RESUMEN
The development and progression of bladder cancer is associated with multiple alterations in the genome, including loss of chromosome 10. Recently, MMAC1/PTEN, a phosphatidylinositol phosphatase, has been mapped to chromosome 10q23. We previously demonstrated that MMAC1/PTEN has tumor suppressive properties in glioblastoma and prostate cancer. To investigate the efficacy of gene therapy with MMAC1/PTEN, we examined whether the exogenous introduction of MMAC1/PTEN via an adenoviral vector (Ad-MMAC) can inhibit tumor growth and reverse drug resistance to doxorubicin in human bladder cancer cells. Human bladder cancer cell lines UM-UC-3 and T24 were infected with Ad-MMAC to induce exogenous expression of MMAC1/PTEN. The cells were then analysed for cell growth and expression of phosphorylated protein kinase B (Akt/PKB) and MMAC1/PTEN. UM-UC-6dox, a doxorubicin resistant subline, was infected with Ad-MMAC to evaluate its role in reversing drug resistance to doxorubicin. We found that MMAC1/PTEN suppressed tumor growth in UM-UC-3 and T24 cells with arrest in the G1 phase of the cell cycle. We also showed that gene therapy with MMAC1/PTEN abrogated phosphorylated Akt/PKB expression in UM-UC-3, T24 and UMUC-6dox cells, and restored doxorubicin sensitivity in UM-UC-6dox. These data demonstrate that MMAC1/PTEN can induce growth suppression and increase sensitivity to doxorubicin in bladder cancer cells and suggest that the MMAC1/PTEN gene and its pathways can be therapeutic targets for bladder cancer.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Genes Supresores de Tumor/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Neoplasias de la Vejiga Urinaria/terapia , Adenovirus Humanos , Ciclo Celular , División Celular , Expresión Génica , Vectores Genéticos , Humanos , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Células Tumorales CultivadasRESUMEN
We recently limited the location of a candidate tumor suppressor gene in invasive (T3a/b) bladder transitional-cell carcinoma (TCC) to a 2.5-cM region at chromosome 10q23.3. This region harbors the MMAC1/PTEN/TEP1 gene (referred to hereafter as MMAC1), a dual-phosphatase tumor-suppressor gene frequently inactivated in variety of malignant tumors. In the present study, we examined whether MMAC1 is a target for inactivation by mutations and deletions in bladder TCC cell lines and specimens. MMAC1 was inactivated by homozygous deletions and mutations in three (27%) of 11 bladder cancer cell lines. One cell line, UC-3, had homozygous deletions, and two other cell lines, T-24 and UC-9, had missense mutations. T-24 had also a nonsense mutation. However, none of the 33 bladder TCC specimens examined had a mutation or deletion in the coding region. These results suggest that MMAC1 is not the primary target for inactivation in bladder TCC and that another gene, in close proximity to the MMAC1 locus, within this region of frequent allelic losses, may be the target for inactivation.
Asunto(s)
Carcinoma de Células Transicionales/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Monoéster Fosfórico Hidrolasas/genética , Proteínas Supresoras de Tumor , Neoplasias de la Vejiga Urinaria/genética , Carcinoma de Células Transicionales/metabolismo , Análisis Mutacional de ADN , Exones , Eliminación de Gen , Reordenamiento Génico , Humanos , Mutación , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/biosíntesis , Polimorfismo Conformacional Retorcido-Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
The effects of transforming growth factor-alpha (TGF-alpha) on cell growth were studied in human glioma U251 cells transfected with antisense TGF-alpha vectors (pcDNAI.neo). Several antisense clones showed a marked decrease in growth rate in serum-free medium but not in medium containing 10% FBS, compared with those of parental cells and clones from sense or vector transfectants. Antisense clones also produced fewer and smaller colonies in anchorage-independent growth assays. Moreover, there was a reduction in TGF-alpha expression in these antisense clones at both the protein and mRNA levels, as determined by enzyme linked immuno-sorbent assay and reverse transcriptase polymerase chain reaction analysis. A U251 clone transfected by TGF-alpha antisense in a different vector (pMT/Ep) also showed a marked suppression in cell growth and TGF-alpha mRNA level. Finally, transfected clones with either vector system, showed decreased tumorigenicity in nude mice. In summary, a strong correlation between the inhibition of glioma cell growth and TGF-alpha expression was obtained from two different plasmid vectors, indicating that the expression of TGF-alpha could be specifically and effectively down-regulated by TGF-alpha antisense vector, which in turn led to growth inhibition. These studies suggests that TGF-alpha plays an essential role in controlling human glioma cell proliferation and may serve as a potential target for treatment of malignant glioma.
