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Delineating the cytogenomic and epigenomic landscapes of glioma stem cell lines.

Baronchelli S, Bentivegna A, Redaelli S, Riva G, Butta V, Paoletta L, Isimbaldi G, Miozzo M, Tabano S, Daga A, Marubbi D, Cattaneo M, Biunno I, Dalprà L - PLoS ONE (2013)

Bottom Line: We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population.Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes.This new overview could have a huge importance in therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Translational Medicine, University of Milan-Bicocca, Monza, Italy.

ABSTRACT
Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term "multiforme" describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common "signature" of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomic-signature (gain of chromosome 7- EGFR gene, loss of chromosome 13- RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes. This new overview could have a huge importance in therapy.

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Related in: MedlinePlus

Cytogenomic profiles of GSCs.(A) Frequency of gains and losses of whole chromosomes in the six GSC lines analyzed by QFQ-banding. The frequencies of numerical aberrations specific for each chromosome were calculated from the total of the analyzed metaphases of the six cell lines and represented as mean values. (B) Composite array CGH profiles of GSC lines. (C) Detailed 1p LOH mapping of GSC lines. A common region of LOH was identified in all the six GSC lines, involving D1S214 microsatellite, located at 1p36.31 and highlighted by the square box.
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pone-0057462-g001: Cytogenomic profiles of GSCs.(A) Frequency of gains and losses of whole chromosomes in the six GSC lines analyzed by QFQ-banding. The frequencies of numerical aberrations specific for each chromosome were calculated from the total of the analyzed metaphases of the six cell lines and represented as mean values. (B) Composite array CGH profiles of GSC lines. (C) Detailed 1p LOH mapping of GSC lines. A common region of LOH was identified in all the six GSC lines, involving D1S214 microsatellite, located at 1p36.31 and highlighted by the square box.

Mentions: All the clonal chromosomal abnormalities pointed out by this analysis are listed in Table 1. As expected, each cell line showed a certain degree of karyotype complexity which ranges from a high number of structural abnormalities, as the GBM2 cell line, to the presence of two subpopulations with a different modal number of chromosomes, as the G179 cell line (G179 and G179*). The modal number of chromosomes varied from near-diploid (G166, GliNS2), near-triploid (GBM2, G179), near-tetraploid (G144, GBM7), to near-pentaploid (G179*). All the chromosomes were involved in numerical alterations (Figure 1A): the most common (73% of the total analyzed metaphases) was gain of chromosome 7, with at least two supernumerary copies in four cell lines (GBM2, GBM7, G179 and G144, Table 1). Other commonly observed numerical changes were: loss of chromosome 13 (43%); loss or gain of chromosome X (28% or 21% of cases, respectively); loss of chromosome Y (39%); loss of chromosome 10 (in three out of six cell lines, frequency of 32%). A total of 59 different clonal chromosomal aberrations were found among the 6 cell lines. Chromosome 1 was the most involved in structural abnormalities: the ever present deletions in 1p36-1p33 were further accompanied by inversions or unbalanced translocations (Table 1 and Figures S2, S3, and S4). Also chromosomes 18, 11 (three out of six cell lines), 12 (four out of six cell lines) were frequently damaged by structural abnormalities. Finally, the long arm of chromosome 6 was affected by loss of genomic material in two cell lines (GBM2 and G166) or by two translocations, involving chromosome 7 (GBM2) or 3 (GliNS2). Lastly, by conventional cytogenetic techniques, it was possible to observe the presence of double minutes, which should not be included in the count of the number of chromosomes. The molecular karyotypes showed some common genomic features of GBM, such as complete loss (isomy) of 9p21.3 locus (GBM2, GBM7, G179 and GliNS2), containing CDKN2A and CDKN2B genes (Figure 1B and Tables S2, S3, S4, S5, and S6). Complete or nearly complete gain of chromosome 7 was evidenced in the same GSC lines, while for G166 cells gain of 7p22.3-q11.2, including EGFR, was observed (Figure 1B and Table S4). Loss of whole chromosome 10 in GBM7 and G179 cell lines led to the inevitable absence of PTEN and DMBT1 genes and the same alteration was obtained in GliNS2 cells through the loss of 10q21.3-q26.3 region (Table S6). Accordingly, loss of PTEN locus (10q23.31) resulted in the lack of detectable PTEN transcriptional expression in G179 and GliNS2 lines and slight expression in GBM7 cells. Considering the absence of genomic alterations at PTEN locus in GBM2 and G166 cell lines, the divergences in gene expression should be ascribed to differences in the methylation levels of PTEN promoter region: G166 cells revealed PTEN expression and lack of promoter methylation, while GBM2 cells displayed no PTEN expression and promoter methylation (Figure S5). Loss of whole chromosome 13, containing RB1 gene, was evidenced in 86% of GBM2 and 29% of G179 cells (Table S2 and S5, respectively). Several CNAs affected chromosome 1: whole p-arm loss nearly in 50% of G179 cells; 1p36-p34 loss in GBM7 and GliNS2 cell lines; 1q21.1-q32.2 gain in G166 (almost 90% of cells); in particular, gain of 1q32.1 locus, containing MDM4 gene, was found also in GBM2 cells. Other aberrations were: gain of 20p and 20q in GBM7 and G166 cells; loss of chromosome Y in four cell lines. In addition, “private” alterations were evidenced, such as the gain of whole chromosome X in G166 line, the amplification of 4q12, containing PDGFR and the loss of TP53 locus (17p13.2-p13.1) in almost 60% of cells in GBM2 line.


