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1.
Oncotarget ; 6(13): 10950-63, 2015 May 10.
Article in English | MEDLINE | ID: mdl-25860932

ABSTRACT

Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133(+), Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy.


Subject(s)
Apoptosis , Bone Morphogenetic Protein 7/administration & dosage , Brain Neoplasms/prevention & control , Glioblastoma/prevention & control , Microspheres , Animals , Blotting, Western , Bone Morphogenetic Protein 7/genetics , Bone Morphogenetic Protein 7/metabolism , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Movement , Cell Proliferation , Delayed-Action Preparations , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Immunoenzyme Techniques , Mice , Mice, Nude , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Stem Cells ; 33(1): 219-29, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25185890

ABSTRACT

Members of the cyclin-dependent kinase (CDK)-inhibitory protein (CIP)/kinase-inhibitory protein (KIP) family of cyclin-dependent kinase inhibitors regulate proliferation and cell cycle exit of mammalian cells. In the adult brain, the CIP/KIP protein p27(kip1) has been related to the regulation of intermediate progenitor cells located in neurogenic niches. Here, we uncover a novel function of p27(kip1) in the adult hippocampus as a dual regulator of stem cell quiescence and of cell-cycle exit of immature neurons. In vivo, p27(kip1) is detected in radial stem cells expressing SOX2 and in newborn neurons of the dentate gyrus. In vitro, the Cdkn1b gene encoding p27(kip1) is transcriptionally upregulated by quiescence signals such as BMP4. The nuclear accumulation of p27(kip1) protein in adult hippocampal stem cells encompasses the BMP4-induced quiescent state and its overexpression is able to block proliferation. p27(kip1) is also expressed in immature neurons upon differentiation of adult hippocampal stem cell cultures. Loss of p27(kip1) leads to an increase in proliferation and neurogenesis in the adult dentate gyrus, which results from both a decrease in the percentage of radial stem cells that are quiescent and a delay in cell cycle exit of immature neurons. Analysis of animals carrying a disruption in the cyclin-CDK interaction domain of p27(kip1) indicates that the CDK inhibitory function of the protein is necessary to control the activity of radial stem cells. Thus, we report that p27(kip1) acts as a central player of the molecular program that keeps adult hippocampal stem cells out of the cell cycle.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Hippocampus/cytology , Neural Stem Cells/cytology , Neurogenesis/physiology , Animals , Apoptosis/physiology , Cell Differentiation/physiology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Cyclin-Dependent Kinase Inhibitor p27/genetics , Hippocampus/metabolism , Humans , Mice , Mice, Knockout , Neural Stem Cells/metabolism
3.
Biomed Res Int ; 2014: 549742, 2014.
Article in English | MEDLINE | ID: mdl-24877113

ABSTRACT

Astrocytic glioma is the most common brain tumor. The glioma initiating cell (GIC) fraction of the tumor is considered as highly chemoresistant, suggesting that GICs are responsible for glioma relapse. A potential treatment for glioma is to induce differentiation of GICs to a more benign and/or druggable cell type. Given BMPs are among the most potent inducers of GIC differentiation, they have been considered as noncytotoxic therapeutic compounds that may be of use to prevent growth and recurrence of glioma. We herein summarize advances made in the understanding of the role of BMP signaling in astrocytic glioma, with a particular emphasis on the effects exerted on GICs. We discuss the prognostic value of BMP signaling components and the implications of BMPs in the differentiation of GICs and in their sensitization to alkylating drugs and oncolytic therapy/chemotherapy. This mechanistic insight may provide new opportunities for therapeutic intervention of brain cancer.


Subject(s)
Astrocytoma/metabolism , Astrocytoma/therapy , Biomarkers, Tumor/metabolism , Bone Morphogenetic Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Animals , Astrocytoma/pathology , Humans , Neoplastic Stem Cells/pathology , Prognosis
4.
Cancer Res ; 73(20): 6264-76, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23950209

ABSTRACT

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.