Asunto(s)
Neoplasias Encefálicas/patología , División Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/patología , Oligodesoxirribonucleótidos Antisentido/farmacología , Factor de Crecimiento Transformador alfa/genética , Animales , Neoplasias Encefálicas/genética , Células Clonales , Glioma/genética , Humanos , Ratones , Ratones Desnudos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Understanding the functional roles of the molecular alterations that are involved in the oncogenesis of prostate cancer, the second most frequent cause of cancer-related deaths among men in the United States is the focus of numerous investigations. To examine the possible significance of alterations associated with the tumor suppressor gene, MMAC/PTEN, in prostate carcinoma, the biological and biochemical effects of MMAC/PTEN expression were examined in LNCaP cells, which are devoid of a functional gene product. Acute expression of MMAC/PTEN via an adenoviral construct resulted in a dose-dependent and specific inhibition of Akt/PKB activation, consistent with the phosphatidylinositol phosphatase activity of MMAC/PTEN. MMAC/PTEN expression induced apoptosis in LNCaP cells, although to a lesser extent than that observed with p53 via an adenoviral construct. However, MMAC/PTEN expression produced a growth inhibition that was significantly greater than that achieved with p53. Overexpression of Bcl-2 in LNCaP cells blocked MMAC/PTEN- and p53-induced apoptosis but not the growth-suppressive effects of MMAC/ PTEN, suggesting that the growth regulatory effects of MMAC/PTEN involve multiple pathways. These studies further implicate the loss of MMAC/PTEN as a significant event in prostate cancer and suggest that reintroduction of MMAC/PTEN into deficient prostate cancer cells may have therapeutic implications.
Asunto(s)
Apoptosis/fisiología , Proteínas de Arabidopsis , Carcinoma/metabolismo , División Celular/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas de Plantas/metabolismo , Canales de Potasio/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Supresoras de Tumor , Carcinoma/genética , Carcinoma/patología , Vectores Genéticos , Humanos , Masculino , Fosfohidrolasa PTEN , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
MMAC/PTEN, a tumor suppressor gene located on chromosome 10q, has recently been shown to act as a phosphatidylinositol 3,4,5-triphosphate phosphatase and to modulate cell growth and apoptosis. Somatic mutations of MMAC/PTEN have been reported in a number of human cancers, especially in glioblastoma multiforme (GBM), although the number of identified mutations (approximately 10-35%) is significantly lower than the frequency of LOH affecting the MMAC/PTEN locus in the specimens (approximately 75-95%). To further investigate the possible alterations that may affect MMAC/PTEN, we examined the expression of the gene by reverse transcription-PCR in a series of gliomas. A significant difference (P < 0.001) was observed between the expression of MMAC/PTEN in GBMs versus lower grades of gliomas, thus mimicking the difference in allelic deletion associated with the locus in these tumors. Furthermore, Kaplan-Meier survival plots, adjusted for age and tumor grade, showed a significantly better prognosis for patients whose tumors expressed high levels of MMAC/PTEN. Additionally, immunostaining of GBMs revealed little or no MMAC/PTEN expression in about two-thirds of the tumors, whereas the other approximately one-third of tumors had significantly higher levels of expression. However, in about two-thirds of the high-expressing specimens, a heterogeneous pattern of expression was observed, indicating that certain cells within the tumor failed to express MMAC/PTEN. The combination of these results suggest that, in addition to molecular alterations affecting the gene, altered expression of MMAC/PTEN may play a significant role in the progression of GBM and patient outcome.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Glioblastoma/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Supresoras de Tumor , Genes Supresores de Tumor , Glioblastoma/diagnóstico , Glioblastoma/patología , Humanos , Inmunohistoquímica , Fosfohidrolasa PTEN , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics.
