Original article
Molecular cytogenetic characterization of four previously established and two newly established Ewing sarcoma cell lines

https://doi.org/10.1016/j.cancergencyto.2005.11.006Get rights and content

Abstract

Most Ewing family tumors are identified by the characteristic translocation t(11;22)(q24;q12), resulting in a fusion protein EWS/FLI1 that acts as an aberrant transcription factor. In a minority of cases, the EWS gene is fused to another member of the ETS gene (ERG, ETV1, E1AF, and FEV). Though the oncogenic transforming capability of the EWS/FLI1 protein is highly suggestive, the exact pathway behind remains to be elucidated. The availability of cell lines may help in the understanding of underlying cellular processes. In this study, we have established two new Ewing sarcoma cell lines and characterized them with molecular cytogenetic tools. This technology was also applied on four other previously published Ewing sarcoma cell lines. Our findings in relation to previous data on similar tumors are discussed.

Introduction

Ewing sarcoma and peripheral primitive neuroectodermal tumors are round-cell sarcomas showing varying degrees of neuroectodermal differentiation [1]. These tumors are cytogenetically well described, and in 85% of these cases, a balanced t(11;22)(q24;12) is found [2], [3]. In the breakpoint of the der(22), a chimeric gene containing the 5' portion of the EWS gene at 22q12 and the 3' portion of the FLI1 gene at 11q24 transcribes a fusion protein that acts as an aberrant transcription factor [4]. In 5–10% of the Ewing sarcomas, a variant translocation t(21;22)(q22;q12) is present, and EWS recombines with ERG at 21q22 [5], [6]. Rare rearrangements are present in the remaining cases, in which the EWS gene recombines with other members of the ETS family genes, namely TEV1 (7p22), E1AF (17q12), and FEV (2q33), respectively [7], [8], [9], [10]. In addition to the primary translocation, several non–tumor specific abnormalities have been observed in Ewing sarcomas. The prognostic significance of these secondary chromosomal abnormalities remains unclear, though the presence of a complex karyotype or additional events, such as p53 mutations, has been associated with an unfavorable outcome [11]. These findings necessitate a thorough cytogenetic evaluation superimposed on a real-time detection of the specific breakpoint to determine prognostic events other than fusion type [1], [12], [13].

A number of Ewing sarcoma cell lines are available [6], [14], but detailed molecular cytogenetic karyotypes of these tumors is limited to a small number of them [15], [16]. Cell lines are useful model systems for understanding oncogenic processes and testing potential new treatment modalities. Documenting their chromosome composition adds valuable genetic data and offers a reference to evaluate genomic changes that may occur “in vitro.”

In this study, we successfully established two new cell lines. In addition to these, we established the molecular karyotype of four previously established cell lines (EW3, EW7, TCC-446, and TC-32). For the identification of numerical and structural chromosomal aberrations, we have used a multicolor fluorescence in situ hybridization (FISH)-based technique (COBRA-FISH) which, in a single experiment, enables the distinction of all 24 different chromosomes and the discrimination of every chromosome arm [17]. Furthermore, we have analyzed the involvement of the EWR region in the translocations by using two cosmid probes spanning the breakpoint region [18].

Section snippets

Samples and cell culture

Primary tumor samples of two cases were collected at our hospital. The clinico-pathologic features were as follows: Patient L872 was a 20-year-old male patient with a primary tumor located at the rib. Patient L1062 was a 16-year-old male patient with primary tumor of the tibia and skeletal metastases. Both tumor cases were CD99 positive and EWS-FLi1 reverse-transcription polymerase chain reaction (RT-PCR) positive.

All samples were obtained by Jamshidi-Trocart biopsy during the diagnostic

RT-PCR for the EWS-FLI1 or EWS-ERG transcript

Two of the six samples (EW3 and TTC-466) showed the presence of the EWS/ERG transcript, and the EWS/FLI1 transcript was detected in all other samples (data not shown).

Primary cultures and establishment of L-872 and L1062 cell lines

Cells from the primary tumors showed excellent growth without contact inhibition. The cells presented initially with a heterogeneous appearance, displaying an adherent, spindle-shaped monolayer cell population that morphologically corresponds to fibroblasts, and a second population with a small and rounded cell morphology and

Discussion

In this study, we have successfully established two new cell lines derived from the primary tumors of Ewing sarcoma samples. We performed molecular karyotyping by COBRA-FISH on these new cell lines and on four previously established cell lines, two of which had the classic EWS/FLI1 and the other two had EWS/ERG rearrangements. Our goal was to determine the karyotype of these cell lines and further characterize the derivative chromosomes involving the EWS region by means of cosmid probes

Acknowledgments

This project was supported by a grant from the Dutch Cancer Foundation (KWF2001-2526). We are grateful to Dr. S.A. Burchill (St. James' University Hospital, Leeds, UK) for kindly donating the TC-32, TTC-466 cell lines, Martine Peter (Laboratoire de Transfert, Institut Curie, Paris, France) for donating cell lines EW3 and EW7, and E. Kerkhof (Leiden University Medical Center, Leiden, the Netherlands) for performing the RT-PCR experiments.

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