Morphological changes in human melanoma cells following irradiation with thermal neutrons

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.     

The effect of infrared laser irradiation on the growth of human melanoma cells in culture

Biophysics - Low-intensity laser irradiation exerts effects on various biological objects and is currently exploited in various branches of medicine. From a practical point of view, irradiation in...     

Selectin ligands on human melanoma cells

Twelve established human melanoma lines were screened for surface expression of the carbohydrate antigens Lewis^a (Le^a), sialyl Lewis^a (SLe^a), dimeric sialyl Lewis^a (diSLe^a), sialyl Lewis^X (SLe^X) and dimeric sialyl Lewis^X (diSLe^X). None of the lines expressed SLe^X, but 11/12 were positive for diSLe^X and 7/12 were positive for SLe^a. Although both diSLe^X and SLe^a have been reported to bind to E-selectin, none of the melanoma lines exhibited E-selectin-dependent adhesion to activated human umbilical vein endothelial cells (HUVECs). Three melanoma lines infected with a retroviral vector carrying the cDNA for the human Lewis fucosyltransferase (FucT-III) subsequently expressed SLe^X at their cell surface and exhibited E-selectin-dependent adhesion to activated HUVECs. Treatment of these transduced cells with inhibitors of O-linked or N-linked protein glycosylation significantly inhibited E-selectin-meiiated adhesion, though fluorescence-activated cell sorter analysis indicated no decrease in cell surface expression of SLe^X, SLe^a or diSLe^X. This suggests that the majority of SLe^X/SLe^a-type glycans endogenously procuded by human melanoma cells are not protein-associated and do not mediate E-selectin-dependent adhesion. These results support the hypothesis that E-selectin-dependent adhesion requires presentation of SLe^X-type moieties on appropriate glycoproteins.     

Protein phosphorylation in irradiated human melanoma cells

In the present study, we examined the response of confluent, primary human fibroblasts and cells of a melanoma (YUSAC2) cell line to ionizing radiation mediated through post-translational protein phosphorylation. Since the purpose of our study was to identify novel radiation-induced phosphoproteins in the DNA damage stress response of melanoma cells, we were primarily interested in changes in protein phosphoserine expression at early times after irradiation. Our rationale was that by examining the overall protein phosphorylation profile (the phosphoproteome) in irradiated cells, we might discover novel radiation-induced phosphoproteins that distinguish fibroblasts from melanoma cells. Cell proteins were separated by gel electrophoresis and phosphoproteins were identified by Western blot analysis using nonspecific anti-phosphoamino acid antibodies. This approach was not pursued previously since adequate antibodies for examining global protein phosphoserine expression were unavailable. While some radiation-induced phosphoprotein changes in high-abundance proteins were identified, in general the sensitivity of this approach was not sufficient to detect changes in low-abundance, regulatory proteins. Characterization of these phosphoproteins will require greater enrichment of low-abundance proteins.     

Generation of cytotoxic effector cells against human melanoma

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2–6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6–10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562, Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.This work was supported by an Elsa, U. Pardee Foundation grant, the Arizona Chronic Disease Research Commission grant and partly by grant CA23074 from the National Institutes of Health, Bethesda, MD, 20892     

Study of motogenic signal in human melanoma cells

The components of the extracellular matrix (ECM) are more than just adhesion sites for migrating tumor cells: following enzymatic degradation of the ECM, the release of sequestrated growth factors increases, thus they become available for tumor cells. In a number of cancers dysfunction of epidermal growth factor receptor (EGFR) or hepatocyte growth factor receptor (c-Met) contribute to the malignant transformation that directly regulates cell proliferation, survival and motility. Furthermore, intracellular calcium level plays an important role in the regulation of the tyrosine kinase pathway. In our preclinical experiments, by administering heparin-derived oligosaccharides we influenced the interaction between human melanoma cells and ECM. In vitro cell migration was inhibited by heparin fragments. Moreover, two of the effective oligosaccharides reduced the number of lung colonies formed in SCID mice. In human melanoma cells an important element of Ca2+ homeostasis, the purinergic Ca2+ channel P2X7 proved to be an anti-apoptotic protein. EGFR and c-Met showed constitutive activity in human melanoma cells, and their inhibition in vitro caused decreased proliferation, migration and elevated apoptosis. Administration of a selective c-Met-TKI significantly decreased primary tumor growth in vivo as well as the capacity for liver colony formation in SCID mice. Selective EGFR-TKI had less inhibitory effect on metastasis formation, and had no effect on the primary tumor. Our results suggest the necessity of a rational dual-specific drug design for the purpose in the therapy of malignant melanoma.     

Terminal transferase-like activity in human melanoma cells.

An enzyme activity, capable of attaching deoxynucleotides to single-stranded oligonucleotide primers, has been detected in extracts from cultured melanoma cells. The substrate preferences of this activity show it to be distinct from the calf thymus terminal transferase (EC 2.7.7.31).     