Asunto(s)
Genes Supresores de Tumor , Glioma/genética , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Supresoras de Tumor , Dominio Catalítico/genética , Adhesión Celular , División Celular , Transformación Celular Neoplásica , Glioma/enzimología , Humanos , Mutación , Fosfohidrolasa PTEN , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Proteínas Recombinantes/biosíntesis , Células Tumorales CultivadasRESUMEN
Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Angiogenic factors are potentially optimal targets for therapeutic strategies because they are essential for tumor growth and progression. In this study, we sought a strategy for efficiently delivering an antisense cDNA molecule of the vascular endothelial growth factor (VEGF) to glioma cells. The recombinant adenoviral vector Ad5CMV-alphaVEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-alphaVEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of s.c. human glioma tumors established in nude mice with intralesional injection of Ad5CMV-alphaVEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumoral cells using antisense strategies has an antitumor effect in vivo. This is the first time that an adenoviral vector is used to transfer antisense VEGF sequence into glioma cells in an animal model, and our results suggest that this system may have clinical and therapeutic utility.
Asunto(s)
ADN sin Sentido/farmacología , Factores de Crecimiento Endotelial/genética , Terapia Genética , Glioma/terapia , Linfocinas/genética , Neovascularización Patológica/terapia , Adenoviridae/genética , Animales , Regulación hacia Abajo , Glioma/irrigación sanguínea , Humanos , Ratones , Ratones Desnudos , ARN Mensajero/análisis , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Extensive genomic deletions involving chromosome 10 are the most common genetic alteration in glioblastoma multiforme (GBM). To localize and examine the potential roles of two chromosome arm 10q tumor suppressor regions, we used two independent strategies: mapping of allelic deletions, and functional analysis of phenotypic suppression after transfer of chromosome 10 fragments. By allelic deletion analysis, the region of 10q surrounding the MMAC/PTEN locus was shown to be frequently lost in GBMs but maintained in most low-grade astrocytic tumors. An additional region at 10q25 containing the DMBT1 locus was lost in all grades of gliomas examined. The potential biological significance of these two regions was further assessed by examining microcell hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have a less transformed phenotype with clones exhibiting an inability to grow in soft agarose. However, presence or absence of DMBT1 did not correlate with any in vitro phenotype assessed in our model system. These results support a model of molecular progression in gliomas in which the frequent deletion of 10q25-26 is an early event and is followed by the deletion of the MMAC/PTEN during the progression to high-grade GBMs.
Asunto(s)
Aglutininas , Deleción Cromosómica , Cromosomas Humanos Par 10/genética , Glioma/genética , Proteínas Supresoras de Tumor , Western Blotting , Neoplasias Encefálicas/genética , Proteínas de Unión al Calcio , ADN de Neoplasias/análisis , Proteínas de Unión al ADN , Genes Supresores de Tumor/genética , Glioma/química , Glioma/metabolismo , Humanos , Hibridación Fluorescente in Situ , Pérdida de Heterocigocidad/genética , Repeticiones de Microsatélite/genética , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/biosíntesis , Células Tumorales CultivadasRESUMEN
The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to have phosphotidylinositol phosphatase activity, implicating its possible involvement in phosphatidylinositol 3'-kinase-mediated signaling. To investigate possible alterations in growth factor-mediated signal transduction, an adenovirus containing MMAC/PTEN, Ad-MMAC, previously shown to inhibit growth and tumorigenicity in glioma cells, was used to acutely express the transgene. Human glioma cells infected with Ad-MMAC but not with control adenoviruses exhibited an inhibition of phosphorylation of both activating residues of Akt, Ser-473, and Thr-308, along with Akt's serine/threonine kinase activity, without significantly altering Akt expression. The effects of functional MMAC/PTEN expression were relatively specific, because members of several other growth factor-mediated signaling pathways showed no altered responses. The presence of MMAC/PTEN also inhibited phosphorylation of BAD, although no evidence of apoptosis in the in situ treated cells was observed. However, U251 glioma cells infected with Ad-MMAC were induced to undergo anoikis at a significantly higher rate than U251 cels treated with control viruses or mock infected with media. These results demonstrate that the acute administration of MMAC/PTEN results in the inhibition of Akt-mediated signaling, growth inhibition, and anoikis, implying that loss of MMAC/PTEN increases cellular proliferation and significantly augments a cell's survival potential during cellular processes that are associated with malignancy.
