Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19

Abstract

Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65-80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q were used for chromosome 19 microcell-mediated transfer, to assess the effect of replacing the deleted segment. Complementation with chromosome 19 significantly reduced the growth rate of the hybrid cells compared with the parental cell lines. Affymetrix U133 Plus 2.0 Gene Chip analysis was performed to measure and compare the expression of the chromosome 19 genes in the chromosome 19 hybrid cell lines to the parental cell line. Probes were considered significantly different when a P value <0.01 was seen in all of the cell line comparisons. Of 345 probes within the commonly deleted 19q region, seven genes (APOE, RCN3, FLJ10781, SAE1, STRN4, CCDC8, and BCL2L12) were identified as potential candidate genes. RT-PCR analysis of primary tumor specimens showed that several genes had significant differences when stratified by tumor morphology or deletion status. This suggests that one or more of these candidates may play a role in glioma formation or progression.

Figure 1

Proliferation of cell lines, assessed by Trypan Blue Exclusion assay. Cells were plated on day 0 on tissue culture treated plates, with 50,000 cells per well. Values listed in the legends are the P value when compared with the appropriated parental pSV control cell line on day 6. (A) A172 cell lines and (B) U87 cell lines; 227 × 97 mm (72 × 72 DPI).

Figure 2

Schematic of the cell line comparisons used for the Affymetrix U133 Plus 2.0 Array. Data from each of the hybrid cell lines was compared with the data collected for the parental control cell line containing the pSVneo vector; 254 × 190 mm (96 × 96 DPI).

Figure 3

RTPCR examining RNA copy number variation in primary tumor specimens for candidate genes. Individual tumors are represented by the individual spots. The black bars indicated the mean ± 2 SEM. (A) Test set of tumors (n = 51) stratified by deletion status of chromosome 19q. (B) Validation set of tumors (n = 26) stratified by tumor deletion status of chromosome 19q. (C) Combined set of tumors (n = 77) stratified by deletion status of chromosome 19q. The table beneath the graph show the P values (Wilcoxon test); 213 × 324 mm (72 × 72 DPI).