Telomere loss in cells treated with cisplatin

Abstract

Telomeres play an important role in the immortalization of proliferating cells. The long tandem repeats of 5'-TTAGGG-3' sequences in human telomeres are potential targets for the anticancer drug cisplatin, which forms mainly intrastrand d(GpG) and d(ApG) cross-links on DNA. The present study reveals that telomeres in cisplatin-treated HeLa cells are markedly shortened and degraded. A dose that killed 61% of the cells but allowed one round of cell division resulted in shortened telomeres before the induction of apoptosis. Higher doses of cisplatin halted cell cycle progression during the first S phase and triggered apoptosis followed by degradation of telomere repeats. A model in which both cell division with incomplete replication and induction of apoptosis by cisplatin could occur was devised to explain the drug-induced telomere loss.

Figure 1

Degradation of telomeric DNA in cisplatin-treated HeLa cells. (A) Southern assay of TRF in HeLa cells cultured with or without cisplatin. Lanes: 1, untreated; 2, 0.5 μM; 3, 1 μM; 4, 2 μM; 5, 5 μM. (B) The hybridization signal from 4.4 to 14.6 kbp was quantitated in each fraction. The relative amount of shortened telomeric DNA in each fraction is shown normalized to the signal from untreated cells. Each signal was quantitated by PhosphorImager analysis. The average of the three independent experiments is shown. Error bars represent one standard deviation.

Figure 2

Cell cycle progression of HeLa cells treated with cisplatin. Histograms of DNA content per cell number of HeLa cells untreated and treated with 0.5, 1, 2, or 5 μM are shown. Cells were maintained in the presence of cisplatin for 24, 48, 72, or 96 h without a change in medium and collected at the times indicated. The DNA content at G₁ (▵) and G₂ (▴) in untreated cells is indicated.

Figure 3

DAPI staining of HeLa cells cultured with or without cisplatin. DAPI-stained cells were viewed by fluorescence microscopy using iplab spectrum p software. (A) Percentage of cells having a condensed nucleus. A total of 200 cells was scored in each view, and three views were obtained for each sample. The average from three independent experiments is shown. Error bars represent one standard deviation. (B) Representative cells 24 h (Upper) and 48 h (Lower) after treatment. (Left) Untreated; (Center) 0.5 μM cisplatin; (Right) 5 μM cisplatin.

Figure 4

Schematic representation of the TRF loss in the first 24 h, caused by high doses (1, 2, and 5 μM, A) and a low dose (0.5 μM, B) of cisplatin. Linear DNA double strands are shown as lines, and the bubbles indicate replication folks.