RESUMEN
PURPOSE: Prostate cancer (PCa) is a leading cause of cancer-related death. Upon androgen-deprivation therapy, the disease may progress further to castration-resistant PCa (CRPC) with a poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs, which play crucial roles in gene regulation. The aim of our study is to find CRPC-associated miRNAs and to evaluate their functional role. METHODS: In this study, 23 benign prostatic hyperplasia (BPH), 76 primary PCa, and 35 CRPC specimens were included. Total RNA extracted from tissue sections was used for miRNA profiling on the Affymetrix GSC 3000 platform. Subsequently, stem-loop RT-qPCR analysis was performed to validate the expression levels of selected miRNAs. PCa cell lines were transfected with miRNA mimics or inhibitors to evaluate the effects on cell proliferation, cell migration and cell invasion. RESULTS: In our profiling study, several miRNAs were found to be deregulated in CRPC compared to primary PCa tissue, of which miR-205 (- 4.5-fold; p = 0.0009), miR-92b (- 3.1 fold; p < 0.0001) were downregulated and miR-3195 (5.6-fold; p < 0.0001), miR-3687 (8.7-fold; p = 0.0006) and miR-4417 (5.0-fold; p = 0.0005) were most upregulated. While KLK3, miR-21 and miR-141 expression levels in androgen-treated VCaP and LNCaP cells were increased, the expression levels of miR-3687 and miR-4417 were reduced. None of the miRNAs were androgen-regulated in the AR-negative PC3 cell line. Overexpression of miR-3687 reduced cell migration and cell invasion, whilst miR-3195 enhanced cell migration. CONCLUSION: We have identified several novel deregulated miRNAs in CRPC tissue, including two microRNAs that are potentially involved in tumor invasion. Our data support the hypothesized involvement of miRNAs in PCa tumorigenesis and progression to CRPC. The applicability of these miRNAs as novel biomarkers for CRPC remains to be further investigated.
Asunto(s)
MicroARNs , Hiperplasia Prostática , Neoplasias de la Próstata Resistentes a la Castración , Anciano , Humanos , Masculino , Persona de Mediana Edad , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , MicroARNs/genética , Invasividad Neoplásica , Células PC-3 , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Regulación hacia ArribaRESUMEN
BACKGROUND: Salivary duct carcinoma (SDC) is an aggressive subtype of salivary gland cancer. Approximately half of SDC patients will develop recurrences or metastases. Therapeutic palliative therapy is therefore often needed. The majority of SDC tumors expresses the androgen receptor (AR) and one-third expresses human epidermal growth factor receptor 2 (HER2), both are potential therapeutic targets. The aim of this paper is to systematically review and summarize the evidence on systemic palliative therapy for SDC and to provide treatment recommendations. MATERIALS AND METHODS: Electronic libraries were systematically searched with a broad search strategy to identify studies where SDC patients received systemic therapy. Due to the rarity of SDC no restrictions were placed on study designs. RESULTS: The search resulted in 2014 articles of which 153 were full-text analyzed. Forty-five studies were included in the analysis, which included in total 256 SDC patients receiving systemic therapy. Two phase 2 trials primarily including SDC patients were identified. The majority of the studies were case series or case reports, resulting in an overall low quality of available evidence. Based on studies including ≥ 5 SDC patients, objective responses to HER2 targeting agents were observed in 60-70%, to AR pathway agents in 18-53% and to chemotherapy in 10-50%. CONCLUSION: For AR or HER2 positive SDC, agents targeting these pathways are the cornerstone for palliative treatment. Regarding chemotherapy, the combination of carboplatin combined with a taxane is best studied. Regarding other targeted agents and immunotherapy evidence is anecdotal, limiting formulation of treatment recommendations for these antineoplastic agents.
