Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR) deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and thus, are likely to be misclassified by STR-profiling. Based on the high-throughput Luminex platform, we developed a 24-plex SNP-profiling assay, called Multiplex Cell Authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analysing a collection of 436 human cell lines from the DSMZ, previously characterised by eight loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10−8 for STR-profiling and ~1 × 10−9 for MCA). MCA enabled the detection of less than 3% contaminating human cells. Analysing MMR deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI 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. © 2002 Wiley-Liss, Inc. Key words: receptor tyrosine kinase; ALK, translocation; t(2;5)(p23; q35); ALCL; tumor cell linesThe genesis of lymphoproliferative disorders and of solid tumors is caused in part by chromosomal translocations, when new hybrid genes are generated and the expression of the fusion gene products possess severe oncogenic properties. 1 Anaplastic large cell lymphoma (ALCL) is associated with a (2;5)(p23;q35) chromosomal translocation, 2 which fuses the gene encoding the ALK receptor tyrosine kinase localized at 2p23 with the housekeeping gene nucleophosmin (NPM) at 5q35. 3 The resultant hybrid gene harbors the NH 2 -terminus of NPM linked to the entire intracytoplasmatic portion of ALK encoding a constitutively activated 80 kDa nuclear phosphoprotein, 4 which could be shown to be sufficient for malignant transformation of murine fibroblasts in vitro or for induction of transplantable lymphoma in mice. 5,6 Recently, a number of investigations have shown that genes other than NPM could provide similar structural and functional prerequisites, since different ALK products have been identified in cases of ALCLs as...
Recently, we have presented a scheme, termed “NKL-code”, which describes physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis including main stages of T-, B- and NK-cell development. Aberrant activity of these genes underlies the generation of hematological malignancies notably T-cell leukemia. Here, we searched for deregulated NKL homeobox genes in main entities of T-cell lymphomas comprising angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (HSTL), NK/T-cell lymphoma (NKTL) and peripheral T-cell lymphoma (PTCL). Our data revealed altogether 19 aberrantly overexpressed genes in these types, demonstrating deregulated NKL homeobox genes involvement in T-cell lymphomas as well. For detailed analysis we focused on NKL homeobox gene MSX1 which is normally expressed in NK-cells. MSX1 was overexpressed in subsets of HSTL patients and HSTL-derived sister cell lines DERL-2 and DERL-7 which served as models to characterize mechanisms of deregulation. We performed karyotyping, genomic and expression profiling, and whole genome sequencing to reveal mutated and deregulated gene candidates, including the fusion gene CD53-PDGFRB. Subsequent knockdown experiments allowed the reconstruction of an aberrant network involved in MSX1 deregulation, including chromatin factors AUTS2 and mutated histone HIST1H3B(K27M). The gene encoding AUTS2 is located at chromosome 7q11 and may represent a basic target of the HSTL hallmark aberration i(7q). Taken together, our findings highlight an oncogenic role for deregulated NKL homeobox genes in T-cell lymphoma and identify MSX1 as a novel player in HSTL, implicated in aberrant NK- and T-cell differentiation.
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1‐PMS2), MutLβ (MLH1‐PMS1), and MutLγ (MLH1‐MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα‐deficient conditions. Here, we report stable expression of GFP‐tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro‐irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation‐directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ‐heterodimers at UVA‐induced DNA lesions. However, we demonstrate that in a MutLα‐deficient background no DNA repair‐specific function carried out by MutLγ can be detected in living cells. J. Cell. Biochem. 114: 2405–2414, 2013. © 2013 Wiley Periodicals, Inc.
A human cell line of neuroblastic tissue, which was believed to have been lost to science due to its unavailability in public repositories, is revived and reclassified. In the 1970s, a triple set of neuroblastoma (NB) cell lines became available for research as MYCN‐amplified vs nonamplified models (CHP‐126/‐134 and CHP‐100, respectively). Confusingly, CHP‐100 was used in subsequent years as a model for NB and, since the 1990s, as a model for neuroepithelioma and later as a model for Ewing's sarcoma (ES), which inevitably led to non‐reproducible results. A deposit at a bioresource center revealed that globally available stocks of CHP‐100 were identical to the prominent NB cell line IMR‐32 and CHP‐100 was included into the list of misidentified cell lines. Now we report on the rediscovery of an authentic CHP‐100 cell line and provide evidence of incorrect classification during establishment. We show that CHP‐100 cells carry a t(11;22)(q24;q12) type II EWSR1‐FLI1 fusion and identify it as a classic ES. Although the question of whether CHP‐100 was a virtual and never existing cell line from the beginning is now clarified, the results of all relevant publications should be considered questionable. Neither the time of the cross‐contamination event with IMR‐32 is known nor was the final classification as a model for Ewing family of tumors available with an associated short tandem repeat profile. After a long road of errors and confusion, authentic CHP‐100 is now characterized as a type II EWSR1‐FLI1 fusion model 44 years after its establishment.
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