Subject(s)
Melanoma/metabolism , Melanoma/pathology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins , Cell Differentiation/physiology , Cell Growth Processes/physiology , Cell Line, Tumor , Epigenomics , Heterografts , Humans , Immunohistochemistry , Melanoma/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Random Allocation , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transfection
5.
Stem Cells ; 30(12): 2796-809, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22987443

ABSTRACT

Adult neural stem cells (NSCs) located in the subventricular zone (SVZ) persistently produce new neurons destined to the olfactory bulb (OB). Recent research suggests that the OB is also a source of NSCs that remains largely unexplored. Using single/dual-labeling procedures, we address the existence of NSCs in the innermost layers of the OB. In vivo, these cells are more quiescent that their SVZ counterparts, but after in vitro expansion, they behave similarly. Self-renewal and proliferation assays in co-culture with niche astrocytes indicate that OB-glia restricts NSC activity whereas SVZ-glia has the opposite effect. Gene expression profiling identifies WNT7A as a key SVZ-glial factor lacking in OB-glia that enhances self-renewal, thereby improving the propagation of OB-NSC cultures. These data demonstrate that region-specific glial factors account for in vivo differences in NSC activity and point to WNT7A as a tool that may be instrumental for the NSC expansion phase that precedes grafting.


Subject(s)
Astrocytes/cytology , Neural Stem Cells/cytology , Olfactory Bulb/cytology , Wnt Proteins/metabolism , Animals , Cell Differentiation/physiology , Cell Growth Processes/physiology , Humans , Mice , Wnt Proteins/genetics
6.
Curr Protoc Stem Cell Biol ; Chapter 2: Unit 2D.10, 2012 May.
Article in English | MEDLINE | ID: mdl-22605645

ABSTRACT

The factors that regulate the switch from adult neural stem cell (aNSC) quiescence to active proliferation are poorly understood. Here we describe a method to study the in vivo effect of a soluble factor on cell cycle entry and proliferation of aNSCs located in the brain neurogenic niches. First, we provide information for implanting osmotic minipumps that will deliver the compound of interest directly into the mouse brain. When combined with the administration of the thymidine analog bromodeoxyuridine (BrdU), this technique is the most basic procedure to study the effects of a soluble factor on aNSC proliferation. We also describe a dual replication labeling protocol using two different halogenated thymidine analogs, chloro- and iododeoxyuridine (CldU and IdU), that allows tracking of proliferating cells and assessing cell cycle re-entry of aNSCs at different time points.


Subject(s)
Adult Stem Cells/drug effects , Cell Division/drug effects , Halogenation/drug effects , Intercellular Signaling Peptides and Proteins/administration & dosage , Intercellular Signaling Peptides and Proteins/pharmacology , Neural Stem Cells/drug effects , Thymidine/analogs & derivatives , Thymidine/pharmacology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Brain/cytology , Brain/drug effects , Cell Proliferation/drug effects , Infusions, Intraventricular , Male , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Solubility/drug effects , Staining and Labeling , Thymidine/administration & dosage , Time Factors
7.
Mol Neurobiol ; 44(3): 235-49, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21728042

ABSTRACT

MicroRNAs are endogenous non-coding small RNAs that have been described as highly conserved regulators of gene expression. They are involved in cancer and in the regulation of neural development and stem cell function. Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate solid tumours such as glioblastoma (GBM), drive malignant progression and mediate radio- and chemoresistance. GBM-derived CSCs share the fundamental stem cell properties of self-renewal and multipotency with neural stem cells (NSCs) and may be regulated by miRNAs. In this review, we will summarize the current knowledge regarding the role of miRNAs in GBM development with a focus on the regulation of GBM-CSCs. We propose a list of miRNAs that could serve as molecular classifiers for GBMs and/or as promising therapeutic targets for such brain tumours.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/physiopathology , Glioblastoma/genetics , Glioblastoma/physiopathology , MicroRNAs/metabolism , Neural Stem Cells/physiology , Signal Transduction/physiology , Cell Differentiation/genetics , Cell Proliferation , Gene Expression Profiling , Humans , MicroRNAs/genetics , Microarray Analysis , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/physiology , Neural Stem Cells/cytology
8.
Genet. mol. biol ; 29(3): 413-422, 2006. tab
Article in English | LILACS | ID: lil-450272

