Modulation of Melanotransferrin and Transferrin Receptor 1 (TFRC)- and CD44-based Signaling for TFRC Up-regulation in Human Melanoma Cells.

BACKGROUND: The human melanoma cell line IGR-1 was used for the detection and regulation of both melanotransferrin (MTf) and transferrin receptor 1 (TFRC, CD71). While the function in iron transport of the TFRC is well documented the functional importance of MTf is not yet fully understood. Due to the up-regulation of TFRC by hyaluronan (HA) some components and aspects of CD44 signaling were investigated. MATERIALS AND METHODS: The cell-surface proteins MTf, TFRC and ERBB2 receptor tyrosine kinase 2 (ERBB2) were detected by immunoluminescent technique using different polyclonal and monoclonal antibodies. Ionomycin was used to inhibit ß-catenin/T-cell-specific transcription factor (TCF) association, essential in HA-CD44-ERBB2 signaling. RESULTS: MTf, was found to be resistant to phosphatidylinositol-specific phospholipase C. However, MTf as well as TFRC were sensitive to partial proteolytic degradation by pronase E and trypsin. The expression of MTf was shown to be up-regulated by mannose-6-phosphate and that of TFRC by HA. Ionomycin at 10 µM inhibited TFRC up-regulation. However, at 50 µM it induced a 7.5-fold increase of TFRC concentration. CONCLUSION: Our results suggest that human melanoma cells are able to up-regulate TFRC expression using HA/CD44 signaling. The whole pathway comprises of the sequence: HA/CD44, neural Wiskott-Aldrich syndrome protein (N-WASP), ERBB2, ß-catenin/TCF, c-MYC and TFRC. Since ß-catenin is also known to be a component of wingless/Int-1-Frizzled signaling that also leads to transcriptional c-MYC activation, the pathway found here might be alternatively used by melanoma cells for iron supply, necessary for cell proliferation.

Mannose-6-phosphate/insulin-like growth factor-II receptor in human melanoma cells: effect of ligands and antibodies on the receptor expression.

BACKGROUND: The M6P/IGF-II receptor belongs to the IGF system which plays a crucial role in tumorigenicity. While the role of the IGF-I receptor in signal transduction is well documented, previous experiments failed to uncover a clear signalling function for the M6P/IGF-II receptor. However, more recent studies have shown the capability of M6P/IGF-II receptor to initiate transmembrane signalling. MATERIALS AND METHODS: Human melanoma cells were used to detect the cell surface expression of the M6P/IGF-II receptor and its modulation by different effectors and monoclonal anti-receptor antibodies. RESULTS: M6P (5 mM) caused an increase of the luminescent receptor signal of about 50% . Pre-incubation of cells with Act-D (5 microg/mL) or CHI (10 microg/mL) following M6P stimulation in the presence of the inhibitors caused a reduction of receptor cell surface expression of 27% or 31%, respectively. The monoclonal antibody (mAb) 2G11 was able to mimic the M6P effect on the receptor up-regulation but the mAb MEM-238 did not. The synergistic effect detected with the combination of M6P and the mAb 2G11 and the failure of 2G11 to compete with the M6P action suggests that both effectors have different binding sites on the receptor. Unlike 2G11 the mAb MEM-238 prevented the M6P effect on receptor up-regulation confirming partially overlapping binding epitopes of both effectors. Brefeldin A was shown to have an inhibiting effect on the vesicular transport of the receptor protein to the plasma membrane and forskolin had an activating effect on the receptor exocytosis with the following enhanced integration into the plasma membrane. CONCLUSION: Up-regulation of the tumour suppressor M6P/IGF-II receptor might represent an approach for anticancer therapy. In addition, results support recent data on the receptor's capability of signal transduction.

Immunoluminescent detection of intercellular adhesion molecule-1 and aminopeptidase N on human melanoma cells.

BACKGROUND: During the complex process of melanoma cell detachment from the primary tumor and subsequent metastasis, different cell surface proteins are involved in tumor cell interactions with extracellular matrix (ECM) components and surrounding cells. Melanoma cells are able to vary their expression patterns of adhesion proteins and proteases or peptidases in different stages of tumor development. MATERIALS AND METHODS: This paper describes the detection of intercellular adhesion molecule-1 (ICAM-1) and aminopeptidase N (APN) on melanoma cells by immunoluminescence. The marker enzyme horseradish peroxidase (HRP) of the secondary antibody (Ab) was used to release luminescence. In addition, APN was detected on intact cells with an activity assay using alanine-p-nitroanilide (Ala-pNA) as substrate including inhibition experiments. RESULTS: The cell surface expression of ICAM-1 and APN on melanoma cells was shown by indirect labelling using two different antibodies for each antigen. The activity and inhibition assays confirmed the immunoluminescent result for APN. CONCLUSION: Both cell surface proteins have a strong impact on tumor cell behaviour and are typical cell markers indicating a high metastatic tumor stage in melanoma. The extensive characterization of melanoma cells offers the possibility for therapeutic approaches with more than one target molecule. Moreover, increasing insights into the components involved in signal transduction could help to develop specific reagents that inhibit tumor-specific pathways.

Re-programming of newt cardiomyocytes is induced by tissue regeneration.

Newt hearts are able to repair substantial cardiac injuries without functional impairment, whereas mammalian hearts cannot regenerate. The cellular and molecular mechanisms that control the regenerative capacity of the newt heart are unknown. Here, we show that the ability of newt cardiomyocytes to regenerate cardiac injuries correlates with their ability to transdifferentiate into different cell types. Mechanical injury of the heart led to a severe reduction of sarcomeric proteins in the myocardium, indicating a partial de-differentiation of adult newt cardiomyocytes during regeneration. Newt cardiomyocytes implanted into regenerating limbs lost their cardiac phenotype and acquired skeletal muscle or chondrocyte fates. Reprogramming of cardiomyocytes depended on contact with the limb blastema because cardiomyocytes implanted into intact, non-regenerating limbs or cultured in vitro retained their original identity. We reason that signals from the limb blastema led to de-differentiation of cardiomyocytes, cell proliferation and re-differentiation into specialized cells and propose that the ability of cardiomyocytes to transdifferentiate into different cell types reflects the cellular program that enables heart regeneration.