Jesse Chung Sean Pang

Antigen presentation and T cell development in H2-M-deficient mice.

HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.

Heterogeneous responses of aquaporin-4 in oedema formation in a replicated severe traumatic brain injury model in rats

Aquaporin-4 (AQP4) is the most abundant water channel in the rat brain. In this study, the distribution pattern and mRNA expression levels of AQP4 were examined in a severe traumatic brain injury model by immunohistochemistry and reverse transcription-polymerase chain reaction. Oedema formation and blood-brain barrier (BBB) integrity were assessed by wet-dry weight measurements and immunostaining of endogenous IgG respectively. In the oedematous contusional cortex with impaired BBB integrity, negative immunostaining of AQP4 and down-regulation of its mRNA level were identified (P<0.05) at 1 day post-injury, while in other oedematous regions of the injured brain where BBB was intact, there was no significant change in the AQP4 expression level. This heterogeneous pattern of AQP4 responses can be interpreted as follows: focal brain injury (such as a contusion) with impaired BBB resulting in vasogenic oedema is associated with reduction of AQP4 expression, whereas, in cytotoxic oedema, associated with diffuse brain injury with intact BBB, changes in AQP4 expression are not significant. This study provides basic information for investigating new treatments for traumatic brain oedema.

Genetic alterations in pediatric high-grade astrocytomas

High-grade astrocytomas are tumors that are uncommon in children. Relatively few studies have been performed on their molecular properties and so it is not certain whether they follow different genetic pathways from those described in adult diffuse astrocytomas. In this study, we evaluated 24 pediatric high-grade astrocytomas (11 anaplastic astrocytomas and 13 glioblastomas) all of which were sporadic and primary. We studied mutations of p53, phosphatase and tensin homolog (PTEN), loss of heterozygosity (LOH) of chromosomes 17p13, 9p21 and 10q23-25, amplification of epidermal growth factor receptor (EGFR), and overexpression of EGFR and p53 protein. In addition, we searched for microsatellite instability (MSI) by using MSI sensitive and specific microsatellite markers. p53 mutations were found in 38% (9/24) of the high-grade astrocytomas and all brain stem tumors except 2 (71%, 5/7) had p53 mutations. PTEN mutations were found in 8% (2/24) of high-grade astrocytomas. However, no EGFR amplification was found in any of them. LOH was found at 17p13.1 in 50% (3/6 informative tumors), 9p21 in 83% (5/6 informative tumors), and 10q23-25 in 78% (7/9 informative tumors). Four tumors showed MSI, and 2 of them that showed widespread MSI were regarded as tumors with replication error (RER+) phenotype. All 4 tumors with MSI showed concurrent LOH of 9p21 and 10q23-25. Combining gene alterations, LOH, MSI, and gene mutations, inactivation of both alleles of PTEN and p53 was found in 57% (4/7 informative tumors) and 50% (3/6 informative tumors) of the cases respectively. We conclude that development of pediatric high-grade astrocytomas may follow pathways different from the primary or secondary paradigm of adult glioblastomas. In a subset of these tumors, genomic instability was also implicated.

Oncogenic role of microRNAs in brain tumors.

MicroRNAs (miRNAs) are short non-protein-coding RNAs that function as key regulators of diverse biological processes through negative control on gene expression at the post-transcriptional level. Emerging evidence indicates that miRNAs play an important role in the development of human cancers, with their deregulation resulting in altered activity of downstream tumor suppressors, oncogenes and other signaling molecules. Recent years have seen considerable progress in miRNA research in brain tumors, particularly in glioblastomas and medulloblastomas, providing novel insights into the pathogenesis of these malignant lesions. Expression profiling has unveiled miRNA signatures that not only distinguish brain tumors from normal tissues, but can also differentiate histotypes or molecular subtypes with altered genetic pathways. Moreover, specific miRNA subsets may have potential diagnostic and prognostic values in some brain tumors. Several deregulated miRNAs uncovered in glioblastomas and medulloblastomas have their gene targets and the associated genetic pathways identified. This review summarizes recent findings of miRNA study in brain tumors.

