Leukemia
SEARCH     advanced search my account e-alerts subscribe register
Journal home
Advance online publication
Current issue
Archive
Press releases
For authors
For referees
Contact editorial office
About the journal
For librarians
Subscribe
Advertising
naturereprints
Contact Springer Nature
Customer services
Site features
NPG Subject areas
Access material from all our publications in your subject area:
Biotechnology Biotechnology
Cancer Cancer
Chemistry Chemistry
Dentistry Dentistry
Development Development
Drug Discovery Drug Discovery
Earth Sciences Earth Sciences
Evolution & Ecology Evolution & Ecology
Genetics Genetics
Immunology Immunology
Materials Materials Science
Medical Research Medical Research
Microbiology Microbiology
Molecular Cell Biology Molecular Cell Biology
Neuroscience Neuroscience
Pharmacology Pharmacology
Physics Physics
Browse all publications
 
Journal home
Advance online publication
Current issue
Archive
Press releases
For authors
For referees
Contact editorial office
About the journal
For librarians
Subscribe
Advertising
naturereprints
Contact Springer Nature
Customer services
Site features
NPG Subject areas
Access material from all our publications in your subject area:
Biotechnology Biotechnology
Cancer Cancer
Chemistry Chemistry
Dentistry Dentistry
Development Development
Drug Discovery Drug Discovery
Earth Sciences Earth Sciences
Evolution & Ecology Evolution & Ecology
Genetics Genetics
Immunology Immunology
Materials Materials Science
Medical Research Medical Research
Microbiology Microbiology
Molecular Cell Biology Molecular Cell Biology
Neuroscience Neuroscience
Pharmacology Pharmacology
Physics Physics
Browse all publications
 
September 2002, Volume 16, Number 9, Pages 1868-1870
Table of contents    Previous  Article  Next   [PDF]
Correspondence
DNA profiling and cytogenetic analysis of cell line WSU-CLL reveal cross-contamination with cell line REH (pre B-ALL)
H G Drexler, H Quentmeier, W G Dirks, C C Uphoff and R A F MacLeod

DSMZ-German Collection of Micro-organisms and Cell Cultures, Braunschweig, Germany

Correspondence to: H G Drexler, DSMZ-German Collection of Micro-organisms and Cell Cultures, Mascheroder Weg 1 B, D-38124 Braunschweig, Germany; Fax: 49-531-2616.150

Abstract

Leukemia (2002) 16, 1868-1870. doi:10.1038/sj.leu.2402610

TO THE EDITOR

Leukemia-lymphoma cell lines represent invaluable resources for the investigation of an endless variety of cellular and molecular processes in vitro. While certain experimental results obtained in cell culture lend themselves to a global interpretation applicable for a whole class of cells (eg for all hematopoietic cells), in other investigations where experimental data are specific for rather narrowly defined types of cells (eg a defined maturation stage within one hematopoietic cell lineage or a specific subtype of hematopoietic malignancy), it is of the utmost importance that the in vitro model cell line corresponds as closely as possible to its original tumor. In other words, it is inappropriate to extrapolate B cell-specific findings to monocytes. Otherwise, why bother with complex cell lines which are difficult to grow and not just simply use some easier cell lines such as HL-60, K-562 or U-937 and boldly extrapolate the results across cell lineages to the desired target cells?

Hence, it is absolutely necessary to identify precisely the cell lineage and stage of arrested differentiation of a leukemia-lymphoma cell line. However, besides the exact characterization it is also mandatory to verify the derivation of that cell line from the presumed patient by unequivocal methods such as forensic-type DNA profiling, cytogenetic karyotyping, etc.

Unfortunately, a large percentage of leukemia-lymphoma cell lines are cross-contaminated1 with the consequence that in many instances these cell lines cannot be used as the specific in vitro models which they are supposed to be. To add fuel to the fire, original investigators who have generated the cross-contaminations or researchers who have used the false cell lines, when informed about the true nature of the cell line, are all too often reluctant to believe these facts or simply ignore them, a phenomenon which we termed the 'false cell line denial syndrome'. Clearly, the widespread and consistent use of misidentified cell lines leads to inaccurate data.2

A small, but unique class of cell lines are those derived from B cell chronic lymphocytic leukemia (B-CLL). About three dozen such cell lines have been described in the literature.2 Except for cell line WSU-CLL, all B-CLL-derived cell lines are Epstein-Barr virus (EBV)-positive. One must discern so-called B-lymphoblastoid cell lines (B-LCLs) whereby normal residual B cells are immortalized from bona fide B-CLL cell lines in which the malignant cells were immortalized. The neoplasticity of the cell line can be verified by the detection of pathognomonic or disease-associated chromosome alterations (but occasionally B-LCLs also carry rearrangements).

