Differentiation Of Melanoma Cells Research Articles

Preclinical evaluation of the antineoplastic efficacy of 7-(2-hydroxyethyl)theophylline on melanoma cancer cells

Differentiation-based therapeutics are an underutilized but potentially a significant option for cancer treatment. The effect of methylxanthines on melanoma cell differentiation has been well documented. We report the in-vitro and in-vivo anticancer potential of a theophylline analogue, 7-(2-hydroxyethyl)theophylline (HET), on murine B16-F10 and human Sk-Mel 110 metastatic melanoma cell lines. The effects on cell proliferation were related to the induction of differentiation, demonstrated as increased intracellular transglutaminase activity. The involvement of this methylxanthine in the control of the in-vitro adhesion and in the in-vivo metastastic spread of melanoma cells was further investigated. HET oral administration of C57BL6/N mice intravenously injected with B16-F10 cells markedly reduced lung metastases frequency. The overall results demonstrated that HET possesses a remarkable in-vivo antimetastatic capability.

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

BackgroundMelanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis.ObjectiveThe involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated.MethodsIn cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot.ResultsIn rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone.ConclusionRapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells.

Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation

Aloin, a natural anthracycline from aloe plant, is a hydroxyanthraquinone derivative shown to have antitumor properties. This study demonstrated that aloin exerted inhibition of cell proliferation, adhesion and invasion abilities of B16-F10 melanoma cells under non-cytotoxic concentrations. Furthermore, aloin induced melanoma cell differentiation through the enhancement of melanogenesis and transglutaminase activity. To improve the growth-inhibiting effect of anticancer agents, we found that the combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin. The results suggest that aloin possesses antineoplastic and antimetastatic properties, exerted likely through the induction of melanoma cell differentiation.

DHA induces apoptosis and differentiation in human melanoma cells in vitro : involvement of HuR-mediated COX-2 mRNA stabilization and β-catenin nuclear translocation

The pro-inflammatory phenotype accompanying melanoma progression includes an enhanced expression of cyclooxygenase-2 (COX-2), which plays an important role in the acquisition of apoptosis resistance, and is a suitable target for melanoma prevention and therapy. We observed that the WM266-4 metastatic melanoma cell line showed a constitutive COX-2 expression higher than that of the primary WM115 cells, an increased cytosolic level of the COX-2 messenger RNA (mRNA)-stabilizer human antigen R (HuR) and a lower susceptibility to basal apoptosis. The transfection of HuR siRNA induced apoptosis and reduced COX-2 protein abundance in both the cells. The same effects were observed treating the cells with the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), which reduced the cytoplasmic location and expression of HuR and, correspondently, decreased COX-2 protein expression and induced apoptosis. DHA also decreased the expression and stability of COX-2 mRNA, increased the β-catenin expression in the nuclei and reduced it in the cytosol, where it forms a complex with HuR and COX-2 mRNA. DHA had also a pro-differentiating effect, which is compatible with the nuclear translocation of β-catenin. These findings allow us to associate for the first time the constitutive expression of COX-2 in melanoma cells to the HuR-mediated stabilization of its mRNA and suggest that also β-catenin may play a role in HuR-mediated COX-2 stabilization in these cells. The data demonstrate that the HuR-mediated stabilization of COX-2 may represent a target of DHA action in melanoma cells and suggest the application of DHA in the prevention and therapy of melanoma.

Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo.

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo.

Nobiletin Induces Differentiation of Murine B16/F10 Melanoma Cells

A previous report demonstrated that nobiletin can induce melanogenesis via up-regulation of extracellular kinase signaling pathways at the later phases; however its ability to induce differentiation in melanoma cells has not been investigated. Nobiletin induced differentiation in murine B16 melanoma cells by increasing tyrosinase activity, melanin content, dendrite formation, and intracellular cAMP, and inhibited cell proliferation in a dose- and time-dependent manners, induced G1 cell cycle arrest, and suppressed phosphorylated protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) protein expressions, which are involved in proliferation. However, nobiletin did not induce DNA fragmentation or condensation indicating potential use of nobiletin to induce differentiation in melanoma cells.

