Adenosine deaminase and adenosine kinase expression in human glioma and their correlation with glioma‑associated epilepsy

Abstract

The aim of the present study was to investigate adenosine deaminase (ADA) and adenosine kinase (ADK) expression in human glioma and to explore its correlation with glioma‑associated epilepsy. Tumor tissues (n=45) and peritumoral tissues (n=14) were obtained from glioma patients undergoing surgery. Normal control tissues (n=8) were obtained from brain trauma patients. The disease grade was determined by histological evaluation and the degree of tumor invasion was evaluated using immunofluorescence analyses. mRNA and protein expression of ADA and ADK were evaluated using reverse transcription quantitative polymerase chain reaction or western blot analysis, respectively. Based on histological evaluations, four cases were classified as Grade I gliomas, 18 cases as Grade II, 17 cases as Grade III and six cases were considered Grade IV. Increased ADA and ADK expression was observed in tumor tissues. ADA was predominantly distributed in the cytoplasm of tumor cells, whereas ADK was detected in the cytoplasm as well as in the nuclei. ADA and ADK levels were upregulated in patients with Grade II and Grade III gliomas compared to those in control subjects (p<0.05). In addition, tumor invasion was detected in peritumoral tissues. The number of ADA‑positive or ADK‑positive cells in tumor tissues was similar between glioma patients with and without epilepsy (p>0.05). However, ADA and ADK expression was upregulated in peritumoral tissues derived from patients with epilepsy compared to that in glioma patients without epilepsy. The results of the present study suggested that ADA and ADK are involved in glioma progression, and that increased ADA and ADK levels in peritumoral tissues may be associated with epilepsy in glioma patients.

Figure 1

Histological examination of gliomas derived from patients. Samples from patients with Grade I, II, III or IV disease were stained with HE or with specific antibodies against GFAP, Ki67, p53 and MGMT (magnification, ×400). Immunopositive cells are indicated by arrows. HE, hematoxylin and eosin; GFAP, glial fibrillary acidic protein; MGMT, O⁶-methylguanine-DNA methyltransferase.

Figure 2

ADA and ADK expression in tissues from patients with various grades of glioma or from non-glioma and non-epileptic patients (normal controls). Normal brain tissues were stained with either (A) anti-ADA or (B) anti-ADK antibodies. Tumor tissues derived from patients with (C and D) Grade II, (E and F) Grade III or (G and H) Grade IV gliomas were stained with (C, E and G) anti-ADA or (D, F and H) anti-ADK antibodies (scale bars: 50 µm for A–F; 100 µm for G and H for. Magnification: A, B, C, D, E, and F, and magnified boxes in C, D, and G x400; G and H, x200). (I) Average number of immunopositive cells per field of view. Values are expressed as the mean ± standard deviation. ^*p<0.05 vs. control. Peri, peritumoral tissues derived from patients with glioma; ADA, adenosine deaminase; ADK, adenosine kinase.

Figure 3

ADA and ADK mRNA and protein expression in brain tissues derived from patients with different glioma disease grades or from non-glioma and non-epileptic patients (normal controls). Samples were obtained from control subjects (n=8) or patients with Grade I (n=4), Grade II (n=18), Grade III (n=17), or Grade IV (n=6) glimoas. (A) Relative ADA and ADK mRNA expression was examined using reverse-transcription quantitative polymerase chain reaction analysis. (B) Protein expression of ADA and ADK was examined by western blot analyses. (C) Representative western blots. Values are expressed as the mean ± standard deviation. ^*p<0.05 vs. control. ADA, adenosine deaminase; ADK, adenosine kinase.

Figure 4

Immunostaining of peritumoral tissues with anti-p53, anti-ADA, and anti-ADK antibodies (magnification, ×200; magnified boxes, ×400). Peritumoral tissues derived from 14 patients with glioma were immunostained with the anti-p53 antibody alone (red) or co-stained with anti-p53 (red) and anti-ADA or anti-ADK (green) antibodies. Nuclei were counterstained using DAPI (blue). Representative immunostaining images for (A) p53 alone (red), (B) ADA (green) and p53 (red), and (C) ADK (green) and p53 (red). Left-hand column, cell nuclei; middle column, immunostaining; right-hand column, merged image, containing magnified window (scale bars, 50 µm). ADA, adenosine deaminase; ADK, adenosine kinase.

Figure 5

ADA and ADK expression in glioma patients with or without epilepsy. (A) Peritumoral tissues were immunostained with anti-ADA or anti-ADK antibodies (magnification A1, A2, A3, and A4, ×400). Boxed images are magnified from the original images. Positive staining is indicated by arrows. (B) Number of immunopositive cells per area. (C) Western blot analysis of ADA and ADK protein expression. (D) Representative western blot. Values are expressed as the mean ± standard deviation. Tumor tissues derived from patients without epilepsy (n=29); tumor tissues derived from patients associated with epilepsy (n=16); peritumoral tissues derived from patients without epilepsy (n=6); peritumoral tissues derived from patients with epilepsy (n=8). ^*p<0.05 vs. peritumoral tissues derived from patients without epilepsy. ADA, adenosine deaminase; ADK, adenosine kinase.