Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. for glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold.Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAEPA-FMRUniv São Paulo, Fac Med, Dept Surg & Anat, BR-14049090 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Med, Dept Cellular & Mol Biol, BR-14049090 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Med, Dept Pathol, BR-14049090 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, BrazilUniv São Paulo, Sch Med, Dept Neurol, BR-01246903 São Paulo, BrazilUniv São Paulo, Fac Med, Dept Pediat, BR-14049090 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, BrazilFAPESP: 04/12133-6FAPESP: 06/57602-9CNPq: 485342/2006Web of Scienc

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas

<p>Abstract</p> <p>Background</p> <p>Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global <it>HDAC </it>expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas.</p> <p>Methods</p> <p>Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV <it>HDACs </it>was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene <it>β-glucuronidase</it>. Protein levels were evaluated by western blotting.</p> <p>Results</p> <p>We found that mRNA levels of class II and IV <it>HDACs </it>were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, <it>p </it>< 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue.</p> <p>Conclusion</p> <p>Our study establishes a negative correlation between <it>HDAC </it>gene expression and the glioma grade suggesting that class II and IV <it>HDACs </it>might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of <it>HDAC </it>mRNA in glioblastomas.</p

Monoclonal B-cell lymphocytosis in first-degree relatives of patients with sporadic (non-familial) chronic lymphocytic leukaemia

Although biological similarities have been described among monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukaemia (CLL), the relationships between these two conditions are not fully understood, and new epidemiological studies in different populations and different countries continue to be reported. Here, we investigated 167 first-degree relatives from 42 families of patients with non-familial (sporadic) CLL, using four-colour flow cytometry. MBL was found in seven of 167 subjects (4.1%). Monoclonality was detected in all cases either by light-chain restriction or by polymerase chain reaction. Fluourescence in situ hybridization did not show any chromosomal abnormality. The prevalence of MBL according to age was 0 (0/54) in individuals aged less than 40 years, 2.5% (2/81) between 40 and 60 years, and 15.6% (5/32) in individuals over 60 years. The prevalence of MBL cases in individuals over 60 years was similar to that found in familial CLL relatives at the same age group. This suggests that in older first-degree relatives of patients with sporadic CLL, the risk of MBL detection is as high as in older first-degree relatives from CLL families, which could render these individuals belonging to `sporadic CLL families` as susceptible as individuals from `familial CLL` to the development of clinical CLL

Indisulam Reduces Viability and Regulates Apoptotic Gene Expression in Pediatric High-Grade Glioma Cells

Pediatric high-grade glioma (pHGG) is one of the most aggressive brain tumors. Treatment includes surgery, radiotherapy, chemotherapy, or combination therapy in children older than 3–5 years of age. These devastating tumors are influenced by the hypoxic microenvironment that coordinatively increases the expression of carbonic anhydrases (CA9 and CA12) that are involved in pH regulation, metabolism, cell invasion, and resistance to therapy. The synthetic sulphonamide Indisulam is a potent inhibitor of CAs. The aim of this study was to evaluate the effects of Indisulam on CA9 and CA12 enzymes in pHGG cell lines. Our results indicated that, under hypoxia, the gene and protein expression of CA9 and CA12 are increased in pHGG cells. The functional effects of Indisulam on cell proliferation, clonogenic capacity, and apoptosis were measured in vitro. CA9 and CA12 gene and protein expression were analyzed by RT-PCR and western blot. The treatment with Indisulam significantly reduced cell proliferation (dose-time-dependent) and clonogenic capacity (p p < 0.01). Indisulam promoted an imbalance in the anti-apoptotic BCL2 and pro-apoptotic BAX protein expression. Our results demonstrate that Indisulam contributes to apoptosis via imbalance of apoptotic proteins (BAX/BCL2) and suggests a potential to overcome chemotherapy resistance caused by the regulation these proteins

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Abstract Background Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression