RESUMEN
Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eµ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.
Asunto(s)
ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Glicoproteínas de Membrana/antagonistas & inhibidores , MicroARNs/uso terapéutico , ADP-Ribosil Ciclasa 1/genética , Animales , Anticuerpos Monoclonales de Origen Murino/química , Caspasa 7/metabolismo , Línea Celular Tumoral , Quinasa 6 Dependiente de la Ciclina/genética , Regulación hacia Abajo , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Lípidos/química , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , MicroARNs/administración & dosificación , MicroARNs/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Nanopartículas/química , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
MicroRNAs (miRNAs) constitute a large class of short RNAs (e.g., 20-24 nucleotides in length), whose main function is to posttranscriptionally regulate the expression of protein-coding genes. Their importance in tumorigenesis has been demonstrated over the past decade, and correspondingly, they have emerged as potential therapeutic molecules and targets. Liver cancer is one of the most common neoplastic diseases worldwide, and it currently has a poor prognosis owing to largely ineffective therapeutic options. Liver cancer is also an excellent model for testing miRNA-based therapy approaches as it can be easily targeted with the systemic delivery of oligonucleotides. In recent years, the role of miRNAs in hepatocellular carcinoma (HCC) has been established with molecular studies and the development of animal models. These studies have also provided the basis for evaluating the therapeutic potential of miRNAs, or anti-miRNAs. In general, the safety of miRNAs has been proven and antitumor activity has been observed. Moreover, because of the absence or presence of mild side effects, the prophylactic use of miRNA-based approaches may be foreseen.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Oligodesoxirribonucleótidos Antisentido , ARN Neoplásico , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , ARN Neoplásico/antagonistas & inhibidores , ARN Neoplásico/genética , ARN Neoplásico/metabolismoRESUMEN
The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/metabolismo , MicroARNs/fisiología , Regiones no Traducidas 3' , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/patología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana EdadRESUMEN
The CpG island methylator phenotype (CIMP) in colorectal tumours can be recognized by an increased frequency of aberrant methylation in a specific set of genomic loci. Because of the strong association of CIMP with high microsatellite instability (MSI-H), the identification of CIMP+ tumours within microsatellite stable (MSS) colorectal cancers may not be straightforward. To overcome this potential limitation, we have built an improved seven-locus set of methylation markers that includes CACNA1G, IGF2, RUNX3, HTR6, RIZ1, MINT31, and MAP1B. This new set of CIMP markers revealed a bimodal distribution of methylation frequencies in a group of 95 MSS colorectal cancers, which allowed a clearer separation between CIMP classes. Correlation of MSS CIMP+ tumours with bio-pathological traits revealed significant associations with location to the proximal colon, mucinous histology, BRAF mutation, and chromosomal stability. A potential trend towards an adverse prognosis of CIMP+ cases was associated with the high frequency of BRAF mutations present within this cohort of tumours. Microarray analysis revealed that CIMP+ tumours are characterized by a unique expression profile, a result that confirms that CIMP+ tumours represent a truly distinct molecular class within MSS colorectal cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Metilación de ADN , ADN de Neoplasias/genética , Repeticiones de Microsatélite/genética , Anciano , Análisis por Conglomerados , Neoplasias Colorrectales/patología , Islas de CpG/genética , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Marcadores Genéticos , Humanos , Masculino , Inestabilidad de Microsatélites , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
The human polyomavirus BK (BKV) is oncogenic in rodents and induces malignant transformation of rodent cells in vitro. Although its role in human tumorigenesis is still debated, BKV represents an excellent model to evaluate molecularly targeted antineoplastic approaches. Here, we have tested whether stable suppression of the T antigen (T-ag) oncogene expression could inhibit the in vitro and in vivo malignant phenotype of BKV-transformed mouse cells. An adenovirus vector system that expresses small hairpin RNAs (shRNAs), which are converted into active small interfering RNAs (siRNA) molecules against the BKV T-ag, was developed. This vector was able to inhibit the expression of BKV T-ag through a highly efficient in vitro and in vivo delivery of the siRNA molecule. In addition, it allowed a stable expression of siRNA for a period of time sufficient to elicit a biological effect. Inhibition of T-ag expression results in reduction of the in vitro growth rate of BKV-transformed cells, which is, at least in part, caused by restoration of p53 activity and induction of apoptosis. In vivo studies proved that adenovirus vectors expressing anti-T-ag siRNA were able to suppress tumorigenicity of BKV-transformed cells. Moreover, adenovirus vector direct treatment of growing tumors resulted in a significant reduction of tumor growth. This study indicates that siRNAs delivery via a viral vector have a potential usefulness as in vivo anticancer tool against viral and cellular oncogenes.
