RESUMEN
PURPOSE: Ribonucleotide reductase is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleotide triphosphates, which are utilized in both DNA synthesis and DNA repair. We reported previously that RR enzyme activity and R2 (catalytic subunit of RR) protein levels were increased after exposure to ionizing radiation (IR) in growth-arrested human tumor cells, suggesting that R2 protein expression regulates RR activity to allow for IR damage repair. Using isogenic human nasopharyngeal carcinoma cells in this study, we examine the relationship of overexpression of either the R1 regulatory subunit or the R2 catalytic subunit of RR to the cellular response of IR damage. MATERIALS AND METHODS: We used three isogenic human nasopharyngeal cancer cell lines previously derived by Zhou et al, including KB, the parental tumor cell line; KB/M1, an R1 protein-overexpressing clone stably transfected with human R1 complementary DNA; and KB/M2, a R2 protein-overexpressing clone stably transfected with human R2 complementary DNA. We initially characterized these isogenic human tumor cell lines in exponential growth for R2 protein expression, RR enzyme activity, and R2 protein changes during the cell cycle by flow cytometry. Subsequently, the IR response in these cell lines was determined by clonogenic survival, cell cycle changes occurring after IR, and an analysis of IR DNA damage determined by pulsed field gel electrophoresis. The effect of combining IR and hydroxyurea, a RR (R2) inhibitor, was also studied in KB and KB/M2 cells. RESULTS: KB/M2 cells were found to have 4.5-fold higher R2 protein expression and a threefold higher RR enzyme activity in exponential growth than KB and KB/M1. Although R2 protein levels increased at the G1/S transition in all cell lines, KB/M2 cells also demonstrated consistently higher R2 protein levels throughout the cell cycle. Using a linear-quadratic analysis of IR clonogenic survival data, KB/M2 cells were more radioresistant than KB and KB/M1 cells, including both decreased alpha and decreased beta values, a finding that correlates with increased reparable IR damage. KB/M2 cells also show a reduced G2 cell cycle arrest and fewer DNA double strand breaks 18 hours after IR (6 Gy). Exposure of KB/M2 cells to hydroxyurea (300 microM) after exposure to IR restored in vitro radiosensitivity in a manner similar to that found in KB and KB/M1 cells. DISCUSSION: An increase in R2 protein levels and RR activity in KB/M2 cells results in IR resistance, which appears mediated by enhanced IR damage repair during G2. R1 protein overexpression in these isogenic human tumor cells (KB/M1) did not affect RR activity or IR response.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma , Reparación del ADN , Inhibidores Enzimáticos/farmacología , Hidroxiurea/farmacología , Neoplasias Nasofaríngeas , Fármacos Sensibilizantes a Radiaciones/farmacología , Ribonucleósido Difosfato Reductasa/metabolismo , Western Blotting , Carcinoma/tratamiento farmacológico , Carcinoma/enzimología , Carcinoma/radioterapia , Ciclo Celular , Línea Celular Tumoral , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de la radiación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Técnicas In Vitro , Células KB/efectos de los fármacos , Células KB/efectos de la radiación , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/enzimología , Neoplasias Nasofaríngeas/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Ribonucleósido Difosfato Reductasa/efectos de los fármacos , Ribonucleósido Difosfato Reductasa/genética , Transfección , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
We previously reported that enhanced active efflux of cisplatin and increased GSH level were observed in KCP-4 cells. In the present study, KCP-4 cells were found to be cross-resistant to ultraviolet (UV) compared with parental KB-3-1 cells. Enhanced nucleotide excision repair (NER) was verified by time-dependent repair of UV-induced DNA damage. In addition, the amount of platinum bound to DNA after exposure to cisplatin decreased in a time-dependent manner in KCP-4 cells and this was reversed by aphidicolin, a DNA polymerase inhibitor. In stationary phase cultures, aphidicolin increased the sensitivity of KCP-4 cells to cisplatin. The expression of xeroderma pigmentosum complementation group F (XPF), an endonuclease involved in NER, was upregulated in KCP-4 cells. In KCP-4 cells the expression of hMSH6, one of the mismatch repair (MMR) factors, was decreased compared to parental KB-3-1 and revertant KCP-4R cells. However, KCP-4 cells were cross-resistant to oxaliplatin, and microsatellite instability was not observed in them. These findings suggest that the enhanced NER activity for DNA damage caused by cisplatin may be involved in cisplatin resistance in KCP-4 cells.