Characterization and comparison of the properties of sarcoma cell lines in vitro and in vivo

To expand the available tools for investigating human sarcomas, we characterized the primary properties of 22 common, uncommon, and newly characterized sarcoma cell lines representing eight different histological subtypes. Throughout the characterization process we noticed that in vitro markers and assays are poor indicators of tumorigenicity and that generated xenografts often bear little resemblance to the original histopathology. In vitro properties examined included morphology, proliferation rate, cell cycle characteristics, invasiveness, and immunohistochemical expression of p53 and phospho-AKT. In vivo properties examined included days to tumor formation in NOD/SCID mice, xenograft morphology in several locations and immunohistochemical expression of Ki67, p53 and phospho-AKT. We believe that such an in depth comparison of a large cohort of sarcoma cell lines will be useful in both designing and interpreting experiments aimed at elucidating both the molecular biology and efficacy of therapeutic agents in sarcomas. However, that data generated also suggests a small set of sarcoma cell lines may be inappropriate for generalizations regarding biological behavior of specific sarcoma subtypes. Integration of functional genomics or other more sophisticated assays of cell lines may help bridge the differences in vitro and in vivo .     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Summary Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the nμ/nμ mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologicaly transformed cells both contained the feline concornativirus-associated cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline rerovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated. This work was supported in part by NIH-NCI RO1-259007, NO1-CP-3571 and CPV08 103563, and Air Force F49620-77-C-110.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Influence of decreased intracellular glutathione level on the mutagenicity of patulin in cultured mouse lymphoma cells

The mutagenicity of the mycotoxin patulin was assessed by the thymidine kinase mutation assay, which is based on point mutations and deletions. Patulin was mutagenic in cultured mouse lymphoma cells and the mutagenicity was significantly increased in cells pretreated with buthionine sulfoximine, which reduces intracellular glutathione levels. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004 Financial support Deutsche Forschungsgemeinschaft (Grant Me 574/14-2)     

Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK^+/+). TK-deficient (TK^?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK^+/? revertants arising in TK^?/? cultures should possess TK enzyme identical with one of those present in the original TK^+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK^+/? lines, each derived in turn from separate TK^?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) K_m′s; and (4) ATP-Mg²⁺ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.     

Spindle cells and their role in Kaposi's sarcoma

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.     

Arsenic trioxide concentration determines the fate of Ewing's sarcoma family tumors and neuroblastoma cells in vitro

Arsenic trioxide (As(2)O(3)) induces both the differentiation and apoptosis of acute promyelocytic leukemia cells in a concentration dependent manner. We assessed the effects of As(2)O(3) in CADO-ES Ewing's sarcoma (ES), JK-GMS peripheral primitive neuroectodermal tumor (PNET), and SH-SY5Y neuroblastoma cells, as they share common histogenetic backgrounds. As(2)O(3) at low concentrations (0.1-1 microM) induced SH-SY5Y differentiation, and whereas PNET cells acquired a slightly differentiated phenotype, change was minimal in ES cells. Extracellular signal-regulated kinase 2 (ERK2) was activated at low As(2)O(3) concentrations, and PD98059, an inhibitor of MEK-1, blocked SH-SY5Y cell differentiation by As(2)O(3). High concentrations (2-10 microM) of As(2)O(3) induced the apoptosis in all three cell lines, and this was accompanied by the activation of c-jun N-terminal kinase. The generation of H(2)O(2) and activation of caspase 3 were identified as critical components of As(2)O(3)-induced apoptosis in all of the above cell lines. Fibroblast growth factor 2 enhanced As(2)O(3)-induced apoptosis in JK-GMS cells. The overall effects of As(2)O(3) strongly suggest that it has therapeutic potential for the treatment of ES/PNET.     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.     

Hiv infection of human gastrointestinal submucosal cells: An in vitro model that mimics kaposi’s sarcoma

Summary Infection of human gastrointestinal submucosal mesenchymal cells with HIV-1 led to cell populations with abnormal growth properties, increased synthesis of endothelial cell and angioblast markers, and release of angiogenic factors. This system may be the first in vitro model for HIV-induced Kaposi’s sarcoma. Research partially supported by NIH grant DK40625.     

Expression profiles of mouse dendritic cell sarcoma are similar to those of hematopoietic stem cells or progenitors by clustering and principal component analyses

We isolated and screened two tumor cell clones DD1 and DG6 with different capacity of metastasis from the same parent cell line, a mouse dendritic cell (DC) sarcoma, using limited dilution method. The genome-wide expressions of DD1 and DG6 cells were detected by Affymetrix's MOE-430A microarray. The expression profiles related with mouse DC development were downloaded from GEO at NCBI and ArrayExpress at EBI database. In order to compare the expression of DC sarcoma and DC developmental arrays which was performed by MG-U74av2, we had screened the best matched probesets between MOE-430A and MG-U74av2 according to the probe identities from Affymetrix technical annotation. After the normalization of 11 housekeeping genes across the 34 arrays (2 DC sarcoma and 32 DC developmental arrays), all these expression profiles were analyzed by the methods of hierarchical clustering, principal component analysis, nearest-neighborhood, and self-organizing maps. The results indicate that expression profiles of DC sarcoma are closer to those of the DC progenitors and hematopoietic stem cells from bone marrow compared with the sorted DCs from spleen. The results support the hypothesis that cancers (tumors or sarcomas) arise from stem cells. It is suggested that the DC sarcomas are more similar to the DC progenitors and hematopoietic stem cells than the relative mature DCs in gene expressions on the large-scale.     