Delineating the cytogenomic and epigenomic landscapes of glioma stem cell lines.

Baronchelli S, Bentivegna A, Redaelli S, Riva G, Butta V, Paoletta L, Isimbaldi G, Miozzo M, Tabano S, Daga A, Marubbi D, Cattaneo M, Biunno I, Dalprà L - PLoS ONE (2013)

Cytogenomic profiles of GSCs.(A) Frequency of gains and losses of whole chromosomes in the six GSC lines analyzed by QFQ-banding. The frequencies of numerical aberrations specific for each chromosome were calculated from the total of the analyzed metaphases of the six cell lines and represented as mean values. (B) Composite array CGH profiles of GSC lines. (C) Detailed 1p LOH mapping of GSC lines. A common region of LOH was identified in all the six GSC lines, involving D1S214 microsatellite, located at 1p36.31 and highlighted by the square box.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585345&req=5

pone-0057462-g001: Cytogenomic profiles of GSCs.(A) Frequency of gains and losses of whole chromosomes in the six GSC lines analyzed by QFQ-banding. The frequencies of numerical aberrations specific for each chromosome were calculated from the total of the analyzed metaphases of the six cell lines and represented as mean values. (B) Composite array CGH profiles of GSC lines. (C) Detailed 1p LOH mapping of GSC lines. A common region of LOH was identified in all the six GSC lines, involving D1S214 microsatellite, located at 1p36.31 and highlighted by the square box.
Mentions: All the clonal chromosomal abnormalities pointed out by this analysis are listed in Table 1. As expected, each cell line showed a certain degree of karyotype complexity which ranges from a high number of structural abnormalities, as the GBM2 cell line, to the presence of two subpopulations with a different modal number of chromosomes, as the G179 cell line (G179 and G179*). The modal number of chromosomes varied from near-diploid (G166, GliNS2), near-triploid (GBM2, G179), near-tetraploid (G144, GBM7), to near-pentaploid (G179*). All the chromosomes were involved in numerical alterations (Figure 1A): the most common (73% of the total analyzed metaphases) was gain of chromosome 7, with at least two supernumerary copies in four cell lines (GBM2, GBM7, G179 and G144, Table 1). Other commonly observed numerical changes were: loss of chromosome 13 (43%); loss or gain of chromosome X (28% or 21% of cases, respectively); loss of chromosome Y (39%); loss of chromosome 10 (in three out of six cell lines, frequency of 32%). A total of 59 different clonal chromosomal aberrations were found among the 6 cell lines. Chromosome 1 was the most involved in structural abnormalities: the ever present deletions in 1p36-1p33 were further accompanied by inversions or unbalanced translocations (Table 1 and Figures S2, S3, and S4). Also chromosomes 18, 11 (three out of six cell lines), 12 (four out of six cell lines) were frequently damaged by structural abnormalities. Finally, the long arm of chromosome 6 was affected by loss of genomic material in two cell lines (GBM2 and G166) or by two translocations, involving chromosome 7 (GBM2) or 3 (GliNS2). Lastly, by conventional cytogenetic techniques, it was possible to observe the presence of double minutes, which should not be included in the count of the number of chromosomes. The molecular karyotypes showed some common genomic features of GBM, such as complete loss (isomy) of 9p21.3 locus (GBM2, GBM7, G179 and GliNS2), containing CDKN2A and CDKN2B genes (Figure 1B and Tables S2, S3, S4, S5, and S6). Complete or nearly complete gain of chromosome 7 was evidenced in the same GSC lines, while for G166 cells gain of 7p22.3-q11.2, including EGFR, was observed (Figure 1B and Table S4). Loss of whole chromosome 10 in GBM7 and G179 cell lines led to the inevitable absence of PTEN and DMBT1 genes and the same alteration was obtained in GliNS2 cells through the loss of 10q21.3-q26.3 region (Table S6). Accordingly, loss of PTEN locus (10q23.31) resulted in the lack of detectable PTEN transcriptional expression in G179 and GliNS2 lines and slight expression in GBM7 cells. Considering the absence of genomic alterations at PTEN locus in GBM2 and G166 cell lines, the divergences in gene expression should be ascribed to differences in the methylation levels of PTEN promoter region: G166 cells revealed PTEN expression and lack of promoter methylation, while GBM2 cells displayed no PTEN expression and promoter methylation (Figure S5). Loss of whole chromosome 13, containing RB1 gene, was evidenced in 86% of GBM2 and 29% of G179 cells (Table S2 and S5, respectively). Several CNAs affected chromosome 1: whole p-arm loss nearly in 50% of G179 cells; 1p36-p34 loss in GBM7 and GliNS2 cell lines; 1q21.1-q32.2 gain in G166 (almost 90% of cells); in particular, gain of 1q32.1 locus, containing MDM4 gene, was found also in GBM2 cells. Other aberrations were: gain of 20p and 20q in GBM7 and G166 cells; loss of chromosome Y in four cell lines. In addition, “private” alterations were evidenced, such as the gain of whole chromosome X in G166 line, the amplification of 4q12, containing PDGFR and the loss of TP53 locus (17p13.2-p13.1) in almost 60% of cells in GBM2 line.

Bottom Line: We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population.Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes.This new overview could have a huge importance in therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Translational Medicine, University of Milan-Bicocca, Monza, Italy.

ABSTRACT
Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term "multiforme" describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common "signature" of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomic-signature (gain of chromosome 7- EGFR gene, loss of chromosome 13- RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes. This new overview could have a huge importance in therapy.

Show MeSH
Related in: MedlinePlus