Asunto(s)
Adenoviridae/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Virales de Tumores/genética , Virus BK/inmunología , Terapia Genética , Vectores Genéticos , Neoplasias Experimentales/terapia , ARN Interferente Pequeño/genética , Animales , Antígenos Transformadores de Poliomavirus/química , Antígenos Transformadores de Poliomavirus/genética , Transformación Celular Viral/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/genética , Neoplasias Experimentales/virología , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Herpesvirus Humano 1/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción Genética/métodos , Animales , Antígenos Virales de Tumores/genética , Virus BK/inmunología , Línea Celular Transformada , Ingeniería Genética , Vectores Genéticos/genética , Herpesvirus Humano 1/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Neoplasias/terapia , ARN Interferente Pequeño/metabolismo , Replicación ViralRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Tat protein stimulates cell proliferation, inhibits apoptosis, displays angiogenic functions and is believed to be involved in the pathogenesis of Kaposi's sarcoma (KS) and other tumours arising in AIDS patients. Tat-transgenic (TT) mice, which constitutively express Tat in all tissues and organs, may therefore be predisposed to tumorigenesis. To test this hypothesis, we treated TT mice with urethane, a general carcinogen inducing tumours of various organs. The results indicate that, after injection of urethane, the incidence of lung tumours and lymphomas is not significantly different in the TT and control (CC) mice, whereas liver preneoplastic lesions and tumours show a significantly greater incidence in TT than in CC mice. This remarkable carcinogenic effect of urethane for the liver may be due to a tat-induced predisposition, manifested as a liver cell dysplasia (LCD), spontaneously affecting most of the TT mice. LCD may exert a promoting effect by stimulating proliferation of cell clones initiated by the mutagenic effect of urethane. In addition, LCD, which is associated with aneuploidy and chromosome instability, may enhance the progression to malignancy of the preneoplastic lesions induced by urethane. Interestingly, a significantly greater incidence of vascular ectasias and haemangiomas was detected in the liver of urethane-treated TT mice, most likely due to the marked angiogenic properties of Tat. This study suggests a role for Tat in the promotion and progression of tumours initiated by exogenous and endogenous carcinogens in HIV-1-infected patients, thereby contributing to the tumorigenesis in the course of AIDS.
Asunto(s)
Carcinógenos/toxicidad , Infecciones por VIH , VIH-1 , Neoplasias Hepáticas/inducido químicamente , Uretano/toxicidad , Animales , División Celular , ADN Complementario/análisis , Femenino , Productos del Gen tat , Humanos , Inmunohistoquímica , Hígado/química , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/inducido químicamente , Linfoma/inducido químicamente , Masculino , Ratones , Ratones Transgénicos , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Two cases of T acute lymphoblastic leukaemia (T-ALL) with an identical t(4;11)(q21;p15) translocation were identified within a prospective study on the biological and clinical features of adult ALL patients enrolled into the therapeutic protocol ALL0496 of the GIMEMA Italian Group. In both cases, the molecular characterization showed an involvement of the NUP98 gene on 11p15 which rearranges with the RAP1GDS1 gene on 4q21. The morphological and immunological features of the leukaemic cells, as well as the clinical behaviour and response to induction therapy, were the same in both patients. Based on the available data, the t(4;11)(q21;p15) translocation involving the NUP98-RAP1GDS1 fusion gene emerges as a new highly specific genetic abnormality that characterizes a subset of T-ALL.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 4 , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas de la Membrana/genética , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Translocación Genética , Adolescente , Adulto , Secuencia de Aminoácidos , Fusión Artificial Génica , Secuencia de Bases , Cromosomas Humanos Par 8 , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Cariotipificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TrisomíaRESUMEN
PURPOSE: To study the clinicobiologic significance of acquired 11q deletions involving the ataxia teleangiectasia locus (ATM+/-) in B-cell non-Hodgkin's lymphomas (NHL). PATIENTS AND METHODS: Fifty-three indolent lymphomas and 82 aggressive lymphomas were studied by conventional cytogenetic analysis and by fluorescence in situ hybridization using an 11q22-23 probe recognizing ATM sequences. Pertinent clinical data were collected. RESULTS: A hemizygous ATM deletion was seen in 44% to 88% of the interphase cells in 15 cases (11.1%); four patients had an indolent lymphoma (follicular center cell lymphoma), and 11 patients had an aggressive lymphoma (five mantle-cell lymphomas [MCLs] and six diffuse large-cell lymphomas). Dual-color hybridization studies showed ATM deletion to be possibly a secondary aberration in three patients with MCL. Ten out of 15 ATM+/- patients had a complex karyotype, 11 out of 15 had more than 90% abnormal metaphases (AA karyotype status), and +12, 13q14 deletion, and 17p13 deletion were seen in seven, four, and five cases, respectively. Patients