Insulin receptors on cultured murine lymphoid tumor cell lines

Summary Eight murine lymphoid tumor cell lines have been examined for the presence of high-affinity insulin receptors. The eight cell lines included two Abelson murine leukemia virus-transformed pre-B cell lines, three plasmacytoma cell lines, and three spontaneous T-cell lymphomas from AKR mice. All of the cell lines in the B-cell series had high-affinity insulin binding sites. The apparent equilibrium association constant (K_a) for the high-affinity binding sites on these cells was 1.3–3.3 × 109 M^–1. Two of the T-cell lymphomas had high-affinity receptor levels so low as to be undetectable in the whole cell binding assay under the conditions used for assaying the other cell lines, although in binding assays performed at very high cell densities, these two cell lines did appear to have a small number of high-affinity insulin binding sites. These results indicate that the growth stimulus provided by the tumor virus in neoplastic transformation of the AKR thymic lymphocytes differs from that provided by lectins in blast transformation of lymphocytes in that the neoplastic transforming event does not always result in the emergence of large numbers of high-affinity insulin receptors. In addition, the existence of cell lines such as the T-cell lymphomas that have nearly exclusively low-affinity binding sites suggests that the low-affinity sites may represent a distinct receptor that is not freely interconvertible with the high-affinity receptor.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium

Summary Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C₃H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Epithelial-to-mesenchymal transition confers resistance to apoptosis in three murine mammary epithelial cell lines

Epithelial-to-mesenchymal transition (EMT) is an essential embryogenic and developmental process, characterized by altered cellular morphology, loss of cell adhesion, and gain of migratory ability. Dysregulation of this process has been implicated in tumorigenesis, mediating the acquisition of migratory and invasive phenotypes by tumor cells. Mammary epithelial cells provide an excellent model in which to study the process, being derived from mammary gland tissue that utilizes EMT to facilitate branching morphogenesis through which the developing gland migrates into and invades the fat pad. Inappropriate EMT has been heavily implicated in the progression of ductal hyperplasia and mammary tumor metastasis. We examined the morphological and molecular changes of three murine mammary epithelial cell lines following EMT induction. EMT was induced in the EpH-4 and NMuMG cell lines by transforming growth factor (TGF)-beta1 but not by ethanol, while the KIM-2 cell line was partially resistant to TGF-beta1 but responded fully to ethanol. The response to EMT-inducing reagent was shown to be critically dependent on the time of treatment, with confluent cells failing to respond. Timelapse photography identified increased motility during wound healing in cells pre-treated with EMT-inducing reagent compared with untreated controls. Furthermore, EMT conferred resistance to UV-induced apoptosis. Our data indicate that evaluation of characteristics other than loss and gain of phenotypic markers may be of benefit when assessing EMT, and contribute to the evidence suggesting that inappropriate EMT facilitates the acquisition of resistance to apoptosis, a key characteristic required for tumor survival.     

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.     

Effects of Taxotere on murine and human tumor cell lines.

Taxotere (RP 56976, NSC 628503), an analog of taxol, is an inhibitor of depolymerisation of microtubules and is currently in Phase I clinical trials. Comparisons of the cytotoxicities of Taxotere and taxol have been studied on several murine (P388, SVras) and human cell lines (Calc18, HCT116, T24, N417, KB). Taxotere was found more potent than taxol (1.3-12 fold), a result which could be explained by its higher affinity than taxol for microtubules. In agreement with its postulated mechanism of action, Taxotere is more cytotoxic on proliferating than on non proliferating N417 cells and does not inhibit cellular DNA, RNA and protein synthesis. Taxotere gives partial cross resistance on P-glycoprotein resistant P388/DOX cell line, in contrast to taxol which gives a complete cross resistance. On the other hand, no cross resistances were observed on Calc18/AM and P388/CPT5 cell lines, bearing modified activities of topoisomerase II and topoisomerase I, respectively. These results underline the higher cytotoxic activity of Taxotere compared to taxol, and the lack of cross resistance of that class of agent with the topoisomerase I and II-related multidrug resistance phenotypes.     