Pilocytic astrocytomas do not show most of the genetic changes commonly seen in diffuse astrocytomas

Aims: While it is well known that pilocytic astrocytomas are clinically distinct from diffuse astrocytomas, few comprehensive studies have focused on their genetic differences. The aim of this study was to examine pilocytic astrocytomas for genetic alterations that are commonly seen in diffuse astrocytomas.

Molecular Analysis of Microdissected de novo Glioblastomas and Paired Astrocytic Tumors

Glioblastoma multiforme (GBM) often displays morphological heterogeneity in that low-grade (LG) area with well-differentiated cells are commonly found adjacent to high-grade (HG) area with poorly-differentiated cells. This heterogeneity may cause difficulty in obtaining representative tumor samples. Nevertheless, the genetic composition of these cells has only been occasionally examined. In the present study, we examined 29 de novo glioblastomas in which distinct LG and HG areas of sufficient volumes could be identified. These areas were microdissected from paraffin-embedded tissues and analyzed for genetic alterations: p53 mutations and immunohistochemistry; allelic losses at 17p13.1, 9p21, and 10q23-25; and amplification of the epidermal growth factor receptor (EGFR) gene and immunohistochemistry. We also examined 14 paired astrocytic tumors, in which a primary Grade II astrocytoma progressed over a period of time to a Grade III or Grade IV tumor. Our findings showed that the LG areas of the de novo glioblastomas exhibited numerous genetic aberrations, the proportion of which was increased in the HG areas. Genetic abnormalities seen in the LG areas were conserved in the HG areas suggesting that these morphologically different cellular subsets were derived from a common transformed clone. Also, the LG areas were genetically different from Grade II astrocytomas of the paired tumor group, in spite of their morphological similarity. In particular, the LG areas had more deletions on 10q23-25 (75% vs 20%, p = 0.04), but fewer p53 mutations (24% vs 71%, p = 0.003) and less p53 protein labeling (45% vs 79%, p = 0.04). These differences suggest that LG and HG areas in de novo glioblastoma are genetically closer to each other compared with paired low- and high-grade tumors that have progressed over time. Moreover, only a small proportion (17%) of our de novo glioblastomas exhibited EGFR amplification while a high proportion (62%) showed either p53 mutations or allelic loss of 17p13.1. We speculate that some de novo GBMs with copious LG areas may constitute a separate group with rapid progression from Grade II astrocytomas.

Transcriptional inactivation of TP73 expression in oligodendroglial tumors.

The TP73 gene, located on chromosome 1p36.3, encodes a product that shares significant structural homology with the tumor suppressor TP53. The aim of this study was to investigate whether TP73 is involved in the development of oligodendroglial tumors, which frequently carry deletions involving 1p36.3. Semi‐quantitative reverse transcription‐polymerase chain reaction was used to determine TP73 transcript levels. Ten of 24 (42%) tumors showed negligible to more than 5‐fold reduction in TP73 expression when compared to normal brain level. To identify potential mechanisms that may modulate TP73 transcription in oligodendroglial tumors, we performed mutation analysis on the TP73 gene. No somatic mutations were however detected in the gene sequence. We then evaluated the possible involvement of epigenetic change in TP73 expression. Bisulfite genomic sequencing detected aberrant hypermethylation at the 5′ region upstream and including the first exon of the TP73 gene in 17 of 44 (39%) oligodendroglial tumors, whereas normal brain tissues showed no methylation in the same region examined. Moreover, 6 of 10 (60%) tumors with negligible or decreased levels of TP73 transcripts were methylation‐positive. In conclusion, our results showed that inactivation of TP73 occurs at the transcriptional level and is associated with promotor hypermethylation. Loss of or reduced TP73 transcript expression may contribute to the tumorigenesis of oligodendroglial tumors.