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Daño del ADN/efectos de la radiación , Reparación del ADN/fisiología , ADN de Neoplasias/fisiología , Resistencia a Antineoplásicos , Células KB/efectos de los fármacos , Afidicolina/farmacología , Disparidad de Par Base/genética , ADN Polimerasa II/antagonistas & inhibidores , ADN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Células KB/metabolismo , Células KB/efectos de la radiación , Repeticiones de Microsatélite , Compuestos Organoplatinos/farmacología , Oxaliplatino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio , Tiazoles , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
Folate-diplasmenylcholine (1,2-di-O-(Z-1'-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC) liposomes have been shown to greatly enhance the potency of water-soluble antitumor agents via a selective folate-mediated uptake and acid-catalyzed endosomal escape mechanism (Rui et al. J. Am. Chem. Soc., 1998; 120:11213--18). This study describes an adaptation of this strategy for the delivery of chloroaluminum phthalocyanine tetrasulfonate ([AlPcS(4)](4-)), a water-soluble sensitizer used in photodynamic therapy, in a binary targeting scheme designed to enhance both its tumor selectivity and phototoxicity. [AlPcS(4)](4-)/DPPlsC:folate liposomes (9.8 microM bulk concentration, 2.5 mM intraliposomal concentration) were substantially more phototoxic to folate-deficient KB cells than 12.5 microM free [AlPcS(4)](4-) after a 30 min irradiation (630-910 nm). Considerable differences in phototoxicity were observed, however, between the commercially-available AlPcS(4)(4-) and an HPLC purified sample of [AlPcS(4)](4-) due to an increased tendency for the latter to aggregate. Experiments with [AlPcS(4)](4-)/DPPC:folate and folate-free [AlPcS(4)](4-)/DPPlsC liposomes (acid-insensitive and non-targeted controls, respectively) showed significantly reduced phototoxicities under the same illumination conditions. Our results imply that higher concentrations of water-soluble sensitizers can be delivered to target cells using the folate receptor-mediated pathway, which can change both the biodistribution and intracellular localization of the sensitizer when acid-labile DPPlsC liposomes are used as the delivery vehicle. Potential advantages of this approach include the use of lower bulk [AlPcS(4)](4-) concentrations, rapid plasma clearance of free [AlPcS(4)](4-), and better phototoxic responses, due to higher intracellular [AlPcS(4)](4-) concentrations combined with reduced collateral photodamage arising from misguided sensitizer accumulation, thereby enhancing the selective phototoxicity of PDT treatments.
Asunto(s)
Sistemas de Liberación de Medicamentos , Ácido Fólico/química , Indoles/administración & dosificación , Células KB/efectos de los fármacos , Compuestos Organometálicos/administración & dosificación , Fotoquimioterapia , Fármacos Fotosensibilizantes/administración & dosificación , Plasmalógenos/química , Aluminio , Transporte Biológico/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Dermatitis Fototóxica , Portadores de Fármacos , Humanos , Indoles/metabolismo , Células KB/metabolismo , Células KB/efectos de la radiación , Liposomas , Luciferasas/metabolismo , Microscopía Confocal , Estructura Molecular , Compuestos Organometálicos/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismoRESUMEN
DNA damage induced by irradiation causes overexpression of the p53 gene, and subsequently the upregulation of p53 downstream genes involved in cell cycle modification. Irradiated malignant cells which possess wild-type p53 have been known to undergo G1 arrest due to p21/Cip1/Waf1 upregulation. Other p53 downstream genes related to the modification of the cell cycle such as gadd45 may cause G2 arrest. Many of the genes which regulate the cell cycle progression have been identified, including the G1 phase specific ink4 family of cyclin-dependent kinase inhibitors (CDK-I), another group of CDK-Is, which affect the cyclin-CDK complexes ubiquitously, and S/G2 accelerator genes. The sequential changes in these cell cycle regulator genes after irradiation has not been clarified. We analyzed the appearance of the apoptotic fraction and cell cycle perturbation after irradiation using KB, a human squamous cell carcinoma line derived from oral floor, and examined the alteration of gene expression for cell cycle regulator genes. The KB cells proceeded to undergo apoptosis in a time and dose dependent manner after irradiation and showed G2 arrest accompanied by upregulation of p53, ubiquitous CDK-Is, and S and G2 accelerator genes.
