RESUMO
The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.
Assuntos
Apoptose , Pontos de Checagem do Ciclo Celular , Mitocôndrias/genética , RNA Longo não Codificante/metabolismo , Animais , Antagomirs/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismoRESUMO
Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3-7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.
Assuntos
Proteínas de Artrópodes/química , Proteína Quinase CDC2/química , Quinases Ciclina-Dependentes/química , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Rhipicephalus/efeitos dos fármacos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/classificação , Proteína Quinase CDC2/metabolismo , Caspases/química , Caspases/metabolismo , Domínio Catalítico , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/classificação , Quinases Ciclina-Dependentes/metabolismo , Escherichia coli/química , Escherichia coli/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Purinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/metabolismo , Rhipicephalus/citologia , Rhipicephalus/enzimologia , Roscovitina , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDK1 and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors.
Assuntos
Proteína Quinase CDC2/química , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Proteína Quinase CDC2/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28/química , Quinase 2 Dependente de Ciclina , Desenho de Fármacos , Flavonoides/farmacologia , Humanos , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Piperidinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Purinas/farmacologia , Roscovitina , Homologia de Sequência de AminoácidosRESUMO
Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.
Assuntos
Ciclinas/metabolismo , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Células COS , Clonagem Molecular , Genes de Protozoários , Teste de Complementação Genética , Histonas/metabolismo , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Proteína do Retinoblastoma/metabolismo , Alinhamento de Sequência , Transfecção , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks). The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1.p12cks1 was detected in the three life-cycle stages of L. mexicana by immunoblotting. Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts. Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L. mexicana p12cks1 and yeast p13suc1. Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L. mexicana, but at a low level in the non-dividing metacyclic form. These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously [Mottram, Kinnaird, Shiels, Tait and Barry (1993) J. Biol. Chem. 268, 21044-21051]. A distinct cdc2-related kinase, L. mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts. The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.