ABSTRACT

Aberrant methylation of CpG islands located in promoter regions represents one of the major mechanisms for silencing cancer-related genes in tumor cells. We determined the frequency of aberrant CpG island methylation for several tumor-associated genes: DAPK, MGMT, p14ARF, p16INK4a, TP73, RB1 and TIMP-3 in 55 brain tumors, consisting of 26 neuroepithelial tumors, 6 peripheral nerve tumors, 13 meningeal tumors and 10 metastatic brain tumors. Aberrant methylation of at least one of the seven genes studied was detected in 83.6 percent of the cases. The frequencies of aberrant methylation were: 40 percent for p14ARF, 38.2 percent for MGMT, 30.9 percent for, p16INK4a, 14.6 percent for TP73 and for TIMP-3, 12.7 percent for DAPK and 1.8 percent for RB1. These data suggest that the hypermethylation observed in the genes p14ARF, MGMT and p16INK4a is a very important event in the formation or progression of brain tumors, since the inactivation of these genes directly interferes with the cell cycle or DNA repair. The altered methylation rate of the other genes has already been reported to be related to tumorigenesis, but the low methylation rate of RB1 found in tumors in our sample is different from that so far reported in the literature, suggesting that perhaps hypermethylation of the promoter is not the main event in the inactivation of this gene. Our results suggest that hypermethylation of the promoter region is a very common event in nervous system tumors.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Brain Neoplasms , CpG Islands , Epigenesis, Genetic , Brazil , Chromosome Deletion , Gene Expression , Methylation , Polymerase Chain Reaction
9.
Oncol Rep ; 13(3): 539-42, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15711769

ABSTRACT

Loss of 1p heterozygosity is one of the most characteristic events in oligodendrogliomas. Several genes located in this region have been previously studied to find the target gene implicated in the development of this tumor without success. Patched-2, RIZ1 and KIF1B are novel oncosuppressor genes located at 1p and involved in different kinds of tumors. We have studied these genes and p18(ink4c) using PCR/SSCP methods to detect sequence variations in a series of 40 oligodendrogliomas in which the allelic status at 1p was analyzed. Polymorphisms or no sequence changes were detected in all four genes analyzed. None of the genes analyzed seem to be the target-gene mapped at 1p involved by mutation in oligodendroglioma development.


Subject(s)
Brain Neoplasms/genetics , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Kinesins/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Oligodendroglioma/genetics , Polymorphism, Genetic , Retinoblastoma Protein/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Brain Neoplasms/physiopathology , Cell Transformation, Neoplastic , Cyclin-Dependent Kinase Inhibitor p18 , DNA Mutational Analysis , Histone-Lysine N-Methyltransferase , Humans , Loss of Heterozygosity , Oligodendroglioma/physiopathology , Patched Receptors , Patched-2 Receptor , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Protein Kinase Inhibitors , Receptors, Cell Surface
10.
Int J Oncol ; 25(5): 1489-94, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15492843

