Molecular characterization of putative chordoma cell lines

Abstract

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Figure 1

Morphological appearance of putative chordoma cell lines in culture. Phase contrast photomicrographs of cultured CCL4B, CM319, GB60, U-CH1, and U-CH2 show the chordoma tumor-like physaliphorous phenotype in only U-CH1 and U-CH2 cells.

Figure 2

Gene expression analysis of chordoma tumors, putative chordoma cell lines, and normal tissues including intervertebral disk (IVD). Five chordoma-specific genes from among the 2351 probe sets used for cluster analysis are included; T: brachyury, KRT19: keratin19, Col2A1: collagen 2A1, CA3: carbonic anhydrase 3, and CD24. Genes with decreased relative expression are shown in green while those with increased relative expression are shown in red. Chordoma tumors (Chordoma and Chordoma-MK) and cell lines U-CH1, U-CH2, and K001 have similar expression patterns distinct from the other putative chordoma cell lines and other tissues.

Figure 3

Gene expression analysis of chordoma tumors, cell lines, and mesenchymal tumors. Five chordoma-specific genes from among the 1208 probe sets used for cluster analysis are included; T: brachyury, KRT19: keratin19, Col2A1: collagen 2A1, CA3: carbonic anhydrase 3, and CD24. Genes with decreased relative expression are shown in green while those with increased relative expression are shown in red. Chordoma tumors (chordoma and chordoma-MK) and cell lines U-CH1, U-CH2, and K001 have a similar expression patterns distinct from the other putative chordoma cell lines and other tissues.

Figure 4

Brachyury expression in putative chordoma cell lines. (a) Immunoblot analysis of Brachyury of CCL4B, CM319, GB60, U-CH1, and U-CH2 cells reveals significant expression of the ∼50 kDa Brachyury protein only in U-CH1 and U-CH2 cells. Molecular weight standard lane is denoted by M. (b) Immunofluorescent staining for Brachyury revealed nuclear localization of the protein in both U-CH1 and U-CH2. (c) Immunoblot analysis of cytokeratin protein expression in CCL4, CM319, GB60, U-CH1, and U-CH2. (d) Immunoblot analysis of PTEN protein expression in MRC-5 primary human fibroblasts, U-CH1 and U-CH2. (e) Immunoblot analysis of activation of Akt in normal human fibroblasts (growing, serum starved for 48 hours then with, or without, 10% added serum for 45 minutes), U-CH1 and U-CH2. Phospho-Akt- (Serine 473-) specific antibodies show Akt activation while similar protein loading was confirmed by staining for total Akt protein levels. For A, C, and E similar protein loading was confirmed by staining for β-actin.

Figure 5

Chromosome 9p21.3 region from array CGH analysis of U-CH1 (a) and U-CH2 (b) cell lines. Both cell lines show loss of the 9p21.3 region encompassing CDKNA2, encoding the cyclin dependent kinase inhibitor p16, MTAP, encoding methylthioadenosine phosphatase, and for U-CH2, DMRTA1, encoding the transcription factor DMRT-family A1 protein. Each dot represents the copy number for the array probe for that region, and a line shows the copy number averaged across the region. Relative signal intensity values are shown on the y-axis; threshold log₂ ratio of  .2 or  .6 was used for gain or large gain, and −.2 and −1 were used for loss and large loss.

Figure 6

Species analysis of putative chordoma cell lines. Agarose gel electrophoresis of ethidium-stained DNA of aldolase PCR analysis of genomic DNA reveals murine origin for CCL3 as shown by similar amplicon fragment sizes found in the murine cell line, NIH 3T3, as compared to those generated from the normal human genomic sample, CEPH 1331-01. The other cell lines (CCL4, CM319, GB60, U-CH1, and U-CH2) gave similar amplicon sizes as those for the human control genomic sample CEPH 1331-01.