Asunto(s)
Apoptosis/fisiología , Glioblastoma/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor , Adenoviridae/genética , Adenoviridae/metabolismo , Activación Enzimática , Receptores ErbB/biosíntesis , Receptores ErbB/fisiología , Expresión Génica , Genes Supresores de Tumor , Glioblastoma/enzimología , Glioblastoma/genética , Humanos , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/fisiología , Transgenes , Células Tumorales CultivadasRESUMEN
The frequency of loss of heterozygosity (LOH) around MMAC/PTEN and DMBT1 loci and survival analyses based on the LOH status were assessed in 110 patients with different histological groups of gliomas. Twenty-six of the patients had anaplastic oligodendrogliomas, 31 had anaplastic astrocytomas, and 53 had glioblastomas multiforme (GM). At the DMBT1 locus, LOH was observed very frequently in all three histological groups, with no significant difference in the frequency of LOH among the three histological groups. At the MMAC/PTEN locus, patients with GM exhibited a significantly increased frequency of LOH (72%) compared with patients with anaplastic astrocytomas (29%) or anaplastic oligodendrogliomas (31%) (P < 0.0001). Kaplan-Meier survival plots showed that patients with LOH at the MMAC/PTEN locus had a significantly worse prognosis than did patients without LOH at the MMAC/PTEN locus [hazard ratio (LOH versus non-LOH), 2.65; 95% confidence interval (CI), 1.69-4.46; P < 0.0001]. Cox proportional hazards regression analysis, adjusted for age at surgery and histological grades (GM and non-GM), showed that LOH at the MMAC/PTEN locus was a significant predictor of shorter survival [hazard ratio (LOH versus non-LOH), 2.01; 95% CI, 1.1-3.5; P = 0.018). Our analysis failed to indicate a similar association between the frequency of LOH at the DMBT1 locus and patient survival [hazard ratio (LOH versus non-LOH), 2; 95% CI, 0.37-3.13; P = 0.2]. These results suggest that the DMBT1 gene may be involved early in the oncogenesis of gliomas, whereas alterations in the MMAC/PTEN gene may be a late event in the oncogenesis related to progression of gliomas and provide a significant prognostic marker for patient survival.
Asunto(s)
Aglutininas , Mapeo Cromosómico , Genes Supresores de Tumor , Glioma/genética , Pérdida de Heterocigocidad , Monoéster Fosfórico Hidrolasas/genética , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor , Proteínas de Unión al Calcio , Proteínas de Unión al ADN , Glioma/mortalidad , Humanos , Fosfohidrolasa PTEN , Pronóstico , Tasa de SupervivenciaRESUMEN
Glioblastoma multiforme is one of the most highly vascularized solid neoplasms, therefore treatments that target neovascularization process would be of great clinical importance. Studies of glioblastoma angiogenesis have revealed that expression of the vascular endothelial growth factor (VEGF) is up-regulated in these tumors. Previous reports have shown that down-regulation of VEGF correlates with modification in the glioma growth. To examine this phenomenon further, in this study we constructed two hammerhead ribozymes (RZI and RZII) to target the 5' common region of VEGF mRNA. Both ribozymes exhibited site-specific cleavage to a 318-nucleotide VEGF transcript and showed a high digestion efficiency in vitro (65-95%). After the transfection of glioma cells with two expression vectors carrying the ribozyme sequence, Northern blot analyses detected high levels of ribozyme expression. Treatment of the glioma cells with the ribozymes resulted in a reduction in VEGF mRNA in six of eight clones. Furthermore, the anti-VEGF effect was confirmed at protein level. Thus, enzyme-linked immunoabsorbent analyses (ELISA) showed a >70% reduction in the VEGF165 expression level. These results indicate that hammerhead ribozymes may be useful in down-regulating VEGF expression and suggest that anti-VEGF strategies may be used to potentiate other gene therapies targeting tumor suppressor genes.
