CORRESPONDENCE

Uncertain Identity of Doxorubicin-Resistant MCF-7 Cell Lines Expressing Mutated p53

Farzaneh Pirnia, Madlaina Breuleux, Erasmus Schneider, Manfred Hochmeister, Susan E. Bates, Andreas Marti, Michel A. Hotz, Daniel C. Betticher, Markus M. Borner

Affiliations of authors: F. Pirnia, M. Breuleux, D. C. Betticher, M. M. Borner (Institute of Medical Oncology), M. A. Hotz (Department of Ear, Nose and Throat Surgery), Inselspital, Bern, Switzerland; M. Hochmeister (Department of Forensic Medicine), A. Marti (Department for Clinical Research), University of Bern; E. Schneider, Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY; S. E. Bates, Medicine Branch, National Cancer Institute, Bethesda, MD.

Correspondence to: Markus M. Borner, M.D., Institute of Medical Oncology, Inselspital, 3010 Bern, Switzerland (e-mail: mborner{at}insel.ch).

The doxorubicin (ADR)-selected human breast cancer cell line (MCF-7/ADR-RES) has been a useful model for a multidrug-resistant subline in cancer research. However, a recent letter to the Journal has alerted the scientific community about the finding that MCF-7/ADR-RES is not a MCF-7-derived cell line. This report led to a change of nomenclature from MCF-7/ADR-RES to NCI/ADR-RES (1).

Another cell line (MCF-7 TH) has been independently selected by doxorubicin treatment at the National Cancer Institute (NCI) and demonstrated some of the same, interesting characteristics of NCI/ADR-RES, such as high levels of MDR-1 and P-glycoprotein expression and a mutated p53 tumor suppressor protein (2). Of interest, both cell lines exhibit the same 21-base-pair deletion beginning at exon 5 spanning codons 126–133 (3). We used this cell line to analyze the effect of the multidrug-resistant phenotype and p53 on the efficacy of anticancer drugs with different mechanisms of action. Various anticancer drugs were more effective in the MCF-7 TH subline than in the parental MCF-7 line, despite a dysfunctional p53 and multidrug-resistant phenotype. These agents induced apoptosis in MCF-7 TH cells as demonstrated by the characteristic morphologic features. Because the activity of specific caspases might affect the propensity of a cell to undergo apoptosis, we examined expression level and cleavage pattern of different caspases in drug-treated MCF-7 TH and parental cells. The MCF-7 TH cell line strongly expressed caspase-3 (Fig. 1Go, A) and cisplatin treatment led to a significant increase of caspase-3-like activity as measured by use of the fluorogenic caspase-3-specific substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-afc) (Fig. 1Go, B). The MCF-7 cell line has lost caspase-3 because of a 47-base-pair deletion within exon 3 of the CASP-3 gene (4). These findings thus suggest that MCF-7 TH also is not derived from MCF-7. This hypothesis was confirmed by DNA fingerprinting, which demonstrated that parental MCF-7 and MCF-7 TH cannot have the same donor, because they differ in all loci except the sex chromosome locus. The analyses were repeated with several different batches of parental MCF-7 and TH cells. Furthermore, MCF-7 parental cells from different sources were compared and all showed an identical allelic distribution profile of the short tandem repeat loci by DNA fingerprinting. The only cell line showing identity with MCF-7 TH by DNA fingerprinting was NCI/ADR-RES. Thus, MCF-7 TH, like NCI/ADR-RES, is not a MCF-7-related cell line.




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Fig. 1. Analysis of caspase-3 protein expression and activity in MCF-7 and MCF-7 TH cell lines. A) Immunoblot analysis of baseline caspase-3 expression in Jurkat (positive control), MCF-7, and MCF-7 TH cells using a caspase-3 polyclonal antibody (6). B) Activation of caspase-3 by cisplatin. MCF-7 TH cells were treated with 10 µg/mL cisplatin. At the indicated time points, caspase-3 activity was measured with the specific fluorogenic substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-afc) (7).

 
This finding is important because doxorubicin-resistant cell lines are a popular tool for new drug development. In a recent article in the Journal, Leoni et al. (5) used NCI/ADR-RES to investigate a novel microtubule-binding indanone. Despite the fact that these authors mislabeled the cell line MCF-7/ADR-RES (1), they did not realize that their observation of caspase-3 activation by the drug would not have been possible in a MCF-7-derived cell line, which contains no caspase-3 (4) (Fig. 1Go, A). The use of a mislabeled cell line can clearly introduce a major source of misinterpretation and possibly wrong strategic decisions in drug development. We, thus, strongly suggest that the identity of cell lines be assessed regularly by DNA fingerprinting because this is a simple and reliable method for identity validation.

REFERENCES

1 Scudiero DA, Monks A, Sausville EA. Cell line designation change: multidrug-resistant cell line in the NCI anticancer screen [letter]. J Natl Cancer Inst 1998;90:862.

2 Wosikowski K, Regis JT, Robey RW, Alvarez M, Buters JT, Gudas JM, et al. Normal p53 status and function despite the development of drug resistance in human breast cancer cells. Cell Growth Differ 1995;6:1395–403.[Abstract]

3 Ogretmen B, Safa AR. Expression of the mutated p53 tumor suppressor protein and its molecular and biochemical characterization in multidrug resistant MCF- 7/Adr human breast cancer cells. Oncogene 1997;14:499–506.[Medline]

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5 Leoni LM, Hamel E, Genini D, Shih H, Carrera CJ, Cottam HB, et al. Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells. J Natl Cancer Inst 2000;92:217–24.[Abstract/Free Full Text]

6 Krajewska M, Wang HG, Krajewski S, Zapata JM, Shabaik A, Gascoyne R, et al. Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease. Cancer Res 1997;57:1605–13.[Abstract]

7 Marti A, Jaggi R, Vallan C, Ritter PM, Baltzer A, Srinivasan A, et al. Physiological apoptosis in hormone-dependent tissues: involvement of caspases. Cell Death Differ 1999;6:1190–200.[Medline]


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