RESUMEN
Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC(50) toxicity values seven- to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABC-transporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol-water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/farmacología , Doxorrubicina/farmacocinética , Esfingomielinas/farmacología , Adenocarcinoma , Animales , Neoplasias de la Mama/patología , Muerte Celular , Membrana Celular , Difusión , Sistemas de Liberación de Medicamentos , Interacciones Farmacológicas , Sinergismo Farmacológico , Células Endoteliales , Fibroblastos , Humanos , Ratones , Ratones Noqueados , Células Tumorales CultivadasRESUMEN
BACKGROUND AND PURPOSE: In a recent study on patients with transitional cell cancer of the bladder treated with curative radiotherapy following TUR-T, we demonstrated that a low apoptotic index and p53 positivity were associated with poor local control. The purpose of this study was to assess the prognostic significance of additional markers implicated in regulation of cell cycle and apoptosis. PATIENTS AND METHODS: Bcl-2, Bax and p21 positivity were detected immunohistochemically on paraffin-embedded pre-treatment biopsies from 83 patients with invasive transitional cell cancer (TCC) of the bladder, treated with radiotherapy. In addition, markers determined in an earlier analysis, i.e.: p53, apoptotic index, cyclin D1, retinoblastoma protein and Ki-67 were included in the multivariate analysis. A stepwise proportional hazard analysis was performed, adjusting for classic prognostic factors (T-stage, grade, multifocality and macroscopic completeness of the TUR). Positivity was defined as >10% of tumor cells staining positive for Bcl-2, Bax and p21, and >20% for p53. RESULTS: Bcl-2 positivity was found in 63%, Bax was positive in 52% and p21 in 55% of cases. In the PH analysis Bcl-2 positivity was found to be related to poor local control (36 vs. 72% at 3 years; P=0.003), as well as to shorter disease-specific survival (74 vs. 94% at 3 years; P=0.017). Evidence for an adverse effect of p53 positivity was also found (local control: 32 vs. 69% at 3 years;P=0.037, disease-specific survival: 76 vs. 92% at 3 years; P=0.043). In an additional PH analysis, we found poor local control rates for bladder cancers with combined Bcl-2 and p53 positivity (17 vs. 65% at 3 years; P=0.0017), and lower disease specific survival (60 vs. 92%; P=0.0024), disease-free survival (7 vs.35%, P=0.0023) and overall survival (39 vs. 80%; P=0.0018). CONCLUSION: This study provides evidence for a poor outcome in patients treated with radiotherapy for TCC of the bladder expressing both Bcl-2 and p53. This relationship was found for local control and disease-free, disease-specific and overall survival.
Asunto(s)
Carcinoma de Células Transicionales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/radioterapia , Ciclo Celular , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Estudios Retrospectivos , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/radioterapia , Proteína X Asociada a bcl-2RESUMEN
Synthetic alkyl-lysophospholipids (ALPs, also referred to as ether-phospholipids) have been studied as antitumor agents for more than a decade. Classical examples of these ALPs include 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); Edelfosine) and hexadecylphosphocholine (HePC; Miltefosine). Unlike most currently available chemotherapeutic drugs that target the nuclear DNA, ALPs exert their action at the plasma membrane level, where they interfere with mitogenic signal transduction pathways. Whereas malignant cells are highly sensitive to the lethal action of ALPs, normal cells remain relatively unaffected, illustrating the potential selective antitumor properties of this class of drugs. Recently, ALPs have regained interest because of their capacity to induce apoptosis in various tumor cell lines. Moreover, in combination with other (conventional) anticancer regimens, ALPs seem to cause an additive and sometimes synergistic cytotoxic effect. These biologic properties make ALPs attractive drugs for further clinical evaluation. The present review discusses recent insights into the mode(s) of action of ALPs, their interaction with ionizing radiation, and clinical application.