Asunto(s)
Carcinoma Ductal/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Conductos Salivales/patología , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Carcinoma Ductal/metabolismo , Carcinoma Ductal/patología , Ensayos Clínicos como Asunto , Humanos , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Cuidados Paliativos/métodos , Receptor ErbB-2/metabolismo , Conductos Salivales/metabolismo , Neoplasias de las Glándulas Salivales/metabolismo , Neoplasias de las Glándulas Salivales/patologíaRESUMEN
AIM: Salivary duct carcinoma (SDC), an aggressive subtype of salivary gland cancer, is androgen receptor (AR)-positive in 67-96% of cases. In patients with locally recurrent and metastatic (R/M) AR-positive SDC, androgen deprivation therapy (ADT) has an overall response rate of 18-64.7%. In this study, we describe the efficacy of adjuvant ADT in patients with poor-risk (stage 4a) AR-positive SDC. METHODS: This is a retrospective cohort study in which patients with stage 4a AR-positive SDC were offered adjuvant ADT, i.e. bicalutamide, luteinizing hormone-releasing hormone (LHRH) analogue or a combination of these after tumour resection. In the control group, data were collected on patients with stage 4a SDC who underwent a tumour resection but did not receive adjuvant ADT. RESULTS: Twenty-two AR-positive SDC patients were treated with adjuvant ADT for a median duration of 12 months. The control group consisted of 111 SDC patients. After a median follow-up of 20 months in the ADT-treated patients and 26 months in the control group, the 3-year disease-free survival (DFS) was estimated as 48.2% (95% confidence interval [CI] 14.0-82.4%) and 27.7% (95% CI 18.5-36.9%) (P = 0.037). Multivariable Cox regression analysis showed a hazard ratio of 0.138 (95% CI 0.025-0.751, P = 0.022) for DFS and 0.064 (95% CI 0.005-0.764, P = 0.030) for overall survival (OS) in favour of the ADT-treated patients. CONCLUSION: Poor-risk, AR-positive SDC patients who received adjuvant ADT have a significantly longer DFS compared with patients in the control group, who did not receive adjuvant ADT. For OS, this was just below and above the significance level, in case there was or was no correction for confounders.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Quimioterapia Adyuvante , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores Androgénicos/metabolismo , Factores de Riesgo , Conductos Salivales , Resultado del TratamientoRESUMEN
AIM: The molecular interactions between transient receptor potential vanilloid subtype 4 channels (TRPV4) and cell junction formation were investigated in the human and mouse urogenital tract. MATERIALS AND METHODS: A qualitative study was performed to investigate TRPV4 channels, adherence junctions (AJs) and tight junctions (TJs) in kidney, ureter and bladder tissues from humans and wild-type and transgenic TRPV4 knockout (-/-) mice with immunohistochemistry, Western blotting, immunoprecipitation and reverse trasnscription-PCR. Cell junction formation in the wild-type and TRPV4 knockout (-/-) mouse was evaluated with immunohistochemistry and transmission electron microscope (TEM) techniques. RESULTS: TRPV4 channels are predominantly located in membranes of epithelial cells of the bladder, ureter and the collecting ducts of the kidney. There is a molecular interaction between the TRPV4 channel and the AJ. TEM evaluation showed that AJ formation is disrupted in the TRPV4 -/- mouse resulting in deficient intercellular connections and integrity of the epithelium. CONCLUSIONS: TRPV4 is believed to be a mechanoreceptor in the bladder. This study demonstrates that TRPV4 is also involved in intercellular connectivity and structural integrity of the epithelium.