ABSTRACT

Primarily involved in cell proliferation and differentiation processes, the plasma membrane-bound ErbB tyrosine kinase receptor family is formed by four members: erbB1/EGFR, erbB2/HER2/Neu, erbB3/HER3 and erbB4/HER4. Calmodulin (CaM) is a Ca2+-binding protein involved in the regulation of multiple intracellular processes that binds directly to EGFR in the presence of Ca2+, inhibiting its tyrosine kinase activity. Two main regions in the receptor have been implicated in this relationship: the calmodulin-binding domain (CaM-BD) and the calmodulin-like domain (CaM-LD); their sequences are highly conserved in other members of this family of receptors. The presence of mutations, amplification and/or overexpression and genomic rearrangement of these domains was investigated for all four erbB family genes in a series of 89 glial tumors, including 44 WHO grade IV glioblastomas, 21 WHO grade III anaplastic astrocytomas, and 24 WHO grade II astrocytomas. Gene alterations were only found in the regions of interest in EGFR. One glioblastoma showed an in frame tandem duplication of the intracellular region including CaM-LD (exons 18-25). CaM-BD gene overdose was evidenced in 18 tumors that showed EGFR amplification in other domains. Over-expression of CaM-BD and CaM-LD was detected in 6 and 17 cases, respectively, of the 19 tumors in which this study was performed. The other three genes coding for the ErbB receptors did not present point mutations, or rearrangements, and only a very low amplification rate was found for erbB2 (1 case) and erbB3 (4 cases). No overexpression of erbB2, erbB3 or erbB4 was detected. These findings suggest that EGFR is the main erbB gene family member non-randomly involved in malignant glioma development, and that the two domains under study, due to their high conservation and wide separation in the EGFR sequence, are good marker regions for evaluating EGFR/erbB1 gene amplification, as well as for analysing the presence of transcripts corresponding to truncated cytosolic forms of the receptor in these tumors.


Subject(s)
Astrocytoma/genetics , Brain Neoplasms/genetics , Calmodulin-Binding Proteins/genetics , Gene Amplification , Genes, erbB/genetics , Glioblastoma/genetics , Astrocytoma/pathology , Biopsy , Brain Neoplasms/pathology , Calmodulin-Binding Proteins/pharmacology , Cell Transformation, Neoplastic , DNA Mutational Analysis , Gene Rearrangement , Glioblastoma/pathology , Humans
11.
Acta Neuropathol ; 108(5): 413-21, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15365725

ABSTRACT

The purpose of this research was to examine the DNA methylation profile of meningiomas. Accordingly, we examined the DNA methylation status of ten tumor-related genes (RB1, p16(INK4a), p73, MGMT, ER, DAPK, TIMP-3, p14(ARF), THBS1, and Caspase-8) in 98 meningiomas (68 grade I; 27 grade II; and 3 grade III samples) using methylation-specific PCR and sequencing. The most frequently methylated genes were THBS1 (30%), TIMP-3 (24%), p16(INK4a) (17%), MGMT (16%), p73 (15%), ER (15%), and p14(ARF) (13%), whereas methylation was relatively rare in the other genes (<10%). Methylation occurred in at least one gene in 77.5% of the cases and in three or more genes in 25.5%. Methylation was tumor specific since it was absent in the controls: two non-neoplastic meningeal samples and two non-neoplastic brain samples. The frequency of aberrant gene methylation in grade I versus grade II-III tumors showed some differences for TIMP-3, THBS1, MGMT, p16(INK4a) and p73; these differences reached statistical significance for TIMP-3: 18% in grade I versus 40% in grade II-III (P < 0.02). Our previous loss of heterozygosity studies provided the allelic constitution at 1p and 22q for 60 of the 98 meningiomas included in this report. The level of aberrant promoter methylation increased in tumors (30 samples) displaying 1p loss (either isolated or as concurrent deletion at 1p/22q; P = 0.014). These meningiomas primarily accumulated the epigenetic changes of THBS1 (14/30; 47%; P < 0.005), TIMP-3 (12/30; 40%; P < 0.05), p73 (10/30; 26%; P < 0.02) and p14(ARF) /p16(INK4a)(7/30 each one; 23%; not significant). Our findings indicate that aberrant DNA methylation of promoter-associated CpG islands in meningiomas contributes to the development of these tumors.


