RESUMO
As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 µM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 µM or 2 µM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells.
Assuntos
Antineoplásicos/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Neoplasias Pulmonares/patologia , Naftalimidas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pirazóis/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Benzoxazóis/química , Benzoxazóis/farmacologia , Sinalização do Cálcio , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Naftalimidas/química , Pirazóis/químicaRESUMO
Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Reparo de DNA por Recombinação , Fase S/genética , Proteínas Supressoras de Tumor/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular , Endonucleases , Humanos , Rad51 Recombinase/análise , Tolerância a Radiação , Fase S/efeitos da radiaçãoRESUMO
During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase α-primase (hypo-Polα), which is, unlike AK-phosphorylated Polα (P-Polα), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-Δp53-p21 signaling significantly reduced the T-Ag-interacting hypo-Polα population and, accordingly, SV40 replication efficiency. Moreover, the ATR-Δp53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Polα, giving rise to T-Ag-interacting hypo-Polα. Thus, the purpose of activating the ATR-Δp53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Vírus 40 dos Símios/patogenicidade , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA , Primers do DNA/genética , Replicação do DNA , Interações Hospedeiro-Patógeno/genética , Humanos , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/fisiologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Replicação ViralRESUMO
In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients, which statistically correlated with poor prognosis and residual disease. These observations implied that SPOC1 might play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 is dynamically regulated during mitosis with increased expression levels and biphasic localization to mitotic chromosomes indicating a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show, using micrococcal nuclease (MNase) chromatin-digestion assays that SPOC1 expression levels proportionally influence the degree of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.
Assuntos
Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Mitose , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Células Eucarióticas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Metáfase , Prófase , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/metabolismoRESUMO
The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.