Establishment in monolayer culture and characterization of four human melanoma cell lines

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (T_c) during exponential growth ranged from 15 to 21 h. The differences among the lines in T_c were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90–100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies

In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.Subject terms: Melanoma, Cell death     

The role of adoptively transferred CD8 T cells and host cells in the control of the growth of the EG7 thymoma: factors that determine the relative effectiveness and homing properties of Tc1 and Tc2 effectors

We had previously examined the factors that regulate the response of OVA-specific TCR-transgenic CD8 T cells to the B16 OVA melanoma, growing as lung metastases. We examine here whether the same parameters operate for EG7, growing intradermally. Tc1 or Tc2 CD8 effector cells from OT-1 mice were injected either mixed with the tumor or i.v. at day 0 or 7. Tc2 were one-fifth to one-tenth as effective as Tc1 when injected with the tumor, in controlling tumor growth, but were only 1/20 to 1/100 injected i.v. Tc1 injected i.v. entered the draining lymph nodes faster than Tc2 and caused a faster accumulation of host cells. Both caused an abrupt termination of host cell entry into lymph nodes and spleen after tumor elimination, but this occurred earlier for Tc1 than for Tc2. Host responses were ineffective in the absence of adoptive transfer but were essential after transfer. Perforin expression in the donor cells plays no role in adoptively transferred Tc1 or Tc2 control of the tumor, and neither IL-4 nor IL5 is needed for Tc1 or Tc2 function. Tc1 cells from mice lacking IFN-gamma, however, control tumor growth less well, whereas Tc2 effectors lacking IFN-gamma are unaffected. Tc1 from IFN-gamma-deficient mice attract fewer host cells to the draining lymph node, whereas Tc1 cells from perforin-deficient donors are unimpaired. We conclude that host cell recruitment is a crucial element in adoptive immunotherapy. The differences between the EG7 and the previous B16 melanoma model are discussed.     

Growth characteristics of human melanoma multicellular spheroids in liquid-overlay culture: comparisons with the parent tumour xenografts

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 X 10(7) microns 3 (a diameter of 270-340 microns) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 microns. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 +/- 0.2 to 2.7 +/- 0.4 days and the fraction of cells in S from 13 +/- 1 to 28 +/- 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G1. The spheroid volume-doubling times did not correlate with those of the parent xenografts (Td = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G1/G0, S and G2 + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts--i.e. 50-80% (G1/G0), 10-30% (S) and 10-20% (G2 + M).     

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014).In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.     

Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines

Background

Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag.

Results

Expression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G₁ fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential).

Conclusions

These data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G₁ phase fraction, were introduced to simplify measures of expression potential.

     

Heat response of human melanoma multicellular spheroids: comparisons with single cells and xenografted tumors

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.     

Growth Characteristics of Human Melanoma Multicellular Spheroids In Liquid-Overlay Culture: Comparisons With the Parent Tumour Xenografts

Abstract. The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 × lo7 pm3 (a diameter of 270-340 μ) before the growth rate tapered off. the morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200, μ. the histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. the sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. the volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 → 0.2 to 2.7 + 0.4 days and the fraction of cells in S from 13 → 1 to 28 → 2%. the volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G,. the spheroid volume-doubling times did not correlate with those of the parent xenografts (T_d= 4.2-22.5 days at V= 200 mm³), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. the fractions of cells in G₁/G₀, S and G₂+ M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts—i.e. 50-80% (G₁/G_o), 10-30% (S) and 10-20% (G₂+ M).     

Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence

Flow cytometry was used to measure intracellular pH (pHi) on an individual cell basis during exponential and plateau phases of growth. In all three cell lines examined a range of pHi values was associated with exponential growth. When cells from the extremes of the pHi distribution were sorted using a fluorescence-activated cell sorter and then restained for cellular DNA content, it was found that the higher pHi values were associated with enrichment of the S, G2, and M phases of the cell cycle, with a corresponding increase in the percentage of G1 cells at the lower pH1 range, suggesting cell-cycle dependence of pHi. It has been shown previously (I. W. Taylor and P. Hodson, 1984, J. Cell Physiol. 121, 517) that PMC-22 human melanoma cells are capable of entering a distinct pH-dependent quiescent state in response to the acidification of the growth medium which occurs naturally during growth to plateau phase. Simultaneous measurement of pHi and external pH showed that under these conditions pHi was maintained at control values down to an external pH of approximately 6.5, below which cytoplasmic acidification took place. This fall in pHi coincided with the onset of the transition to quiescence. Individual quiescent cells (defined by failure to incorporate bromodeoxyuridine during a 24-h exposure) could not be identified as such on the basis of a low pHi, suggesting that the probability of cell cycling is reduced by lowering pHi. Those cells which remained in cycle showed a markedly reduced rate of DNA synthesis, but a cell-cycle phase distribution similar to that in exponential growth, indicating that prolongation of all cell-cycle phases is an additional factor influencing overall population growth. The external pH at which both of these effects on cell proliferation kinetics took place in vitro is similar to that which occurs regionally within solid tumors, suggesting that pH effects could play a significant role in determining tumor cell growth in vivo.     

