Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.     

Enhanced natural killer sensitivity with concomitant clonal selection for cells bearing homogeneously staining regions in the human melanoma cell line MeWo upon induction of differentiation with theophylline

Culture of the human melanoma cell line MeWo in the presence of 1 mM theophylline was associated with an increase in susceptibility to natural killer (NK)-mediated cytolysis. The phenomenon was detected as early as 72 hours after initiation of theophylline treatment, reaching maximum values at 3-4 weeks and remaining stable for longer than 3 months of testing, provided the cells were maintained in the presence of theophylline. The alteration in target sensitivity was selective for NK-mediated cytolysis, since other mechanisms of cell-mediated cytolysis, including antibody-dependent cell-mediated cytotoxicity and monocyte-mediated and lectin-induced cytolysis, were comparable between untreated and treated cells. The enhanced susceptibility of theophylline-treated cultures to NK lysis, as compared to NK lysis susceptibility of untreated MeWo cells, was not significantly changed by pretreatment of effector lymphocytes with interferon. Evidence for differentiation in theophylline-treated cultures was obtained. In addition, however, cytofluorometric and karyologic analysis revealed the existence of two subpopulations of differing ploidy in the MeWo line. The hypodiploid, NK-sensitive subpopulation, bearing homogeneously staining regions on two chromosomes, could be selected by growth in theophylline. Therefore, selection of subpopulations in heterogeneous tumor cell lines by chemical inducers suggests an alternative and novel mechanism for enhancement of NK sensitivity.     

A case of erythroleukaemia with homogeneously staining regions on chromosomes

A case of erythroleukaemia with homogeneously staining regions on chromosomes is described. The number of chromosomes in bone marrow cells varied from 41 to 46 with a mode of 43. A variety of structural and numerical chromosomal abnormalities was observed. Two marker chromosomes with homogeneously staining regions were found in all aneuploid cells examined. Intensive chemotherapy failed to induce a remission and the patient died 3.9 months after diagnosis. The relation of the homogeneously staining regions on chromosomes to the two courses of chemotherapy prior to cytogenetic study is discussed.     

Cell lines from human colon carcinoma with unusual cell products, double minutes, and homogeneously staining regions.

Two human colon carcinoma cell lines derived from the same tumor specimen were characterized. The cell lines, COLO 320 and COLO 321, have amine precursor uptake and decarboxylation cell properties, such as ectopic production of norepinephrine, epinephrine, serotonin, adrenocorticotropic hormone, and parathyroid hormone. The cells were morphologically different from most colon cell lines. Double minutes (DM) were initially present in nearly 100% of the metaphases. In a few subcultures of COLO 320, DM have persisted for 1.5 years. However, in COLO 321 and some subcultures of COLO 320, a loss of DM was observed and new marker chromosomes with homogeneously staining regions were observed. These unusual cell lines should be valuable for studies of apudomas of the colon and the cytogenetic phenomena of DM and homogeneously staining regions.     

Stimulation of melanogenesis in a human melanoma cell line by retinoids

Retinoic acid was found to be a potent stimulant of pigmentation in human Hs939 melanoma cells. Exposure to 1 microM retinoic acid for longer than four days caused both a decrease in the rate of cell proliferation and a concomitant increase in melanogenesis. These effects of retinoic acid progressed lin-early in a time-dependent and a dose-dependent fashion such that at the end of a seven-day treatment cell growth was inhibited by approximately 65%, and both melanin content and tyrosinase activity increased more than three-fold over the control. Interpolation of the dose-response curves indicated that 3 nM retinoic acid would cause half-maximal melanogenesis stimulation. No elevation in the level of cyclic adenosine 3':5'-monophosphate could be detected in the melanoma cells following various periods of exposure to retinoic acid, and the cells were unresponsive to alpha-melanocyte-stimulating hormone. In the presence of the tyrosinase inhibitor phenylthiocarbamate, retinoic acid was capable of inhibiting cell proliferation without enhancing melanin synthesis. The tumor promoter phorbol myristate acetate did not affect either the proliferation or the differentiation of the Hs939 melanoma cells. However, the enhancement of melanogenesis by 1 microM retinoic acid was inhibited by 66% in the presence of 0.1 microM phorbol myristate acetate. The tumor promoter did not reverse the growth-inhibitory effect of retinoic acid. Phorbol, a non-tumor promoter, was effective. Other retinoids, such as 13-cis-retinoic acid, retinyl acetate, nd the trimethylmethoxyphenyl analog of retinoic acid, also inhibited the proliferation and enhanced melanin production in the Hs939 cells. In contrast, retinyl palmitate, the phenyl analog of retinoic acid, and the pyridyl analog of retinoic acid were ineffective.     

