora B kinase phosphorylates kinetochore substrates to correct improper kinetochore-microtubule (KT-MT) attachments, whereas tension across the centromeres inactivates Aurora B kinase, and PP2A phosphatase dephosphorylates the kinetochore proteins to stabilize the attachments. However, the molecular entity of the tension sensing mechanism remains elusive. In a previous report, we showed that centromeric SET/TAF1 on Sgo2 up-regulates Aurora B kinase activity via PP2A inhibition in prometaphase. Here we show that Aurora B and Bub1 at the centromere/kinetochore regulate both kinase activities one another in an inter-kinetochore distance-dependent manner, indicating a positive feedback loop. We further show that the centromeric pool of SET on Sgo2 depends on Bub1 kinase activity, and the centromeric localization of SET decreases in a distance-dependent manner, thereby inactivating Aurora B in metaphase. Consistently, ectopic targeting of SET to the kinetochores during metaphase hyperactivates Aurora B via PP2A inhibition, and thereby rescues the feedback loop. Thus, we propose that SET, Aurora B and Bub1 form a distance-dependent positive feedback loop, which spatiotemporally may act as a tension sensor at centromeres.
Subject(s)
Aurora Kinase B/metabolism , Centromere/metabolism , DNA-Binding Proteins/metabolism , Feedback, Physiological , Histone Acetyltransferases/metabolism , Histone Chaperones/metabolism , Protein Serine-Threonine Kinases/metabolism , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/metabolism , Biomechanical Phenomena , Enzyme Activation , HeLa Cells , HumansABSTRACT
An Amendment to this Article has been published and is linked from the HTML version of this paper.
ABSTRACT
Chromosomal instability (CIN) is a major trait of cancer cells and a potent driver of tumor progression. However, the molecular mechanisms underlying CIN still remain elusive. We found that a number of CIN(+) cell lines have impairments in the integrity of the conserved inner centromere-shugoshin (ICS) network, which coordinates sister chromatid cohesion and kinetochore-microtubule attachment. These defects are caused mostly by the loss of histone H3 lysine 9 trimethylation at centromeres and sometimes by a reduction in chromatin-associated cohesin; both pathways separately sustain centromeric shugoshin stability. Artificial restoration of the ICS network suppresses chromosome segregation errors in a wide range of CIN(+) cells, including RB- and BRCA1-deficient cells. Thus, dysfunction of the ICS network might be a key mechanism underlying CIN in human tumorigenesis.
Subject(s)
Carcinogenesis/metabolism , Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromosomal Instability , Chromosome Segregation , BRCA1 Protein/genetics , Carcinogenesis/genetics , Cell Cycle Proteins/genetics , Centromere/genetics , Chromatids/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , HeLa Cells , Histones/metabolism , Humans , Kinetochores/metabolism , Lysine/metabolism , Methylation , Microtubules/metabolism , Retinoblastoma Protein/genetics , CohesinsABSTRACT
Chromosome structure is dynamically regulated during cell division, and this regulation is dependent, in part, on condensin. The localization of condensin at chromosome arms is crucial for chromosome partitioning during anaphase. Condensin is also enriched at kinetochores but its precise role and loading machinery remain unclear. Here we show that fission yeast (Schizosaccharomyces pombe) kinetochore proteins Pcs1 and Mde4--homologues of budding yeast (Saccharomyces cerevisiae) monopolin subunits and known to prevent merotelic kinetochore orientation--act as a condensin 'recruiter' at kinetochores, and that condensin itself may act to clamp microtubule binding sites during metaphase. In addition to the regional recruitment factors, overall condensin association with chromatin is governed by the chromosomal passenger kinase Aurora B. Aurora-B-dependent phosphorylation of condensin promotes its association with histone H2A and H2A.Z, which we identify as conserved chromatin 'receptors' of condensin. Condensin phosphorylation and its deposition onto chromosome arms reach a peak during anaphase, when Aurora B kinase relocates from centromeres to the spindle midzone, where the separating chromosome arms are positioned. Our results elucidate the molecular basis for the spatiotemporal regulation of mitotic chromosome architecture, which is crucial for chromosome partitioning.
