Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with α-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or α-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.     

Modulation of the Antigenic Phenotype of Human Melanoma Cells by Differentiation-inducing and Growth-suppressing Agents

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-β), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-β results in an induction and/or increased expression of ICAM-1. HLA Class I antigens and HLA Class II antigens. IFN-β and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-γ), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-β plus IFN-γ which synergistically but reversibly suppresses HO-1 growth. to induce melanin synthesis or terminal differentiation in HO-1 cells. The inhibitor of protein kinase C, H-7, only marginally alters 72 hr growth suppression induced by MEZ or the interferons, used alone or in combination. In several experiments, H-7 only partially and variably inhibited the enhanced expression of ICAM-1, HLA Class I antigens and HLA Class II antigens in HO-1 cells treated with MEZ. IFN-β or IFN-γ, used alone or in various combinations. This model system will be useful in defining the biochemical, genomic and antigenic changes associated with the chemical induction of terminal differentiation and the loss of proliferative capacity in human melanoma cells.     

In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-α and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-α showed strong PKC-α mRNA expression in cultured melanocytes, whereas PKC-α mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-α mRNA and approximately 6-fold more PKC-α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-α provides further evidence for cell type specificity in the balance of PKC-α expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.     

免疫毒素Luffin B-Ng76对人黑色素瘤细胞的体外抑制作用

用ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６Ｂ凝胶亲和层析法从丝瓜籽中分离纯化了单链致核糖体失活蛋白（ｒｉｂｏｓｏｍｅｉｎａｃ－ｔｉｖａｔｉｎｇｐｒｏｔｅｉｎ,ＲＩＰ）——ｌｕｆｉｎＢ。并将ｌｕｆｉｎＢ与抗人黑色素瘤细胞单抗Ｎｇ７６制成了免疫毒素,命名为ｌｕｆｉｎＢ－Ｎｇ７６,它对体外培养的黑色素瘤细胞Ｍ２１有很强的抑制作用,ＩＣ５０为２．５×１０－１１ｍｏｌ／Ｌ,毒性比游离的ｌｕｆｉｎＢ提高４０００倍,而它对非靶的ＨｅＬａ细胞的毒性较Ｍ２１细胞低１２００倍。结果提示ｌｕｆｉｎＢ用于制备免疫毒素具有良好的应用前景。     

Expression and In Vitro Regulation of Integrins by Normal Human Urothelial Cells

Integrins are thought to be essential adhesion receptors for the maintenance of tissue hisr tioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed α2β1 and α3β1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, α6β4 and occasionally αvβ4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca⁺⁺serum-free medium, the monolayer cultures expressed α2β1, α3β1 and α5β1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas α6β4 was distributed in a random pattern over the substratum. Increasing exogenous Ca⁺⁺concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, α6β4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of α6β4 in high Ca⁺⁺media, and also of α3 and α5, but not α2, subunits. These results suggest that α2β1 and α3β1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas α6β4 is the principal integrin involved in substratum adhesion.     

Onconase对B16黑色素瘤细胞的体内外生长抑制作用

Onconase(Onc)是一种从林蛙(Rana pipiens)卵细胞内提取的核酸酶,实验证实其在体外和体内对多种肿瘤细胞都具有显著的杀伤效果。在大肠杆菌中表达纯化的重组Onc和天然提取蛋白质具有相似的活性,通过测定该蛋白质对黑色素瘤B16细胞的IC50和建立荷瘤小鼠模型探讨了Onc体内外的抗肿瘤效果。实验结果表明:B16细胞在体外对Onc敏感性较K562细胞低,其IC50为6.37μmol/L;但体内每次每只小鼠给予5mg/kg Onc也可显著地抑制B16细胞的生长,延长小鼠的生存时间。实验提供了一种简化高效获得具有天然活性Onc的方法,同时通过Onc对低敏感性肿瘤黑色素瘤细胞的杀伤研究,丰富了对Onc抗肿瘤作用的认识,为治疗黑色素瘤提供了线索。     

Effects of Some Growth Factors and Cytokines on the Expression of the Repair Enzyme MGMT and Protein MARP in Human Cells In Vitro

The inducible repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) eliminates O⁶-methylguanine adducts in DNA and protects the cells from damaging effects of alkylating agents. We have found that anti-MGMT antibodies recognize both the MGMT protein with a mol. weight?~?24 kDa and a protein with a mol. weight?~?48 kDa, which was named MARP (anti-methyltransferase antibody recognizable protein). A number of growth factors and cytokines were shown to regulate the expression of MGMT and MARP proteins. The ranges of concentrations of several growth factors and cytokines that caused increasing or decreasing protein amounts in human cell cultures were determined. The results of special biological experiments have allowed us to assume a possible role of MARP in the repair of alkyl adducts in human cells.     

Effect of Pituitary and Ovarian Hormones on Human Melanocytes In Vitro

Normal human melanocytes in culture became enlarged and dendritic after a 2-day incubation with either the pituitary (β-MSH, a potent analog of α-MSH, ACTH, FSH and LH) or the ovarian (estradiol, estriol and progesterone) hormones. Under the same experimental conditions, pituitary hormones also increased both the tyrosinase activity and tyrosinase-related protein-1 (TRP-1) while ovarian hormones increased TRP-1 but not tyrosinase activity. The results suggest that pituitary and ovarian hormones possibly induce hyperpigmentation of the skin by stimulating the melanogenesis in epidermal melanocytes, and that estradiol and progesterone may be involved in the pathogenesis of melasma (chloasma) usually developing between early adulthood and menopause in which a high concentration of serum ovarian hormones was maintained.     