P047. Heme oxygenase-1 is a key player in differentiation of melanoma cells

Was, H.a; Tejchman, A.a; Dubiel, M.a; Dominik, P.a; Kotlinowski, J.a; Zebzda, A.b; Dulak, J.a; Jozkowicz, A.a Author Information

Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared with adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression, respectively. This links BRN2 as an activator, and conversely, MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense-ablated cell lines decreased the melanoma sphere-forming capability, cell adhesion during 3D-spheroid formation and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere-culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than the growth as adherent melanoma cells.

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.

Chrysin-induced apoptosis is mediated through p38 and Bax activation in B16-F1 and A375 melanoma cells

Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound extracted from honey, plants and propolis. It possesses anti-inflammatory activity, anti-oxidant properties and promotes cell death by perturbing cell cycle progression. In this study, our attention focused on the possible role that chrysin may have as a potential anti-cancer agent, and we tested its biological activity in murine and human melanoma cell lines (B16-F1 and A375). This study demonstrated that chrysin reduced melanoma cell proliferation and induced cell differentiation in both human and murine melanoma cells through synthesis increase and intracellular accumulation of protoporphirin IX (PpIX). Furthermore, following treatments with chrysin an increase in the expression of porphobilinogen deaminase (PBG-D) was noted. This study demontrated also that chrysin induces cell death in human and murine melanoma cells through caspase-dependent mechanisms, involving down-regulation of ERK 1/2, and activation of p38 MAP kinases. Induction of cell death may be a promising therapeutic approach in cancer therapy. Our results suggest that chrysin may be considered a potential candidate for both cancer prevention and treatment.

APAF-1 is related to an undifferentiated state in the testicular germ cell tumor pathway

Apoptotic protease activating factor-1 (APAF-1) is a key regulator gene of apoptosis, located downstream from p53. Loss of APAF-1 expression is associated with chemorefractory malignant melanoma and neuronal cell differentiation. In order to make clear the function of APAF-1 in the carcinogenesis of germ cell tumors, we evaluated the expression levels of APAF-1 and several apoptosis and differentiation markers by immunohistochemistry in formalin-fixed paraffin-embedded samples from 43 cases of testicular germ cell tumor (TGCT) and six specimens of normal testis tissue. Expression of cleaved caspase-3, Oct-3/4, and Ki-67 were also examined by immunohistochemistry to evaluate apoptotic reactivity, tumor differentiation, and proliferation activity, respectively. APAF-1 was downregulated in two TGCT cell lines by siRNA transfection, and subsequent expression of the Ki-67 and Oct-3/4 genes and differentiation markers of three embryonic germ layers including keratin16 (KRT16) for ectoderm, vimentin (VIM) for mesoderm and GATA4 for endoderm were then tested. No significant relationship was found between APAF-1 expression and apoptotic activity in TGCTs. Expression of APAF-1, Oct-3/4, and Ki-67 was significantly higher in seminomas than in non-seminomas. In TGCTs, higher APAF-1 expression was correlated with higher proliferation (high Ki-67) and a lower degree of differentiation (high Oct-3/4). Interestingly, the expression of APAF-1 gradually decreased in accordance with tumor differentiation (seminoma and embryonal carcinoma > teratoma). Downregulation of APAF-1 in TGCT cell lines resulted in a decrease of Ki-67 and Oct-3/4 and an increase of VIM and KRT16 gene expression. These data show that higher expression of APAF-1 is related to an undifferentiated state in the TGCT pathway.