Asunto(s)
Terapia Genética , Glioma/terapia , Northern Blotting , ADN Recombinante/genética , Regulación hacia Abajo , Factores de Crecimiento Endotelial/genética , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Glioma/genética , Humanos , Linfocinas/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , ARN/genética , ARN/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Transcripción Genética/genética , Transfección/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The loss of large segments or an entire copy of chromosome 10 is the most common genetic alteration in human glioblastomas. To address the biological and molecular consequences of this chromosomal alteration, we transferred a human chromosome 10 into a glioma cell clone devoid of an intact copy. The hybrid cells exhibited an altered cellular morphology, a decreased saturation density, and a suppression of both anchorage-independent growth and tumor formation in nude mice. The hybrids also expressed the recently identified candidate tumor suppressor gene MMAC1/PTEN. To further identify gene products that may be involved in glioma progression, a subtractive hybridization was performed between the human glioblastoma cells and the phenotypically suppressed hybrid cells to identify differentially expressed gene products. Sixty-one clones were identified, with nine clones being preferentially expressed in the hybrid cells. Four cDNA clones represented markers of differentiation in glial cells. Two cDNA clones shared homology with platelet derived growth factor-alpha and the insulin receptor, respectively, both genes previously implicated in glioma progression. A novel gene product that was expressed predominantly in the brain, but which did not map to chromosome 10, was also identified. This clone contained an element that was also present in three additional clones, two of which also exhibited differential expression. Consequently, the presence of a functional copy of chromosome 10 in the glioma cells results in differential expression of a number of gene products, including novel genes as well as those associated with glial cell differentiation.
Asunto(s)
Neoplasias Encefálicas/genética , Cromosomas Humanos Par 10 , Genes Supresores de Tumor/genética , Glioblastoma/genética , Northern Blotting , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Deleción Cromosómica , Cromosomas Humanos Par 10/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/genética , Humanos , Células Híbridas , Cariotipificación , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
A candidate tumor suppressor gene, MMAC1/PTEN, located in human chromosome band 10q23, was recently identified based on sequence alterations observed in several glioma, breast, prostate, and kidney tumor specimens or cell lines. To further investigate the mutational profile of this gene in human cancers, we examined a large set of human tumor specimens and cancer cell lines of many types for 10q23 allelic losses and MMAC1 sequence alterations. Loss of heterozygosity (LOH) at the MMAC1 locus was observed in approximately one-half of the samples examined, consistent with the high frequency of 10q allelic loss reported for many cancers. Of 124 tumor specimens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (approximately 10%) of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (approximately 23%). Novel alterations identified in this gene include a missense variant in a melanoma sample and a splicing variant and a nonsense mutation in pediatric glioblastomas. Of 76 tumor cell lines prescreened for probable LOH, microsequence alterations of MMAC1 were detected in 12 (approximately 16%) of the lines, including those derived from astrocytoma, leukemia, and melanoma tumors, as well as bladder, breast, lung, prostate, submaxillary gland, and testis carcinomas. In addition, in this set of tumor cell lines, we detected 11 (approximately 14%) homozygous deletions that eliminated coding portions of MMAC1, a class of abnormality not detected by our methods in primary tumors. These data support the occurrence of inactivating MMAC1 alterations in multiple human cancer types. In addition, we report the discovery of a putative pseudogene of MMAC1 localized on chromosome 9.
Asunto(s)
Cromosomas Humanos Par 10 , Mutación , Neoplasias/genética , Monoéster Fosfórico Hidrolasas , Proteínas Tirosina Fosfatasas/genética , Proteínas Supresoras de Tumor , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Niño , Mapeo Cromosómico , Exones , Femenino , Eliminación de Gen , Marcadores Genéticos , Variación Genética , Glioblastoma/genética , Glioblastoma/patología , Glioma/genética , Glioma/patología , Humanos , Intrones , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Neoplasias/patología , Fosfohidrolasa PTEN , Mutación Puntual , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/biosíntesis , Eliminación de Secuencia , Neoplasias Testiculares/genética , Neoplasias Testiculares/patología , Células Tumorales CultivadasRESUMEN
The development of primary human brain tumors, particularly glioblastoma multiforme (GBM), has been associated with a number of molecular and chromosomal abnormalities. In this study, a novel tumor suppressor locus was identified and localized after the transfer of a human chromosome 4 into U251 human GBM cells. Hybrid clones containing a transferred neomycin-resistance tagged chromosome 4 revealed an inability to form tumors in nude mice and a greatly decreased efficiency of soft agarose colony formation. As a control, clones containing a transferred chromosome 2 were generated, and these retained the tumorigenic phenotype of the parental U251 cells. The presence of the transferred chromosomes was demonstrated by gain of polymorphic loci and FISH analyses. Several suppressed hybrid clones were shown to contain spontaneously reduced versions of the transferred chromosome 4. A common region of the fragmented chromosome 4 was retained among these clones that included the epidermal growth factor locus at 4q24-26 and several adjacent markers. The identification of a common fragment in the suppressed clones suggests the presence of a tumor suppressor gene or genes in this region, involved in glioma oncogenesis.