Comparative genomic hybridization detects losses of chromosomes 22 and 16 as the most common recurrent genetic alterations in primary ependymomas

In this study, we used comparative genomic hybridization to provide an overview of chromosomal imbalances in a series of 20 adult and 8 childhood ependymomas. All tumors displayed multiple genomic imbalances. Loss of genetic material was observed in chromosomes 22q (71%), 16 (57%), 17 (46%), 6 (39%), 19q (32%), 20q (32%), and 1p (29%), with the overlapped deletion regions determined at 16p13.1-13.3, 16q22-q24, 19q13.1-13.4, 20q13.1-13.2 and 1p36.1-36.3. Gain of DNA was commonly detected on chromosomes 5q (46%), 12q (39%), 7q (36%), 9q (36%), and 4q (32%), with overlapped regions of gain mapped to 5q21-22, 12q15-24.1, 7q11.2-31.2, 9q12-32, and 4q23-28, respectively. These findings suggest a greater degree of genomic imbalance in ependymomas than has been recognized previously and highlight chromosomal loci likely to contain oncogenes or tumor suppressor genes that may contribute to the molecular pathogenesis of this tumor. Our study also confirmed previous findings on frequent losses of 17 and 22q in ependymomas and further identified chromosome 16 loss as a common recurrent genetic aberration in ependymomas.

Restoration of Wild-type PTEN Expression Leads to Apoptosis, Induces Differentiation, and Reduces Telomerase Activity in Human Glioma Cells

PTEN is a candidate tumor suppressor gene identified on human chromosome 10q23.3 that is frequently mutated or deleted in 30% to 44% of glioblastomas. Transient expression study of PTEN in glioma cells indicates that PTEN plays an important role in cellular proliferation, tumorigenicity, cell migration, and focal adhesions. In this study, we examined the biological consequences on U87MG glioma cells after stable gene transfer of wild-type PTEN. Cells stably expressing wild-type PTEN protein were found to have suppressed proliferation, as determined by cell counting and Ki-67 staining, as well as inhibited anchorage-independent growth. The PTEN-expressing cells also showed higher expression of glial fibrillary acidic protein and changed morphologically from spindle-shaped to elongated cell bodies with multiple slender processes, suggesting that these cells have undergone differentiation. In addition, telomerase activity decreased more than 10-fold in PTEN-expressing cells when compared with control cells. More importantly, apoptosis was detected in about 5% of PTEN-expressing cells, representing a 17-fold (p < 0.01) increase over the control cells. Taken together, these results suggest that PTEN plays an important role in regulation of cell homeostasis by maintaining a balance between proliferation, differentiation, and apoptosis.

Expression of p19 INK4d, CDK4, CDK6 in glioblastoma multiforme

Deregulation of the G1/S checkpoint is a frequent event in the development of glioblastoma multiforme (GBM). Previous studies have shown more than 50% of primary GBM tumours contain either complete loss of the p16INK4a locus or amplification of the CDK4 gene. Moreover, many heterozygosity studies have shown deletion on human chromosome 19p13.2, where the p19INK4d gene has been localized. We examined the expression of p19INK4d and its two CDK substrates in a series of glioma-derived cell lines and tumours. No gene rearrangement or deletion was observed in the p19INK4d gene in these cell lines; however, expression of CDK4 and CDK6 was elevated relative to matched normal brain tissue in eight of 18 GBM tumours (44%). Furthermore, CDK6 expression level was increased in 12/14 glioblastomas, but undetectable in tumour samples of a previous lower grade tumour from the same patient. These data attest to the functional importance of both CDK4 and CDK6 in astrocytic tumourigenesis, particularly during the later stages of tumour progression.Deregulation of the G1/S checkpoint is a frequent event in the development of glioblastoma multiforme (GBM). Previous studies have shown more than 50% of primary GBM tumours contain either complete loss of the p16 INK4a locus or amplification of the CDK4 gene. Moreover, many heterozygosity studies have shown deletion on human chromosome 19p13.2, where the p19 INK4d gene has been localized. We examined the expression of p19 INK4d and its two CDK substrates in a series of glioma-derived cell lines and tumours. No gene rearrangement or deletion was observed in the p19 INK4d gene in these cell lines; however, expression of CDK4 and CDK6 was elevated relative to matched normal brain tissue in eight of 18 GBM tumours (44%). Furthermore, CDK6 expression level was increased in 12/14 glioblastomas, but undetectable in tumour samples of a previous lower grade tumour from the same patient. These data attest to the functional importance of both CDK4 and CDK6 in astrocytic tumourigenesis, particularly during the later stages of tumour progression.