Asunto(s)
Barrera Hematonerviosa/fisiología , Uniones Intercelulares/fisiología , Canales Catiónicos TRPV/fisiología , Sistema Urogenital/metabolismo , Animales , Barrera Hematonerviosa/ultraestructura , Humanos , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Riñón/fisiología , Riñón/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Canales Catiónicos TRPV/metabolismo , Vejiga Urinaria/fisiología , Vejiga Urinaria/ultraestructura , Sistema Urogenital/ultraestructura , Urotelio/fisiología , Urotelio/ultraestructuraRESUMEN
Prostate cancer (PCa) is the second most common diagnosed malignant disease in men worldwide. Although serum PSA test dramatically improved the early diagnosis of PCa, it also led to an overdiagnosis and as a consequence to an overtreatment of patients with an indolent disease. New biomarkers for diagnosis, prediction, and monitoring of the disease are needed. These biomarkers would enable the selection of patients with aggressive or progressive disease and, hence, would contribute to the implementation of individualized therapy of the cancer patient. Since the FDA approval of the long noncoding PCA3 RNA-based urine test for the diagnosis of PCa patients, many new noncoding RNAs (ncRNAs) associated with PCa have been discovered. According to their size and function, ncRNAs can be divided into small and long ncRNAs. NcRNAs are expressed in (tumor) tissue, but many are also found in circulating tumor cells and in all body fluids as protein-bound or incorporated in extracellular vesicles. In these protected forms they are stable and so they can be easily analyzed, even in archival specimens. In this review, the authors will focus on ncRNAs as novel biomarker candidates for PCa diagnosis, prediction, prognosis, and monitoring of therapeutic response and discuss their potential for an implementation into clinical practice.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de la Próstata , ARN no Traducido , Humanos , Masculino , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismoRESUMEN
Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50 <75 ng) was found for the CMV promoter in all cell lines, which is in agreement with the dual luciferase assay findings. Unlike the dual luciferase findings, however, the DT-tox176 assay showed protein inhibition of CN65 (PSA promoter/enhancer) and PSE-hK2 (PSA enhancer and basal human kallikrein 2 promoter) in HEK293 and DLD cells indicating "leakiness" of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT-tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay.
Asunto(s)
Bioensayo/métodos , Perfilación de la Expresión Génica/métodos , Terapia Genética , Regiones Promotoras Genéticas/genética , Toxina Diftérica , Expresión Génica , Vectores Genéticos , Humanos , Masculino , Especificidad de Órganos , Próstata , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Células Tumorales CultivadasRESUMEN
Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.
Asunto(s)
Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Secuencia de Bases , Huella de ADN , ADN de Neoplasias , Humanos , Masculino , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the Western male population. Despite the tremendous efforts that have been made to improve the early detection of this disease and to design new treatment modalities, there is still an urgent need for new markers and therapeutic targets for the management of prostate cancer patients. Using differential display analysis to compare the mRNA expression patterns of normal versus tumor tissue of the human prostate, we identified a cDNA, DD3, which is highly overexpressed in 53 of 56 prostatic tumors in comparison to nonneoplastic prostatic tissue of the same patients. Reverse transcription-PCR analysis using DD3-specific primers indicated that the expression of DD3 is very prostate specific because no product could be amplified in 18 different normal human tissues studied. Also, in a sampling of other tumor types and a large number of cell lines, no expression of DD3 could be detected. Molecular characterization of the DD3 transcription unit revealed that alternative splicing and alternative polyadenylation occur. The fact that no extensive open reading frame could be found suggests that DD3 may function as a noncoding RNA. The DD3 gene was mapped to chromosome 9q21-22, and no homology of DD3 to any gene present in the computer databases was found. Our data indicate that DD3 is one of the most prostate cancer-specific genes yet described, and this makes DD3 a promising marker for the early diagnosis of prostate cancer and provides a powerful tool for the development of new treatment strategies for prostate cancer patients.
Asunto(s)
Cromosomas Humanos Par 9 , Regulación Neoplásica de la Expresión Génica , Próstata/metabolismo , Neoplasias de la Próstata/genética , Transcripción Genética , Secuencia de Bases , Mapeo Cromosómico , Exones , Humanos , Intrones , Cariotipificación , Masculino , Datos de Secuencia Molecular , Neoplasias de la Próstata/cirugía , ARN Mensajero/genética , Valores de Referencia , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.
