Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest.

Departments of Pharmacology & Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610-0267, USA.
Differentiation (Impact Factor: 2.84). 06/2009; 78(1):35-44. DOI: 10.1016/j.diff.2009.04.001
Source: PubMed

ABSTRACT The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10–150 μM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.
    Cellular & Molecular Biology Letters 12/2012; 17(4). · 1.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Urodele amphibians such as Japanese common newts have a remarkable ability to regenerate their injured neural retina, even as adults. We found that hematological- and neurological-expressed sequence 1 (Hn1) gene was induced in depigmented retinal pigment epithelial (RPE) cells, and its expression was maintained at later stages of newt retinal regeneration. In this study, we investigated the distribution of the HN1 protein, the product of the Hn1 gene, in the developing retinas. Our immunohistochemical analyses suggested that the HN1 protein was highly expressed in an immature retina, and the subcellular localization changed during this retinogenesis as observed in newt retinal regeneration. We also found that the expression of Hn1 gene was not induced in mouse after retinal removal. Our results showed that Hn1 gene can be useful for detection of undifferentiated and dedifferentiated cells during both newt retinal development and regeneration.
    Stem cells international. 01/2012; 2012:436042.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.
    PLoS ONE 05/2014; 9(5):e95919. · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 20, 2014