Biosynthesis and secretion of thrombospondin in human melanoma cells

A polyclonal antisera specific for human platelet thrombospondin (TSP) has been utilized to study the biosynthesis and secretion of TSP in the M21 human melanoma cell line. Pulse-chase indirect immunoprecipitation analysis reveal that human melanoma cells rapidly synthesize and secrete this platelet alpha-granule associated glycoprotein. Topographical analysis of the melanoma cell surface by indirect immunofluorescence indicate that the TSP molecules have no obvious extracellular organization. The implications of thrombospondin synthesis in the metastatic process of melanoma are discussed.     

Biosynthesis and secretion of fibronectin in human melanoma cells

The biosynthesis and secretion of cellular fibronectin from human melanoma cells have been investigated by pulse-chase/immunoprecipitation analysis. Melanoma cells synthesize endoglycosidase H (Endo H)-sensitive glycoprotein precursors of fibronectin glycoproteins which chase to an Endo H-resistant monomer with an apparent Mr of 240,000 (240 K). This molecule, which has a significantly higher molecular weight than normal plasma or cellular fibronectin, is rapidly secreted by melanoma cells, resulting in the secretion of 80% of newly synthesized fibronectin in 120 min, following a 10-min biosynthetic pulse. This active secretory process can be inhibited by brief exposure of melanoma cells to sodium monensin (10(-7) M), which also results in a modified fibronectin of lower apparent Mr. Monosaccharide-incorporation studies of melanoma fibronectin reveal that monensin significantly inhibits galactose and fucose incorporation into this glycoprotein, correlating with reported effects of monensin on Golgi apparatus functions. These studies indicate that this tumor-associated and biosynthetically altered cellular fibronectin is a rapidly secreted major N-linked glycoprotein of metastatic human melanoma cells.     

Cytotoxicity studies of human melanoma cells and fibroblasts.

In seven human melanoma cell lines and one human fibroblast strain some correlation of resistance to cell killing was found with two bifunctional alkylating agents (melphalan, chlorambucil) and three monofunctional agents (4(5)-(3,3-dimethyl-l-triazeno)imidazole-5(4)-carboxamide (DTIC), methylmethane sulphonate (MMS) and N-methyl-N1-nitro-N-nitrosoguanidine (MNNG), but little cross-resistance was found between these two groups of agents or with cytosine arabinoside (ara-C). In contrast to previous studies with rodent tumours, potentially synergistic (chloroquine, arginine) or antagonistic (ascorbic acid, leucine) compounds did not affect the toxicity of melphalan in a human melanoma cell line. In two melanoma lines DTIC induced patterns of DNA damage (inhibition of semi-conservative synthesis) and repair (strand breaks and repair synthesis) similar to, but not identical with, those induced by the methylating agent MMNG. These results suggest that a methylating species is derived from DTIC but has a different reactivity toward DNA compared with MNNG.     

Biological properties of human melanoma cells in culture.

Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and that metastatic melanoma seems to display a higher degree of malignant transformation than the primary.     

Effect of UV-B irradiation on release of arachidonic acid from B-16 melanoma cells

B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 microM) and calmodulin inhibitor W7 (0.5 microM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca2+. The release decreased significantly at lower Ca2+ concentrations. These results suggest that Ca2+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release of radiolabeled materials, possibly arachidonic acid and its metabolites, from the cells.     

Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells

The influence of environmental factors (cytokines, matrix components, serum factors and O(2) level) on expression of receptors for angiogenic versus angiostatic CXC chemokines in human microvascular endothelial cells has not been extensively investigated. Our semi-quantitative RT-PCR analysis demonstrated that TNF-alpha and IFN-gamma repressed CXCR4 mRNA levels in immortalized human microvascular endothelial HMEC-1 cells after 4 h, whereas only TNF-alpha displayed inhibitory activity in primary human microvascular endothelial cells (HMVEC). CXCR4 mRNA expression was not affected by VEGF, GM-CSF, IL-1beta or various basal membrane matrix components, but was significantly up-regulated after serum starvation and/or hypoxic treatment of the microvascular endothelial cells. The alternative CXCL12 receptor, CXCR7/RDC1, was also up-regulated by hypoxia in HMEC-1 cells, although less consistently than CXCR4. Furthermore, hypoxia and serum starvation were required for cell surface display of CXCR4 and CXCL12 induction of ERK activation in HMEC-1 cells. In contrast, CXCR2 and CXCR3 mRNA levels remained, respectively, low and undetectable under all the conditions tested, and surface expression of CXCR2, CXCR3 and CXCR7 on the HMEC- 1 cells could not be demonstrated by FACS. In the human SK-MEL-5 melanoma cell line, CXCR4 mRNA expression was also increased under hypoxic conditions, whereas CXCR2 mRNA levels remained low and levels of CXCR3 and CXCR7 were undetectable. However, immunohistochemical staining of human metastatic melanoma sections demonstrated that CXCR2, CXCR3, CXCR4 and CXCR7 are expressed on tumor cells and, to a lesser extent, on endothelial cells. These results demonstrate that the tumor microenvironment regulates chemokine receptor expression through both cytokine and oxygen levels.