Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.     

Divergen properties of two human lymphocytic cell lines isolated from a single specimen of peripheral blood

Summary Two lymphocytic cell lines were obtained from a single specimen of peripheral blood buffy coat from a patient with acute lymphoblastic leukemia who concurrently developed classical infectious mononucleosis. These two cell lines exhibit distinguishing differences in culture in parallel with reviously described differences in the cells when implanted into eonatal Syrian hamsters. These observations suggest that the cell lines were derived from two pre-existing different classes or “stelines” of cells—one of which resembles other cell lines derived from patients with acute lymphoblastic leukemia, whereas the other resembles cell lines derived from other, patients with infecttious mononucleosis. These studies were suported in part by research Grants C-6516 from the National Cancer Institute and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health.     

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.     

Flow karyotyping of human melanoma cell lines

Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

Studies on isolated cloned populations from irradiated human embryonic cell cultures.

The recovery of substrains with stable chromosome aberrations from irradiated fibroblast culture are reported. Four human fetal cell strains were exposed to 600 rad of gamma rays at 200 rad/min. The efficiency of recovering viable cloned subpopulations was approximately 87%, and the frequency of clones with abnormal chromosomes was 40/100 colonies. G-band chromosome analyses for 34 abnormal substrains are described. Karyotypes of some of the clones with complex rearrangements are also presented. Analyses of a total of 47 aberrant events in the 34 abnormal substrains revealed at 7:1 and a 9:1 translocation-inversion and translocation-deletion ratios, respectively. Five of the abnormal substrains were continuously cultured; all except one showed signs of sensecence toward the end of 44 +/- 10 doublings. Unusual prolonged proliferation capacity was observed in substrain FFS-1-9. The significance of this finding is discussed.     

Cancer-testis gene expression profile in human melanoma cell lines

A growing number of evidence indicates that cancer-testis antigens (CTA) can be used as specific targets for immune therapy of malignant melanoma. The aim of this study was to provide a basis for selecting the most suitable CTA by analyzing the mRNA expression profile of genes encoding CTA in melanoma cell lines. We used a real-time quantitative PCR to measure the expression level for the following genes: GAGE1, NY-ESO-1, MAGEA1, PASD, SCP1, SEMG1, SPANXA, SSX1, and PRAME. The objects of study were cell lines mel P, mel Si, mel Mtp, mel Il, mel Hn, mel Ibr, and mel Kor obtained from patients diagnosed with disseminated melanoma. We established that the highest frequency of occurrence and the highest expression level had the following genes: GAGE1, NY-ESO-1, MAGEA1, SCP1, SPANXA, SSX1, and PRAME. Their mRNA translation products can be promising candidates for immunotherapy.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

Calmodulin content and distribution in six human melanoma cell lines

Calmodulin content and distribution between soluble and particulate fractions were determined by radioimmunoassay in six human melanoma cell lines exhibiting differences in tumor origin (primary or metastatic), degree of tumorigenicity and of pigmentation (amelanotic or melanotic). The results indicate that a) total, soluble and particulate calmodulin levels expressed as ng/10(6) cells or ng/micrograms of proteins remained constant for five out of six cell lines when cells grew from subconfluency to confluency. For IGR 37 line, derived from metastatic melanoma, the calmodulin content decreases from 2.39 to 1.27 ng/micrograms protein for total calmodulin, from 2.17 to 1.52 ng/micrograms protein for soluble calmodulin and from 2.61 to 1.02 ng/micrograms protein for particulate calmodulin, b) total, soluble and particulate calmodulin levels expressed as ng/microgram proteins were twofold (at confluency) to fourfold (at subconfluency) higher in the two cell lines from metastatic origin, IGR 37 and IPC 167. As for example, for total calmodulin, values in IGR 37 and IPC 167 cell lines, were, respectively at subconfluency, 2.39 and 2.31 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.96 ng/micrograms protein and at confluency: 1.27 and 1.98 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.90 ng/micrograms protein, c) ratio of calmodulin between soluble and particulate fractions was about 1 for the two autologous cell lines IGR 37 and IGR 39 and varies from 2 to 3 for the four other cell lines.     

Radiosensitivity of the cells of an established human melanoma cell line and the parent melanoma xenograft

Survival curves of cells from a human melanoma xenograft (E.F.) and a cell line (FME) established from this xenograft were determined. The cells of the established line were harvested from exponentially growing cultures, plateau phase cultures or solid tumours in athymic mice (FME-X) before irradiation. During irradiation the cells were kept suspended in culture medium. The colony forming ability of the cells was assayed in soft agar. The Do-value was significantly higher for the parent xenograft than for the established line, whether grown in vitro or in vivo (p less than 0.0001). In addition, the Dq-value was significantly lower for the xenograft than for exponentially growing cultures of the established line (p less than 0.05). Thus the radiation response of the cells of the established line was not representative for that of the cells from the parent xenograft. It is concluded that survival curves for established cell lines should be used with great caution in attempts to predict the radiocurability of human tumours of corresponding histological type.     

Double immunoglobulin production in cloned somatic cell hybrids between two human lymphoid cell lines.

Several clones of independently established somatic cell hybrids between two human lymphoid cell lines, Raji and Namalwa, were examined for surface immunoglobulin expression. Double-antibody radioimmunoassays were established for kappa and lambda light chains. Immunoglobulins were detergent-extrated by Triton X-100 and quantified by radioimmunoassay. The Raji parent expressed small amounts of kappa chains on its surface, and the Namalwa parent a 10 fold greater amount of lambda chains. We show that the majority of the hybrid clones co-express both parental phenotypes.     

Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma

Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumours are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high-grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracerebral cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI -0.41, 0.46). Survival analysis of enriched gene sets (P < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis- and glutamate-mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from 'The Cancer Genome Atlas' project (r = 0.62, 95% bootstrap CI: -0.42, 0.43). We then used these data to select and inhibit a novel target (glutamate receptor) and showed that LY341595, a glutamate receptor specific antagonist, could prolong survival in intracerebral tumour-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies.