Roderick A. F. MacLeod

Widespread intraspecies cross‐contamination of human tumor cell lines arising at source

We present a panoptic survey of cell line cross‐contamination (CLCC) among original stocks of human cell lines, investigated using molecular genetic methods. The survey comprised 252 consecutive human cell lines, almost exclusively tumor‐derived, submitted by their originators to the DSMZ and 5 additional cell repositories (CRs), using a combination of DNA profiling (4‐locus minisatellite and multilocus microsatellite probes) and molecular cytogenetics, exploiting an interactive database (http://www.dsmz.de/). Widespread high levels of cross‐contaminants (CCs) were uncovered, affecting 45 cell lines (18%) supplied by 27 of 93 originators (29%). Unlike previous reports, most CCs (42/45) occurred intraspecies, a discrepancy attributable to improved detection of the more insidious intraspecies CCs afforded by molecular methods. The most prolific CCs were classic tumor cell lines, the numbers of CCs they caused being as follows: HeLa (n = 11), T‐24 (n = 4), SK‐HEP‐1 (n = 4), U‐937 (n = 4) and HT‐29 (n = 3). All 5 supposed instances of spontaneous immortalization of normal cells were spurious, due to CLCC, including ECV304, the most cited human endothelial cell line. Although high, our figure for CCs at the source sets a lower limit only as (i) many older tumor cell lines were unavailable for comparison and (ii) circulating cell lines are often obtained indirectly, rather than via originators or CRs. The misidentified cell lines reported here have already been unwittingly used in several hundreds of potentially misleading reports, including use as inappropriate tumor models and subclones masquerading as independent replicates. We believe these findings indicate a grave and chronic problem demanding radical measures, to include extra controls over cell line authentication, provenance and availability. Int. J. Cancer 83:555–563, 1999.

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

Check your cultures! A list of cross-contaminated or misidentified cell lines

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well‐recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handlers knowledge. Cross‐contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross‐contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response—how can a researcher know if their own cell lines are cross‐contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross‐contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross‐contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross‐contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

False leukemia–lymphoma cell lines: an update on over 500 cell lines

Human leukemia–lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines

The initial identification of the ALK gene, expressed as C‐terminal part of the transforming fusion protein NPM‐ALK in the t(2;5)(p23;q35) lymphoma‐associated chromosomal translocation, revealed a novel receptor tyrosine kinase (RTK). In order to expand the knowledge on ALK expression in the human system, we examined a panel of human cell lines for ALK expression and found that transcription is completely repressed in cell lines of entodermal origin (0/21). Furthermore, full length receptor expression is absent in cell lines of the hematopoietic system with the exception of t(2;5)‐associated anaplastic large cell lymphomas lines (ALCL), which are known to express chimeric NPM‐ALK mRNA. Cell lines established from solid tumors of ectodermal origin, including melanoma and breast carcinoma, exhibited widespread mRNA expression of the ALK receptor at a broad range (53/64), an association which was found to be strongest in cell lines derived from neuroblastoma (6/6), glioblastoma (8/8) as well as in cell lines established from Ewing sarcoma (4/4) and retinoblastomas (2/2). Because of the reported involvement of neutrophin tyrosine kinase receptors in autocrine differentiation in neuroblastomas, we analyzed cell lines positive for full length or chimeric ALK protein for the presence of phoshotyrosine residues within the intracellular region of ALK. While the constitutive activation of chimeric NPM‐ALK molecules could be shown, no evidence was found for induced or constitutively activated ALK receptors in neuroblastoma, melanoma or breast carcinoma cell lines. Although the receptor could be shown to be consistently expressed with exclusive specificity in tissues developed from the ectoderm, our results do not support any involvement of ALK in the stimulation of tumorigenic cell growth or differentiation so far, indicating that ALK expression is a physiologic rather than a pathologic phenomenon.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.

A mutation in the JH2 pseudokinase domain of the Janus kinase 2 gene (JAK2 V617F) has been described in chronic myeloproliferative disorders (MPD). We screened 79 acute myeloid leukemia (AML) cell lines and found five positive for JAK2 V617F (HEL, MB-02, MUTZ-8, SET-2, UKE-1), 4/5 with histories of MPD/MDS. While SET-2 expressed both mutant (mu) and wild-type (wt) JAK2, remaining positives carried homo-/hemizygous JAK2 mutations. Microsatellite analysis confirmed losses of heterozygosity (LOH) affecting the JAK2 region on chromosome 9p in MB-02, MUTZ-8 and UKE-1, but also in HEL, the only JAK2mu cell line lacking any reported MPD/MDS history. All five JAK2mu cell lines displayed cytogenetic hallmarks of MDS, namely losses of 5q or 7q, remarkably in 4/5 cases affecting both chromosomes. Our combined FISH and microsatellite analysis uncovered a novel mechanism to supplement mitotic recombination previously proposed to explain JAK2 LOH, namely chromosome deletion with/without selective JAK2mu amplification. Confirming the importance of the mutated JAK2 protein for growth and prevention of apoptosis, JAK2mu cell lines displayed higher sensitivities to JAK2 inhibition than JAK2wt cell lines. In summary, JAK2 V617F cell lines, derived from patients with history of MPD/MDS, represent novel research tools for elucidating the pathobiology of this JAK2 mutation.

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

Continuous hematopoietic cell lines as model systems for leukemia–lymphoma research

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitts lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.

False human hematopoietic cell lines: cross-contaminations and misinterpretations.

The risk of adventitious contamination and subsequent overgrowth of cell lines by unrelated cells is a potential and often recurring problem where cells are grown and studied. This problem of intraspecies and interspecies cross-contamination among human cell lines has been recognized for over 25 years; incidences of cell cross-contamination between 17 and 35% have been reported. The most useful methods to detect human cell cross-contamination are DNA fingerprinting and cytogenetic analysis, each complementing the other. Using this combination, we found that in total 14.8% of the human hematopoietic cell lines received either from the original investigator (n = 117 cell lines) or from secondary sources (n = 72 cell lines) were cross-contaminated with another hematopoietic cell line and were thus false cell cultures. Another problem relates to the fact that not every cell line established from a patient with a hematopoietic malignancy is a malignant cell line; unintended immortalization of non-malignant B cells by ‘passenger’ Epstein–Barr virus (EBV) leads to the establishment of B-lymphoblastoid cell lines (termed EBV+ B-LCLs), an event which is much more frequent than the establishment of a ‘true’ leukemia–lymphoma–myeloma cell line. These EBV+B-LCLs are most often (albeit not always) unrelated to the malignant clone. The misinterpretation of such EBV+ B-LCLs as true malignant hematopoietic cell lines (particularly in research areas investigating B cell-derived neoplasms such as myeloma) and the indiscriminate use of these cell lines may render some of the results of such studies irrelevant to the pathobiology of the disease concerned. However, a combination of markers commonly allows for an accurate determination of the nature of EBV+ B-LCLs: immunoprofile, cellular morphology, EBV status, and karyotype. In summary, the continuous need for vigilant quality and identity control procedures is emphasized by the high incidences of cross-contaminated cell lines. Most laboratories using cells cultured in vitro maintain multiple cell lines. Such cell lines should be monitored regularly for their identity and specific characteristics in order to prevent invalidation of research work due to incidents of cell line cross-contamination or misinterpretation.