U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells

Abstract

It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line.

Keywords: GBM; STR; U251; U373; aCGH; cell lines; cross-contamination; in vitro models.

Figure 1

Array comparative genomic hybridization (CGH) data of U-251 clones reveal accumulation of genetic alterations after long-term culture. (A) The aCGH profile shows complete chromosomal duplications and hemizygous losses (trisomies and monosomies) in U-251MG typical of primary glioblastoma (GBM), while the profiles of the other subclones have a more complex pattern. Several homozygous deletions (arrows) are detected (B) Eucledian distance calculation indicates that U-251-4q12 diverged earlier from an ancestral U-251MG clone than U-251N and U-251-FGA20gain, and that the divergence of U-251 and U-251-FGA20gain is a relatively late event. (C) All subclones show homozygous deletion of CDKN2A (p16/p14 ARF). (D) U-251-4q12 has an amplification at chromosome 4q12. (E) platelet-derived growth factor receptor α (PDGFRα) mRNA expression is increased in all three long-term passaged U-251 subclones compared to U-251MG.

Figure 2

DNA ploidy and karyotyping. Flow cytometric DNA ploidy analyses show that the U-251 subclones differ in their DNA ploidy. Lymphocytes (representing diploid DNA) are shown in gray. (A) Manual count of chromosomes in G-banded metaphases showing different ploidy levels in the U-251 subclones.

Figure 3

Phenotypic characterization and DNA analysis of U-251 clones. (A) Phase contrast images of U-251MG, U-251-4q12, U-251N, and U251-FGA20gain show variation in morphology and growth pattern. Scale bars = 100 μm. (B) α-tubulin staining of U-251 subclones show variation in size and cytoskeleton organization. Scale bars = 50 μm. (C) Flow cytometric histograms of cell surface marker expression profiling of U-251MG, U-251-4q12, and U251N shows high level of CD44 expression and low levels of CD133/1 and CD15. Subpopulations of CD15^high and A2B5^high cells are present only in U-251N cells (boxed area). Expression levels of analyzed markers are presented in comparison to negative controls (“black dotted” profiles).

Figure 4

In vitro and in vivo growth U-251 clones. (A) Growth curves of U-251MG in MEM, U-251MG in DMEM, U-251-4q12, U-251N, and U251-FGA20gain. (B) BrdU incorporation shows various distributions of the S-phase cells. One representative figure is shown, while numbers are the mean from three replicate analyses. (C) Examples of variation in colony formation obtained by U-251MG (left), U-251-4q12 (middle left), U-251N (middle right), and U-251-FGA20gain (right). Single colonies are shown as insets with scale bars = 100 μm. (D) Survival of clonogenic colonies after irradiation at 2, 5, and 10 Gy. U-251-4q12, U-251N, and U-251-FGA20gain. (E) Kaplan-Meier survival curves after intracranial implantation of the U-251 subclones in NOD/SCID mice.