with ATM+/- more frequently had a complex karyotype (P =.01) and the AA karyotype (P =.04) compared with patients without ATM+/-. With the exception of a poor performance status (P =.001), no correlation was found between ATM+/-, initial clinical variables, and complete remission rate; whereas a highly significant association was found with shorter survival (P <.0001). This cytogenetic lesion maintained its prognostic importance in multivariate analysis (P =.0004), along with performance status (P =.0006), serum lactate dehydrogenase level (P =.03), splenomegaly (P =.01), and histologic grade (P =.03). When analyzing indolent lymphomas and aggressive lymphomas separately, ATM+/- maintained its prognostic importance as an independent variable in both histologic groups (P =.0001 and P =.016, respectively). CONCLUSION: Though possibly not representing a primary genetic lesion in the majority of cases, the acquired ATM+/- status has clinicobiologic importance in NHL, possibly representing a major cytogenetic determinant of outcome.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11/genética , Linfoma de Células B/genética , Proteínas Serina-Treonina Quinasas/genética , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Femenino , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Interfase , Cariotipificación , Linfoma de Células B/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Proteínas Supresoras de TumorRESUMEN
The stromal interaction molecular 1 gene (STIM1) encodes a type I trans-membrane protein of unknown function, which induces growth arrest and degeneration of the human tumor cell lines G401 and RD but not HBL100 and CaLu-6, suggesting a role in the pathogenesis of rhabdomyosarcomas and rhabdoid tumors. Here, we describe the STIM1 genomic organization including the identification of the promoter region. The gene consists of 12 exons that span a region larger than 250 kb between the genes RRM1 and NUP98. Nucleotide sequences of all exon-intron boundaries were determined and oligonucleotide primers for the amplification of individual exons were designed. The promoter region was identified within a 1.8-kb SacI fragment at the 5' end of the gene. In vitro CpG methylation of the promoter region indicated that transcription can be downregulated by this mechanism. The genetic tools developed in the present work will help to determine whether pathogenetic mechanisms that associate STIM1 with tumorigenesis involve mutations in coding sequences and/or promoter, and whether methylation could determine STIM1 transcriptional down-regulation in tumor samples.
Asunto(s)
Cromosomas Humanos Par 11 , Exones , Proteínas de la Membrana , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , División Celular/genética , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Cósmidos , Regulación Neoplásica de la Expresión Génica , Humanos , Intrones , Reacción en Cadena de la Polimerasa , Tumor Rabdoide/genética , Rabdomiosarcoma/genética , Molécula de Interacción Estromal 1 , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2, CDKN1C, and KVLQT1, are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A, BWR1B, and BWR1C. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O'Keefe, D., Dao, D. , Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021-2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith-Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Mutación , Neoplasias/genética , Proteínas de Transporte de Catión Orgánico , Proteínas/genética , Análisis Mutacional de ADN , Femenino , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Células Tumorales CultivadasAsunto(s)
Proteínas Bacterianas , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor/genética , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Southern Blotting , Cromosomas Artificiales de Levadura/genética , Cartilla de ADN , Humanos , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1RESUMEN
The expression of GOK, a gene recently identified at 11p15.5, was studied in breast cancer, rhabdomyosarcoma, and rhabdoid tumor cell lines. In these neoplasms, deletions at 11p15 and suppression of tumorigenicity induced by a normal human chromosome 11 were previously demonstrated. Whereas breast cancer cell lines express readily detectable levels of GOK mRNA, expression is absent in rhabdomyosarcoma and rhabdoid tumor cell lines. This is in contrast with the high expression of GOK in skeletal muscle, the normal tissue of origin of rhabdomyosarcomas, suggesting that down-regulation of GOK expression could be involved in tumor development. In agreement with this hypothesis, transfection of GOK cDNA into G401 derived from a rhabdoid tumor and RD cells derived from a rhabdomyosarcoma that do not express detectable levels of GOK mRNA, induced cell death. Because GOK expression is not compatible with growth of these tumor cells, these results support the hypothesis that loss of GOK expression plays a role in tumor establishment or progression and suggest that GOK may act as a recessive tumor suppressor gene in rhabdomyosarcomas and rhabdoid tumors. On the contrary, transfection of GOK cDNA into the breast cancer cell line HBL100 produced no detectable effects, indicating that the growth-suppressive effect of GOK in RD and G401 cells was specific. Because rhabdomyosarcomas have been observed in cases of Beckwith-Wiedemann syndrome, a genetic disorder linked to 11p15, a role of GOK in this disease cannot be excluded.