Asunto(s)
Antineoplásicos/uso terapéutico , Éteres Fosfolípidos/uso terapéutico , Fosforilcolina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Inhibidores de Fosfodiesterasa/uso terapéutico , Fosfolípidos/metabolismo , Fosforilcolina/uso terapéuticoRESUMEN
Alkyl-lysophospholipids (ALPs) represent a new class of antitumor drugs that induce apoptotic cell death in a variety of tumor cell lines. Although their precise mechanism of action is unknown, ALPs primarily act on the cell membrane, where they inhibit signaling through the mitogen-activated protein kinase (MAPK) pathway. Because stimulation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway is essential for radiation-induced apoptosis in certain cell types, we tested the effect of ALPs in combination with ionizing radiation on MAPK/SAPK signaling and apoptosis induction. Here, we present data showing that three ALPs, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, hexadecylphosphocholine, and the novel compound octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate (D-21266) induce time- and dose-dependent apoptosis in the human leukemia cell lines U937 and Jurkat T but not in normal vascular endothelial cells. Moreover, in combination with radiation, ALPs strongly enhance the induction of apoptosis in both leukemic cell lines. All tested ALPs not only prevented MAPK activation, but, like radiation, stimulated the SAPK/JNK cascade within minutes. A dominant-negative mutant of c-Jun inhibited radiation- and ALP-induced apoptosis, indicating a requirement for the SAPK/JNK pathway. Our data support the view that ALPs and ionizing radiation cause an enhanced apoptotic effect by modulating the balance between the mitogenic, antiapoptotic MAPK, and the apoptotic SAPK/JNK pathways. This type of modulation of specific signal transduction pathways in tumor cells may lead to the development of new therapeutic strategies.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Éteres Fosfolípidos/farmacología , Fosforilcolina/análogos & derivados , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/efectos de la radiación , Rayos gamma , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Células Jurkat/efectos de los fármacos , Células Jurkat/efectos de la radiación , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 12 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilcolina/farmacología , Transducción de Señal/efectos de la radiación , Células U937/efectos de los fármacos , Células U937/efectos de la radiaciónRESUMEN
In melanoma, the relationship between sun exposure and the origin of mutations in either the N-ras oncogene or the p53 tumour-suppressor gene is not as clear as in other types of skin cancer. We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to TA or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour.
Asunto(s)
Mutación del Sistema de Lectura , Genes p53 , Melanoma/genética , Neoplasias Inducidas por Radiación/genética , Mutación Puntual , Neoplasias Cutáneas/genética , Luz Solar/efectos adversos , Rayos Ultravioleta/efectos adversos , Sustitución de Aminoácidos , Exones , Genes ras , Humanos , Melanoma/etiología , Melanoma/patología , Metástasis de la Neoplasia , Neoplasias Inducidas por Radiación/patología , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Eliminación de Secuencia , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ionizing radiation, like a variety of other cellular stress factors, initiates apoptosis, or programmed cell death, in many cell systems. This mode of radiation-induced cell kill should be distinguished from clonogenic cell death due to unrepaired DNA damage. Ionizing radiation not only exerts its effect on the nuclear DNA, but also at the plasma membrane level where it may activate multiple signal transduction pathways. One of these pathways is the stress-activated protein kinase (SAPK) cascade which transduces death signals from the cell membrane to the nucleus. This review discusses recent evidence on the critical role of this signaling system in radiation- and stress-induced apoptosis. An improved understanding of the mechanisms involved in radiation-induced apoptosis may ultimately provide novel strategies of intervention in specific signal transduction pathways to favorably alter the therapeutic ratio in the treatment of human malignancies.
Asunto(s)
Apoptosis/fisiología , Apoptosis/efectos de la radiación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Transducción de Señal , Endotelio Vascular/patología , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Células Tumorales Cultivadas/patologíaRESUMEN
Human cutaneous melanoma is heterogeneous with respect to the genetic aberrations involved and the genes altered are potential targets for the immune system. The incidence of cutaneous melanoma is known to be linked to UV peak exposure, and the N-ras oncogene is clearly one of the genes involved in the UV carcinogenesis in melanoma. It is mutated in a significant proportion of melanomas and therefore may serve as a target for T cells. Here, we report that an human leukocyte antigen-A2 binding peptide CLLDILDTAGL, encompassing the frequently found 61-Leu mutation in N-ras, induces cytotoxic T lymphocytes from healthy donor blood that lyse 61-Leu N-ras transfected melanoma cells. Furthermore, we have found an association between the presence of N-ras mutations and clinical response to immunotherapy with interleukin-2 plus interferon in a group of stage IV melanoma patients. Although the overall survival of these patients was not affected by the N-ras status of their melanomas, these studies suggest that mutated N-ras may provide a target for cytotoxic T lymphocytes in melanoma patients.
