Andrea Caricasole

Human growth-differentiation factor 3 (hGDF3) : developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFβ superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is downregulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

A luciferase-reporter vector with blue-white selection for rapid subcloning and mutational analysis of eukaryotic promoters ☆

A luciferase-reporter plasmid vector has been constructed for the rapid subcloning and analysis of eukaryotic promoters. Identification of recombinants, when using this vector, is greatly facilitated by the retention of blue-white selection (alpha-complementation).

Analysis of the Response of Human Embryonal Carcinoma Cells to Activin A

The activins and inhibins are dimeric polypeptide hormones belonging to the transforming growth factor-beta (TGF-β) superfamily of growth factors. Three separate genes encode for inhibin a, β A and β B subunits: inhibin β dimers give rise to the activins, while inhibin αβ heterodimers form inhibin. Initially discovered as modulators of pituitary follicle-stimulating hormone (FSH) production, the activins were later shown to elicit biological responses ranging from mesoderm induction in amphibians to the control of proliferation, differentiation, and apoptosis in mammalian systems (1, 2). According to the model proposed for TGF-β signaling, activin effects are mediated by a receptor complex comprising at least two types (I and II) of transmembrane Ser-Thr kinase receptors (3). Most activin effects can be negatively modulated by inhibin and the activin-binding protein follistatin.

Bone morphogenetic proteins and retinoic acid induce human endogenous retrovirus HERV-K expression in NT2D1 human embryonal carcinoma cells.

Expression of the HERV‐K human endogenous retrovirus is very low in normal and tumor tissue, but is readily detected in testicular germ cell tumors (TGCT). NT2D1 human embryonal carcinoma cells represent in vitro models for the stem cells of TGCT, and can be differentiated by treatment with bone morphogenetic proteins (BMP) or retinoic acid (RA). In a search for BMP target genes in NT2D1 cells, HERV‐K was identified as an early BMP and RA target. It was shown that HERV‐K expression was induced upon treatment of NT2D1 cells with BMP or with RA, but not with activin or transforming growth factor (TGF)‐β. Induction of HERV‐K expression was rapid but transient, with transcripts becoming undetectable in differentiated NT2D1 cultures. Thus NT2D1 cells provide a suitable in vitro system for the study of the factors controlling HERV‐K expression during cellular differentiation, which may play a role in HERV‐K expression in TGCT.

Expression of Inhibin Subunits, Follistatin, and Activin Receptors in Normal Testicular Cells and Testicular Tumors

Inhibin has been defined as a gonadal glycoprotein that can suppress the secretion of gonadotrophins, preferentially that of follicle-stimulating hormone (FSH) from the pituitary gland (1). It consists of an α-subunit, which is covalently linked by a disulfide bond to either of two β-subunits, β A or β B, giving rise to inhibin A and B, respectively. Alternatively, β-subunits can dimerize to form activins: homodimers of β A- β B-subunits are designated activin A or B, respectively, whereas β A β B -dimers are named activin AB (2, 3). All subunits are synthesized as larger precursor proteins; the factors that influence combination of subunits and cleavage of the precursors are scarcely known.