RESUMEN
T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/efectos de los fármacos , Neoplasias Pulmonares/radioterapia , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Fosfatasas cdc25/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/efectos de la radiación , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Inhibidores de Proteínas Quinasas/farmacología , Quinolonas/farmacología , Tolerancia a Radiación/genética , Transducción de Señal , Tasa de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto , Fosfatasas cdc25/genética , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-ras(Ha) transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser(280). Since Akt phosphorylates Chk1-Ser(280), the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Genes cdc/efectos de la radiación , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de la radiación , Neoplasias Cutáneas/metabolismo , Animales , Transformación Celular Neoplásica/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN/efectos de la radiación , Immunoblotting , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de la radiación , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Quinasas/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de la radiación , Neoplasias Cutáneas/genética , Rayos Ultravioleta , Fosfatasas cdc25/metabolismo , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
The rapid cell cycle arrest in response to DNA damage is mediated by degradation of the Cdc25A phosphatase, a proto-oncogene whose mRNA is frequently overexpressed in human tumours. Here, we study the occurrence and mechanisms of Cdc25A deregulation in human breast cancer cell lines. We demonstrate aberrantly elevated Cdc25A protein abundance and phosphatase activity in eight out of 15 cell lines, in some cases resulting in abrogation of the Cdc25A-mediated checkpoint response to ionizing radiation (IR), and this defect correlated with hypersensitivity to IR. Furthermore, we present evidence that deregulation of Cdc25A occurs predominantly on the post-transcriptional level, as overabundant Cdc25A protein was usually not accompanied by adequate mRNA overexpression. Instead, we demonstrate that aberrantly enhanced protein stability is an important mechanism underlying Cdc25A overabundance in a subset of breast cancer cell lines. Given the frequency of this mechanism, we propose that the DNA integrity checkpoint controlling Cdc25A protein stability might be a common target for deregulation in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Fosfatasas cdc25/metabolismo , Femenino , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Proto-Oncogenes Mas , Regulación hacia Arriba , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
Induction of interstrand crosslinks (ICLs) in chromosomal DNA is considered a major reason for the antiproliferative effect of psoralen plus ultraviolet A (PUVA). It is unclear as to whether PUVA-induced cell cycle arrest is caused by ICLs mechanically stalling replication forks or by triggering cell cycle checkpoints. Cell cycle checkpoints serve to maintain genomic stability by halting cell cycle progression to prevent replication of damaged DNA templates or segregation of broken chromosomes. Here, we show that HaCaT keratinocytes treated with PUVA arrest with S-phase DNA content. Cells that had completed DNA replication were not perturbed by PUVA and passed through mitosis. Cells treated with PUVA during G1-phase continued traversing G1 until arresting in early S-phase. PUVA induced rapid phosphorylation of the Chk1 checkpoint kinase at Ser345 and a concomitant decrease in Cdc25A levels. Chk1 phosphorylation, decrease of Cdc25 A levels and S-phase arrest were abolished by caffeine, demonstrating that active checkpoint signaling rather than passive mechanical blockage by ICLs causes the PUVA-induced replication arrest. Overexpression of Cdc25A only partially overrode the S-phase arrest, suggesting that additional signaling events implement PUVA-induced S-phase arrest.
Asunto(s)
Cafeína/metabolismo , Ciclo Celular/efectos de la radiación , Reactivos de Enlaces Cruzados/farmacología , Ficusina/farmacología , Rayos Ultravioleta , Animales , Ciclo Celular/fisiología , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Humanos , Queratinocitos/efectos de la radiación , Mitosis/efectos de la radiación , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Quinasas/efectos de la radiación , Ratas , Fase S/efectos de la radiación , Serina/metabolismo , Serina/efectos de la radiación , Fosfatasas cdc25/metabolismo , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Quinasas/metabolismo , Fosfatasas cdc25/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN , Activación Enzimática , Células HeLa , Humanos , Cinética , Modelos Biológicos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Radiación Ionizante , Fase S/efectos de la radiación , Serina/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.
Asunto(s)
Ciclo Celular/genética , Replicación del ADN/efectos de la radiación , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Fosfatasas cdc25/fisiología , Alelos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Línea Celular , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN , Humanos , Ratones , Fosforilación , Proteínas Quinasas/genética , Tolerancia a Radiación , Radiación Ionizante , Fase S/efectos de la radiación , Serina/metabolismo , Transducción de Señal , Transfección , Proteínas Supresoras de Tumor , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
The activation of cell cycle checkpoints in response to genotoxic stressors is essential for the maintenance of genomic integrity. Although most prior studies of cell cycle effects of UV irradiation have used UVC, this UV range does not penetrate the earth's atmosphere. Thus, we have investigated the mechanisms of ultraviolet B (UVB) irradiation-induced cell cycle arrest in a biologically relevant target cell type, the early stage human melanoma cell line, WM35. Irradiation of WM35 cells with UVB resulted in arrests throughout the cell cycle: at the G1/S transition, in S phase and in G2. G1 arrest was accompanied by increased association of p21 with cyclin E/cdk2 and cyclin A/cdk2, increased binding of p27 to cyclin E/cdk2 and inhibition of these kinases. A loss of Cdc25A expression was associated with an increased inhibitory phosphotyrosine content of cyclin E- and cyclin A-associated cdk2 and may also contribute to G1 arrest following UVB irradiation. The association of Cdc25A with 14-3-3 was increased by UVB. Reduced cyclin D1 protein and increased binding of p21 and p27 to cyclin D1/cdk4 complexes were also observed. The loss of cyclin D1 could not be attributed to inhibition of either MAPK or PI3K/PKB pathways, since both were activated by UVB. Cdc25B levels fell and the remaining protein showed an increased association with 14-3-3 in response to UVB. Losses in cyclin B1 expression and an increased binding of p21 to cyclin B1/cdk1 complexes also contributed to inhibition of this kinase activity, and G2/M arrest. Oncogene (2000) 19, 4480 - 4490.
Asunto(s)
Ciclo Celular/efectos de la radiación , Melanoma/patología , Proteínas Musculares , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/efectos de la radiación , Ciclinas/metabolismo , Ciclinas/efectos de la radiación , Humanos , Melanoma/metabolismo , Melanoma/radioterapia , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/efectos de la radiación , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de la radiación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de la radiación , Proteínas Quinasas/metabolismo , Proteínas Quinasas/efectos de la radiación , Proteínas/metabolismo , Proteínas/efectos de la radiación , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/efectos de la radiación , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/efectos de la radiación , Rayos Ultravioleta , Fosfatasas cdc25/metabolismo , Fosfatasas cdc25/efectos de la radiaciónRESUMEN
The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.