Asunto(s)
Cromosomas Humanos Par 4/genética , Genes Supresores de Tumor/genética , Glioma/genética , Animales , Southern Blotting , Mapeo Cromosómico , ADN/análisis , Factor de Crecimiento Epidérmico/genética , Glioma/patología , Humanos , Células Híbridas , Ratones , Repeticiones de Microsatélite , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
BACKGROUND: Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors. PURPOSE: We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis. METHODS: Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. RESULTS: p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G2 phase before undergoing cell death. Transfer of p21 induced a G2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G2 phase, or apoptosis. CONCLUSIONS AND IMPLICATIONS: Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that over expression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Ciclinas/fisiología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/fisiopatología , Proteína p53 Supresora de Tumor/fisiología , Adenoviridae , Apoptosis , Western Blotting , Neoplasias Encefálicas/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , ADN de Neoplasias/genética , Citometría de Flujo , Vectores Genéticos , Glioblastoma/genética , Humanos , Fenotipo , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia ArribaRESUMEN
Deletions involving regions of chromosome 10 occur in the vast majority (> 90%) of human glioblastoma multiformes. A region at chromosome 10q23-24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.
Asunto(s)
Cromosomas Humanos Par 10/genética , Genes Supresores de Tumor/genética , Glioblastoma/genética , Mutación/genética , Monoéster Fosfórico Hidrolasas , Proteínas Tirosina Fosfatasas/genética , Proteínas Supresoras de Tumor , Secuencia de Aminoácidos , Animales , Células Cultivadas , Análisis Mutacional de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Neoplasias/genética , Fosfohidrolasa PTEN , ARN Mensajero/análisis , ARN Neoplásico/análisis , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Genetic deletions to chromosome 10 have been extensively documented for human glioblastomas (GBMs). To identify gene products that may be involved in malignant progression, a subtractive hybridization was performed between GBM cells and hybrid cells suppressed for tumorigenicity following microcell transfer of chromosome 10. One novel cDNA isolated from this subtraction showed consistent upregulation (approximately 4 to 10-fold) that correlated with the nontumorigenic phenotype of the hybrid cells. Subsequent analysis resulted in the identification of a full length cDNA (2,569 bp) termed RIG (regulated in glioma). RIG expression was either not detected or detected only at low levels in cultured glioma cells and primary glioblastoma specimens compared to normal brain cells. The 2.6 kb RIG mRNA was expressed predominantly in normal brain with lower levels in heart and lung. Sequence analysis showed no significant homology to known gene products. Genomic alterations of RIG were present in approximately 25% of glioma cell lines examined. Also, RIG mapped to chromosome 11p15.1, a region that is known to be altered in malignant astrocytomas. The differential expression pattern, tissue distribution and chromosomal location of RIG suggests it serves as a molecular marker for or may play a role in the malignant progression of GBMs.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Astrocitoma/metabolismo , Secuencia de Bases , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/metabolismo , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Regulación hacia Abajo , GTP Fosfohidrolasas , Glioblastoma/genética , Humanos , Pulmón/metabolismo , Datos de Secuencia Molecular , Miocardio/metabolismo , ARN Mensajero/metabolismo , Distribución Tisular , Trasplante Homólogo , Proteínas Supresoras de TumorRESUMEN
Genomic deletions involving chromosome 4 have recently been implicated in several human cancers. To identify and characterize genetic events associated with the development of head and neck squamous cell carcinoma (HNSCC), a fine mapping of allelic losses associated with chromosome 4 was performed on DNA isolated from 27 matched primary tumor specimens and normal tissues. Loss of heterozygosity (LOH) of at least one chromosome 4 polymorphic allele was seen in the majority of tumors (92%). Allelic deletions were confined to short arm loci in four tumors and to the long arm loci in 12 tumors, suggesting the presence of two regions of common deletion. One region of frequent deletion was centered at D4S405 on 4p and included the loci D4S1546 to D4S428 in approximately 41% of the tumors. The common region of deletion on 4q was more complex and extended from D4S1571 to D4S1573. Frequent genetic alterations were observed within this region (4q25) and one marker, D4S407, exhibited a high frequency of LOH (>75%). These results indicate that alterations of chromosome 4 regions are associated with HNSCC tumorigenesis and further localizes the regions that may harbor tumor suppressor genes.