Subject(s)
CpG Islands/genetics , DNA Methylation , DNA, Neoplasm/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Alleles , Female , Humans , Loss of Heterozygosity , Male , Middle Aged , Polymerase Chain Reaction , Promoter Regions, Genetic
12.
Mutat Res ; 554(1-2): 23-32, 2004 Oct 04.
Article in English | MEDLINE | ID: mdl-15450401

ABSTRACT

O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing DNA damage from alkylating agents. By removing alkyl groups from the O6-position in guanine, MGMT can prevent G:C to A:T transition mutations, a type of variation frequently involving TP53 mutations in brain tumors. Promoter hypermethylation of CpG islands in tumor-related genes can lead to their transcriptional inactivation, and this epigenetic mechanism has been shown to participate in MGMT silencing in some cancers, including those affecting the nervous system. Accordingly, a link between both genetic and epigenetic anomalies may exist in these neoplasms. To determine the relevance of defective MGMT function due to aberrant methylation in relation to the presence of TP53 mutations, we studied 469 nervous system tumors (including all major histological subtypes) for MGMT promoter methylation and TP53 mutations at exons 5-8. Overall, aberrant methylation occurred in 38% of the samples (180/469), with values higher than 50% in the more malignant forms such as glioblastomas and anaplastic gliomas including those with astrocytic, oligodendroglial and ependymal differentiation. In contrast, the non-glial tumors displayed an overall aberrant MGMT promoter methylation of 26%, even though this group includes highly malignant tumors such as neuroblastomas, medulloblastomas and brain metastases. Overall, TP53 mutations were found in 25% of the methylated MGMT tumors (45/180), whereas only 10% of the unmethylated MGMT tumors (30/289) showed TP53 changes (P < 0.001). G:C to A:T changes occurred at CpG sites in 9% of methylated tumors, and in 0.7% of the unmethylated samples. This type of transition at non-CpG dinucleotides was also more frequent in the tumors with aberrant MGMT methylation (5%) than the unmethylated tumors (0.7%). These data suggest that MGMT silencing as a result of promoter hypermethylation may lead to G:C to A:T transition mutations in the TP53 gene of some histological nervous system tumor subtypes.


Subject(s)
DNA Methylation , DNA Repair/genetics , Genes, p53 , Nervous System Neoplasms/genetics , O(6)-Methylguanine-DNA Methyltransferase/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Child , Child, Preschool , DNA Primers , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational
13.
Oncol Rep ; 12(3): 663-6, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15289853

ABSTRACT

Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.


Subject(s)
Brain Neoplasms/genetics , Caspases/biosynthesis , Caspases/genetics , CpG Islands , DNA Methylation , Medulloblastoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Alleles , Base Sequence , Brain Neoplasms/metabolism , Caspase 8 , Cell Line, Tumor , Child , Child, Preschool , DNA/metabolism , DNA Primers/chemistry , DNA Primers/pharmacology , Female , Gene Deletion , Gene Expression Regulation, Neoplastic , Gene Silencing , Homozygote , Humans , Male , Medulloblastoma/metabolism , Middle Aged , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Nucleic Acid , Transcription, Genetic
14.
J Neurooncol ; 67(1-2): 159-65, 2004.
Article in English | MEDLINE | ID: mdl-15072463

ABSTRACT

Aberrant methylation of promoter CpG islands in human genes represents an alternative mechanism for genetic inactivation, and contributes to the development of human tumors. Nevertheless, thus far, few reports have analyzed methylation in ependymomas. We determined the frequency of aberrant CpG island methylation of several tumor-associated genes: p16(INK4a), RB1, MGMT, DAPK, TIMP3, THBS1, TP73, NF2 and Caspase 8 in a group of 27 ependymomas, consisting of 22 WHO grade II samples and five anaplastic WHO grade III tumors. The respective methylation indices (number of genes methylated/total genes analyzed) for both tumor groups was 0.195 and 0.198. Overall methylation rates greater than 20% were detected in MGMT, TIMP3, THBS1 and TP73. NF2 and Caspase 8 each presented hypermethylation in less than 10% of cases, and the cell-cycle regulators RB1/p16(INK4a) were hypermethylated in 4% and 18% of the samples, respectively, mostly affecting the low-grade forms. Our findings suggest that methylation commonly contributes to the inactivation of cancer-related genes in ependymomas.


