P39/Tsugane cells are a false cell line contaminated with HL-60 cells and are not suitable for mechanistic studies in myelodysplastic syndromes

The P39/Tsugane myelomonocytoid cell line was generated in 1983 by Nagai and colleagues from a 69-year old man with chronic myelomonocytic leukemia (CMML) that had progressed to acute myeloid leukemia (AML), French-American-British classification subtype M2.1 This cell line was deposited in the Japanese Cell Repository Bank (JCRB, now known as the Japanese Collection of Research Bioresources), in July 1986, where its unique identifier is JCRB0092.

After DNA fingerprinting technology became widely available in the 1990s, the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and the JCRB applied fingerprinting techniques to repository cell lines, and both cell banks found that their P39/Tsugane cells share genetic identity with HL-60 cells, which were derived at the National Cancer Institute (NCI) from a woman with suspected acute promyelocytic leukemia (later found to be AML FAB M2), and first reported in 1976.2

The JCRB now lists P39/Tsugane as a misidentified or false cell line (http://cellbank.nibio.go.jp/cellbank_e.html), stating that evidence of cross-contamination with HL-60 cells was “found by DNA fingerprinting first, later confirmed by STR-PCR [short tandem repeat-polymerase chain reaction].”

Likewise, using Affymetrix 10K Single Nucleotide Polymorphism (SNP) arrays as part of the Cancer Genome Project, the Wellcome Trust Sanger Institute in the United Kingdom observed 97% genetic identity between P39/Tsugane cells and HL-60 cells from the NCI60 cell line set (http://www.sanger.ac.uk/genetics/CGP/Genotyping/nci60.shtml). As Hans Drexler of the DSMZ has observed, “a distressingly large percentage of purported MDS cell lines had been cross-contaminated”, and the list of cell lines recognized as false or contaminated by the DSMZ includes P39/Tsugane cells.3,4

Despite these findings, P39/Tsugane cells continue to be widely used as an MDS cell line, and I recently received several manuscripts for review in which generalizations about MDS pathobiology were made on the basis of in vitro experiments performed in P39/Tsugane cells. In order to determine whether an aliquot of unique P39/Tsugane cells exists and to confirm the findings of the JCBR, DSMZ, and Sanger Institute, I obtained P39/Tsugane cells from two laboratory groups with publications that made use of these cells. (These laboratories graciously supplied the cells despite knowing that I intended to evaluate them for cross-contamination with HL-60 cells.)

In a laboratory area that had never worked with HL-60 cells, I grew the putative P39/Tsugane cells to confluence in RPMI 1640 media supplemented with 10% fetal calf serum, and compared them to HL-60 cells from the NCI (HL-60 cells courtesy of Scott. H. Kaufmann, MD PhD, Mayo Clinic, Rochester, MA, USA). Methods of comparison included G-banded karyotyping supplemented by fluorescent in situ hybridization (FISH, courtesy of Rhett Ketterling, MD, Cytogenetics Laboratory, Mayo Clinic) and a 12-marker DNA Variable Nucleotide Tandem Repeat (VNTR) panel used for forensic work (courtesy of W. Edward Highsmith, PhD, Division of Laboratory Genetics, Mayo Clinic).

Both of the P39/Tsugane aliquots tested exhibited karyotyping/FISH and VNTR results identical to the HL-60 cells. The published karyotype of P39/Tsugane cells is “45,XY,+del(6)(q15),9q+, t(14;16)-(q24;q21),-16,-17”.

In contrast, among the aliquots I examined, there were no metaphases with this karyotype, FISH results for chromosome 17p deletion were normal, and the karyotype was instead female as for HL-60 cells (other investigators who are working with P39/Tsugane cells and wish to easily determine whether their aliquot is actually HL-60 cells might begin by assessing for presence of a Y chromosome by FISH, since P39/Tsugane cells were obtained from a man and HL-60 cells from a woman).

While it is possible that an aliquot of a unique P39/Tsugane cell line exists, and the published results might still retain some relevance even though the experiments were actually performed in HL-60 cells, MDS investigators should be aware that the P39/Tsugane cells available from major cell banks and in widespread use in research laboratories are contaminated by HL-60 cells. Drexler recently reviewed all putative MDS cell lines and found only 3 of them (MDS92, M-TAT, and TER-3) that had both been established in the MDS phase (i.e. rather than after AML transformation, as for P39/Tsugane) and might be valid, although more analysis at the genome and proteome levels is required.4

Obtaining a suitable cell line that mimics characteristics of MDS and is suitable for mechanistic biological analysis remains a research priority.

P39/Tsugane cells cannot provide any special insight into MDS and should not be used for this purpose.