Effect of FMdC on the cell cycle of some leukemia cell lines.

BACKGROUND: (E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), an irreversible inhibitor of ribonucleotide reductase, displays a strong toxicity towards many cell lines derived from human solid tumors, while its activity on leukemia lines is less well-known. The aim of this study was to assess the effect of FMdC on the cell cycle and cell death of human leukemia lines HL-60 and MOLT-4, and murine leukemia L-1210 in vitro. It has been assumed that a prerequisite of FMdC cytotoxicity is intracellular phosphorylation by deoxycytidine kinase (dCK). METHODS:Cell cultures in the exponential phase of growth were exposed to different concentrations of FMdC (10 nM to 10 microM) for 6 and 24 hours. In a parallel set of experiments 1 mM deoxycytidine was added to prevent phosphorylation of the drug by dCK. The DNA and protein content in the cells, as well as Annexin V/PI binding were assessed by flow cytometry. The cell cycle was analyzed by the MacCycle software. RESULTS: The cytotoxic effects of FMdC, i.e., G(1)/S block and cell death were observed, associated with pronounced changes in the protein content. These effects were of variable intensity among the cell lines studied (HL-60 being the most susceptible), and in some cases, were not completely reversed by deoxycytidine excess. CONCLUSIONS: FMdC is a potent cytotoxic/cytostatic agent against human leukemia cell lines in vitro. It also changes the cellular protein content. Unphosphorylated FMdC may slightly influence the cell cycle of some leukemic lines.     

Specificity of growth inhibition of melanoma by 4-hydroxyanisole

An experimental study using human melanoma (NEL-MI), rat hepatoma (Fu5-5), and human kidney (293-31) cell lines was undertaken in order to evaluate the antitumor activity of 4-hydroxyanisole (4-OHA) in vitro. Prior reports have indicated highly specific antitumor activity of 4-OHA against melanoma cells in vitro. This specific antitumor activity has been proposed to be due to the oxidation of 4-OHA by tyrosinase to cytotoxic oxidation products. Dose-dependent cytotoxicity was observed when cells were cultured for 72 h in the presence of 4-OHA. At 100 microM, 4-OHA produced growth inhibition of 62%, 32%, and 55% in melanoma, hepatoma, and kidney cell lines, respectively. No effect was seen at 10 microM 4-OHA. 1,000 microM 4-OHA produced 100% kill. Tyrosinase activity was detected only in melanoma cells. The effect of 100 microM 4-OHA on the incorporation of 3H DNA precursors in melanoma, hepatoma, and kidney cells was also studied. Thymidine incorporation was inhibited in all three cell lines at the lowest cell density tested, with the greatest inhibition seen on melanoma cells. As cell density increased, the effect of 4-OHA on thymidine incorporation decreased. With respect to RNA synthesis, 4-OHA significantly reduced the incorporation of uridine in all three cell lines, with the greatest effect in melanoma cells. Cell density also affected the inhibition of uridine incorporation, but to a lesser extent than that observed on thymidine incorporation. The effect of 4-OHA on leucine incorporation was modest and uninfluenced by cell density. Thus, cytotoxicity of 4-OHA may involve two different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of novel melanoma cell lines

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.     

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.     

Regulation of Bacterial Cell Walls: Turnover of Cell Wall in Staphylococcus aureus

The cell wall of Staphylococcus aureus was shown to undergo turnover during exponential growth. The rate of turnover, about 15% per generation, was identical for both cell wall polymers, peptidoglycan and teichoic acid. Both the old and newly synthesized wall material appeared to undergo turnover at similar rates. The rate of turnover followed first-order kinetics until more than 90% of the original wall was lost. Cell wall turnover was completely blocked under conditions of unbalanced synthesis known to inhibit cellular autolysis, e.g., addition of chloramphenicol. Cell wall turnover was shown to occur in a number of different strains of S. aureus and appears to be widely distributed in this species.     

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.     

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.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

溶瘤腺病毒靶向杀灭葡萄膜黑色素瘤的实验研究

葡萄膜黑色素瘤是成人最严重的原发性恶性肿瘤之一.传统的治疗方法,包括手术、放射治疗和化学治疗效果都不是很理想.溶瘤腺病毒H101,能够特异性地在p53突变的肿瘤细胞中复制并杀伤肿瘤细胞,同时对正常细胞影响较少,且已由中国国家食品药品监督管理总局批准上市.为了研究H101对葡萄膜黑色素瘤的治疗效果,通过体外感染葡萄膜黑色素瘤细胞,发现H101能够显著抑制葡萄膜黑色素瘤细胞的增殖并促进细胞凋亡,抑制细胞周期,而对正常的ARPE-19细胞没有影响.在体内实验中,建立了SP6.5细胞的荷瘤小鼠模型,在H101治疗后抑制了肿瘤的生长,延长了动物寿命.上述结果表明,溶瘤腺病毒H101治疗葡萄膜黑色素瘤是一种可行的方法.     

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.