RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Genes Supressores de Tumor/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor , Neoplasias da Bexiga Urinária/terapia , Adenovírus Humanos , Ciclo Celular , Divisão Celular , Expressão Gênica , Vetores Genéticos , Humanos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Células Tumorais CultivadasRESUMO
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.
Assuntos
Carcinoma de Células de Transição/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Neoplasias da Bexiga Urinária/genética , Carcinoma de Células de Transição/metabolismo , Análise Mutacional de DNA , Éxons , Deleção de Genes , Rearranjo Gênico , Humanos , Mutação , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/biossíntese , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismoRESUMO
The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
Assuntos
Apoptose/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Divisão Celular/genética , Genes Supressores de Tumor , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Neoplasias da Mama/genética , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Proteínas Associadas aos Microtúbulos/genética , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Cell-matrix interactions exert a profound influence on cell function and behavior. Our earlier observations suggested that disruption of the actin cytoskeleton results in the inhibition of phorbol ester-induced matrix metalloproteinase (MMP)-9 expression. In this study, to understand the role of protein tyrosine phosphatases in matrix metalloproteinase-9 expression, we treated glioblastoma cells with vanadate and phenylarsine oxide (PAO), which are inhibitors of protein tyrosine phosphatases. Vanadate and PAO inhibited expression of phorbol ester-induced MMP-9 as well as constitutive expression of matrix metalloproteinase-2 in a dose- and time-dependent fashion. An assay of the activity of phosphotyrosine phosphatase (PTPase) indicated that vanadate-treated cells had reduced PTPase activity compared with that of untreated controls. Vanadate and PAO also inhibited actin polymerization, cell spreading, migration, and invasion of glioma cells. Furthermore, elevated levels of protein tyrosine phosphorylation were observed in vanadate- and PAO-treated cells in both a concentration- and time-dependent fashion and were seen to have an inverse correlation with focal adhesion kinase protein expression. These results suggest that vanadate and PAO inhibited migration and invasion of glioma cells by their effect on the cytoskeleton and inhibition of MMP expression.
Assuntos
Actinas/efeitos dos fármacos , Arsenicais/farmacologia , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Invasividade Neoplásica/prevenção & controle , Proteínas Tirosina Fosfatases/metabolismo , Vanadatos/farmacologia , Moléculas de Adesão Celular/genética , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glioma , Humanos , Fosfoproteínas/metabolismo , Fosfotirosina/análise , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fator de Crescimento Transformador alfa/genética , Animais , Neoplasias Encefálicas/genética , Células Clonais , Glioma/genética , Humanos , Camundongos , Camundongos Nus , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Apoptose/fisiologia , Proteínas de Arabidopsis , Carcinoma/metabolismo , Divisão Celular/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Supressoras de Tumor , Carcinoma/genética , Carcinoma/patologia , Vetores Genéticos , Humanos , Masculino , PTEN Fosfo-Hidrolase , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Biomarcadores Tumorais/biossíntese , Glioblastoma/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Proteínas Supressoras de Tumor , Genes Supressores de Tumor , Glioblastoma/diagnóstico , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , PTEN Fosfo-Hidrolase , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
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.
Assuntos
DNA Antissenso/farmacologia , Fatores de Crescimento Endotelial/genética , Terapia Genética , Glioma/terapia , Linfocinas/genética , Neovascularização Patológica/terapia , Adenoviridae/genética , Animais , Regulação para Baixo , Glioma/irrigação sanguínea , Humanos , Camundongos , Camundongos Nus , RNA Mensageiro/análise , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Genes Supressores de Tumor , Glioma/genética , Monoéster Fosfórico Hidrolases/biossíntese , Proteínas Supressoras de Tumor , Domínio Catalítico/genética , Adesão Celular , Divisão Celular , Transformação Celular Neoplásica , Glioma/enzimologia , Humanos , Mutação , PTEN Fosfo-Hidrolase , Fenótipo , Monoéster Fosfórico Hidrolases/genética , Proteínas Recombinantes/biossíntese , Células Tumorais CultivadasRESUMO
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.
Assuntos
Aglutininas , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Glioma/genética , Proteínas Supressoras de Tumor , Western Blotting , Neoplasias Encefálicas/genética , Proteínas de Ligação ao Cálcio , DNA de Neoplasias/análise , Proteínas de Ligação a DNA , Genes Supressores de Tumor/genética , Glioma/química , Glioma/metabolismo , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade/genética , Repetições de Microssatélites/genética , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/biossíntese , Células Tumorais CultivadasRESUMO
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.
Assuntos
Apoptose/fisiologia , Glioblastoma/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor , Adenoviridae/genética , Adenoviridae/metabolismo , Ativação Enzimática , Receptores ErbB/biossíntese , Receptores ErbB/fisiologia , Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/fisiologia , Transgenes , Células Tumorais CultivadasRESUMO
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.
Assuntos
Aglutininas , Mapeamento Cromossômico , Genes Supressores de Tumor , Glioma/genética , Perda de Heterozigosidade , Monoéster Fosfórico Hidrolases/genética , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Glioma/mortalidade , Humanos , PTEN Fosfo-Hidrolase , Prognóstico , Taxa de SobrevidaRESUMO
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.
