We investigated six established and widely-used ACC cell lines and found that all six had been replaced with other cells (). The cells labeled ACC2, ACC3, and ACCM were HeLa cells, while the ACCS cells were human bladder cancer cells T24 (intraspecies cross-contamination). The putative ACCNS and CAC2 cells were actually derived from mice (ACCNS) or rats (CAC2; interspecies cross-contamination). Our ACC cells were never cultured simultaneously with HeLa cells, and no T24, rat or mouse cells were cultured in our incubators. Thus, we did not cause the contamination. None of these cells were derived from ACC patients. These results unfortunately call into question the validity of results from prior studies using these cell lines.
Misidentified and cross-contaminated ACC cell lines.
Cell lines established from human tissues are extensively used to study human diseases and other conditions. Lines derived from malignant neoplasms are particularly useful tools for investigating mechanisms of tumor initiation and development. These cells are often used as pre-clinical models for the disease, as sources for biomarker or drug target identification, and as vessels to screen for efficacy hits and for early toxicity indicators. Because of the increasing importance of cultured cells in cancer research and other biomedical studies, cross-contamination of cell lines is becoming a more frequently encountered problem. It has been reported that approximately 20% of cell lines are incorrectly designated 
. Nevertheless, researchers infrequently authenticate cell lines prior to initiating studies or before freezing stocks. The NIH has recently recognized the problem and has issued a notice that cell line authentication must accompany all grant applications and all publications of research findings (http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html
HeLa cells were the first established human cancer cell line 
. They were quickly distributed worldwide with attendant contamination problems. Cross-contamination of cell lines with HeLa cells was first reported in 1968 
. Among the first established 20 human cell lines, 18 were eventually identified as HeLa cells 
. In the past, various methods were developed to identify intraspecies contamination of cells in culture. Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) initially played a significant role in identifying HeLa contamination 
. Phosphoglucomutase (PGM) electrophoretic polymorphisms also supported HeLa cell identification 
. HeLa cells show Type A G6PD and Type1 PGM profiling. Besides these biochemical methods, a genetic approach has also been applied: HeLa cells display characteristic trypsin-Giemsa stained chromosome band patterns 
. In addition, the presence or absence of a Y chromosome can be assessed by fluorescent staining. The expression of HLA antigen on the cell surface was an alternative approach to verify cell identification. This approach revealed that EJ-1 cells share the same genetic profile with T24 bladder cancer cells 
. Currently STR profiling of polymorphic markers has replaced these methods. It is an efficient and reliable method for detecting cross-contamination of human cell lines. An earlier usage of STR showed that ECV304 was a contaminant of T24 cells 
Cell line contamination is a significant problem. For example, one study analyzed 550 leukemia-lymphoma cell lines and reported that 82 (14.9%) were cross-contaminated with different cells; some were redundant but others were not of leukemia-lymphoma origin 
. Another study reported that the MDA-MB-435 human breast cancer cell line was thoroughly contaminated with the M14 human melanoma cell line 
. M14 cross-contaminants have also been found in the thyroid cancer cell line NPA87 and its sublines, which make it the most frequent contaminant group in thyroid cancer cells, followed by HT-29 colon cancer contaminants 
Despite these facts, there is continued use of the above-mentioned cell lines, as reported in recent publications 
. As a consequence, reports with invalid hypotheses and incorrect results have been published. Thus, use of contaminated cell lines may cause significant delays in the development of new treatments or new biomarkers. They may also add significant costs to the process if researchers spend time and money investigating false leads.
ACC studies are no exception to this problem 
. We performed a PubMed search and found that 75 original research papers using the cell lines examined in this study were published from 1991 to 2008 (). Notably, ACC2 and ACCM were used in more than 30 published studies. Many of these reports proposed new therapeutic applications based on results derived from use of the cell lines. Our study suggests that ACC cell lines should be authenticated before research involving their use is performed.
Number of articles citing 6 ACC cell lines.
If the contamination identified here is widespread among other cell lines that are identified as ACC cells, it will be necessary to establish new ACC cell lines. It will be essential to ensure that the new cell lines do not become contaminated.
As a first, step prior to the creation of cell lines, researchers should freeze and store multiple samples of the original material and then, when a cell line is established, they should perform STR profiling to confirm that the cells are identical to the donor tissue. Sporadic ACC tumors often overexpress c-Kit, keratin, S-100 protein, and actin 
. Detection of these ACC-specific markers is an alternative approach to quality control of ACC cells. It should be performed in all new ACC cell lines. After verification, cell line stocks should be deposited in one or more BRCs before information about them is published. The quality control procedures at BRCs may reduce contamination problems. Additionally, because ACC is composed of duct-type epithelial cells and myoepithelial cells, it may be prudent to establish each component separately to avoid one of the cellular components becoming dominant during passaging.
The genotypes, karyotypes and phenotypes of new cell lines must be documented by the originators and/or the BRCs using cells with low passage numbers. This approach will avoid the effects of extended passage number on selective pressure and genetic instability in cell lines 
. Researchers should also include data concerning STR profiling of donor tissue and the cell line in publications of studies that use the new cell line. An additional important measure would be to ensure that all personnel working with cultured cells are given thorough instructions on how to avoid contamination. Finally, publications of any ACC research using ACC cell lines should include cell line authentication. We anticipate that these efforts will considerably reduce cell contamination and thus improve the quality of ACC research.