The cell line WSU-CLL was reported to be derived from the peripheral blood sample of a 68-year-old black male with therapy-resistant B-CLL in terminal phase in 1992.3 In comparison with other bona fide B-CLL cell lines, WSU-CLL presented the following remarkable features: (1) the cells were EBV-negative; all other B-CLL cell lines are EBV+; (2) the cells were CD5- CD10+; this immunoprofile pattern has not been reported in other B-CLL cell lines. Of particular importance are two facts: (1) the cell line was not authenticated, that means it was not proven by DNA profiling or other unequivocal genetic approaches (marker chromosomes, Southern blot banding patterns of IGH rearrangements, etc) that the cell line was indeed established from this patient; (2) the complete karyotype of the cell line was presented in written and pictorial form in the original publication.3

We observed the following consensus karyotype for WSU-CLL cells held at the DSMZ: 45<2n>X-Y, del(3)(p21p24.2), der(4)t(4;21)(q31;q22), der(5)t(5;12)(q31;p13), der(12)t(5;12) (q31;p13)dup(12)(q14q21), der(12)t(12;21)(p13;q22)inv(12)(p13q22), der(16)t(16;21) (q23-24;q21-22)x2, der(18;21)(q10;q10), der(21)t(12;21)(p13;q22), ie closely resembling the original karyotype published for this cell line.3 Thus, DSMZ stocks of the WSU-CLL cell line fairly represent that described by their originators. However, this karyotype also resembles in detail that which we previously determined for REH.4 In particular, both cell lines carry complex four-way translocations involving breakpoints at 4q31, 12p13 (ETV6), 16q23-24 and 21q22 (AML1), together with parallel rearrangement of band p13 of the homologous chromosome 12. This series of chromosome changes is anything other than random, effecting t(12;21) together with microdeletion of the residual 12p13/ETV6 region, ie as commonly observed in primary BCP-ALL. However, the underlying cytogenetic mechanism is unique to REH where its occurrence is appropriate, and WSU-CLL where it is not. Importantly, our data revealed a karyotype essentially identical to the karyotype published for WSU-CLL, allowing us to rule out that these aberrations occurred during in vitro propagation in our laboratory.

We have had the opportunity to compare the DNA profile of WSU-CLL cells which we had obtained directly from the original investigators with those of over 500 human leukemia-lymphoma cell lines stored in our DNA profiles databank.5 We found identical single-locus and multi-locus DNA profiles (Figure 1) for WSU-CLL and two independently sourced stocks of cell line REH, respectively, deposited at the DSMZ cell lines bank,5 and obtained from the American Type Culture Collection (ATCC). REH was established in 1974 from the peripheral blood of a 15-year-old girl with acute lymphoblastic leukemia at relapse.2 The lineage and stage of arrested differentiation of cell line REH corresponds to that of a B cell precursor (BCP). Further evidence was provided by the detection of the TEl-AML1 (now known as EVT6-RUNX1) fusion gene which results from the t(12;21)(p13;q22) and which occurs only in primary and cultured BCP cells (including cell line REH);4 all three cell cultures examined showed this genetic abnormality (Figure 2). Cytogenetic analysis has shown that the TEL-AML1 fusion is generated in REH by a unique four-way translocation, t(4;12;21;16)4 which we have now confirmed in WSU-CLL. Taken together, WSU-CLL is definitely only a subclone of cell line REH and is hence a BCP cell line.

It has been reported that there are significant phenotypic differences between WSU-CLL and REH. For instance, while WSU-CLL grows very well in SCID mice, REH cells allegedly do not grow in these animals. However, a literature search shows that different investigators were able successfully to heterotransplant REH cells into nude and SCID mice.6 Nevertheless, it is well known that subclones derived explicitly or inadvertently from their parental cultures can develop quite divergent phenotypic features. A good case in point is the widely distributed cell line K-562 for which quite dramatic phenotypic differences between subcultures with regard to growth kinetics, cloning efficiency and hemoglobin production have been noted.7 A large panel of K-562 sublines shared several common surface antigens but expressed a marked diversity and variability of other markers.8 In this context, we examined the expression of a panel of immunomarkers on the cell lines WSU-CLL, REH (ATCC) and REH (DSMZ) (Table 1). It became evident that these genotypically identical cell lines express some phenotypic markers to various extents, eg CD20, CD34, CD138 (Figure 3).

