The killing effect of 4-S-cysteaminylphenol, a newly synthesised melanin precursor, on B16 melanoma cell lines

We have examined the killing effect of 4-S-cysteaminylphenol (4-S-CAP), a newly synthesised melanin precursor, on B16 melanoma cell lines possessing different melanin-producing activities and found it to be particularly effective in heavily melanised melanoma cells, but less so in moderately melanised melanoma cells, and having no effect on amelanotic melanoma cells and nonmelanoma cells. Thus, it was found that the killing effect of 4-S-CAP is highly dependent upon the synthesis of melanin and tyrosinase in melanoma cells, suggesting that 4-S-CAP may become toxic to melanoma cells only after oxidation by tyrosinase. The killing activity of 4-S-CAP also was found to be associated with a profound inhibition of the thymidine incorporation in pigmented melanoma cells, as compared to the uridine and leucine incorporation. Further, the inhibition of DNA synthesis was most pronounced in heavily melanised melanoma cells, less so in moderately melanised melanoma cells, and not seen in amelanotic melanoma cells. As a possible mechanism that might account for this action, it may be that 4-S-CAP is oxidised by tyrosinase to the o-quinone form via the catechol derivative and that some of the quinones then conjugate with sulfhydryl enzymes including DNA polymerase, thus exerting a killing activity for pigmented melanoma cells. Thus, 4-S-CAP appears to provide a new, effective cytotoxic agent for rational chemotherapy of malignant melanomas.     

稳定表达人MAGE-1基因小鼠黑色素瘤细胞系的建立

目的:构建真核表达载体,并在小鼠黑色素瘤B16细胞中稳定表达。方法:用PCR方法扩增质粒pUC19-MAGE-1中目的基因MAGE-1,连接到真核表达载体pCDNA3.1中,构建真核表达pCDNA3.1-MAGE-1,脂质体法转染小鼠黑色素瘤B16细胞。G418筛选阳性克隆(B16-MAGE-1),用RT-PCR和Western blot检测阳性克隆中mRNA和蛋白质的表达;分别以2×105个B16细胞和B16-MAGE-1接种于C57BL/6小鼠右侧背部皮下,观察B16-MAGE-1细胞的致瘤能力。结果:用PT-PCR与Western bolt分别在B16-MAGE-1细胞中检测到人MAGE-1基因的mNRA和蛋白质的表达,两组共20只小鼠均皮下成瘤,且肿瘤大小没有明显差异。结论:成功构建了真核表达载体pCDNA3.1-MAGE-1,获得了稳定表达人MAGE-1基因的小鼠黑色素瘤B16细胞系,并保持很好的致瘤能力,为研究MAGE家族在肿瘤免疫治疗中的应用奠定了基础。     

Mutation and expression of the p53 gene in malignant melanoma cell lines

Three monoclonal antibodies (MAbs) against p53 protein (PAb 24o, DO-I and Pab1801) were used to define the immunophenotype of 13 melanoma cell lines. Immunoreactions could be detected in 12 out of 13 cell lines by using the indirect immunofluorescence technique. In 7 of these the majority of cells displayed cytoplasmic staining whereas positive nuclei were detected in only a few cells. Two cell lines had predominantly nuclear reactivity, while the remaining 3 cell lines showed signals in both locations. Despite identical nuclear staining patterns, the 3 MAbs produced qualitatively distinct cytoplasmic immunoreactions. PAb240 and DO- I, which showed similar staining frequencies, appeared more sensitive in the detection of p53 protein than did PAb 1801. Immunoprecipita-tions of lysates from each of the cell lines with MAbs DO-I and 1801 (which bind to both wild-type and mutant p53 species) detected 53–kDa proteins, whereas PAb240 (which recognizes the mutant conformation of the protein in this type of assay) detected 53–kDa proteins in only 4 cell lines. Nucleotide sequencing of exons 5 to 9 of TP53 in these latter cell lines showed that each has homozygous point mutations in the locus, whereas in the others no TP53 alterations were found. Three of the 4 mutations were C-to-T transversions, alterations possibly caused by damage from UV-light. Our findings indicate that immunostaining with p53 antibodies, although common in malignant melanoma, results from the presence of mutant p53 protein in about 30% of the cases tested. Neither immunostaining with PAb240 nor the patterns of intracellular distributions of the signals are sufficient to detect TP53 mutations.     

