ABSTRACT
To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.
Subject(s)
Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyridines/pharmacology , Animals , CDC2 Protein Kinase/antagonists & inhibitors , Cell Line, Tumor , Cyclin-Dependent Kinases/antagonists & inhibitors , Down-Regulation/drug effects , Gene Amplification , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , N-Myc Proto-Oncogene Protein , Neuroblastoma/pathology , Roscovitine , Xenograft Model Antitumor AssaysABSTRACT
The green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex-inducing pheromone at a concentration of < 10-16 M. Early biochemical responses to the pheromone involve structural modifications within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromone-treated Volvox, four novel genes were identified that encode four closely related Volvox metalloproteinases that we use to define a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12-25%, and Ser(Pro)2-4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex-inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc-dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline-rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modifications of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.
Subject(s)
Chlorophyta/genetics , Glycoproteins/genetics , Metalloendopeptidases/genetics , Multigene Family , Plant Proteins , Sex Attractants/physiology , Transcriptional Activation/physiology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary , Glycoproteins/chemistry , Metalloendopeptidases/chemistry , Molecular Sequence Data , RNA, Messenger/genetics , Sequence Homology, Amino AcidABSTRACT
The green alga Volvox is one of the simplest multicellular organisms and is capable of both asexual and sexual reproduction. Sexual development is initiated by a glycoprotein pheromone that acts at a concentration below 10(-16) M. The extracellular matrix (ECM) appears to play a key role in signal amplification: several ECM proteins contain a domain with homology to the sex-inducing pheromone.
Subject(s)
Chlorophyta/physiology , Sex Attractants/physiologyABSTRACT
The alga Volvox carteri represents one of the simplest multicellular organisms. Its extracellular matrix (ECM) is modified under developmental control, e.g. under the influence of the sex-inducing pheromone that triggers development of males and females at a concentration below 10(-16) M. A novel ECM glycoprotein (pherophorin-S) synthesized in response to this pheromone was identified and characterized. Although being a typical member of the pherophorins, which are identified by a C-terminal domain with sequence homology to the sex-inducing pheromone, pherophorin-S exhibits a completely novel set of properties. In contrast to the other members of the family, which are found as part of the insoluble ECM structures of the cellular zone, pherophorin-S is targeted to the cell-free interior of the spherical organism and remains in a soluble state. A main structural difference is the presence of a polyhydroxyproline spacer in pherophorin-S that is linked to a saccharide containing a phosphodiester bridge between two arabinose residues. Sequence comparisons indicate that the self-assembling proteins that create the main parts of the complex Volvox ECM have evolved from a common ancestral gene.
Subject(s)
Chlorophyta/metabolism , Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Algal Proteins , Amino Acid Sequence , Arabinose/chemistry , Biological Transport , Cloning, Molecular , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/isolation & purification , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/isolation & purification , Molecular Sequence Data , Peptides/chemistry , Pheromones/metabolism , Phosphoric Diester Hydrolases/chemistry , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
Pherophorins are extracellular matrix (ECM) glycoproteins from Volvox that share homology with the sex-inducing pheromone. A novel pherophorin (pherophorin III) was characterized both with respect to expression pattern and proteolytic processing in vivo. Furthermore, it was shown that the pherophorins represent a protein family of ECM glycoproteins exhibiting a modular composition: their N-terminally located domain is a homolog of a domain found in the ECM glycoprotein SSG 185. Together with SSG 185, pherophorin I is a main component of the cellular zone within the ECM. The Volvox genome contains a tandem arrangement of genes encoding pherophorin II-related polypeptides. Inhibition of proteolytic processing of pherophorin II and III in vivo appears to result in the suppression of sexual induction.
Subject(s)
Chlorophyta/chemistry , Extracellular Matrix Proteins/chemistry , Glycoproteins/chemistry , Algal Proteins , Amino Acid Sequence , Chlorophyta/genetics , Chlorophyta/physiology , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Hydrolysis , Molecular Sequence Data , Multigene Family , Sequence Homology, Amino Acid , Sex Attractants , Sodium Dodecyl Sulfate , SolubilityABSTRACT
The sex-inducing pheromone of Volvox carteri is a glycoprotein that triggers development of males and females at a concentration below 10(-16) M. Evidence is presented for the existence of a novel mechanism of signal amplification operating within the extracellular matrix of this multicellular organism. A family of 70 kDa matrix glycoproteins denoted pherophorins bear a C-terminal domain being homologous to the sex-inducing pheromone. Under the influence of the pheromone, this domain is liberated by highly specific proteolysis.
