Differentially expressed genes in association with in vitro invasiveness of human epithelioid sarcoma

A Weber

R Engers

S Nockemann

L Gohr

A Hausen

H Gabbert

Institute of Pathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Duesseldorf, Germany

Dr Engers ed.frodlesseud-inu.dem@sregne

Accepted 2001 Apr 17.

Abstract

Aims—Differential display reverse transcription polymerase chain reaction (RT-PCR) was performed to identify genes associated with the invasive potential of human epithelioid sarcoma.

Methods—Two different clonal subpopulations, GRU-1A and GRU-1B, derived from the same human epithelioid sarcoma cell line GRU-1 and known to differ greatly in their invasive potential were compared by means of mRNA fingerprinting.

Results—Using a set of 10 arbitrary upstream primers and nine anchored oligo-dT primers, 22 candidate gene fragments were identified; differential expression was confirmed in four of these fragments by northern blot analysis. At the mRNA level, apoferritin light chain was predominantly expressed by the highly invasive cell line GRU-1A. In contrast, the mitochondrial gene M1, encoding cytochrome c oxidase I, and the TI-227H gene were expressed more strongly by the low invasive cell line GRU-1B. Furthermore, a novel gene fragment was identified and cloned that was preferentially expressed in the low invasive cell line GRU-1B, and therefore might have an inhibitory role in invasion. Consequently, this gene fragment was designated as expressed in low invasive sarcoma cells (ELISC-1).

Conclusions—A novel gene fragment (ELISC-1) and three known genes were identified as potential regulators of tumour invasiveness. Cloning of the entire sequence of ELISC-1 and subsequent investigations are required to establish its biological role.

Keywords: invasion, epithelioid sarcoma, differential gene expression, heterogeneity

Abstract

Metastasis is the major cause of death in patients with cancer. Many patients suffering from malignant tumours already have metastases at the time of diagnosis. It is of fundamental importance to elucidate the regulatory mechanisms underlying the complex process of metastasis to improve therapeutic strategies.^1,² The first step in metastasis formation is the process of invasion, during which tumour cells leave the primary tumour and actively infiltrate the surrounding tissue.¹ However, malignant tumour cells arising from the same original tumour often show a broad heterogeneity with respect to both morphology and biological properties, such as invasion and metastasis. This intratumoral heterogeneity probably reflects different steps of progression of an individual tumour cell from a normal towards a malignant invasive and metastatic phenotype.^3–⁵ These differences are probably parallelled by differences in the repertoire of genes expressed at a given time.

Recently, we successfully established an in vitro model for studying the mechanisms involved in the regulation of tumour cell invasiveness. The GRU-1 cell line was isolated from a human epithelioid sarcoma, which is a highly malignant soft tissue tumour, typically arising in the extremities of young adults and presenting an extremely aggressive clinical course with frequent metastasis and treatment failure.⁶ Subsequently, the clonal subpopulations GRU-1A and GRU-1B were isolated from GRU-1 and characterised with respect to morphological features, growth kinetics, drug sensitivities, cell motility, cell substrate adhesion, and in vitro invasiveness.^7–⁹ In these studies, GRU-1A had a significantly higher invasive potential than GRU-1B.⁷ Because GRU-1A and GRU-1B cells have the same genetic background, but differ with respect to their invasive behaviour, differences in the pattern of gene expression should be responsible for the phenotypic diversity of both clonal cell lines.¹⁰ Hence, GRU-1A and GRU-1B provide a valuable system for the identification of candidate invasion related genes.

Differential display reverse transcription polymerase chain reaction (RT-PCR), originally introduced by Liang and Pardee in 1992,¹¹ provides a useful tool for identifying differences in gene expression between related cell populations, and has been applied successfully in an increasing number of studies investigating the molecular nature of cancer.^12,¹³ A comparative analysis has shown that differential display RT-PCR has many advantages (identification of low abundant mRNA, need for only small amounts of starting RNA, and ability to identify increases and decrease in messages) compared with related techniques, such as electronic subtraction or subtractive hybridisation.¹⁴

In our present study, differential display RT-PCR was used to identify and isolate genes whose expression is associated with the invasive phenotype of two different clonal human sarcoma cell lines. At the RNA level, four different genes were found to be differentially expressed between the two cell lines. Apoferritin light chain was predominantly expressed by the highly invasive cell line GRU-1A, whereas cytochrome c oxidase I, TI-227H, and a novel gene fragment (ELISC-1) were more strongly expressed by the low invasive cell line GRU-1B.

Acknowledgments

We thank Mrs S Sliwka, Mrs A Florange-Heinrichs, and Mr M Ringler for their excellent technical assistance and Dr BM Ghadimi for helpful discussions and critically reviewing the manuscript.

Acknowledgments

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