Metastasis of a human melanoma cell line in the nude mouse

A human melanoma cell line has been established which when inoculated subcutaneously into nude mice, is consistently metastatic. In order to document blood-borne spread, it was necessary to excise the primary tumour so prolonging the life of the animal and allowing metastases to become apparent. Macroscopically detectable metastatic spread at autopsy was reliably indicated by weight loss of the animals. Metastases were widespread and involved the lungs, abdominal cavity and organs and the gonads. The size of the primary tumour at the time of its removal, and not the period of s.c. growth, determined the incidence of metastatic disease. Removal of tumours weighing less than 0.6 g prevented metastasis, whereas all of the animals showed widely disseminated disease if the tumour was allowed to attain a size of 1.6 g before excision.     

Characterization of a cathepsin-H-like enzyme from a human melanoma cell line

A cathepsin-H-like enzyme has been isolated from cultured human melanoma cells (G 361 cell line). The enzyme is similar to cathepsin H(s) of normal tissues in molecular weight, enzymatic characteristics (substrates, inhibitors, pH optima, Km values), and immunoreactivity. The inactive form of the enzyme with a molecular mass of 40 kDa has been found in the culture medium. The inactive enzyme is activated by acid pH, pepsin, and cathepsin-D-like enzyme treatments and converted into a form with a molecular mass of 28 kDa. The activated extracellular cathepsin-H-like enzyme and the active intracellular enzyme exhibit the same characteristics. The melanoma-derived cathepsin-H-like enzyme degrade fibrinogen and fibronectin, but not laminin or type-IV collagen. We conclude that the extracellular cathepsin-H-like enzyme may have important functions, together with other proteinases, in the destruction of extracellular matrix components, thus enabling proliferation, migration, and metastasis to occur.     

Effects of steroids on laminin-binding integrins in a human melanoma cell line

The MEL-85 human melanoma cell line was used to investigate the effects of both estradiol and dexamethasone on expression of laminin (LM) receptors and cell adhesion capacity. Immunoblotting of eluates from whole-cell extracts applied to LM Sepharose indicates the presence of an LM-binding protein of 116-130 kDa that reacted with an anti-beta 1 integrin antibody, suggesting that the putative LM receptor of MEL-85 cells is a member of the integrin family. Analysis of 125I-LM binding to whole cells indicates the existence of low-affinity components which display positive co-operativity. LM-fragment-8 competes for this binding to the same extent as unlabelled LM (75%), while fragment PI is inactive and fibronectin (FN) competes by about 30% only. Binding of labelled fragment-8 exhibits a pattern similar to that of intact LM. Cell adhesion to substrates coated with LM and LM fragments closely parallels binding to cells in suspension. MEL-85 cells were estradiol-receptor-negative. Estradiol treatment did not stimulate LM receptor levels or attachment to LM. Growth rate also remained unaltered. To characterize the glucocorticoid dependence of MEL-85 cells, we first established the presence of glucocorticoid receptors and an inhibitory effect on growth rate. Dexamethasone treatment resulted in marked enhancement of adhesion to LM, without altering LM receptor number or affinity. In addition, dexamethasone changed the morphology of MEL-85 cells in conjunction with higher LM expression as evaluated by immunofluorescence.     

Altered methionine metabolism in metastatic variants of a human melanoma cell line

In order to identify the biochemical defect(s) responsible for the reduced levels of DNA 5-methylcytosine (5-mCyt) found within highly metastatic (in athymic "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo, the ability of these cells to grow in culture medium devoid of exogenous methionine but containing either homocysteine (Hcy) or 5'-deoxy-5'-methylthioadenosine (MeSAdo) was determined. In contrast to the parental MeWo tumor line, many (but not all) of these malignant variants were completely unable to proliferate in methionine-free homocysteine-containing medium. Many of these malignant variants also exhibited a reduced ability to proliferate in methionine-free MeSAdo-containing medium. Cell lines established from "artificial" metastases of MeWo or its cloned sublines, exhibited no consistent reduction in their ability to grow in methionine-free medium containing either Hcy or MeSAdo. These observations suggest that alterations in S-adenosylmethionine(AdoMet)-dependent transmethylation reactions may contribute to "progression" of the MeWo tumor from a relatively benign to a highly autonomous and malignant state.     

DNA methylating capacity in metastatic variants of a human melanoma cell line

Certain highly metastatic (in athymic immunosuppressed "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo have been found to contain dramatically reduced levels of DNA 5-methylcytosine compared to the parental cell line. To identify the underlying biochemical defect which could be responsible for the reduced DNA methylation within these cells, the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine and level of extractable DNA-methyltransferase were examined. No significant difference in the ratio of S-adenosylmethionine/S-adenosylhomocysteine or extractable DNA-methyltransferase activity were found between the highly malignant variants and the parental cell line. Thus, stable alterations in these cellular parameters are not likely responsible for the reduction in DNA 5-methylcytosine content which appears to occur during "progression" of the MeWo tumor line from a relatively benign to highly malignant state.     

