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. 2012;7(2):e30804.
doi: 10.1371/journal.pone.0030804. Epub 2012 Feb 2.

Parallel evolution under chemotherapy pressure in 29 breast cancer cell lines results in dissimilar mechanisms of resistance

Bálint Tegze  1 , Zoltán Szállási, Irén Haltrich, Zsófia Pénzváltó, Zsuzsa Tóth, István Likó, Balázs Gyorffy
Affiliations

Parallel evolution under chemotherapy pressure in 29 breast cancer cell lines results in dissimilar mechanisms of resistance

Bálint Tegze et al. PLoS One. 2012.

Abstract

Background: Developing chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines.

Methods: Parallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7) and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS.

Results: All together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2-46 fold and 3-28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps (ABCB1, ABCG2). Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells.

Conclusion: We surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance.

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Conflict of interest statement

Competing Interests: Dr. Istvan Liko is employed by Gedeon Richter Plc. No other conflicts of interest exist. The employment of Dr. Liko does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Overview of the generation of cell lines.
The original cell lines were split and new cell lines were generated parallel by treatment with gradually increasing concentration of doxorubicin or paclitaxel (detailed description of the treatment protocol is in the Methods section).
Figure 2
Figure 2. Circos plots showing the relative cross-resistance for MCF-7 derivatives (A,) and MDA-MB-231 derivatives (B,) against four chemotherapy agents.
The ribbon thickness corresponds to the relative resistance. Yellow cell lines were treated with doxorubicin and orange cell lines with paclitaxel. Note the high cross-resistance against 5-fluorouracil in three paclitaxel-treated and one doxorubicin-treated cell line. In contrast, minimal cross-resistance against cisplatin can be observed. 5FU: 5-fluorouracil, CISP: cisplatin, DOX: doxorubicin, PAX: paclitaxel.
Figure 3
Figure 3. Hierarchical clustering image of the samples using the RT-PCR measured genes in both doxorubicin- and paclitaxel-resistant cell lines.
Red and green: up- and down- regulated as compared to the mean of all experiments. The white dots represent the sample with the highest absolute expression. Overall only a few mechanisms are activated in one cell line to achieve resistance.
Figure 4
Figure 4. Flow cytometric analysis of rhodamin 123 stained resistant and parental MDA-MB-231 and MCF7 cell lines.
(black: parental cell line, red: resistant cell line showing altered Pgp function, green: resistant cell line with normal Pgp function). A, MDA-MB-231-R19 (red), MDA-MB-231-R11 (green); B, MDA-MB-231-R1 (red), MDA-MB-231-R8 (green); C, MCF7-R7 (red), MCF7-R2 (green); D, MCF7-R20 (red), MCF7-R12 (green). Differences between the Pgp function of the resistant cell lines suggest, that alteration of the Pgp function can only explain the resistance in a few cell lines.
Figure 5
Figure 5. Complete overview of cytogenetic aberrations of the resistant derivatives of the MDA-MB-231 cell line.
White background: normal chromosomes, green background: chromosomes of the parental MDA-MB-231 cell lines, red background: chromosomal changes in the resistant cell lines (A,). Position of the most important genes are marked by blue arrows. Representative karyotype of the MDA-MB-231-R18 cell line (B,); ploidity (C,) and number of new structural aberrations (D,) on each chromosome across the parental (blue) and all resistant (red) cell lines. Chromosomal instability (CIN) was measured computing the Shannon Diversity Index for chromosomes 3, 17 and 21 in ten different metaphases. Threshold for chromosomal instability (CIN+) was set above 2.1 (E,).
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