Asunto(s)
Genes ras/efectos de la radiación , Inmunoterapia Adoptiva/métodos , Melanoma/genética , Melanoma/terapia , Mutación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/inmunología , Rayos Ultravioleta/efectos adversos , Proteínas ras/genética , Proteínas ras/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas ras/efectos de la radiaciónRESUMEN
Intermittent or recreational exposure to sunlight is thought to contribute to development of human cutaneous melanoma. We investigated the incidence of ras oncogene mutation in human cutaneous melanoma in connection to sun-exposed body sites in the patient, using a large series of DNA samples derived from paraffin-embedded material as well as from fresh tumor samples and cell lines. We first show that, of the ras family, predominantly N-ras is activated (15%), whereas rarely H-ras or K-ras are mutated. The occurrence of N-ras mutations correlates with continuous exposure to sunlight of the primary tumor site. Of all tumors initiated on chronically sun-exposed body sites, 26% contained mutated N-ras, in contrast to 0% of sun-protected melanomas. Melanoma lesions obtained from patients from North or Central Europe contained fewer N-ras mutations (12%) as compared with patients from Australia (24%). Mutations were specifically associated with nodular melanoma and to a lesser extent with lentigo malignant melanoma. N-ras mutations did not correlate with metastasis or survival parameters. This study identifies a subset of cutaneous melanomas that contain in the primary lesion ultraviolet-induced N-ras mutations, which are maintained through further progression.
Asunto(s)
Genes ras/efectos de la radiación , Melanoma/genética , Mutación Puntual/efectos de la radiación , Neoplasias Cutáneas/genética , Secuencia de Bases , ADN de Neoplasias/química , ADN de Neoplasias/aislamiento & purificación , Genes ras/genética , Humanos , Melanoma/patología , Datos de Secuencia Molecular , Adhesión en Parafina , Mutación Puntual/genética , Pronóstico , Estudios Retrospectivos , Neoplasias Cutáneas/patología , Luz Solar/efectos adversos , Rayos UltravioletaRESUMEN
Determination of the activation state of oncogenes as well as tumor suppressor genes is a main subject of interest in the analysis of the mechanism of tumor initiation. In human melanoma, the c-myc and N-ras oncogenes have been found to be activated in approximately 50% and 15% of the analyzed material, respectively. These studies have mostly been done on fresh tumor material or cell lines. Only in a few cases has an attempt been made to look at tumor heterogeneity or clonality with respect to the activation of oncogenes. We have adjusted the polymerase chain reaction (PCR)/single-stranded conformation polymorphism analysis (SSCP) technique to screen paraffin-embedded melanoma material for the presence of N-ras mutations and found genetic defects at particular progression stages. In one melanoma of the skin, we were able to sublocalize an N-ras mutation in the intraepidermal tumor part, that was absent in the part deeply invading the dermal layer. We conclude that a thorough investigation of N-ras activation in human melanoma should include analysis of histologically different parts of the tumor.
Asunto(s)
ADN de Neoplasias/genética , Genes ras , Melanoma/genética , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Neoplasias Cutáneas/genética , Secuencia de Bases , Análisis Mutacional de ADN , Humanos , Datos de Secuencia Molecular , Adhesión en ParafinaRESUMEN
Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.
Asunto(s)
ADN de Hongos/química , Precursores del ARN/metabolismo , ARN de Hongos/metabolismo , ARN Ribosómico/biosíntesis , Saccharomyces cerevisiae/genética , Secuencia de Bases , Northern Blotting , Análisis Mutacional de ADN , ADN de Hongos/genética , Escherichia coli/genética , Eliminación de Gen , Técnicas de Transferencia de Gen , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Precursores del ARN/química , ARN de Hongos/química , Relación Estructura-ActividadRESUMEN
The rate of ribosomal protein gene (rp-gene) transcription in yeast is accurately adjusted to the cellular requirement for ribosomes under various growth conditions. However, the molecular mechanisms underlying this co-ordinated transcriptional control have not yet been elucidated. Transcriptional activation of rp-genes is mediated through two different multifunctional transacting factors, ABF1 and RAP1. In this report, we demonstrate that changes in cellular rp-mRNA levels during varying growth conditions are not parallelled by changes in the in vitro binding capacity of ABF1 or RAP1 for their cognate sequences. In addition, the nutritional upshift response of rp-genes observed after addition of glucose to a culture growing on a non-fermentative carbon source turns out not to be the result of increased expression of the ABF1 and RAP1 genes or of elevated DNA-binding activity of these factors. Therefore, growth rate-dependent transcription regulation of rp-genes is most probably not mediated by changes in the efficiency of binding of ABF1 and RAP1 to the upstream activation sites of these genes, but rather through other alterations in the efficiency of transcription activation. Furthermore, we tested the possibility that cAMP may play a role in elevating rp-gene expression during a nutritional shift-up. We found that the nutritional upshift response occurs normally in several mutants defective in cAMP metabolism.