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

Abstract

Background: 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 HDAC 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.

Methods: 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 HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting.

Results: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 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.

Conclusion: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs 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 HDAC mRNA in glioblastomas.

Figure 1

Classification of classes I, II, and IV HDACs by structure and cellular localization.[2,6,44,45].

Figure 2

Relative expression of HDAC genes in astrocytomas and normal brain tissue. Class I in purple, class II in orange, and class IV in green. Data obtained by qRT-PCR analysis using Taqman^® probes for each gene using the GUS β gene as housekeeping. Standard curves were constructed for each gene by considering at least 3 points in triplicate of 10-fold serial dilution of cDNA in water, starting from 1:10 of a volume of undiluted cDNA transcribed from 1.0 μg of total RNA. The slopes of standard curves ranged from -3.17 to -3.87. Blank and standard controls (calibrators) were run in parallel to verify amplification efficiency within each experiment. To obtain the Ct (cycle) values, we established a threshold of 0.1. *AI, AII and AIII mean astrocytoma grade I, II and III respectively. GBM means glioblastoma.

Figure 3

Protein analysis. (A) HDAC9 and (B) acetyl H3 and acetyl H4 in glioblastoma (GBM), astrocytoma grade III (AIII), astrocytoma grades I and II (AI and II), and normal brain. Actin was used as endogenous control.