Subject(s)
Central Nervous System Neoplasms/genetics , CpG Islands/genetics , DNA Methylation , Ependymoma/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Gene Silencing , Humans , Male , Middle Aged , Polymerase Chain Reaction
15.
Cancer Genet Cytogenet ; 148(2): 148-51, 2004 Jan 15.
Article in English | MEDLINE | ID: mdl-14734228

ABSTRACT

Deletions at 1p are frequent in meningioma and represent a genetic marker associated with the genesis of atypical WHO grade II forms. Previous mutational analysis of TP73, a structurally and functionally TP53 homologous gene located at 1p36.33, failed to demonstrate a significant rate of sequence variations linked to gene inactivation in meningiomas with 1p loss. As an alternative, TP73 may be inactivated through aberrant 5' CpG island methylation, a primary mechanism participating in the inactivation of tumor suppressor genes during tumorigenesis. We determined the methylation status of the TP73 gene in a series of 60 meningiomas (33 grade I, 24 grade II, and 3 grade III samples), including tumors with deletion at 1p (n=30) and with intact 1p (n=30). Aberrant methylation was detected in 10 cases (33%) with 1p deletion and in 3 tumors (10%) with retention of alleles at this chromosome arm. The distribution of the 13 cases of methylation according to malignancy grade was 7 grade I, 5 grade II, and 1 grade III tumor. Accordingly, although TP73 aberrant methylation was more frequent in meningiomas with 1p deletion (P<0.05), no association with the grade of malignancy could be established. These findings, together with the previously reported increased TP73 expression in malignant meningiomas suggest that opposing functions of this gene may characterize distinct subsets of tumors: suppressed or reduced expression as a result of CpG methylation in some grade I-grade II tumors, and enhanced expression in some more malignant forms.


Subject(s)
DNA Methylation , Meningioma/genetics , Phosphoproteins/genetics , Trans-Activators/genetics , Chromosomes, Human, Pair 1 , Humans , Sequence Analysis, DNA
16.
Int J Mol Med ; 13(1): 93-8, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14654977

ABSTRACT

The aberrant methylation of the CpG island promoter regions acquired by tumor cells is one mechanism for loss of gene function. The high methylation rate for RB1 and death-associated protein-kinase gene (DAP-kinase) (60 and 90%, respectively) previously found in brain metastases suggests this mechanism could be non-randomly associated to tumor progression and metastasis. Thus, in addition to these two genes, we determined the methylation status of the genes p16INK4a, glutathione S-transferase P1 (GSTP1), O6-methylguanine DNA methyltransferase (MGMT), thrombospondin-1 (THBS1), p14ARF, TP53, p73, and tissue inhibitor of metalloproteinase 3 (TIMP-3), in 18 brain metastases of solid tumors, with methylation specific PCR. The metastases were derived from malignant melanoma (three cases), lung carcinoma (six cases), breast carcinoma (three cases), ovarian carcinoma (two cases) and one each from colon, kidney, bladder and undifferentiated carcinoma. We detected methylation levels in the tumor samples of 83% in p16INK4a, 72% in DAP-kinase, 56% in THBS1, 50% in RB1, 39% in MGMT, 33% in GSTP1 and p14ARF each, 22% in p73 and TIMP-3 each, and 11% in TP53. The methylation index (number of genes methylated/number of genes tested) varied between 0.1 and 0.6, with an average of 0.42, indicating that a high grade of gene methylation accumulates parallel to the tumor metastasis process. Our data suggest an important role for gene methylation in the development of brain metastases, primarily involving epigenetic silencing of DAP-kinase, THBS1 and the cell-cycle regulators RB1/p16INK4a.