Radiation-induced cell cycle delays and p53 status of early passage melanoma cell lines

Cell cultures exposed to DNA-damaging agents such as gamma radiation respond by arresting at cell cycle checkpoints, and the p53 tumor suppressor protein is strongly implicated in this behavior. We have investigated the TP53 status and cell cycle response to ionizing radiation of a series of early passage cell lines (designated NZM1 to NZM15) previously developed from patients with metastatic melanoma. The TP53 status of each of the cell lines was determined by single-strand conformation polymorphism and DNA sequence analysis. The majority of the lines appeared to have a wild-type TP53 gene sequence, consistent with published studies. Two lines (NZM4 and NZM7.2) were found to have an identical T-->C transition mutation in nucleotide 721 (exon 7) of the coding region. NZM7.2 (mutant) and NZM7.4 (wild-type) were clonally derived from the same line (NZM7). The existence of radiation-induced cell cycle arrest in G and/or G2M phase was determined 16 h after irradiation (6.3 Gy) by DNA staining and flow cytometric analysis. The mitotic inhibitor paclitaxel was used as a reference compound, with or without irradiation, to assess the efficiency of radiation-induced cell cycle arrest. G1 phase arrest was associated only with the presence of the wild-type TP53 gene, but the efficiency of induced arrest varied among the cell lines and the period of G phase arrest appeared to be short. A significant difference (P < 0.002) was also found between the efficiency of induction of G2 phase arrest and the presence of wild-type TP53 gene. The results provide evidence that although the melanoma cell lines generally had an intact TP53 gene, the efficiency of p53-mediated cycle arrest might be deficient and contribute to the resistance of this tumor to treatment.     

纤维连接蛋白双结构域重组多肽对B16黑色素瘤生长的抑制作用

目的 研究纤维连接蛋白双结构域重组多肽（ＣＨ５０）抑制体内肿瘤细胞形成转移灶的作用。方法 采用皮下和腹腔接种黑色素瘤Ｂ１６／Ｆ１细胞,在治疗后测定肿瘤大小及转移灶数量。结果 对于皮下接种肿瘤,局部注射较远距离注射更为有效,对肿瘤生长抑制可达５０％：对腹腔仙形成的微小肿瘤转移灶的抑制达８０％;     

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

Effects of several flavonoids on the growth of B16F10 and SK-MEL-1 melanoma cell lines: relationship between structure and activity

Although flavonoids seem to be capable of acting at all stages of the carcinogenic process, little information is available on their action in melanoma cell lines. The aim of this study was to assess the response of B16F10 and SK-MEL-1 melanoma cell lines to treatment with six different flavonoids after 24 and 72 h of exposure and to relate the response to their structure. We then compared the findings with those for melphalan treatment. When cultures were treated for 24 h, only slight inhibition at the highest concentrations (25 and 50 microM) of tangeretin and luteolin were observed, whereas melphalan caused a dose-related inhibition of growth at all concentrations. Quercetin, hesperetin, 7,3'-dimethylhesperetin and eriodictyol did not produce any effect at 24 h on B16F10 or SK-MEL-1 cells, results which point to the low toxicity of flavonoids. After 72 h of exposure culture growth was inhibited by 7,3'-dimethylhesperetin at 50 microM, but lower concentrations had no effect. Tangeretin was the most effective of the flavonoids in inhibiting B16F10 and SK-MEL-1 cell growth, showing a clear dose-response curve after 72 h. These results suggest that the absence of the C2-C3 double bond on hydroxylated flavonoids results in a loss of effect on both the cell lines, while the higher activity of tangeretin compared with 7,3'-dimethylhesperetin suggests that the presence of at least three adjacent methoxyl groups confers a more potent antiproliferative effect.     

The Inhibitory Effect of Oridonin on the Growth of Fifteen Human Cancer Cell Lines

OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate of cell growth was measured by the MTT assay, and compared with a negative control and 5-Fu-positive control. RESULTS The 50% inhibiting concentration （IC50） and maximal inhibition （Imax） of oridonin shown by studying the growth of the cancer cell lines were as follows： leukemias （HL60 cells： 3.9 μg/ml and 73.8%, K562 cells： 4.3 μg/ml and 76.2%）; esophageal cancers（SHEEC cells： 15.4 μg/ml and 99.2%, Eca109 cells： 15.1 μg/ml and 84.6%, TEl cells： 4.0 μg/ml and 70.2%）; gastric cancers （BGC823 cells： 7.6 μg/ml and 98.7%, SGC7901 cells： 12.3 μg/ml and 85.7%）; colon cancers （HT29 cells： 13.6 μg/ml and 97.2%, HCT cells： 14.5 μg/ml and 96.5%）; liver cancers （Bel7402 cells： 15.2 μg/ml and 89.2%, HepG2 cells： 7.1 μg/ml and 88.3%）; pancreatic cancer （PC3 cells： 11.3 μg/ml and 68.4%）; lung cancer （A549 cells： 18.6 μg/ml and 98.0% ）; breast cancer （MCF7 cells： 18.4 μg/ml and 84.7%）; uterine cervix cancer （Hela cells： 13.7 μg/ml and 98.5%）. CONCLUSION Oridonin had a relatively wide anti-tumor spectrum, and a relatively strong inhibitory effect on the growth of the 15 human cancer cells. Inhibitory effects were concentration dependent.     

人黑色素瘤细胞系基因突变的研究

目的　揭示金属蛋白酶类 (MMPs) ,层粘连蛋白受体 (LN R)与人黑色素瘤细胞侵袭转移的关系 ,并探讨MMPs ,LN R用以判断肿瘤细胞侵袭转移的可能性。方法　通过流式细胞术 (FCM )定量研究和蛋白酶活性分析 (zy mography) ,对具有不同潜在转移能力的人黑色素瘤细胞进行研究。 结果　早期WM 3 5不产生MMPs ;WM 13 4 1B仅产生MMP 2不产生MMP 9;进展期WM 983A和远处转移瘤株WM 45 1既产生MMP 2又产生MMP 9。瘤细胞表面67KDLN R的荧光阳性率和全部细胞的平均荧光强度大小顺序为WM 45 1>WM 983A >WM 13 4 1B >WM 3 5。结论　MMPs和LN R与人黑色素瘤细胞系的侵袭转移能力获得之间存在着密切关系 ,并可作为一种较特异的肿瘤侵袭转移标记物被应用于肿瘤研究与治疗中。     

B16-F10-luc-G5黑色素瘤细胞生物学特性的研究

目的：探讨B16-F10-luc-G5黑色素瘤细胞的生物学特性.方法：倒置荧光显微镜下连续观察B16-F10-luc-G5细胞生长动态.流式细胞术检测冻存复苏后、培养基漂浮细胞、胰酶消化损伤后的细胞死亡率以评价其对实验损伤的耐受性.1×104/孔B16-F10-luc-G5细胞按1∶2梯度稀释至0.78×102/孔,加入底物荧光素后检测其生物发光特性.Babl/C和C57 bl/6雄性小鼠各10只接种该细胞,目测肿瘤生长速度和活体成像监测Babl/C小鼠移植瘤生长动态以评价其成瘤特性.结果：B16-Fl0-luc-G5细胞生长动态符合典型B16-F10细胞特性,无自发及激发荧光.B16-F10-luc-G5细胞冻存、漂浮细胞及对数生长期细胞消化后死亡率分别为23.8％、35.8％和4.8％.B16-F10-luc-G5细胞数与实测平均光子数之间存在线性回归关系,检测光子数可反映细胞数目.两组小鼠移植成瘤率均为100％,平均肿瘤出现时间差异显著（t =9.05,P＜0.05）,移植瘤病理符合B16黑色素瘤典型生长特点;活体成像监测可灵敏监测移植瘤生长动态.结论：B16-F10-luc-G5细胞具有生长快、对各种实验操作耐受性好、标记生物发光基因易追踪等优点,且其一般特性与B16-F10细胞基本相同,是肿瘤学研究良好的实验材料.     

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.     

The effect of blue light exposure and use of intraocular lenses on human uveal melanoma cell lines

Little is known about the effect of blue light on inducing melanocytic malignant transformation. We chose to investigate the effect of blue light (475 nm wavelength) on the proliferation rates of uveal melanoma cells. In addition, we tested two different intraocular lenses to determine the possible effects of ultraviolet absorbing and blue light filtering intraocular lenses on the changes in proliferation. Four human uveal melanoma cell lines (92.1, MKT-BR, OCM-1, SP6.5) were exposed to blue light with and without the presence of ultraviolet absorbing and blue light filtering intraocular lenses. Cells covered by aluminum foil were used as a control. The proliferation rate of the cells compared with the control was then assessed using the Sulforhodamine-B proliferation assay. Cells exposed to blue light showed a statistically significant (P<0.05) increase in proliferation. Those exposed to blue light through a standard ultraviolet absorbing intraocular lens showed a smaller increase in proliferation, whereas those exposed with a blue light filtering intraocular lens showed no increase in proliferation than the control in all four cell lines. The exposure of cells to blue light led to an increase in proliferation in all cell lines compared with the control. The use of blue light filtering intraocular lenses abolished these increases in proliferation in the four cell lines. These results indicate that blue light filtering intraocular lenses may have a protective effect on the proliferation rates of uveal melanoma cells exposed to blue light.     