Asunto(s)
Anomalías Múltiples/genética , Transformación Celular Viral , Rotura Cromosómica , Fibroblastos/virología , Anomalías Múltiples/patología , Antineoplásicos/farmacología , Bleomicina/farmacología , Camptotecina/farmacología , Línea Celular , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Preescolar , ADN/efectos de los fármacos , ADN/genética , ADN/efectos de la radiación , Daño del ADN , Reparación del ADN , Etopósido/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Células HeLa , Humanos , Mitomicina/farmacología , Mutación , Síndrome , Rayos XRESUMEN
The transcription factor p53 controls the proliferation and survival of cells exposed to DNA damage. The specific DNA-binding domain of p53 (residues 102-292) has a complex tertiary structure that is stabilized by zinc. In this study, we showed that exposure of cultured cells to the membrane-permeable chelator N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine induced wild-type p53 to accumulate in an immunologically "mutant" form (PAb240+, PAb1620-) with decreased DNA-binding activity. Removal of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine from culture medium allowed p53 to refold into the immunologically wild-type form, followed by a transient increase in DNA binding, expression of the cyclin-dependent kinase inhibitor p21WAF1, and cell-cycle delay in the G1 phase. Thus, modulation of intracellular zinc induced conformational changes in p53 that activated wild-type function, suggesting that metalloregulation may play a role in controlling p53.
Asunto(s)
Quelantes/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Etilenodiaminas/farmacología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Zinc/química , Zinc/metabolismo , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Cicloheximida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Células Tumorales CultivadasRESUMEN
Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.
Asunto(s)
Antioxidantes/farmacología , Quelantes/farmacología , Cobre/metabolismo , Proteínas Serina-Treonina Quinasas , Tiocarbamatos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Acetilcisteína/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Proteínas de Ciclo Celular , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN , Humanos , Peróxido de Hidrógeno/farmacología , Sustancias Intercalantes/farmacología , Transporte Iónico/efectos de los fármacos , Peroxidación de Lípido , Oxidación-Reducción , Estrés Oxidativo , Fenantrolinas/farmacología , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Proteínas/fisiología , Pirrolidinas/farmacocinética , Tiocarbamatos/farmacocinética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteínas Supresoras de TumorRESUMEN
X-ray sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following ionizing radiation. To localize the defective hamster gene (XRCC8) on the human genome, human chromosomes were introduced into the AT-like hamster mutants, by microcell mediated chromosome transfer. Although, none of the human chromosomes corrected the defect in these mutants, the defect was corrected by a single mouse chromosome, derived from the A9 microcell donor cell line. In four independent X-ray-resistant microcell hybrid clones of V-E5, the presence of the mouse chromosome was determined by fluorescent in situ hybridization, using a mouse cot-1 probe. By PCR analysis with primers specific for different mouse chromosomes and Southern blot analysis with the mouse Ldlr probe, the mouse chromosome 9, was identified in all four X-ray-resistant hybrid clones. Segregation of the mouse chromosome 9 from these hamster-mouse microcell hybrids led to the loss of the regained X-ray-resistance, confirming that mouse chromosome 9 is responsible for complementation of the defect in V-E5 cells. The assignment of the mouse homolog of the ATM gene to mouse chromosome 9, and the presence of this mouse chromosome only in the radioresistant hamster cell hybrids suggest that the hamster AT-like mutant are homologous to AT, although they are not complemented by hamster chromosome 11.
Asunto(s)
Ataxia Telangiectasia/genética , Cricetinae/genética , Mutación , Tolerancia a Radiación/genética , Animales , Supervivencia Celular , Células Cultivadas , Aberraciones Cromosómicas , Cromosomas , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Prueba de Complementación Genética , Humanos , Ratones , Especificidad de la Especie , Rayos XRESUMEN
Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.
