Original article
Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques

https://doi.org/10.1016/S0165-4608(02)00670-2Get rights and content

Abstract

The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found.

Introduction

Cell lines are useful tools in the field of carcinogenesis for studying biologic, biochemical, and molecular aspects of malignant transformation. Many cell lines have been characterized using molecular cytogenetic methods such as fluorescence in situ hybridization (FISH), multicolor FISH, and spectral karyotyping (SKY), and the results have contributed to a better knowledge of neoplasia 1, 2, 3, 4, 5, 6.

The A673 cell line was established from a patient with a possible primary rhabdomyosarcoma (RMS) [7]. This cell line has been widely used as an important tool for improving our knowledge of tumor biology. These cells are known to produce several growth factors with oncogenic potential, as well as cell growth-inhibitory factors [8]. These cells can also induce tumors in nude mice 9, 10 and contain some cancer-related genes with hypermethylated promoters 11, 12. The A673 cell line is referred to in the literature both as Ewing tumor (ET) or sarcoma (ES) and as RMS. Although both these tumor types have specific and well-characterized translocations, no cytogenetic report describing the complete karyotype has been published. Ewing tumor presents a t(11;22)(q24;q12) recurrent chromosomal translocation in about 85% of the cases. At the molecular level, this translocation shows that the EWS and FLI1 genes are fused 13, 14, 15. The most frequent chromosome rearrangements identified in RMS are t(2;13)(q35;q14) and its variant t(1;13)(p36;q14), which give rise to two fusion genes, PAX3/FKHR and PAX7/FKHR, respectively 16, 17, 18. At the molecular level, A673 displays fusion of EWS and FLI1 genes as a consequence of the t(11;22) translocation, but does not present the fusions of PAX3 and PAX7 with FKHR resulting from t(2;13) and t(1;13), respectively 19, 20.

These discrepancies prompted us to characterize the A673 cell line by conventional and molecular cytogenetic techniques. The combination of SKY, FISH, and reverse transcriptase polymerase chain reaction (RT-PCR) has revealed only the presence of a complex translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes. Both these events are specific to ET.

Section snippets

Cell line

The A673 cell line (American Type Culture Collection [ATCC], Manassas, VA, USA) and the alveolar rhabdomyosarcoma cell line RC2 (a gift from a Dr. Lollini, Cancer Institute, University of Bologna, Italy), with the t(1;13) [20], were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine and 13% fetal bovine serum. The alveolar rhabdomyosarcoma cell line Rh30 [19], which contains the t(2;13), was maintained in RPMI 1640 medium supplemented with 2 mM l-glutamine and

Conventional cytogenetics

Cells of the cell line were exposed to colcemid (0.1 μg/mL) for 1.5 hours at 37°C and harvested routinely. Metaphase chromosomes were GTG-banded by a conventional trypsin–Giemsa technique and karyotyped according to the International System for Human Cytogenetic Nomenclature, 1995 revision [21].

SKY study

Slide preparation and hybridization, using commercial probes, were carried out according to the manufacturer's instructions (Applied Spectral Imaging [ASI], Migdal Ha'Emek, Israel). Images were acquired

Molecular studies

After cDNA synthesis, PCR was performed to detect chimeric transcripts derived from the t(11;22), t(2;13), and t(1;13). All samples were analyzed according to standardized primers, protocols, and criteria 18, 22, 23, 24. To verify the integrity of the isolated RNA and the correct synthesis of the cDNA, the ubiquitously expressed ABL gene was amplified in a separate PCR reaction. The PCR was performed in two steps: the first round was performed with the external primers and the second with

ET studies

A conventional cytogenetic study was carried out on the A673 cell line. This revealed a complex karyotype with multiple rearrangements affecting chromosomes 3, 5, 8, 9, and 16. Additional unbalanced translocations were found [der(1)t(1;19), t(5;8), der(16)t(3;16), der(9)t(9;13), and +der(11)t(11;13)] and loss of material was observed in chromosomes 3 and 4 (Fig. 1). Neither the t(11;22) nor its variants, characteristic of ET, were detected in the karyotype.

By means of SKY analysis we

Discussion

Since its establishment, the A673 cell line, which was derived from a possible primary RMS [7], has been widely used to extend our knowledge of tumor biology in RMS. This cell line is defined as one of RMS in the ATCC and has been used in different studies during the 1980s to obtain and purify tumor growth inhibitory factors 25, 26 or to investigate the mechanisms of human peripheral blood monocyte-mediated cytotoxicity in tumor cells [27]. It is currently being used as an RMS to evaluate tumor

Acknowledgements

We would like to thank Blanca Fernández Martı́nez, Teresa López Jiménez, and MaCarmen Guijarro Martı́n for their technical assistance; and to Angel Pestaña for providing the cell line. Angel Martı́nez-Ramı́rez is a fellow of the Instituto de Salud Carlos III. Bárbara Meléndez and Sandra Rodrı́guez-Perales are fellows of the Comunidad Autónoma de Madrid and Centro Nacional de Investigaciones Oncológicas, respectively.

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