A Comparison Between Melanotic and Amelanotic Retinal Pigment Epithelial Cells In Vitro Concerning the Effects of L-Dopa and Oxygen on Cell Cycle

Melanin precursors and free radicals, cytotoxic substances, are produced during melanin synthesis by tyrosinase. We compared these cytotoxic effects of L-dopa and oxygen on the cell cycle of melanotic retinal pigment epithelial (RPE) cells with amelanotic RPE cells because of the differences of tyrosinase activities between melanotic and amelanotic RPE cells. Flow cytometric DNA analysis of RPE cells exposed to L-dopa (100 μM and 250 μM) were conducted at several oxygen concentrations (20%, 10%, and 5%). The dose-dependent effect of L-dopa to arrest the cell cycle (the S phase) was more pronounced in melanotic than in amelanotic RPE cells, and oxygen caused arrest in the G₁ phase.     

Evaluation of the Potential In Vitro Antiproliferative Effects of Millimeter Waves at Some Therapeutic Frequencies on RPMI 7932 Human Skin Malignant Melanoma Cells

The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm² were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were ≤0.3 mW/cm². Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.     

微管相关抗癌药物诱导胃癌细胞自噬性细胞死亡的研究

目的:探讨对微管相关抗癌药物诱导凋亡不敏感的胃癌细胞是否发生非凋亡形式的细胞死亡,并进一步明确自噬和自噬性细胞死亡的存在。方法:Annexin V／PI双染用流式细胞仪和MTT法分别检测紫杉醇、长春新碱诱导SGC-7901及BGC-823细胞的凋亡率和总死亡率,死细胞DAPI染色荧光显微镜检测非凋亡性细胞死亡,吖啶橙染色流式细胞仪和荧光显微镜分别定量、定性检测自噬和自噬性细胞死亡的存在。结果:紫杉醇和长春新碱可以诱导凋亡不敏感胃癌细胞BGC-823出现非凋亡性细胞死亡,处理BGC-823细胞早期(24h内)即可出现明显的细胞自噬性变化,紫杉醇诱导的自噬高峰期出现在药物作用3h-6h,长春新碱诱导的自噬高峰期出现在药物作用24h,自噬性细胞死亡存在并可能是药物诱导的非凋亡性细胞死亡的主要形式。结论:微管相关抗癌药物紫杉醇和长春新碱可以诱导凋亡不敏感胃癌细胞BGC-823自噬及自噬性细胞死亡,可能为提高胃癌的化疗敏感性提供新的思路。     

In Vitro Effects of Film-Forming Polymers on the Growth and Morphology of Pyrenophora Avenae and Pyricularia Oryzae

Ethokem (Midkem Agrochemicals, Northampton, UK) and Bond (Newman Agrochemicals, Cambridge, UK) completely inhibited linear growth of Pyricularia oryzae at 1 and 2% concentrations of the compounds and strongly inhibited growth of Pyrenophora avenae in vitro . Vapor Gard (Miller Chemical & Fertilizer Corporation, Hannover, PA, USA) was less effective with relatively little inhibition of P. oryzae , but was a more effective inhibitor of P. avenae . All three compounds decreased cell lengths of both pathogens and increased cell diameters of P. avenae . Vapor Gard and Ethokem did not significantly alter cell diameters of P. oryzae , but a highly significant increase was observed with Bond. Gross changes in hyphal morphology were observed including swollen shortened cells, granulation of the cytoplasm, increased branching and collapsed empty cells. Ethokem and Bond decreased growth rate and inhibited biomass production of P. avenae .     

siRNA沉默G6PD表达对人皮肤黑色素瘤细胞生长及凋亡的影响

研究表明,葡萄糖-6-磷酸脱氢酶(G6PD)与肿瘤的发生、发展、临床表型及治疗和预后有关.为阐明G6PD与肿瘤的关系及其机理,针对人G6PD基因设计3条siRNA和一条无关序列,再据每一序列,合成两条互补并含siRNA正反义链的DNA,退火后与含GFP的载体pRNAT-u6.2/Lenti连接,转染人皮肤黑色素瘤A375细胞,Real-time PCR筛选有效的一条siRNA,经病毒颗粒包装和病毒生产并感染A375细胞,G418筛选后,挑取单个阳性克隆放大培养,Western blotting检测siRNA干扰G6PD效率为88.83%,构建成G6PD缺陷型A375稳转细胞(A375-G6PDA).与野生型A375细胞(A375-WT)比较,A375-G6PD△的G6PD活性仅为21.53%,细胞倍增时间延长,增殖被抑制,克隆形成率降低25%(P0.05),提示,G6PD缺陷可能通过下调P53蛋白表达和上调Caspase.3的表达,抑制G2/M期向G0/G1期转换的进程,促进A375的凋亡,机理有待于进一步探讨.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.