Assuntos
Terapia Genética , Glioma/terapia , Northern Blotting , DNA Recombinante/genética , Regulação para Baixo , Fatores de Crescimento Endotelial/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica , Glioma/enzimologia , Glioma/genética , Humanos , Linfocinas/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA/genética , RNA/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Gênica/genética , Transfecção/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 10 , Genes Supressores de Tumor/genética , Glioblastoma/genética , Northern Blotting , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Humanos , Células Híbridas , Cariotipagem , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Homologia de Sequência do Ácido Nucleico , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND: Elongation factor-1 (EF-1) is a cellular protein that plays a role in protein synthesis by mediating the transfer of aminoacyl-tRNA to 80S ribosomes. It is comprised of four subunits: alpha, beta, gamma, and delta. EF-1gamma is a substrate for the maturation-promoting factor, which determines entry into the M-phase of the cell cycle in all eukaryotic cells. Previously, the authors showed that EF-1gamma RNA is overexpressed in a high proportion of colorectal carcinomas. At that time, there were no antibodies to EF-1gamma, so the EF-1gamma protein could not be examined. Because levels of RNA do not always parallel the levels of the protein it encodes, it was important to develop antibodies to EF-1gamma to examine its expression at the protein level in colorectal carcinoma. METHODS: Twenty-nine patients undergoing surgical resection for colorectal adenocarcinoma were studied. A polyclonal antibody to EF- 1gamma in rabbit was prepared. Tumors and normal-appearing mucosa distant from the tumor (> or = 10 cm) were obtained from each patient. Cytosolic proteins were extracted from the tissues and examined by Western blot analysis with the EF-1gamma antibody. Colonic tumors also were studied by immunohistochemical analysis with another EF-1gamma polyclonal antibody. RESULTS: Using Western blot analysis, the authors observed greater expression of EF-1gamma in the tumors than in the more distal normal-appearing mucosa. Overexpression was not observed in the patients with the two Dukes Stage A tumors, but was observed in four of ten patients with Dukes Stage B tumors, seven of eight patients with Dukes Stage C tumors, and six of nine patients with Dukes Stage D tumors. Overall, 17 of 29 patients (59%) were found to have overexpression of EF-1gamma. Using immunohistochemical analysis, EF-1gamma protein was shown to be located predominantly in tumor epithelium rather than the stroma or infiltrating mononuclear cells. CONCLUSIONS: Previous studies showed that EF-1gamma mRNA frequently is overexpressed in colorectal adenocarcinoma. This study showed that EF-1gamma also was overexpressed at the protein level in colorectal adenocarcinoma relative to more distal normal-appearing mucosa from the same patient. Immunohistochemical analysis demonstrated that this protein was expressed predominantly in the tumor epithelial cells and therefore was not derived from cells involved in the desmoplastic response.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Fatores de Alongamento de Peptídeos/biossíntese , Neoplasias Retais/genética , Adenocarcinoma/patologia , Adulto , Idoso , Animais , Western Blotting , Neoplasias do Colo/patologia , Feminino , Expressão Gênica , Humanos , Mucosa Intestinal , Masculino , Pessoa de Meia-Idade , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/genética , RNA Mensageiro/metabolismo , Coelhos , Neoplasias Retais/patologiaRESUMO
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.
Assuntos
Cromossomos Humanos Par 10 , Mutação , Neoplasias/genética , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases/genética , Proteínas Supressoras de Tumor , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Criança , Mapeamento Cromossômico , Éxons , Feminino , Deleção de Genes , Marcadores Genéticos , Variação Genética , Glioblastoma/genética , Glioblastoma/patologia , Glioma/genética , Glioma/patologia , Humanos , Íntrons , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Neoplasias/patologia , PTEN Fosfo-Hidrolase , Mutação Puntual , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/biossíntese , Deleção de Sequência , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Cromossomos Humanos Par 4/genética , Genes Supressores de Tumor/genética , Glioma/genética , Animais , Southern Blotting , Mapeamento Cromossômico , DNA/análise , Fator de Crescimento Epidérmico/genética , Glioma/patologia , Humanos , Células Híbridas , Camundongos , Repetições de Microssatélites , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Cowden syndrome (CS) is an autosomal dominant disorder associated with the development of hamartomas and benign tumors in a variety of tissues, including the skin, thyroid, breast, endometrium, and brain. It has been suggested that women with CS are at increased risk for breast cancer. A locus for CS was recently defined on chromosome 10 in 12 families, resulting in the identification of the CS critical interval, between the markers D10S215 and D10S541. More recently, affected individuals in four families with CS have been shown to have germ-line mutations in a gene known as "PTEN," or "MMAC1," which is located in the CS critical interval on chromosome 10. In this study, we report three novel MMAC1 mutations in CS and demonstrate that MMAC1 mutations are associated with CS and breast cancer. Furthermore, we also show that certain families and individuals with CS do not have mutations in the coding sequence of MMAC1. Finally, we did not detect MMAC1 mutations in a subpopulation of individuals with early-onset breast cancer, suggesting that germ-line mutations in this gene do not appear to be common in this group.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Síndrome do Hamartoma Múltiplo/genética , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases/genética , Proteínas Supressoras de Tumor , Cromossomos Humanos Par 10/genética , Feminino , Genes Dominantes , Marcadores Genéticos/genética , Haplótipos/genética , Humanos , Escore Lod , Masculino , Mutação , PTEN Fosfo-Hidrolase , Linhagem , Reação em Cadeia da Polimerase , Fatores de Risco , Análise de Sequência de DNARESUMO
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.
Assuntos
Neoplasias Encefálicas/fisiopatologia , Ciclinas/fisiologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/fisiopatologia , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae , Apoptose , Western Blotting , Neoplasias Encefálicas/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , DNA de Neoplasias/genética , Citometria de Fluxo , Vetores Genéticos , Glioblastoma/genética , Humanos , Fenótipo , Proteína Supressora de Tumor p53/biossíntese , Regulação para CimaRESUMO
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.