Asunto(s)
Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/biosíntesis , ADN de Neoplasias/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células KB/efectos de la radiación , Neoplasias de la Boca/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Saccharomyces cerevisiae , Proteínas Supresoras de Tumor , Apoptosis/genética , Apoptosis/efectos de la radiación , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , Ciclinas/biosíntesis , Ciclinas/genética , Daño del ADN , ADN de Neoplasias/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Fase G2/efectos de la radiación , Genes p16 , Genes p53 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células KB/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Proteínas Motoras Moleculares , Proteínas de Neoplasias/genética , Biosíntesis de Proteínas , Proteínas/genética , Proteínas Quinasas Asociadas a Fase-S , Proteína p53 Supresora de Tumor/biosíntesis , Proteinas GADD45RESUMEN
Exposure to solar ultraviolet (UV) B radiation is responsible for skin inflammation and tumour progression. Cyclooxygenase and ornithine decarboxylase are believed to be involved in such processes since they participate in the synthesis of mediators of inflammation and cell differentiation, respectively. We have investigated the in vitro modulation of expression of such genes by UVB radiation in different skin cell lines. We have observed that accumulation of ornithine decarboxylase mRNA is unaffected by even high UVB doses in both human epidermal keratinocytes and dermal fibroblasts, whereas cyclooxygenase-2 levels were significantly up-regulated by low UVB doses in KB human epidermoid keratinocytes. Depletion of total intracellular glutathione levels in KB cells amplified the activation, revealing a role for an oxidative component of UVB in modulating cyclooxygenase gene expression. Transfer of medium from UVB irradiated keratinocytes to fibroblasts resulted in a significant activation of cyclooxygenase expression and activity, while ornithine decarboxylase levels were unaffected. We conclude that UVB radiation can activate cyclooxygenase gene expression in human skin cells both by direct activation pathways or indirectly by inducing a paracrine mechanism.
Asunto(s)
Fibroblastos/efectos de la radiación , Isoenzimas/biosíntesis , Queratinocitos/efectos de la radiación , Ornitina Descarboxilasa/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Piel/efectos de la radiación , Línea Celular Transformada/enzimología , Línea Celular Transformada/efectos de la radiación , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Ciclooxigenasa 2 , Citocinas/metabolismo , Citocinas/farmacología , Activación Enzimática/efectos de la radiación , Inducción Enzimática/efectos de la radiación , Epidermis/enzimología , Epidermis/efectos de la radiación , Feto , Fibroblastos/enzimología , Fibroblastos/metabolismo , Glutatión/metabolismo , Humanos , Células KB/enzimología , Células KB/efectos de la radiación , Queratinocitos/enzimología , Masculino , Proteínas de la Membrana , Proteínas de Neoplasias/biosíntesis , Estrés Oxidativo , Pene , Prostaglandinas/biosíntesis , ARN Mensajero/análisis , ARN Neoplásico/análisis , Piel/enzimologíaRESUMEN
The radioresistant N10 and parental KB cell lines were examined for the expression of human DNA repair genes which were related to the repair of radiation-induced DNA damage by northern blot analysis using five kinds of DNA probes (XRCC1, XRCC3, XRCC5, RAD51, RAD52). In the unirradiated condition, N10 cells showed higher expression of XRCC1, XRCC3 and RAD51 mRNA than did KB cells. The X-irradiation induced a time-dependent increase in the mRNA levels of XRCC3 and RAD51 in both cell lines with a maximum at 2 h postirradiation. The XRCC1 mRNA in N10 was maintained at the same level even after irradiation, whereas that in KB was decreased after irradiation. There was no difference in the expression of XRCC5 and RAD52 mRNA between N10 and KB cells in both unirradiated and irradiated conditions. From these findings, it was suggested that XRCC1, XRCC3 and RAD51 contribute to the radioresistance in cell line N10.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Northern Blotting/métodos , Línea Celular , ADN Complementario , Humanos , Células KB/patología , Células KB/efectos de la radiación , ARN Neoplásico/aislamiento & purificación , Recombinasa Rad51 , Tolerancia a Radiación , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Mitogen-activated protein kinases (MAPKs) mediate many of the cellular effects of growth factors, cytokines and stress stimuli. Their activation requires the phosphorylation of a threonine and a tyrosine residue located in a Thr-X-Tyr motif (where X is any amino acid) [1]. This phosphorylation is catalysed by MAPK kinases (MKKs), which are all thought to be 'dual specificity' enzymes that phosphorylate both the threonine and the tyrosine residue of the Thr-X-Tyr motif [2]. Here, we report that the MAPK family member known as stress-activated protein kinase-1c (SAPK1c, also known as JNK1) [3] is activated synergistically in vitro by MKK4 ([4] [5] [6]; also called SKK1 and JNKK1) and MKK7 ([7] [8] [9]; also called SKK4 and JNKK2). We found that MKK4 had a preference for the tyrosine residue, and MKK7 for the threonine residue, within the Thr-X-Tyr motif. These observations suggest that the full activation of SAPK1c in vivo may sometimes require phosphorylation by two different MKKs, providing the potential for integrating the effects of different extracellular signals. They also raise the possibility that other MAPK family members may be activated by two or more MKKs and that some MKKs may have gone undetected because they phosphorylate the tyrosine residue only, and therefore do not induce any activation unless the threonine has first been phosphorylated by another MKK.