GRIM-19 Expression and Function in Human Gliomas

Abstract

Objective: We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas.

Methods: Tumor tissues were isolated and frozen at -80 just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a Genefishing DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis.

Results: Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisense-transfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-beta and retinoic acid than U343MG-A cells or antisense-transfection cells; the anti-proliferative activity was related to apoptosis.

Conclusion: GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.

Keywords: Cell line; GRIM-19; Gene Fishing; Glioblastoma; Human glioma.

Fig. 1

Differential display (DD)-PCR for detection of tumor-related genes shows five candidates, including GRIM-19 (lane 1 : normal brain; lanes 2 and 3 : astrocytoma samples; lanes 4 and 5 : anaplastic astrocytoma samples; and lanes 6-9 : glioblastoma samples).

Fig. 2

Reverse transcription (RT)-PCR for GRIM-19 shows the coincidence with DD-PCR data. N : normal brain, A : astrocytoma sample, AA : anaplastic astrocytoma sample, GBM : glioblastoma sample, O : oligodendroglioma sample, AO : anaplastic oligodendroglioma.

Fig. 3

The fluorescence microscopic pictures show that GRIM-19 is highly expressed in U343-G-S and less expressed in U343-G-AS, as compared with U343MG-A (original magnification ×100).

Fig. 4

GRIM-19 is expressed both in the nucleus and cytoplasm in U343-G-S using fluorescence confocal microscopy.

Fig. 5

Immunofluorescence staining shows the cytoskeletal changes after transfection (actin : red; vimentin : green).

Fig. 6

The morphology of U343-G-AS is smaller and rounded compared with U343MG-A (Fig. 5 and 6; staining with 0.1% toluidine blue; original magnification ×200).

Fig. 7

Cell-migration tests by simple scratch technique show that the U343-G-S has more motility in terms of the distance of migration compared with U343MG-A and U343-G-AS, but there was no significant difference in the cell number (p > 0.05; staining with toluidine blue; original magnification ×40).

Fig. 8

In the Matrigel invasion assay, the mean number of invading cells was 110.3 ± 12.4, 122.5 ± 16.1, and 117.3 ± 14.6 for U343-G-S, U343MG-A, and U343-G-AS, respectively. There was no significant difference between the three cells (p > 0.05; staining with hemacolor; original magnification ×40).

Fig. 9

MTT assay was performed to determine whether or not GRIM-19 is associated with the combination of INF and RA in the regulation of cell survival. A : U343-G-S cells treated for 24 hours with various concentrations of INF/RA. B : U343-G-S cells treated for 24 hours with INF/RA, INF, and RA. (I : INF 1,500 U only, R : RA 2 µM only, IR : combination of INF 1,500 U and RA 2 µM). C : U343-G-S, U343MG-A, and U343-G-AS cells treated for 24 hours with INF 1,500 U/RA 2 µM. AS : U343-G-AS, S : U343-G-S.

Fig. 10

FACS analysis shows the anti-proliferative activity of U343-G-S, U343MG-A, and U343-G-AS cells treated for 24 hours with INF/RA.