Chloroform extract from Moricandia arvensis inhibits growth of B16‐F0 melanoma cells and promotes differentiation in vitro

Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study we have investigated inhibitory capacity of ethyl acetate, chloroform (Chl) and methanol extracts from Moricandia arvensis on mouse melanoma (B16-F0) and human keratinocyte (HaCaT) cell proliferation. Influence of Chl extract on percentage distribution in cell cycle phases and melanogenesis was also studied. Cell viability was determined at various periods using the MTT assay, and flow cytometry was used to analyse effects of Chl extract on progression through the cell cycle and apoptosis. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Chl extract exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells. Morphological changes in B16-F0 cells, accompanied by increase of tyrosinase activity, and of melanin synthesis were observed, which are markers of differentiation of malignant melanoma cells. Furthermore, cell cycle analysis revealed that B16-F0 cells treated with Chl extract were arrested predominantly in G(1) phase. Chl extract had the ability to reverse malignant melanoma cells from proliferative to differentiated state, thus providing a new perspective in developing novel strategies for prevention and treatment of malignant melanoma, possibly through consumption of the extract in an appropriate cancer prevention diet. Moreover, there is scope for the extract being introduced into cosmetic products as a natural tanning agent.

Effects of Sesquiterpenes Isolated From Largehead Atractylodes Rhizome on Growth, Migration, and Differentiation of B16 Melanoma Cells

The aims of this study were to isolate sesquiterpene compounds from the largehead atractylodes rhizome (LAR) and to investigate their effects on B16 cancer cells. A total of 8 sesquiterpenes from LAR were identified, of which eudesm-4 (15), 7-diene-9α, 11-diol (7) was isolated for the first time. All 8 compounds inhibited growth of B16 cells, and atractylenolide I (AT-I), atractylenolide II (AT-II), and atractylenolactam (ATR) were the most potent, with IC(50) values of 76.46, 84.02, and 54.88 μΜ, respectively. Monomer lactone or lactam structures in the 8 compounds appeared to be critical for their antiproliferative activities. In addition, AT-I, AT-II, and ATR could induce cell differentiation and inhibit cell migration. Western blot analysis indicated that 2 of the compounds, AT-I and AT-II, could inactivate ERK, where all 3 inhibited AKT activation, suggesting that Ras/ERK and PI3K/AKT signaling pathways are involved in the action mechanisms of the LAR sesquiterpene compounds.

Induction of Terminal Differentiation in Melanoma Cells on Downregulation of β-Amyloid Precursor Protein

The incidence of melanoma, the most aggressive type of skin cancer, is increasing dramatically, and an effective treatment for patients with advanced disease is as yet unavailable. Greater insight into the molecular features of primary and metastatic melanoma is required, particularly the identification of key regulatory genes that shield the tumor cells from terminal differentiation and apoptosis. The beta-amyloid precursor protein (APP) is a cell surface receptor and the transmembrane precursor of the Abeta-peptide, which has an important role in Alzheimer's disease. The study presented here provides evidence that APP is expressed at high levels in advanced-stage melanomas, and that the cells cleave APP and secrete sAPP. We show that blocking the expression of APP by RNA interference impairs the proliferation of metastatic melanoma cells and leads to their terminal and irreversible differentiation. In addition, suppressing APP expression in a metastatic melanoma cell line renders the cells susceptible to several chemotherapeutic agents. Targeting APP may thus constitute a new approach to the treatment of this disease.

Abstract 5456: Preferential targeting of N-Ras mutant and other wild type B-Raf human melanoma cells with c-Met inhibitor: a preclinical promise