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Activación Enzimática , Humanos , Interleucina-1/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Células KB/efectos de los fármacos , Células KB/efectos de la radiación , MAP Quinasa Quinasa 7 , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia , Especificidad por Sustrato , Treonina/metabolismo , Tirosina/metabolismo , Rayos UltravioletaRESUMEN
The Y-box binding protein, YB-1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB-1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB-1 in the nucleus at 20 min and thereafter. This translocation of YB-1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H-7, but not HA-1004. Both green fluorescent protein (GFP)-YB-1 and GFP-YB-1C with the C-terminus (248-317) of YB-1 were located mainly in the cytosol, but GFP-YB-1deltaC with a deletion at the C-terminus of YB-1 was located in the nucleus. YB-1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C-mediated signal transduction pathway, and the C-terminal region of YB-1 might be important for cytoplasmic retention of YB-1.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células KB/efectos de la radiación , Sulfonamidas , Rayos Ultravioleta , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Células COS , Núcleo Celular/efectos de la radiación , Proteínas de Unión al ADN/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes , Humanos , Isoquinolinas/farmacología , Proteínas Luminiscentes/metabolismo , Factores de Transcripción NFI , Proteínas Nucleares , Inhibidores de Proteínas Quinasas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria , Proteína 1 de Unión a la Caja YRESUMEN
Interleukin-1 receptor antagonist (IL-1RA) is a cytokine that acts to antagonize IL-1 activity without agonist function. The expression of IL-1RA has been reported in many cell types, including the keratinocyte that covers the outer most part of the skin. However the modulation of IL-1RA by ultraviolet B (UVB), which is the most biologically active UV, has not been reported yet. We therefore selected a keratinocyte cell line with a cytokine-producing profile similar to that of keratinocytes and tested the effect of UVB on its ability to produce IL-1RA mRNA. IL-1RA mRNA was constitutively expressed in the cell line and began to be suppressed by 3 h after the UVB irradiation with 100 mJ/cm2. The level of IL-1RA expression became lowest by 16 h after the irradiation with 100 mJ/cm2. Simultaneously, IL-1 alpha mRNA started to increase by 1 h and peaked by 3-16 h after the irradiation with 10-100 mJ/cm2. The differential expression of IL-1 alpha and IL-1RA mRNA following exposure to a high dose (100 mJ/cm2) of UVB may markedly potentiate the role of IL-1 in UV-induced inflammation.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Interleucina-1/genética , Interleucina-1/efectos de la radiación , ARN Mensajero/efectos de la radiación , Proteínas Recombinantes/genética , Proteínas Recombinantes/efectos de la radiación , Sialoglicoproteínas/genética , Sialoglicoproteínas/efectos de la radiación , Rayos Ultravioleta , Southern Blotting , Carcinoma de Células Escamosas/genética , Línea Celular , Cartilla de ADN , Sondas de ADN , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/análisis , Células KB/inmunología , Células KB/metabolismo , Células KB/efectos de la radiación , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Piel/metabolismo , Piel/efectos de la radiación , Factores de Tiempo , Rayos Ultravioleta/clasificaciónRESUMEN
Epidermal cells (EC) are well known as a source of cytokines, including interleukin (IL)-6. In the present study, we investigated whether ultraviolet (UV) light and corticosteroids (CS) affect IL-6 production by normal (HNK) or malignant (KB) human keratinocytes. Supernatants derived from UVB (100 J/m2)- but not from UVA (100-1500 kJ/m2)-exposed EC (HNK and KB) contained significantly increased levels of IL-6 activity. This was also confirmed by Western blot analysis, resulting in specific bands at 23 kD and 27 kD. Northern blot analysis revealed an enhanced IL-6 mRNA expression after UVB exposure. Addition of hydrocortisone, prednisolone, or dexamethasone immediately after UVB irradiation significantly blocked UVB or IL-1-induced IL-6 mRNA expression and production by EC. The suppressive effect was observed at doses in the physiologic (10(-7)-10(-9) M) as well as pharmacologic (10(-5)-10(-7) M) range. In contrast, the nonactive steroid prednisone did not affect EC IL-6 mRNA expression. These findings indicate that increased IL-6 production by EC after UVB irradiation may mediate local and systemic inflammatory reactions following extensive sun exposure. Thus, the therapeutic effect of corticosteroids observed in various inflammatory diseases may be partly due to their downregulating capacity of IL-6 production.