Expression Analysis and Potential Functional Role of the CXCR4 Chemokine Receptor in Bladder Cancer

doi:10.1016/j.eururo.2004.10.001

European Urology

Volume 47, Issue 1, January 2005, Pages 111-117

https://doi.org/10.1016/j.eururo.2004.10.001 Get rights and content

Abstract

Objective:

Recent analysis gave evidence for the fact that the chemokine receptor CXCR4 is of functional significance in the multistep procedure of metastasis directing tumor cells to their metastatic target organs. In our study we investigated the expression of the CXCR4 receptor on bladder cancer cells and the functional activation of the CXCR4 receptor in bladder cancer cell lines regarding signal transduction pathways, which are involved in metastasis.

Methods:

Receptor transcript expression was analysed and quantified by reverse transcription PCR with RNA extracted from different samples of native human bladder cancer tissue, normal urothelium and the bladder cancer cell lines J82 and T24. Measurement of intracellular [Ca⁺⁺]_i and analysis of intracellular stress fiber formation, chemotaxis, Matrigel invasion and proliferation were performed in the bladder cancer cell lines J82 and T24 upon stimulation with the specific agonist SDF-1.

Results:

Specific CXCR4 receptor transcripts were detected in normal urothelium, the bladder cancer cell lines J82 and T24 and in all bladder carcinoma specimens. We did not observe a quantitative correlation of transcript expression and tumor stage. While SDF-1 did not evoke increases in [Ca⁺⁺]_i in bladder cancer cell lines, we observed a distinct rise in intracellular stress fiber formation, chemotactic activity, invasion through Matrigel coated membranes and cell growth upon stimulation with SDF-1, which was blocked in the presence of a specific CXCR4 receptor antibody.

Conclusion:

Our results support that the chemokine receptor CXCR4 is an interesting candidate for the future investigation of metastasis of bladder cancer in vivo.

Introduction

Chemokines are small secreted proteins (8–11 kDa) playing a major role in the regulation of leukocyte adherence to endothelial cells, leukocyte migration through transendothelial membranes, and tissue invasion [1]. Based on the position of their NH₂-terminal cysteine residues they are divided into four subfamilies: CC, CXC, C and CX₃C chemokines. The coupling of chemokines to their specific receptors, which predominantly belong to the family of G protein-coupled receptors, results in the activation of numerous signalling pathways. The CXC chemokine ‘stromal derived cell factor-1α’ (SDF-1α) and its receptor, CXCR4, play a role in B-cell lymphopoiesis and myelopoiesis and acts as a chemotactic factor for T-cells and monocytes [2], [3], [4]. Furthermore, tumor-derived chemokines induce the migration of host cells (e.g. monocytes and lymphocytes) into the solid tumor [5], [6]. On the other hand, the chemokine receptor CXCR4 is expressed on the surface of tumor cells and might promote the metastasis of tumor cells into target organs [6], [7]. Expression of the CXCR4 receptor has been demonstrated on tumor cells of breast cancer, melanoma, and some leukemias [6], [7], [8]. Activation of the CXCR4 receptor by SDF-1α induces signal transduction pathways in tumor cells which are key factors for metastasis, e.g. pseudopodia formation, migration, and proliferation [7], [8].

To understand the potential role of the CXCR4 receptor in bladder cancer we analyzed its expression in bladder cancer tissue and normal urothelium. Furthermore, functional effects upon stimulation of the CXCR4 receptor with SDF-1α with regard to intracellular Ca²⁺-transients, migration, pseudopodia formation, proliferation, and invasion of bladder cancer cell lines were investigated.

Section snippets

Materials

Minimal essential medium (MEM) with Earle's salts, McCoys 5A Medium with L-Glutamine, fetal calf serum, streptomycin, penicillin G and trypsin/EDTA were purchased from Invitrogen (Eggenstein, Germany). Recombinant SDF-1α and monoclonal anti-human CXCR4 (fusin) antibody were from R&D systems (Minneapolis, MN, USA). Collagen was from Sigma (Taufkirchen, Germany) and polyvinylpyrrolidone-free polycarbonate filter membranes were from nucleopore (Tübingen, Germany).

Methods

J82 and T24 cells (American tissue

CXCR4 mRNA expression

In a first series of experiments we analysed the CXCR4 expression in normal and malignant urothelium (Fig. 1) RNA from two different B-lymphoblastoid cell lines served as positive controls. Using RT-PCR with CXCR4-specific primers we detected specific transcripts in 2 samples of normal urothelium, 11 different samples of bladder cancer tissue obtained from native tumor and the bladder carcinoma cell lines J82 and T24. We subsequently performed a quantitative RNA expression analysis in 2 samples

Discussion

Chemokine and cytokine receptors play a crucial role in the development of hematopoetic cells and in the homing of hematopoetic cells to the bone marrow [15], [16]. Interestingly, recent findings proposed that the chemokine receptor CXCR4/SDF-1α receptor/ligand interaction is involved in the metastasis of tumor cells. Because a role of the CXCR4/SDF-1α receptor/ligand interaction has not been investigated in the metastasis of bladder cancer we performed CXCR4 expression studies and functional

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Expression Analysis and Potential Functional Role of the CXCR4 Chemokine Receptor in Bladder Cancer

Abstract

Objective:

Methods:

Results:

Conclusion:

Introduction

Section snippets

Materials

Methods

CXCR4 mRNA expression

Discussion

Lancet

Methods

Blood

Chemokines: Agents for the immunotherapy of cancer?

Nat Rev Immunol

Molecular cloning and structure of a pre-B-cell growth-stimulating factor

Proc Natl Acad Sci USA

A highly efficaciuous lymphocyte chemoattractant, stromal cell derived factor1 (SDF-1)

J Exp Med

Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1

Nature

Chemokines in cancer

Cytokine Growth factor Rev

Chemokines: not just leukocyte chemoattractants in the promotion of cancer

Nat Immunol

Involvement of chemokine receptors in breast cancer metastasis

Nature