Glial cell line–derived neurotrophic factor promotes invasive behaviour in testicular seminoma cells

Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology, Fondazione Pasteur Cenci Bolognetti, Sapienza University of Rome, Rome Department of Experimental Medicine, Second University of Naples, Naples Department of Radiological, Oncologic and Pathologic Sciences, Sapienza University of Rome, Rome, Italy.
International Journal of Andrology (Impact Factor: 3.7). 04/2012; 35(5):758-68. DOI: 10.1111/j.1365-2605.2012.01267.x
Source: PubMed


The glial cell line-derived neurotrophic factor (GDNF) has multiple functions that promote cell survival, proliferation and migration in different cell types. The experimental over-expression of GDNF in mouse testis leads to infertility and promotes seminomatous germ cell tumours in older animals, which suggests that deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis. GDNF activates downstream pathways upon binding to its specific co-receptor GDNF family receptor-a 1 (GFRA1). This complex then interacts with Ret and other co-receptors to activate several intracellular signalling cascades. To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell tumours, we analysed GFRA1 and Ret expression patterns in seminoma samples. We demonstrated, via immunohistochemistry, that GFRA1, but not Ret, is over-expressed in in situ carcinoma (CIS) and in intratubular and invasive seminoma cells compared with normal human germ cells. Functional analysis of the GDNF biological activity was performed on TCam-2 seminoma cell line. Reverse transcription-PCR (RT-PCR) and immunohistochemical analyses demonstrate that TCam-2 cells express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level. In TCam-2 cells, although GDNF is not mitogenic, it is able to induce migration, as demonstrated by a Boyden chamber assay, possibly through the Src and MEK pathways. Moreover, GDNF promotes invasive behaviour, an effect dependent on pericellular protease activity, possibly through the activity of matrix metalloproteinases. GFRA1 over-expression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer.

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    • "The cells were assayed for their ability to migrate through a polycarbonate filter (pore size, 8 µm; Whatman International) using Boyden chambers (NeuroProbe) as previously described [25]. Cells were cultured for 72 hours with or without 10% EW in the presence of 10% FBS and then maintained for 16 hours under serum-free conditions. "
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    ABSTRACT: Seminoma is one of the most common Testicular Germ Cell Tumours that originates during embryonic development due to an alteration of the local niche that in turn results in a delayed or blocked differentiation of Primordial Germ Cells. The block of differentiation is actually a common way to develop cancer disease as postulated by the "embryonic rest theory of cancer". In agreement with this theory different studies have demonstrated that embryonic cues display the capacity of reprogramming aggressive cancer cells towards a less aggressive phenotype. Herein we investigate the ability of a culture medium added with 10% egg albumen (EW, Egg White) to modulate seminoma cell phenotype and behaviour, by ensuring a proper set of morphogenetic signals. We chose to use the TCam-2 seminoma cell line that has been established as the only available cell line, obtained from a primary testicular seminoma. EW is able to: 1) modify TCam-2 cell spreading rate and cell-substrate adhesion without affecting proliferation and survival indexes; 2) modulate TCam-2 actin distribution pattern increasing cortical localization of actin filaments; 3) increase TCam-2 cell-cell junction capability; 4) decrease both chemo-sensitive and collective TCam-2 migratory behaviour. According to these observations morphometric fractal analysis revealed the ability of EW to increase Circularity and Solidity parameters and, consequently, to decrease Fractal dimension. Prompted by these observations we hypothesize that EW treatment could rescue, at least in part, the neoplastic-metastatic behaviour of seminoma cells.
    Full-text · Article · Oct 2013 · PLoS ONE
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    • "Immunohistochemistry (IHC) was performed as described previously[22]. Briefly, 3–5-lm-thick paraffin sections from human and mouse testis samples were serially collected and mounted on polylysine-coated slides. Dewaxed and rehydrated sections were incubated in 10 mM citrate buffer (pH 6.0) in a microwave oven for antigen retrieval at 750 W three times for 5 min. "
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    ABSTRACT: To date, in the human seminiferous epithelium only 6 associations of cell types have been distinguished, subdividing the epithelial cycle into 6 stages of very different duration. This hampers comparisons between studies on human and laboratory animals in which the cycle is usually subdivided into 12 stages. We now propose a new stage classification on basis of acrosomal development made visible by immunohistochemistry (IHC) for (pro)acrosin. IHC for acrosin gives results that are comparable to periodic acid Schiff (PAS) staining. In the human too, we now distinguish 12 stages that differ from each other in duration by a factor of two, at most. B spermatogonia are first apparent in stage I, preleptotene spermatocytes are formed in stage V, leptonema starts in stage VII and spermiation takes place at the end of stage VI. A similar timing was previously observed in several monkeys. Stage identification by way of IHC for acrosin appeared possible for tissue fixed in formalin, Bouin fixative, diluted Bouin fixative, Cleland fluid and in modified Davidson fixative, indicating a wide applicability. In addition, it is also possible to distinguish the 12 stages in glutaraldehyde/osmium-tetroxide fixed/plastic embedded testis material, without IHC for acrosin. The new stage classification will greatly facilitate research on human spermatogenesis and enable a much better comparison with results from work on experimental animals than hitherto possible. In addition, it will enable a highly focused approach to evaluate spermatogenic impairments such as germ cell maturation arrests or defects and to study details of germ cell differentiation.
    Preview · Article · Aug 2013 · Biology of Reproduction
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    • "Thus, the extracellular concentration of GDNF in a given area of the seminiferous tubule may vary with time [10]. Intriguingly, GDNF has also been demonstrated to stimulate chemotaxis in both normal, transformed cells [11]–[14] and seminoma cells [15]. Here, we tested the hypotheses that GDNF is a chemoattractant for undifferentiated spermatogonia, including stem/progenitor cells, and that the GDNF pathway may affect proteins involved in actin cytoskeleton rearrangement in target cells. "
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    ABSTRACT: In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.
    Full-text · Article · Apr 2013 · PLoS ONE
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