Elsevier

Leukemia Research

Volume 23, Issue 7, July 1999, Pages 643-651
Leukemia Research

Expression of the growth arrest-specific gene 6 (GAS6) in leukemia and lymphoma cell lines

https://doi.org/10.1016/S0145-2126(99)00075-2Get rights and content

Abstract

The initial identification of GAS6 as a protein expressed in response to growth arrest suggested that it might function as a negative regulator of cell proliferation. Since the transforming activity of the GAS6 receptor (AXL/UFO) was documented, GAS6 might stimulate rather than inhibit proliferation. In order to detect aberrant expression of GAS6 we examined gene expression in 46 cell lines of precursor B-, B- and T-cell origin as well as from Hodgkin’s disease and cell lines established from various myeloproliferative disorders. In our study, the expression of GAS6 reveals a constitutive transcriptional activation in 8/46 cases of proliferating cell lines. The GAS6 mRNA expression could be shown in 4/22 cell lines of the lymphoid arm and in 4/17 of the myeloid lineages of the hematopoietic system. No transcripts could be detected in the CD30+ Hodgkin and anaplastic large cell lymphomas (0/7). Interestingly, the steady state mRNA levels showed neglectable GAS6 expression in precursor B and B-cell lines (1/9), but could be detected in terminally differentiated plasma cell lines (4/4). The predominantly GAS6-expressing cell lines of non-lymphoid origin have been established from acute myeloid leukemias of the M4 subtype (3/4). In order to demonstrate evidence for an autocrine regulation of growth in permanent hematopoietic cell lines, we measured the GAS6 expression in cell lines with strong positivity for the AXL/UFO receptor mRNA. Constitutive basal levels of GAS6 mRNA and protein expression could be only detected in 3/23 AXL/UFO expressing cell lines. Although a general mechanism seems most unlikely, further studies are necessary to demonstrate the involvement of GAS6 in single cases of disordered growth or chemotaxis/adhesion of leukemia and lymphomas.

Introduction

The growth arrest-specific gene 6 (GAS6) was originally isolated from murine and human fibroblast cell lines, which have been kept in proliferative quiescence by serum starvation [1], [2]. GAS6 is a new member of the vitamin K–dependent family of proteins with a significant homology to human protein S, a negative co-regulator of the blood coagulation pathway [3]. Similarly to protein S, GAS6 shows defined structural motifs as four epidermal growth factor (EGF)-like repeats, a large carboxy-terminal region with homology to steroid-binding globulin, but it lacks the consensus domain recognized by thrombin, which is involved in the regulation of the biological activity of protein S.

Recently, GAS6 was identified as the ligand for the AXL/UFO receptor tyrosine kinase, while protein S was identified as the ligand of a related tyrosine kinase, TYRO3 [4], [5]. The members of this new family of transmembrane receptors, namely RSE (also known as SKY, BRT, TIF, TYRO3), AXL (also known as UFO, ARK, TYRO 7) and MER have been isolated using the homology-based cloning of the sequence similarity of the kinase domain [6], [7], [8]. All of them display immunoglobulin G-like and fibronectin type III domains in the extracellular region similar to those found in many cell adhesion molecules. The report of homophilic interactions of ectopically expressed AXL/UFO receptors which lead to cell aggregates, and the finding that GAS6 is able to inhibit the adhesion of granulocytes to endothelial cells have supported the concept of AXL/UFO functioning as a new cell adhesion molecule [9], [10].

AXL/UFO has been described previously to be present in normal and malignant hematopoietic tissue. High levels are expressed in CD34+ hematopoietic progenitors and in neoplastic cells or cell lines established from acute or chronic myeloid leukemias [5], [11]. The AXL/UFO receptor has been isolated from myeloid leukemia cells and the transforming ability of the receptor is observed when the protein is overexpressed in NIH3T3 fibroblasts [7], [12]. GAS6 has been shown to be expressed in whole bone marrow and was found to be present diffusely in myelomonocytic precursors. These findings suggest that the activation of the tyrosine kinase by its ligand, GAS6, might stimulate rather than inhibit the proliferation and prompted us to investigate the expression of GAS6 in tumor cell lines of the hematopoietic tissue. We therefore examined the expression of the GAS6 gene in order to assign an expression pattern to AXL/UFO-expressing cell lines or specific lineages of the hematopoietic system.

Section snippets

Cell lines and cell culture conditions

All cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and have been tested for authenticity by DNA fingerprinting and karyotypic markers. All cell lines are free of viral and mycoplasma contaminations and are grown in appopriate media according to the instructions of the DSMZ catalogue [13]. Cells are subjected to proliferative tests in their logarithmic growth with viabilities of higher than 90% as examined by trypan blue

GAS6 mRNA expression of hematopoietic tumor cell lines

This study was undertaken to expand the data available on the distribution and function of the GAS6 gene in a panel of hematopoietic cell lines. To avoid the induction of GAS6 expression by starvation, the respective cell lines have been harvested in their logarithmic growth with viabilities of higher than 90%. We chose in a first step a highly sensitive RT-PCR method to measure the presence or absence of GAS6 transcripts. In order to detect the recently published splice variants of the GAS6

Discussion

The expression of GAS6 mRNA and protein has been reported to be up-regulated upon serum starvation in human and murine fibroblasts [1], [2]. Recently, GAS6 has been identified as the ligand of the tyrosine kinase receptors AXL/UFO and RSE [5], [6]. The nude mouse tumorigenicity assay and the classical DNA-mediated transformation assay have demonstrated the transforming activity of the AXL/UFO [7], [12]. Because of the broad distribution of the receptor in the hematopoietic system and its

Acknowledgements

We are grateful to Dr Hilmar Quentmeier for helpful discussions and critical reading of the manuscript. We also appreciate the excellent technical assistance of Margarete Zaborski. D. Rome was supported by the European Programme ‘Leonardo’. W. Dirks contributed to the collection, assembly and analysis of data, provided concept and design; drafted and revised the paper and gave full approval. D. Rome, F. Ringel, K. Jager, R. MacLeod collected and assembled the data. H.G. Drexler provided

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