Long-term Culture of EBV-induced Human Lymphoblastoid Cell Lines Reveals Chromosomal Instability

Abstract

To preserve material for future genetic studies, human B-lymphocytes from whole blood samples are routinely transformed into lymphoblastoid cell lines (LCLs) by in vitro infection with Epstein-Barr virus. To determine the rate and frequency of chromosomal changes during long-term culture, we established 10 LCLs (from eight individuals). Before transformation, these cases showed a normal karyotype (three cases), a small supernumerary marker chromosome (three cases), or an aberrant karyotype (four cases). Chromosome analyses were performed at 8-week intervals over a period of at least 1 year, up to 3 years. Surprisingly, we demonstrate that chromosomal instability is the rule, rather than the exception, during long-term culture of LCLs. The most commonly observed acquired clonal aberration was trisomy 12, which emerged in all cell lines within 21 to 49 weeks after infection. Telomeric fusions indicating telomere shortening were found after ~21 weeks. After 1 year of cultivation, the proportion of cells with the original karyotype decreased to ≤10% in 7 of the 10 cell lines. To preserve cells with aberrant genomes, we conclude the cultivation time of LCLs must be restricted to the absolute minimum time required.

Keywords: chromosomal instability (CIN); cytogenetics; immortalization; quantitative fluorescence in situ hybridization (Q-FISH); transformation.

Figure 1.

Temporal course of the median value of characteristics scored from 30 metaphase spreads per 10 lymphoblastoid cell lines as a function of culture time in weeks. Horizontal axis shows culture time divided in 5-week intervals from initiation to 5 weeks (weeks 1 to 5) to 95 weeks (weeks 91 to 95) following culture set-up. (A) Percentage of cells with original karyotype; (B) percentage of cells with trisomy 12 or derived aberrations; (C) percentage of non-clonal aberrations; and (D) number of telomeric fusions.

Figure 2.

Temporal course of characteristics in cell line N1 scored from 30 metaphases. (A) Percentage of cells with karyotype 46,XX; (B) percentage of cells with 47,XX,+12 (filled squares) and 46,XX,add(11q) (triangles); (C) percentage of cells with non-clonal changes; and (D) number of telomeric associations.

Figure 3.

Temporal course of characteristics in cell line N2 scored from 30 metaphases. (A) Percentage of cells with karyotype 46,XX; (B) percentage of cells with 47,XX,+12 (filled squares) and 48,XX,+11,+12 (triangles); (C) percentage of cells with 50,XXX,+11,+12,+15; (D) percentage of cells with non-clonal changes; and (E) number of telomeric associations.

Figure 4.

Chromosomal modifications in lymphoblastoid cell lines: (A) Examples of telomeric fusions with non-involved homologues displayed from cell lines N1, A2 (twice), A1-2, and N3, from left to right. (B) Examples of 16qh undercondensation resulting in elongation of the region (cell lines A1-1 and N2), loss of 16q (line N2), and triradial formation (duplication of 16q; lines A1-1 and A1-2).

Figure 5.

Examples of the dicentric marker dic(13;16)(q34;q11.2) from cell line A1-2 (daughter line): (A) G-banded chromosomes 13 (upper row) and 16 (lower row) from preimmortal diploid cell (first pair) and postimmortal tetraploid state; the rearranged chromosome 13 is always in the right position. After development of the dicentric marker, only three normal homologues of chromosome 16 remained in the 4n state. The second to sixth pairs: 16p deleted from marker; 16qh undercondensation (twice), second centromere of dicentric visible, and second centromere invisible by G-banding. (B) FISH with whole chromosome painting probe for chromosome 13 (wcp13, red) plus centromere-specific probe for chromosome 16 (CEP16, green) demonstrates the presence of chromosome 16 centromere in the dicentric, either with (left) or without (right, one homologue of chromosome 16 was missing in this cell) constriction. (C) FISH with locus-specific probe LSI13 (green, from 13q14) plus wcp16 (red) verifies the composition of the dicentric. Abbreviation: FISH, fluorescence in situ hybridization.

Figure 6.

Average TL as a percentage of internal control (arbitrary units of T/C ± SD) measured by Q-FISH as a function of culture time in weeks in three LCLs: line N1 (blue), line A1-1 (green), and line A2 (red). Telomere length shortens with culture time. Abbreviations: TL, telomere length; LCL, lymphoblastoid cell line; Q-FISH, quantitative fluorescence in situ hybridization.