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Cited by 105Salivary Gland Tumors Diagnosis and TreatmentIdentification and Quantification in FoodGenetic diversity and population structure

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Genetic Profiling Reveals Cross-Contamination and Misidentification of 6 Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS and CAC2

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Phuchareon, Janyaporn, Ohta, Yoshihito, Woo, Jonathan M., Eisele, David W., Tetsu, Osamu·2009·PLoS ONE
DOI 10.1371/journal.pone.0006040PMC PMC2698276
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Abstract

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells.

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Crossref · 10.1371/journal.pone.0006040

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Figure 1 HeLa contaiminated-ACC2, ACC3, and ACCM demonstrate a variation in the von Willebrand factor gene (vWA). Electrophoretic profiles of the vWA marker for HeLa, ACC2/Sa, ACC2/Zh, ACC3, and ACCM are shown. Complete STR profiling is shown in Table 1 and Figures S2, S3, S4, S5, S6, S7, S8, S9, S10. A multiplex PCR reaction was performed using two-color detection fluorescent dye-linked primers. One ng of each genomic DNA was independently amplified in a 25 µl reaction volume. The amplified PCR products were separated by capillary electrophoresis on a 3730xI DNA Analyzer (Applied Biosystems) and analyzed using GeneMapper v4.0 software (Applied Biosystems). Cell lines for the study were generously provided by Drs. Takashi Saku (ACC2/Sa and ACC3), Naishuo Zhu (ACC2/Zh), Kanemitsu Shirasuna (ACCS), Noriaki Tanaka (ACCM and ACCNS), and Ruy Jaeger (CAC2). HeLa cells were purchased from the ATCC.
Figure 1 HeLa contaiminated-ACC2, ACC3, and ACCM demonstrate a variation in the von Willebrand factor gene (vWA). Electrophoretic profiles of the vWA marker for HeLa, ACC2/Sa, ACC2/Zh, ACC3, and ACCM are shown. Complete STR profiling is shown in Table 1 and Figures S2, S3, S4, S5, S6, S7, S8, S9, S10. A multiplex PCR reaction was performed using two-color detection fluorescent dye-linked primers. One ng of each genomic DNA was independently amplified in a 25 µl reaction volume. The amplified PCR products were separated by capillary electrophoresis on a 3730xI DNA Analyzer (Applied Biosystems) and analyzed using GeneMapper v4.0 software (Applied Biosystems). Cell lines for the study were generously provided by Drs. Takashi Saku (ACC2/Sa and ACC3), Naishuo Zhu (ACC2/Zh), Kanemitsu Shirasuna (ACCS), Noriaki Tanaka (ACCM and ACCNS), and Ruy Jaeger (CAC2). HeLa cells were purchased from the ATCC.CC BY · PubMed Central
Figure 2 ACCNS cells were mouse cells and CAC2 cells were rat cells. A cytochrome C oxidase subunit I (COI) DNA barcoding assay was performed using multiplex species-specific primer sets for 12 species (+, lanes 3, 5, 6 and 7) or 11 species-specific sets without mouse primers (−, lanes 2 and 4). The PCR products were separated in a 2% agarose gel and visualized with ethidium bromide following amplification of 5′ end of COI region. Lanes 1 and 8: 100 bp ladders. Lanes 2 and 3: ACCNS. Lanes 4 and 5: CAC2. Lane 6: Mixed DNA template from 12 species. Lane 7: no template DNA. The animals and expected PCR product sizes were as follows; human (391 bp), cat (341 bp), Chinese hamster (315 bp), rhesus monkey (287 bp), horse (243 bp), African green monkey (222 bp), rat (196 bp), dog (172 bp), mouse (150 bp), rabbit (136 bp), goat (117 bp), and cow (102 bp). The PCR products were sequenced to confirm that they contained the species-specific nucleotide sequence.
Figure 2 ACCNS cells were mouse cells and CAC2 cells were rat cells. A cytochrome C oxidase subunit I (COI) DNA barcoding assay was performed using multiplex species-specific primer sets for 12 species (+, lanes 3, 5, 6 and 7) or 11 species-specific sets without mouse primers (−, lanes 2 and 4). The PCR products were separated in a 2% agarose gel and visualized with ethidium bromide following amplification of 5′ end of COI region. Lanes 1 and 8: 100 bp ladders. Lanes 2 and 3: ACCNS. Lanes 4 and 5: CAC2. Lane 6: Mixed DNA template from 12 species. Lane 7: no template DNA. The animals and expected PCR product sizes were as follows; human (391 bp), cat (341 bp), Chinese hamster (315 bp), rhesus monkey (287 bp), horse (243 bp), African green monkey (222 bp), rat (196 bp), dog (172 bp), mouse (150 bp), rabbit (136 bp), goat (117 bp), and cow (102 bp). The PCR products were sequenced to confirm that they contained the species-specific nucleotide sequence.CC BY · PubMed Central

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Research works printed but not linked: 4

  1. [8]Carlson ER Kim j Mirowski GW Stewart JCB Zarbo RJ 2008 Salivary gland diseases. Oral Pathology: Clinical Pathology Correlations. Regezi JA Sciubba JJ Jordan RCK St. Louis, MI, USA Saunders 179 215
  2. [16]Takata T Kudo Y Zhao M Ogawa I Miyauchi M 1999 Reduced expression of p27(Kip1) protein in relation to salivary adenoid cystic carcinoma metastasis. Cancer 86 928 35 10491517
  3. [30]Suda K Rothen-Rutishauser B Günthert M Wunderli-Allenspach H 2001 Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features. In Vitro Cell Dev Biol Anim 37 505 14 11669284
  4. [37]MacLeod RA Dirks WG Matsuo Y Kaufmann M Milch H 1999 Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer 83 555 63 10508494

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Metadata

Authors
Phuchareon, J., Ohta, Y., Woo, J. M., Eisele, D. W., & Tetsu, O.
Affiliations
University of California, San Francisco (US)
Corresponding author
Tetsu, Osamu
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-06-25
Volume
4
Issue
6
Pages
e6040
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0006040
PMC
PMC2698276
OpenAlex
W2000248995
Cited by
105
References
46
Citations by year
2026: 4 · 2025: 4 · 2024: 2 · 2023: 5 · 2022: 3 · 2021: 3 · 2020: 4 · 2019: 2 · 2018: 10 · 2017: 14 · 2016: 10 · 2015: 5 · 2014: 7 · 2013: 10 · 2012: 9
FWCI
7.44
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • Adenoid Cystic Carcinoma Research Foundation
  • Johns Hopkins University
  • University of California, San Francisco
Topics
  • Health Sciences › Medicine › Surgery › Salivary Gland Tumors Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Identification and Quantification in Food
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic diversity and population structure
Keywords
adenoid cystic carcinoma, cell line contamination, cell line authentication, short tandem repeat profiling, DNA fingerprinting, HeLa cells, T24 cells, salivary gland carcinoma
MeSH terms
Alleles, Animals, Urinary Bladder Neoplasms, Carcinoma, Adenoid Cystic, Electron Transport Complex IV, HeLa Cells, Humans, Salivary Gland Neoplasms, Gene Expression Regulation, Neoplastic, Gene Expression Profiling, Cell Line, Tumor, Mice, Rats

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