Subject(s)
Brain Neoplasms/genetics , DNA Methylation , Promoter Regions, Genetic , Adult , Aged , Brain Neoplasms/secondary , Female , Glutathione S-Transferase pi , Glutathione Transferase/genetics , Humans , Isoenzymes/genetics , Male , Middle Aged , Tissue Inhibitor of Metalloproteinase-3/genetics , Tumor Suppressor Protein p14ARF/genetics
17.
Clin Cancer Res ; 9(15): 5601-6, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14654541

ABSTRACT

PURPOSE: The purpose of this research was to examine the DNA methylation profile of schwannomas. EXPERIMENTAL DESIGN: We examined the DNA methylation status of 12 tumor-related genes (NF2, RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1, caspase-8, TP53, and GSTP1) in 44 sporadic and/or NF2-associated schwannomas using methylation-specific PCR. RESULTS: The most frequently methylated genes were THBS1 (36%), p73 (27%), MGMT (20%), NF2 (18%), and TIMP-3 (18%). The RB1/p16INK4a gene pair displayed aberrant methylayed alleles in 15% of cases, whereas methylation was relatively rare in the other genes (<5%). Methylation was tumor specific because it was absent in two nonneoplastic nerve sheath samples and two nonneoplastic brain samples studied as controls. CONCLUSIONS: Our findings indicate that aberrant methylation seems to be a mechanism for NF2 gene inactivation, considered an early step in schwannoma tumorigenesis, and as well, aberrant hypermethylation of other tumor-related genes might represent secondary events that also contribute to the development of these tumors.


Subject(s)
DNA Methylation , Dinucleoside Phosphates/metabolism , Neurilemmoma/genetics , Neurofibromatosis 2/genetics , Adult , Aged , Female , Genes, Neoplasm/genetics , Humans , Male , Middle Aged , Polymerase Chain Reaction
18.
Oncol Rep ; 10(5): 1519-23, 2003.
Article in English | MEDLINE | ID: mdl-12883734

ABSTRACT

Aberrant methylation of the promoter CpG island of human genes is an alternative gene inactivation mechanism that contributes to the carcinogenesis of human tumours. We have determined the methylation status of the CpG island of 11 tumour-related genes (RB1, p14ARF, p16INK4a, p73, TIMP-3, MGMT, DAPK, THBS1, caspase 8, TP53 and GSTP1) in 18 neurofibromas (including one plexiform neurofibroma) and three neurofibrosarcomas, as well as two non-neoplastic peripheral nerve sheath samples, using methylation-specific polymerase chain reaction. The series included sporadic and neurofibromatosis type 1-associated tumours. The incidence of aberrant methylation in the tumour samples was 52% for THBS1, 43% for MGMT, 33% for TIMP-3, 19% each for p16INK4a and p73, 14% for RB1, 5% for p14ARF, and 0% for DAPK, caspase 8, TP53 and GSTP1. No methylation of these genes was detected in the two samples of non-neoplastic peripheral nerve sheath. All but three samples in the study displayed aberrant methylation in at least one of the studied genes, and there was no correlation between methylation status and the patients' clinical parameters. These findings suggest that methylation of some tumour-related genes may play a significant role in the tumourigenesis of neurofibromas/neurofibrosarcomas.