Chromatin bodies in multidrug resistant hybrid cells have centromeres and originate from homogeneously staining regions

Antikinetochore antibodies from patients with the CREST syndrome of scleroderma were used as probes to study homogeneously staining regions (HSRs) in multidrug resistant (MDR) mouse tumor cells, and chromatin bodies (CBs) in MDR mouse-hamster hybrid cells. In one mouse tumor line the C-band positive HSR showed antigenic properties and displayed many weekly fluorescent spots, i.e. it contained kinetochore proteins. The immunofluorescence pattern of the HSR could also be observed in interphase nuclei. The C-band positive CBs of the hybrid cells had active centromeres, as shown by double kinetochore spots. These results support our hypothesis that CBs originate from C-band positive HSRs.     

alpha-Melanocyte-stimulating hormone binding and biological activity in a human melanoma cell line

Synthetic alpha-melanocyte-stimulating hormone (alpha-MSH) was found to bind to the plasma membrane of the HM6A human melanoma cell line, using an immunocytochemical method. When treated with 10(-7) to 10(-9) M alpha-MSH, melanoma cells exhibited an increase of intracellular cyclic adenosine 3':5'-monophosphate, followed by stimulation of tyrosinase activity. Significant inhibition of DNA synthesis measured by [3H]thymidine uptake and inhibition of cell growth was found. A retrovirus expression was detected in the supernatant of HM6A cells as assayed by the KC cell syncytium-forming test. In he presence of 10(-7) M alpha-MSH, the number of syncytium-forming units was increased 15-fold.These results demonstrate that alpha-MSH modulates human melanoma differentiation and virus expression in vitro.     

Receptor-mediated cyotoxicity of a-MSH fragments containing melphalan in a human melanoma cell line

Four α-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep I and 2), the latter being the 4–10 sequence known to be the main α-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize α-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep I and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled a-MSH from its binding sites at concentrations similar to the 4–10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxic/ in terms of IC₅₀ values, Pep I being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep I and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an α-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep I to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with a-MSH central fragments in human melanoma cells due to the presence of α-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.     

Human interferons inhibit experimental metastases of a human melanoma cell line in nude mice

Therapy with human lymphoblastoid interferon HuIFN-alpha(N1), or recombinant human interferon gamma, rHuIFN-gamma, inhibited experimental pulmonary metastases of the human melanoma cell line, DX3-azac, in BALB/c nude mice and significantly prolonged survival. The human IFNs had no effect on nude mouse lung and spleen NK cell activity, lung macrophage activity, haemoglobin or white cell counts. HuIFN-alpha(N1) had no effect on the levels of the IFN induced enzyme 2-5A synthetase in nude mouse lungs although the rHuIFN-gamma caused some elevation. In addition, clearance of radiolabelled DX3-azac cells was identical in control or human IFN treated mice, and there was no histological evidence of an increase in immune effector cells associated with the metastatic lesions in treated mice. Human IFN therapy did not affect the state of differentiation of the melanoma cells in vivo as measured by melanin content, but both IFNs inhibited the development of colonies of DX3-azac cells in vitro. We conclude that in this model system IFNs have direct anti-proliferative effects on metastatic cells.     

Detection of tumor-associated antigen in human melanoma cell line supernatants.

Spent tissue culture medium (CDM-S) removed from a single cell line of human malignant melanoma grown in serum-free CDM, contained tumor-associated antigenic activity. Antibodies to CDM-S measured by complement fixation were detected in 44% (31/70) melanoma, 55% (15/27) sarcoma, 63% (24/38) carcinoma and 15% (11/72) normal sera. Delayed cutaneous hypersensitivity reactions (DCHR) were demonstrated in 4/5 melanoma patients at a 500 mug dose, 3/5 at a 100 mug dose and in 1/7 carcinoma patients at the 500 mug dose. One ml of CDM-S was shown to contain antigen equivalent to that obtained from the membranes of 2.9 X 10(7) tissue-cultured melanoma cells. After purification, 84% (16/19) sera from melanoma patients, 66% (12/18) from sarcoma and carcinoma patients and 8% (2/26) from normal controls were positive to the antigen by complement fixation.     

Stability of cytogenetic alterations in a human melanoma cell line and five clonal derivatives

A cytogenetic study was done on a human malignant melanoma cell line and its 5 clones. Chromosome banding analysis indicated the presence of 7 "shared" markers (M) and 9 unique markers (m) that were present only in the clones. Chromosomes 1, 5, 9, 12, 17 and 21 were involved in M-markers and chromosomes 1, 2, 4, 6, 8, 9, 11, 16, 17, 18 and 21 were involved in m-marker formation. Both parental and clonal lines had near-triploid chromosome numbers. A number of M-markers were isochromosomes of the short (p) and long (q) arms of chromosome 1. Our cytogenetic data indicate that the parental line contained subpopulations of cells that were in different stages of karyotypic evolution.     