Asunto(s)
Cromosomas Humanos Par 4 , Daño del ADN/genética , Replicación del ADN/genética , Animales , Línea Celular , Aberraciones Cromosómicas/genética , Cricetinae , Relación Dosis-Respuesta en la Radiación , Técnicas de Transferencia de Gen , Humanos , Células Híbridas/efectos de la radiación , Hibridación Fluorescente in Situ , RatonesRESUMEN
It has been shown that the Chinese hamster cell mutant V-C8 is sensitive to different DNA damaging agents, such as mitomycin C (MMC), alkylating agents, UV light, and X-rays. We found that V-C8 is also sensitive to the following radiomimetic agents: bleomycin (approximately 2-fold, based on D10 values), H2O2 (approximately 2-fold), streptonigrin (approximately 11-fold), and etoposide (approximately 8-fold). Two independent spontaneous MMC-resistant revertants isolated from V-C8 cells show a level of cell killing by X-rays, EMS, and UV light which is similar to that of wild-type cells, suggesting that the observed pattern of cross-sensitivity of V-C8 cells to a wide spectrum of DNA damaging agents results from a single mutation. V-C8 cells also display radioresistant DNA synthesis following gamma-irradiation which, however, remained almost unchanged in the V-C8 revertants. The measurement of the level and rate of repair of DNA single- and double-strand breaks (SSBs and DSBs, respectively) by the DNA elution technique showed that the V-C8 mutant has a slower repair of DSBs induced by gamma-rays. The described unique phenotype of V-C8 cells suggested that V-C8 represents a novel type of mutant amongst X-ray-sensitive hamster cell mutants. To confirm this, complementation analysis with other X-ray-sensitive mutants was performed. V-C8 cells were fused with EM9, XR-1, xrs5, sxi-1, V-3, V-E5, irs3, and BLM2 mutant cells, representing different complementation groups. All the obtained hybrids regained X-ray resistance (or bleomycin resistance in the case of V-C8/BLM2 hybrids) similar to that of wild-type cells, indicating that V-C8 represents a new complementation group. The results presented indicate that V-C8 is defective in a gene involved in a pathway operating in the responses to different DNA damaging agents in mammalian cells.
Asunto(s)
Daño del ADN , Reparación del ADN , Replicación del ADN , Animales , Bleomicina/farmacología , Células CHO , Supervivencia Celular/efectos de la radiación , Cricetinae , ADN/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Radicales Libres , Rayos gamma , Prueba de Complementación Genética , Mitomicina/farmacología , Mutágenos/farmacología , Radiación Ionizante , Estreptonigrina/farmacología , Inhibidores de Topoisomerasa I , Rayos XRESUMEN
Cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) show an abnormal response to ionizing radiation-induced DNA damage, such as an increased cell killing and a diminished inhibition of DNA synthesis. The enhanced killing of A-T (group D) cells by X-rays can be corrected by multiple cDNAs, mapping to different chromosomes (6, 11, 17, and 18). In order to examine whether genes located on these chromosomes complement AT-D cells, normal neo-tagged chromosomes 6, 11, 17, and 18 were introduced into AT-D cells by microcell-mediated chromosome transfer. However, correction of the enhanced killing of AT-D cells by X-rays could only be achieved by chromosome 11 and by none of the other chromosomes tested. The enhanced killing of A-T (complementation group C) cells was also corrected by chromosome 11. Usually, but not in all microcell hybrid clones, chromosome 11 also corrected the radioresistant DNA synthesis (RDS) phenotype of AT-D and AT-C cells. These results (i) confirm findings by others suggesting assignment of the ATD and ATC genes to chromosome 11, (ii) demonstrate that several genes can modify the cellular radiation response when they are taken out of their normal genomic context and/or control, and (iii) indicate that the RDS phenotype and the enhanced cell killing in A-T are independent pleiotropic features resulting from the primary mutations in A-T. Also, our findings underscore that, in establishing cDNAs as candidate genes for A-T, microcell-mediated chromosome transfer studies are needed to exclude nonspecific correcting effects of these candidate cDNA genes.
Asunto(s)
Ataxia Telangiectasia/genética , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 6 , Prueba de Complementación Genética , Animales , Mapeo Cromosómico , ADN/biosíntesis , Relación Dosis-Respuesta en la Radiación , Humanos , Células Híbridas , Ratones , Fenotipo , Tolerancia a Radiación/genética , Rayos XRESUMEN
In order to isolate a human gene complementing the defect in A-T-like hamster cell mutants, the mutants were used as recipients for genomic DNA transfection, using either HeLa chromosomal DNA or DNA from a human cosmid library. Three primary transformants with an intermediate X-ray sensitivity and almost normal sensitivity to MMS, but retaining radioresistant DNA synthesis (RDS), were obtained. To identify the human chromosome that complements the defect in the A-T-like mutants, and to assess the degree of complementation for survival and RDS, microcell-mediated chromosome transfer was used. At least 20 independent hybrid clones between the mutant and each one of the human chromosomes 1, 2, 4, 5, 15, 17 or 18 were isolated. All hybrid clones remained X-ray sensitive, except one with chromosome 4, and another with chromosome 15, both showing an intermediate X-ray sensitivity. By using in situ hybridization we found that this partial correction was due to the presence of a mouse chromosome. In these two hybrids containing the mouse chromosome together with human chromosome 4 or 15, RDS was fully complemented only in the hybrid with chromosome 4 but not in the one containing chromosome 15, suggesting that RDS and X-ray sensitivity may be complemented independently.