Asunto(s)
Cromosomas Humanos Par 11 , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana , Proteínas de Neoplasias/genética , Tumor Rabdoide/genética , Rabdomiosarcoma/genética , Mapeo Cromosómico , Biblioteca de Genes , Marcadores Genéticos , Humanos , Pérdida de Heterocigocidad , Masculino , Proteínas de Neoplasias/biosíntesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Tumor Rabdoide/patología , Rabdomiosarcoma/patología , Molécula de Interacción Estromal 1 , Testículo/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Twenty-one expressed sequence tags (ESTs) belonging to unidentified transcripts were mapped to 9 intervals at 11p 15.5-p15.3. Thirteen were mapped to a region of an estimated size of 7 Mb with the help of a YAC contig. The remaining eight were mapped outside this region, and the centromeric or telomeric position of the ESTs relative to the YAC contig was defined with the help of a cellular hybrid containing a derivative chromosome 11 with a break-point within the region covered by the contig. The average size of the intervals, where the ESTs were mapped, was estimated to be 0.3-0.4 Mb. The refinement of the chromosomal location of these ESTs could be of great help in the identification of potential candidate genes for disease locus mapping at 11p15.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 11 , ADN Complementario , Animales , Expresión Génica , Humanos , Pérdida de Heterocigocidad , RatonesRESUMEN
We have analyzed genomic DNAs from a patient who developed acute myeloid leukemia 1 year after a myeloproliferative disorder was diagnosed. The development of the acute leukemia was associated with the acquisition of a t(9;11)(p22;q23) chromosome translocation. ALL-1 gene rearrangement, on chromosome 11, was present at the onset of the acute phase, but not during the chronic phase of the myeloproliferative disorder. The genomic rearrangement on chromosome 9 was within an unidentified region. By the use of polymerase chain reaction, we were able to determine that the chromosomal rearrangement was completely absent during the chronic phase of the myeloproliferative disorder, indicating that the ALL-1 gene rearrangement was causally related to the development of the acute phase. The rapid progression into the acute phase suggests that this case might be therapy related. This work provides a clear example of association of a molecular defect with the development of a specific clinical leukemic stage, and supports the indication that ALL-1 gene rearrangement is associated with poor clinical outcome in adult leukemias.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 9 , Leucemia Monocítica Aguda/genética , Trastornos Mieloproliferativos/genética , Translocación Genética , Adulto , Secuencia de Bases , Reordenamiento Génico/genética , Humanos , Leucemia Monocítica Aguda/patología , Masculino , Datos de Secuencia Molecular , Trastornos Mieloproliferativos/patología , Reacción en Cadena de la PolimerasaRESUMEN
Genetic alterations of chromosome region 11p15 have been detected in neoplastic diseases as well as in cancer-predisposing syndromes. The cloning of the entire chromosomal region will be important for the identification and characterization of critical tumor suppressor genes. We have developed a yeast artificial chromosome contig that covers up to 7 Mb of this chromosome band. The most centromeric marker included in the contig is D11S932 and the most telomeric is D11S470. We have developed 18 new STS markers, which have been located in the contig in relation to 16 known markers. One of the yeast artificial chromosome clones was found to span the chromosome 11 breakpoint of the translocation t(11;18), associated with a case of Beckwith-Wiedemann syndrome. Cloning the regions in proximity to this translocation might reveal the presence of a gene altered in association with the development of Beckwith-Wiedemann syndrome.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Cromosomas Humanos Par 11 , Secuencia de Bases , Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 18 , Clonación Molecular , Cartilla de ADN/química , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Translocación GenéticaRESUMEN
Chromosome 11 is frequently altered in several types of human neoplasms. In breast cancer, loss of heterozygosity has been described in two regions of this chromosome, 11p15 and 11q22-23. In this report we have dissected the two regions using high-density polymorphic markers, and have found that there are at least two independent areas of loss of heterozygosity in each region, suggesting that multiple genes on chromosome 11 may be targets of genetic alteration during tumor establishment or progression. The regions defined are: at 11p15, between loci D11S576 and D11S1318 and between D11S988 and D11S1318; at 11q23, between D11S2000 and D11S897 and between D11S528 and D11S990. The narrowing of these regions of loss should facilitate the cloning of the regions in yeast artificial chromosomes to identify the critical tumor suppressor genes.