Transfer of copper from metallothionein to nonmetallothionein proteins in cultured cells

Copper uptake and distribution with time among cytoplasmic proteins were followed in cultured cells under several conditions: (1) CHO cells, which cannot synthesize metallothioneins, were labeled with⁶⁷Cu in the presence of 100 μM ZnCl₂; (2) Cd^r30F9 cells, which contain some constitutive metallothionein (MT), were labeled in the absence of additional ZnCl₂ and; (3) Cd^r30F9 cells were labeled in the presence of ZnCl₂, under which conditions they synthesized additional metallothioneins. The exogenous⁶⁷Cu and ZnCl₂, where present, were then removed, and the distributions of⁶⁷Cu among size fractions of the cellular proteins were observed at intervals for 16 h. In addition, a culture identical to condition (3) above was also treated with 100 μM ZnCl₂ during the redistribution period. The⁶⁷Cu was initially resolved into three peaks by Sephadex G-75 chromatography: high molecular weight, intermediate molecular weight, and MT. The⁶⁷Cu in the MT fraction decreased with at _1/2 of 10–12 h. In contrast to this, generally, in cells with a higher initial⁶⁷Cu bound to metallothionein, there was a progressive increase in the amount of⁶⁷Cu eluting with the high- and intermediate-molecular-weight fractions. Since no other source of⁶⁷Cu was available, these experiments suggest that copper stored in MT can be transferred to other proteins in these cells.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

A dileucine motif targets MCAM-l cell adhesion molecule to the basolateral membrane in MDCK cells

Melanoma cell adhesion molecule (MCAM), an adhesion molecule belonging to the Ig superfamily, is an endothelial marker and is expressed in different epithelia. MCAM is expressed as two isoforms differing by their cytoplasmic domain: MCAM-l and MCAM-s (long and short). In order to identify the respective role of each MCAM isoform, we analyzed MCAM isoform targeting in polarized epithelial Madin-Darby canine kidney (MDCK) cells using MCAM-GFP chimeras. Confocal microscopy revealed that MCAM-s and MCAM-l were addressed to the apical and basolateral membranes, respectively. Transfection of MCAM-l mutants established that a single dileucine motif (41-42) of the cytoplasmic domain was required for MCAM-l basolateral targeting in MDCK cells. Although double labelling experiments showed that MCAM-l is not a component of adherens junctions and focal adhesions, its expression on basolateral membranes suggests that MCAM-l is involved in epithelium insuring.     

Evidence for Ca-dependent protein phosphorylation in vitro in guard cells from Vicia faba L.

Protein phosphorylation in vitro was investigated in guard cells from Vicia faba. A number of proteins with apparent molecular masses of 72, 67, 57, 52, 49, 44, 37, and 26 kDa were phosphorylated when guard-cell extract was incubated with [γ-³²P]ATP under Ca²⁺-free conditions. In the presence of Ca²⁺ at 1 μM, several proteins with apparent molecular masses of 125, 83, 41, 31, and 25 kDa were newly phosphorylated. These Ca²⁺-dependent protein phosphorylations were suppressed by (8R^*,9S^*,11S^*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252a), a wide-range inhibitor of protein kinases, suggesting that the protein phosphorylations were mediated by protein kinases. Several proteins were phosphorylated in vitro in mesophyll extract from Vicia. In contrast to guard cells, there was no detectable Ca²⁺-dependent protein phosphorylation in mesophyll cells. 1-(5-Indonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), an inhibitor of myosin light chain kinase (MLCK), and an antagonist of calmodulin (CaM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins in guard cells. Fractionation experiments revealed that the Ca²⁺-dependent phosphorylated proteins with molecular masses of 41 and 25 kDa were present in the mitochondria, and the 125- and 31-kDa proteins in the cytosol. These results suggest that Ca²⁺-dependent protein phosphorylation occurs markedly in guard cells, and that Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins may be catalyzed by MLCK or MLCK-like protein kinase in guard cells.     

Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells

Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.     

Addition of both platelets and thrombin in combination accelerates tumor cells to adhere to endothelial cells In vitro

Summary Platelets and coagulation are involved in the pathogenesis of blood-borne metastases. The aim of this study is to obtain more information about the mechanisms involved in the initial adhesion of tumor cells to endothelial cells. In short term experiments with tumor cells, suspended in the medium of cultured endothelial cells, we tested whether addition of both platelets and thrombin cause more tumor cell adhesion to endothelial cells, than when either platelets or thrombin are acting alone. HeLa cells or HT29 cells, prelabeled with radioactive ⁵¹Cr, human platelets, and thrombin were added to human endothelial cell cultures. Following 15 min of shaking at 37° C, the percentage of tumor cell adhesion was calculated. The percentages of adhering tumor cells with the presence of both platelets and thrombin were greatly increased compared to controls. Addition of hirudin 2 min before thrombin lowered the adhesion percentage of tumor cells. Hirudin added immediately before and 2 min after thrombin gave only minor effects. When the endothelium was treated with superoxide dismutase, catalase, and mannitol, the adhesion of tumor cells was lowered with catalase and superoxide dismutase. The cause of tumor cell-endothelial cell interaction is probably complex. Our results show that activated platelets enhance the tumor cell adhesion, and that generation of active oxygen species may be important in the initial phase of the interaction.     

Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect onin vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells

Summary We have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis. EDITOR'S STATEMENT Although compelling theoretical arguments sometimes can be made for the likely existence of growth-inhibitory substances of physical relevance in the control of cell proliferation, experiments aimed at identifying and studying such factors often are difficult to design and interpret, and little strong data exists to suggest that growth-inhibitory substances are important regulatorsin vivo. The information presented in this paper represents a start toward developing a useful system for studying growth-inhibitory factor. David W. Barnes     

Tunicamycin-resistant mutations in mouse FM3A cells

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 μg/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1–1.2 μg tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10⁻⁴ to 10⁻⁵ in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 μg N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 × 10⁻⁶ per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D₁₀ value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [³H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.