Asunto(s)
Ataxia Telangiectasia/genética , Células CHO/fisiología , Células CHO/efectos de la radiación , ADN Complementario/genética , Mutación , Tolerancia a Radiación , Animales , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/fisiopatología , Cricetinae , ADN Complementario/biosíntesis , ADN Complementario/efectos de la radiación , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Prueba de Complementación Genética , Células HeLa , Humanos , Ratones , Transfección , Transformación Genética , Rayos XRESUMEN
The radiosensitive Chinese hamster V79 cell mutants (V-C4, V-E5 and V-G8), isolated previously in our laboratory, have been shown to resemble human ataxia telangiectasia (A-T) cells. These hamster cell mutants were further characterized with respect to cross-sensitivity to different radiomimetic agents and to mutation induction by X-rays. The data on cell survival (D10 values) show that they are hypersensitive to adriamycin (2-3-fold increase), etoposide (3-fold for V-G8 and 6-fold for V-E5 and V-C4), calicheamicin gamma 1I (4-fold) and streptonigrin (3-fold for V-G8 and V-C4, and 12-fold for V-E5). The frequency of X-ray-induced hprt mutations is slightly enhanced in the hamster mutant cells treated with the same dose. However, the mutants show similar mutability as parental V79 cells when considering the same survival level. The overall conclusion from these studies is that these hamster cell mutants mimic the phenotypic characteristics observed in cultured cells from A-T patients and, therefore, may be defective in the same repair pathway as their human counterparts.
Asunto(s)
Aminoglicósidos , Ataxia Telangiectasia/patología , Línea Celular , Mutación , Animales , Antibacterianos/farmacología , Ataxia Telangiectasia/genética , Línea Celular/efectos de los fármacos , Línea Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cricetinae , Doxorrubicina/farmacología , Enediinos , Etopósido/farmacología , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Mutágenos/farmacología , Estreptonigrina/farmacologíaRESUMEN
The relation of radiosensitivity and radioresistant DNA synthesis (RDS) was studied in the AT-like Chinese hamster cell mutants V-C4, V-E5, and V-G8 of the same complementation group. Proliferating hybrid cell lines obtained after fusion of the AT-like hamster cell mutants with their parental V79 cells showed only very partial complementation of RDS, while radiosensitivity with respect to cell killing was fully complemented in these hybrid cells. Therefore, RDS behaves as a (co-)dominant feature in hybrids of AT-like mutants and wild-type V79 cells, indicating that RDS cannot be used for complementation analysis of X-ray-sensitive Chinese hamster cells. In contrast to the intraspecies hybrids, a full complementation of RDS was observed in interspecies hybrids between the rodent AT-like mutants and human (HeLa) cells, suggesting a species-specific regulation of DNA synthesis. These results indicate that radiosensitivity and RDS are pleiotropic effects of the same afflicted gene.
Asunto(s)
Ataxia Telangiectasia/genética , Reparación del ADN , ADN/biosíntesis , Genes Dominantes , Animales , Línea Celular , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Rayos gamma , Prueba de Complementación Genética , Técnicas In Vitro , Rayos XRESUMEN
Differential hybridization analysis was used to identify molecular differences between a relative benign and a highly aggressive rat prostatic tumor derived from the Dunning R-3327-H adenocarcinoma. From the several differentially expressed mRNAs identified, we here report the characterization of pBUS51 which encodes a transcript highly expressed in all-anaplastic Dunning tumors. Only a very low expression was detectable in normal rat prostate or in the differentiated tumors of the Dunning system. Nucleotide sequence analysis and computer-assisted database comparison revealed that pBUS51 was highly homologous to vimentin and therefore likely the rat homolog of this protein.