In summary, different approaches have revealed that the WSU-CLL cell line is indeed the REH cell line and most likely resulted from an inadvertent cross-contamination during cell culture at the original source, presumably during the attempts to establish a B-CLL cell line. Hence, WSU-CLL is a subclone of BCP-ALL cell line REH and it is clearly inappropriate to use WSU-CLL as a model for B-CLL.

References

1 Drexler HG, Dirks WG, MacLeod RAF. False human hematopoietic cell lines: cross-contaminations and misinterpretations. Leukemia 1999; 13: 1601-1607. MEDLINE

2 Drexler HG (ed). The Leukemia-Lymphoma Cell Line FactsBook Academic Press: San Diego, 2000.

3 Mohammad RM, Mohamed AN, Hamdan MY, Vo T, Chen B, Katato K, Abubakr YA, Dugan MC, Al-Katib A. Establishment of a human B-CLL xenograft model: utility as a preclinical therapeutic model. Leukemia 1996; 10: 130-137. MEDLINE

4 Uphoff CC, MacLeod RAF, Denkmann SA, Golub TR, Borkhardt A, Janssen JWG, Drexler HG. Occurrence of TEL-AML1 fusion resulting from (12;21) translocation in human early B-lineage leukemia cell lines. Leukemia 1997; 11: 441-447. MEDLINE

5 Drexler HG, Dirks WG, MacLeod RAF, Quentmeier H, Steube KG, Uphoff CC (eds). DSMZ Catalogue of Human and Animal Cell Lines, 8th edn Braunschweig: Germany, 2001.

6 Hara H, Luo Y, Haruta Y, Seon BK. Efficient transplantation of human non-T-leukemia cells into nude mice and induction of complete regression of the transplanted distinct tumors by ricin A-chain conjugates of monoclonal antibodies SN5 and SN6. Cancer Res 1988; 48: 4673-4680. MEDLINE

7 Dimery IW, Ross DD, Testa JR, Gupta SK, Felsted RL, Pollack A, Bachur NR. Variation amongst K562 cell cultures. Exp Hematol 1983; 7: 601-610.

8 Ichiki AT, Bamberger EG, Wust CJ, Lozzio CB. Diversity of cell surface hematopoietic antigens on K-562 sublines identified with monoclonal antibodies. Leukemia Res 1986; 10: 565-574.

Figures

Figure 1 AmpFLP and high-resolution STR DNA profiles. DNA typing of the indicated cell lines was carried out using Apo-B1, D17S5, D1S80, D2S44, PAH and Col2A as polymorphic VNTR markers for PCR amplification as described previously.2 The obtained PCR products of specific VNTRs were run separately on a 1.2% agarose gel for size determination of the amplicons and comparison within the DNA profile database (data not shown) and together in a single lane (upper part). A multiple-locus DNA profile was generated using 10 mug of genomic DNA digested to completion (HinfI), size-separated on a 0.7% agarose gel and blotted on positively charged nylon membranes. After hybridization to a digoxigenin-labelled (GTG)5 oligo as probe, a chemiluminescent detection was carried out and the blot was exposed for 1 h to X-ray film. Molecular weight markers were used for size determination. Cervix carcinoma cell line HeLa was used as positive control in AmpFLP analysis.

Figure 2 Identification of TEL-AML1 fusion by RT-PCR. RNAs derived from REH cell cultures (REH-ATCC and REH-DSMZ), WSU-CLL and human tongue squamous cell carcinoma CAL-33 (negative control) were used in a RT-PCR reaction with the sense primer TEL969 and the antisense primer NAML1.4 The amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The integrity of the RNA and cDNA and the fidelity of the PCR reaction were demonstrated by amplification of the ABL transcript using F-ABL-A2 (5'-TGA CTT TGA GCC TCA GGG TCT GAG TGA AGC C-3') and R-ABL-A3 (5'-CCA TTT TTG GTT TGG GCT TCA CAC CAT TCC-3').

Figure 3 Immunofluorescence analysis of CD138 expression. REH (DSMZ), REH (ATCC) and WSU-CLL cells were stained with a specific antibody (CD138, clone ID4; Dianova, Hamburg, Germany). Cell surface expression was examined by flow cytometry (FACScan, Becton Dickinson, Heidelberg, Germany). Background staining with an isotype control antibody is shown as an open curve.

Tables

Table 1 Immunomarker expression of REH and WSU-CLL

Received 22 February 2002; accepted 26 March 2002
September 2002, Volume 16, Number 9, Pages 1868-1870
Table of contents    Previous  Article  Next    [PDF]