Abstract Melanoma is the deadliest skin cancer known and the incidence continues to rise. The 2009 report from American Cancer Society predicts there would be 68,720 new cases of melanoma this year and deaths amounting to 8650. Melanoma detected early responds well to surgery but metastatic melanoma is highly resistant to conventional radiation and chemotherapy, and most immunotherapy, although this area is improving. Targeted therapies are most attractive as several mutations and altered growth pathways have recently been identified. Approximately 60% of melanoma are known to express mutated B-Raf, about 20% express mutated N-Ras, and the rest a variety of other mutations. The B-Raf mutated tumors are exclusive of the N-Ras, suggesting at least two biologically distinct groups. The c-Met/HGF growth promoting pathway has recently been identified as active in human cutaneous as well as uveal melanoma, with reports showing that HGF may regulate melanoma cell proliferation, migration and differentiation. However, the activation and functional activities of the HGF receptor, c-Met, has been studied only minimally in melanoma. Our studies report that as high as 90% of melanoma cell lines express c-Met and most of these secrete a detectable level of the ligand HGF. Melanoma cells with different somatic mutations, including the mutually exclusive mutated B-Raf or N-Ras genes and other cells with wild type copies of both genes were directly compared for sensitivity to c-Met inhibition using the small molecule c-Met inhibitor PHA665752 from Pfizer. We have previously reported that the N-Ras mutant melanoma cells (SB2, SK-Mel-2 cell lines) are more sensitive compared to the B-Raf mutated lines (A375, WM35, WM793). Subsequently we observed that the majority of cell lines in the non B-Raf mutated populations (including mutant N-Ras) were positive for phospho-c-Met indicating activation of c-Met pathway. After analyses of patient tumors by IHC, we report that 12/19 B-Raf wild type melanoma including N-Ras mutated, show activation of the c-Met pathway by detection of phospho-c-met in situ. Using formalin-fixed paraffin embedded primary cutaneous melanoma tumor tissues that had DNA sequencing after laser capture micro-dissection, we found that most B-Raf wild type tumors have activated c-Met. 5 out of 6 tumors with wild type B-Raf and N-Ras show positive staining for phospho-c-Met and about 83% cells are stained. 7 out of 13 tumors with wild type B-Raf and mutant N-Ras show phospho-c-Met with 54% cells staining positive, but in the group of B-Raf mutant tumors, only 1 out of 5 tumors stained positive for phospho-c-Met with staining in 20% cells. Thus most human melanoma with wild type B-Raf show c-Met activation, as indicated by c-Met phosphorylation, and could be the ideal subgroup for therapeutically targeting c-Met in melanoma. This work was supported by a Melanoma SPORE grant from the NCI (P50 CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5456.

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations

The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

Mechanism of Regulation and Suppression of Melanoma Invasiveness by Novel Retinoic Acid Receptor-γ Target Gene Carbohydrate Sulfotransferase 10

Retinoic acid (RA) induces growth arrest and differentiation of S91 murine melanoma cells and serves as a valuable model for this disease. RA acts through activation of RA receptors (RAR), which are members of the nuclear receptor superfamily of ligand-inducible transcription factors. Interestingly, differentiation is mediated by RARgamma, but not by RARalpha or RARbeta, suggesting that RARgamma possesses unique and uncharacterized molecular properties. To address this question, DNA microarrays in combination with RAR isoform-specific agonists were employed to identify novel RARgamma target genes that may play a role in this process. Here, we identified and validated carbohydrate sulfotransferase 10 (CHST10) as a novel RARgamma target gene in S91 cells. The RARgamma-inducible CHST10 promoter was obtained, and two atypical, independently functioning RA response elements were identified in a 425 bp region. Surprisingly, this fragment is bound by RARgamma, but not by RARalpha or RARbeta, thus providing a mechanism for the observed RARgamma-specific regulation. CHST10 is a sulfotransferase that forms HNK-1 glycan on neural cell adhesion proteins and glycolipids, and HNK-1 is thought to modulate cell adhesion and possibly metastasis. We show that CHST10 is also regulated by RARgamma in a significant subset of human melanoma cells, and three-dimensional cell culture migration assays suggest that CHST10 functions as a suppressor of invasiveness, but not proliferation, in these cells. Induction of CHST10 by RARgamma-activating retinoids may present a novel therapeutic strategy to inhibit invasiveness in a subset of melanoma patients.