Characterization of a human melanoma cell line with non-oestrogen receptor dependent tamoxifen resistance

While investigating the mechanism of synergistic cytotoxicity between tamoxifen (TAM) and cisplatin (DDP) we developed a TAM-resistant variant of the human melanoma cell line T-289. We sought to characterize this cell line with respect to the effect of TAM resistance on a variety of different intracellular cell cycle control and apoptotic pathways. A TAM-resistant variant cell line (289 TAMs) was developed and the technique of Western analysis was to determine the changes in protein expression that occurred as a result of the development of TAM resistance. In this model, TAM resistance resulted in an increase in the detectable basal levels of cyclin E, GADD 153, p16, BAX, Bcl-XL, and wild-type and mutant p53, an increase in TAM induction of p16, and a decrease in the detectable basal levels of cyclin D1, p21 and p27. There were no detectable changes in the levels of pRb. In the TAM-resistant variant, p21 levels were essentially undetectable, while p27 was present and maintained its response to TAM Induction, albeit at a much lower level. This investigation demonstrates that the development of TAM resistance is associated with a change in the detectable levels of a variety of cell cycle control and apoptosis-related proteins. While the exact role that each change plays in the development of tamoxifen resistance remains to be determined, these data suggest that the development of resistance to a particular agent results in changes in multiple, seemingly unrelated pathways. These data have implications for future studies in both the laboratory and the clinic.     

微小RNA-27 a对黑色素瘤WM239细胞增殖和凋亡的影响

目的：探讨微小RNA-27a（ miR-27a）模拟物和抑制物转染黑色素瘤WM239细胞后对细胞增殖和凋亡的影响。方法将miR-27a模拟物、抑制物及其阴性对照转染WM239细胞,荧光显微镜观察转染效率,实时荧光定量PCR检测相应的微小RNA,四甲基偶氮唑盐（ MTT）法检测细胞增殖,流式细胞仪检测细胞凋亡和细胞周期。结果细胞转染效率为80%~90%。转染miR-27a模拟物后,细胞内miR-27a表达量明显上升（2-△△CT值为26.98±0.01）,与正常对照组相比差异有统计学意义（ t＝－1123.67,P＝0.00）;转染miR-27a抑制物后,细胞中miR-27a的表达量下降（2-△△CT值为0.96±0.02）,与正常对照组相比差异无统计学意义（t＝4.04,P＝0.06）。转染miR-27a模拟物后,细胞增殖受到明显抑制,与正常对照组相比差异具有统计学意义[72 h吸光度（0.45±0.02）∶（0.72±0.01）,F＝129.56,P﹤0.05]。miR-27a模拟物组G0-G1期的细胞比例升高[（74.83±1.46）∶（63.73±1.25）,F＝30.33,P﹤0.05],S期和G2-M期细胞比例减少[（21.33±1.75）∶（27.50±1.25）,F＝14.98,P﹤0.05;（3.90±1.31）∶（8.80±2.10）,F＝3.66,P﹤0.05];模拟物组细胞凋亡率与正常对照组相比明显增加[（29.67±0.91）%∶（1.44±0.85）%, F＝530.90,P﹤0.01];而抑制物组对细胞周期和凋亡无明显作用。结论 miR-27a抑制黑色素瘤细胞增殖,具有抑瘤作用,这与其促进细胞凋亡,阻滞细胞周期于G0-G1期相关。     

Recombinant immune interferon down-regulates Ha-ras-1 proto-oncogene products in a human melanoma cell line

The antiproliferative and antineoplastic effects of the interferons may result, at least in part, from changes in the expression and quantity of specific oncogene products. To explore this hypothesis we have determined the effect of interferons, including recombinant leukocyte (IFN-alpha), fibroblast (IFN-beta) and immune (IFN-gamma), on expression of the Ha-ras proto-oncogene in the human melanoma cell line Colo 38. While concentrations of up to 1000 U/ml of either IFN-alpha or IFN-beta did not affect the total amounts of Ha-ras products, IFN-gamma at concentrations ranging from 20 to 200 U/ml caused a dose- and time-dependent (48-96 hr) reduction (approximately 40%) in the accumulation of Ha-ras-1 mRNA and in the synthesis of the specific protein products. Downregulation of this proto-oncogene occurs prior to the antiproliferative effects of IFN-gamma and parallels similar IFN-gamma mediated changes in the expression of certain melanoma associated antigens. The present findings indicate that this experimental model may prove valuable in determining whether a direct relationship exists between the antiproliferative activity of specific interferons and the downregulation of oncogene expression.