Anti-proliferative activity of haloperidol in B16 mouse and human SK-MEL-28 melanoma cell lines

Sigma receptors are present in cancer cell lines. The aim of the present study is to evaluate the anti-tumor activity of a series of sigma 1, sigma 2 and sigma 1/2 ligands in B16 melanoma cell lines. Proliferation, apoptosis, intracellular ATP content, cell cycle and molecular regulators were analyzed. Cell growth was determined using the sulforhodamine B (SRB) colorimetric cytotoxicity assay. Apoptosis was assessed by flow cytometry and DNA fragmentation, using ELISA cell death assay. ATP content was measured spectrofluorometrically and cell cycle analysis was performed by flow cytometry. The cytoplasmic and nuclear expression of cell cycle regulatory molecules, cyclin D and CDK2 (cyclin dependent kinase 2) were determined by Western blot analysis and quantified by densitometry. The sigma ligands in single digit micromolar concentrations inhibited B16 and multidrug-resistant B16 COL/R cell growth, leading to cell death at higher concentrations. The potency order was: haloperidol, reduced-haloperidol, ifenprodil tartrate, opipramol and carbetapentane citrate. B16 COL/R cells were to some extent, less sensitive to sigma ligands. Further studies have shown that the growth inhibitory effect of sigma ligands could be attributed to G1 arrest of the cell cycle, mediated by a marked decrease in cytoplasmic and nuclear cyclin D and CDK2 protein expression, though haloperidol induced loss of cell viability due to apoptosis. Sigma ligands induced an early decrease in ATP content. These data stimulated us to examine the combined anti-proliferative activity of haloperidol and the tyrosine kinase inhibitor imatinib mesylate (STI 571), on SK-MEL-28 human melanoma cells. Preliminary experiments demonstrated a marked synergistic interaction between the two agents.     

胃癌细胞周期不同时相MTS1/p16、p21、p53的表达

进一步明明确不同周期时相肿瘤细胞Ｐ１６基因的表达水平及在细胞周期调控中的作用。方法改进并建立了一个高同步率,高收获率的细胞周期同步化的方法,通过此方法把细胞同步在不同的时期,对ＲＮＡ及蛋白的表达进行研究,确定地不同同量丰中Ｐ１６的表达有何变化,同时分析其它几个重要的抑癌基因如Ｐ５３、Ｐ２１,研究它们在细胞周期中的表达以及与Ｐ１６基因相经关系。结果确定在不同周期时相的胃癌细胞系ＭＧｃ８０－３Ｇ     

Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4

The function of p16^INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16^INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16^INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16^INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16^INK4 independent of Rb. The transfected p16^INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1), p16^INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16^INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16^INK4 or expressed reduced amounts of p16^INK4 protein. Therefore, p16^INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16^INK4 is a tumor suppressor gene. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

The growth promoting effect of transforming growth factor-alpha in human breast cancer cell lines

Two human breast cancer cell lines, BT-20 and ZR-75-1, were examined with the aim of the elucidating the pathological roles of human transforming growth factor (TGF)-alpha in breast cancers. The TGF-alpha receptor was found to be present in both cell lines. A clonogenic assay revealed that concentrations of TGF-alpha greater than 10(10) M induced a significant increase in colony formation, indicating TGF-alpha to be a breast cancer cell growth factor. Northern blot analysis revealed, moreover, that both cell lines expressed TGF-alpha mRNA. Taking these observations together, it is reasonably possible to assume that TGF-alpha is an autocrine growth factor for breast cancer cells. Although it has been proposed that TGF-alpha could be an epidermal growth factor (EGF) superagonist with regard to its colony formation stimulating activity, the present study demonstrated the colony formation stimulating activities of TGF-alpha and EGF not to be all that much different in the two breast cancer cell lines.