Asunto(s)
Neoplasias de la Mama/genética , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Eliminación de Gen , Secuencia de Bases , Neoplasias de la Mama/patología , Clonación Molecular , Genes Supresores de Tumor , Marcadores Genéticos , Heterocigoto , Humanos , Metástasis Linfática , Índice Mitótico , Datos de Secuencia Molecular , Estadificación de NeoplasiasRESUMEN
A BK virus (BKV) episomal vector (pRPneoCMV) was constructed for expression of cDNAs under control of the cytomegalovirus (CMV) immediate-early promoter. Transfection of pRPneoCMV for expression of the chloramphenicol acetyltransferase (CAT) gene in several human cell lines showed that the CMV promoter is more efficient than the HIV-1 and RSV LTRs in directing gene expression from episomal vectors. In 293 human cells pRPneoCMV/CAT is twenty times more active in CAT expression than the well known pSV2CAT vector in COS7 cells. Stable expression of the gene of the herpes simplex virus type 1 and type 2 glycoprotein G, cloned into pRPneoCMV, was obtained in 293 cells. This vector will allow direct cloning of newly synthesized cDNAs whose expression can be monitored in human cells.
Asunto(s)
Virus BK/genética , Clonación Molecular/métodos , ADN Complementario/genética , Vectores Genéticos , Plásmidos , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Citomegalovirus/genética , Expresión Génica , Humanos , Regiones Promotoras Genéticas , Transfección , Proteínas del Envoltorio Viral/genéticaRESUMEN
BK virus (BKV) is a human papovavirus that readily transforms rodent cells, but not human cells, to a neoplastic phenotype, suggesting that tumor-suppressor functions expressed in human cells control BKV oncogenicity. Transfer of a normal human chromosome 11 to BKV-transformed mouse cells suppresses the malignant phenotype. In this report we map the regions of chromosome 11 involved in tumor suppression. Transfer of chromosome 11 to the BKV-transformed hamster cell line HKBK produces monochromosomic hybrids retaining only portions of the transferred human chromosome. We have compared the tumorigenicity of the hybrids with the molecular mapping of chromosome 11 retained regions. This analysis indicated that 3 regions of human chromosome 11, 11p15.5, 11p13 and 11q13, cooperate in tumor suppression. However, 11q13 seems the most important, since all the HKBK/H11-induced tumors analysed had lost this region, whereas 11p15.5 and 11p13 were sometimes retained. The chromosomal regions identified in this study are deleted in several types of human tumors, suggesting that the BKV transformation system specifically detects tumor-suppressor genes on chromosome 11 that are involved in human oncogenesis. This model may be of use in isolating and cloning such genes. The results of this report raise the possibility that BKV may have a synergistic tumorigenic effect in human cells where tumor-suppressor genes controlling its oncogenic potential are inactivated.
Asunto(s)
Virus BK/patogenicidad , Transformación Celular Neoplásica , Cromosomas Humanos Par 11 , Genes Supresores de Tumor , Neoplasias Experimentales/etiología , Animales , Línea Celular , Aberraciones Cromosómicas , Cricetinae , Humanos , Células Híbridas , Ratones , FenotipoRESUMEN
bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins. The result suggested that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down-regulate bcl-2 expression. We found, indeed, that overexpression of a mutant p53 (mut 175) in MCF-7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory element responsible for the down-regulation. If this mechanism will be proved also for the wild-type p53 allele, it may disclose a possible mechanism for p53-induced apoptosis: down-regulation of bcl-2.