Subject(s)
Central Nervous System Neoplasms/genetics , CpG Islands , DNA Methylation , Neurofibroma/genetics , Neurofibrosarcoma/genetics , Adult , Female , Humans , Male , Middle Aged , Polymerase Chain Reaction
19.
Cancer Genet Cytogenet ; 144(2): 134-42, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12850376

ABSTRACT

Promoter hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. To determine the frequency and timing of hypermethylation during carcinogenesis of nonastrocytic tumors, we analyzed promoter methylation status of 10 tumor-associated genes in a series of 41 oligodendrogliomas (22 World Health Organization [WHO] grade II; 13 WHO grade III; 6 WHO grade II-III oligoastrocytomas) and 7 WHO grade II-III ependymomas, as well as 2 nonneoplastic brain samples, by a methylation-specific polymerase chain reaction. Aberrant CpG island methylation was detected in 9 of 10 genes analyzed, and all but one sample displayed anomalies in at least one gene. The frequencies of hypermethylation for the 10 genes were as follows, in oligodendrogliomas and ependymomas, respectively: 80% and 28% for MGMT; 70% and 28% for GSTP1; 66% and 57% for DAPK; 44% and 28% for TP14(ARF); 39% and 0% for THBS1; 24% and 28% for TIMP3; 24% and 14% for TP73; 22% and 0% for TP16(INK4A); 3% and 14% for RB1; and 0% in both neoplasms for TP53. No methylation of these genes was detected in normal brain tissue samples. We conclude that a high frequency of aberrant methylation of the 5' CpG island of the MGMT, GSTP1, TP14(ARF), THBS1, TIMP3, and TP73 genes is observed in nonastrocytic neoplasms. This aberration seems to occur early in the carcinogenesis process (it is already present in the low-grade forms), although in some instances (DAPK, THBS1, and TP73) it appears also associated with the genesis of anaplastic forms.


Subject(s)
DNA Methylation , Ependymoma/genetics , Oligodendroglioma/genetics , Promoter Regions, Genetic , Adult , CpG Islands , Female , Genes, p16 , Genes, p53 , Humans , Loss of Heterozygosity , Male , Middle Aged , O(6)-Methylguanine-DNA Methyltransferase/genetics , Tumor Suppressor Protein p14ARF/genetics
20.
Oncol Rep ; 10(4): 1031-3, 2003.
Article in English | MEDLINE | ID: mdl-12792765

ABSTRACT

Death-associated protein (DAP) kinase is a gene that participates in apoptosis induced by interferon gamma. It appears to play a role in lung cancer metastasis in animal models, suggesting that DAP-kinase may function as a metastasis suppressor by inducing apoptosis. Expression silencing through CpG island methylation of DAP-kinase has been frequently found in connection with adverse survival, as cells lacking DAP-kinase appear to be more invasive and more metastatic in lung cancer. The purpose of this study was to analyze the promoter methylation status of DAP-kinase gene in brain metastases of solid tumors. Methylation-specific PCR was performed on ten brain metastasis samples derived from malignant melanoma (three cases), lung cancer (two), breast carcinoma (two), ovarian carcinoma (two) and colon carcinoma (one case), and in corresponding peripheral blood DNA samples. Two normal brain tissue samples were also analyzed, no promoter hypermethylation was observed in either case. DAP-kinase hypermethylated alleles were identified in nine metastases (90%), and in peripheral blood lymphocytes DNA from four cases. Our data suggest that silencing of DAP-kinase through promoter hypermethylation is a common event in the multistep process of tumor metastasis, including brain involvement.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/secondary , Calcium-Calmodulin-Dependent Protein Kinases/genetics , DNA Methylation , Promoter Regions, Genetic , Adult , Apoptosis Regulatory Proteins , Brain Neoplasms/enzymology , Breast Neoplasms/blood , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Calcium-Calmodulin-Dependent Protein Kinases/blood , Colonic Neoplasms/blood , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , DNA, Neoplasm/genetics , Death-Associated Protein Kinases , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Male , Melanoma/blood , Melanoma/enzymology , Melanoma/genetics , Middle Aged , Ovarian Neoplasms/blood , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/genetics , Polymerase Chain Reaction , Sulfites
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