Melanoma cell differentiation induced by lupeol separates into two stages: morphological and functional changes

Electron microscopic observation revealed that lupeol induced melanosome maturation in B16 2F2 mouse melanoma cells and we therefore studied the effects of lupeol on the intracellular events responsible for melanosome transport. Incubation with lupeol for 8 h attenuated the actin stress fiber assembly in B16 2F2 mouse melanoma cells, resulting in dendritic formation in the cells. Longer exposure to lupeol (48 h) increased the expression of tyrosinase, MITF (a specific transcription factor for tyrosinase), Rab27a, and myosin-Va, which are required for melanosome transport.

Differentiation of B16-BL6 Melanoma Cells into Microtubule-Associated Protein 2-Positive Cells after Treatment with the Histone Deacetylase Inhibitors Butyrate and Trichostatin A

Inhibition of histone deacetylase (HDAC) modulates the expression of many genes and induces cell cycle arrest, apoptosis, and differentiation in several cancer cell lines. Melanoma is a malignant phase of cutaneous melanocytes originally derived from the neural crest and is highly metastatic. A therapeutic agent capable of inducing differentiation of metastatic melanoma cells into a nonmalignant stage would be useful to prevent metastasis. We examined whether HDAC inhibitors can induce differentiation of the murine melanoma cell line B16-BL6 into neural-type cells in vitro. A morphologic change accompanied by extended dendrites was induced in melanoma cells after treatment with the HDAC inhibitors butyrate and trichostatin A (TSA). The altered morphology was similar to that of neural cells. Many of the extended dendrites fused with other dendrites of neighboring cells and had synapse-like knobs on their dendrites. These dendrites showed positive labeling with anti-α/β-tubulin and anti-L1 cell adhesion molecule (L1CAM) antibodies but were seldom stained with phalloidin, suggesting that the neural cell-related proteins were major components of the extended dendrites but that actin stress fibers were not. Furthermore, the mature neuron-specific cytoskeletal protein, microtubule-associated protein 2 (MAP2), was detected with the specific antibody in the extended dendrites of cells treated with butyrate and TSA. Butyrate treatment increased the levels of MAP2 and neural cell adhesion molecule (NCAM) mRNA expression in the cells. However, the treatment did not alter the expression of neurofilament light chain (NF-L) mRNA. The observations suggest that the differentiated cells were neural-type cells but not complete neural cells.

Gene expression analysis of terminal differentiation of human melanoma cells highlights global reductions in cell cycle-associated genes

Defects in differentiation are frequently observed in cancer cells. By appropriate treatment specific tumor cell types can be induced to terminally differentiate. Metastatic HO-1 human melanoma cells treated with IFN-β plus mezerein (MEZ) undergo irreversible growth arrest and terminal differentiation followed by apoptosis. In order to define the molecular changes associated with this process, changes in gene expression were analyzed by cDNA microarray hybridization and by semi-quantitative and quantitative RT-PCRs of representative 44 genes. The expression of 210 genes was changed more than two-fold at either 8 or 24 h post-treatment (166 up and 44 down). Major biological processes associated with the up-regulated genes were response to endogenous/exogenous stimuli (38%), cell proliferation (13%), cell death (16%) and development (30%). Approximately 34% of the down-regulated genes were associated with cell cycle, 9% in DNA replication and 11% in chromosome organization, respectively. Suppression of cell cycle associated genes appeared to directly correlate with growth arrest observed in the terminal differentiation process. Expression of Calpain 3 (CAPN3) variant 6 was suppressed by the combined treatment and maintained high in various melanoma cell lines. However, over-expression of the CAPN3 did not significantly affect growth kinetics and cell viability, suggesting that up-regulation of CAPN3 alone may not be a causative, but an associated change with melanoma development. This analysis provides further insights into the spectrum of up-regulated and the first detailed investigation of down-regulated gene changes associated with and potentially causative of induction of loss of proliferative capacity and terminal differentiation in human melanoma cells.