Asunto(s)
Alelos , Carcinoma de Células Escamosas/genética , Mapeo Cromosómico , Cromosomas Humanos Par 4/genética , Eliminación de Gen , Neoplasias de Cabeza y Cuello/genética , Humanos , Estudios ProspectivosRESUMEN
Malignant human gliomas are the most common forms of primary tumors in the central nerve system. Due to their location and invasive nature, treatment so far has been mainly palliative. Thus, understanding the molecular detail of tumor transformation and progression is crucial for developing effective therapeutic strategy for this fetal tumor. Among the genetic alternations found in these tumors, p53 inactivation and PDGF/PDGFR activation represent the early events, and the loss of chromosome 10 and gene amplification and rearrangement of EGFR represent the late events. Studies with both glioma cell lines and primary tumor tissues have strongly suggested that TGF-alpha and EGFR function as an important autocrine loop in supporting proliferation of human glioma, especially in high grade glioma, since elevated TGF-alpha expression is also found in these high grade tumors. Furthermore, down regulation of the expression of TGF-alpha by antisense constructs has been shown to inhibit several types of human tumor cell growth including glioma. Other means of therapeutic approaches using this autocrine loop as a target also include the use of monoclonal antibodies and their cytotoxic conjugated. Considerable understanding of the EGFR-mediated signal transduction pathways has become available recently, which including GRB2/mSOS1 mediated MAP kinase activation; JAK/STATs pathway; PLC-gamma pathway. However, much work still needs to be done before a specific component of these pathways can be applied for effective control of tumor growth in the clinic.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Factor de Crecimiento Transformador alfa/fisiología , Animales , Neoplasias Encefálicas/metabolismo , HumanosRESUMEN
Glioblastoma multiforme is distinguished from its less malignant astrocytoma precursors by intense angiogenesis and frequent loss of tumor suppressor genes on chromosome 10. Here we link these traits by showing that when a wild-type chromosome 10 was returned to any of three human glioblastoma cell lines U251, U87, or LG11, they lost their ability to form tumors in nude mice and switched to an antiangiogenic phenotype, as measured by the inhibition of capillary endothelial cell migration and of corneal neovascularization. This change in angiogenesis was directly due to the increased secretion of a potent inhibitor of angiogenesis, thrombospondin-1, because: (a) neutralizing thrombospondin completely relieved the inhibition; (b) the inhibitory activity of thrombospondin was not dependent on transforming growth factor beta; and (c) chromosome 10 introduction did not alter secreted inducing activity. The inducing activity was dependent on vascular endothelial cell growth factor and had an ED50 of 10 microg/ml in media conditioned by parental cells and 9-13 microg/ml in media conditioned by chromosome 10 revertants. Normal human astrocytes were also antiangiogenic due to secreted thrombospondin. The effect of chromosome 10 on thrombospondin production in vitro was reflected in patient material. Normal brain and lower grade astrocytomas known to retain chromosome 10 stained strongly for thrombospondin, but 12 of 13 glioblastomas, the majority of which lose chromosome 10, did not. These data indicate that the loss of tumor suppressors on chromosome 10 contributes to the aggressive malignancy of glioblastomas in part by releasing constraints on angiogenesis that are maintained by thrombospondin in lower grade tumors.