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous ¹⁴C-AA (10 μM) to PGE₂ (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B₂ (TXB₂) (7.4%), PGD₂ (4.4%) and PGF_2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF_1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID₅₀=0.6 μM) while the synthesis of TXB₂ and HHT was selectively inhibited by benzylimidazole (ID₅₀=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R_f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.     

Laminin binding and internalization by human and murine mammary gland cell lines in vitro.

We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.     

Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5μM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumour cell lines.     

Conjugated linoleic acid reduction of murine mammary tumor cell growth through 5-hydroxyeicosatetraenoic acid

Conjugated linoleic acid (CLA) is a dietary fatty acid that has been shown to reduce tumorigenesis and metastasis in breast, prostate and colon cancer in animals. However, the mechanism of its action has not been clarified. The goal of this study was to determine whether CLA altered mouse mammary tumor cell growth and whether specific metabolites of the lipoxygenase pathway were involved in CLA action. Both t10, c12-CLA and a lipoxygenase inhibitor, but not c9, t11-CLA or linoleic acid (LA), reduced mouse mammary tumor cell viability and growth by inducing apoptosis and reducing cell proliferation. t10, c12-CLA reduced the production of the 5-lipoxygenase metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE). That effect was not seen with c9, t11-CLA or LA. Adding 5-HETE back to tumor cells reduced the t10, c12-CLA effect on both apoptosis and cell proliferation. These data suggest that t10, c12-CLA reduction of tumor cell growth may involve the suppression of the 5-lipoxygenase metabolite, 5-HETE, with subsequent effects on apoptosis and cell proliferation.     

Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies

We evaluated the capacity of freshly isolated blood monocytes to mediate antibody-dependent cellular-mediated cytotoxicity (ADCC) in cooperation with murine anti-tumor monoclonal antibodies (MAbs). Blood monocytes isolated from most donors by adherence selection to fibronectin-coated plastic surfaces and subsequently depleted of natural killer/killer (NK/K) cells exhibited significant ADCC activity against tumor cell lines in combination with an IgG3 antitumor MAb (BR55-2). However, significant variation in ADCC competence was observed among donors. Culture parameters influencing monocyte ADCC activity were evaluated and optimized. The influence of MAb isotype on ADCC capacity of anti-tumor MAbs was also evaluated using anti-tumor class-switch variant hybridoma proteins and a panel of anti-tumor MAbs. MAbs of the IgG2a and IgG3 subclasses exhibited high ADCC potential, whereas MAbs of the IgG2b subclass exhibited no ADCC activity. One of two IgG1 MAbs tested exhibited high ADCC activity with monocyte effectors. The role of monocytes or macrophages in tumor remission of cancer patients undergoing MAb immunotherapy is not known. However, correlative studies of monocyte ADCC capacity and responsiveness of cancer patients to MAb immunotherapy may help to establish the role of these effectors in MAb-mediated tumor remissions.     

Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice.

Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency.     

Marker chromosomes of murine cell lines

Using the C-method of chromosome staining four marker chromosomes were revealed in the transplanted murine line SC-1, one comparatively rare marker chromosome was shown in RAG line, small marker chromosomes occurred almost in all cells of RVP3 line. Marker chromosomes found in the studied lines by the C-method of chromosome staining make it possible to distinguish these lines from each other.     

Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.     

Synthesis of murine mammary tumor viral proteins in vitro

The coding potential of murine mammary tumor viral genomic RNA was investigated by in vitro translation of various size classes of RNAs isolated from the virions. The major products of translation of full-size 35S polyadenylylated virion RNA were gag-related polyproteins of 75,000, 105,000, and 180,000 daltons (P75, P105, and P180, respectively). Studies on the kinetics of translation of these three proteins established that they were synthesized independently and that the smaller proteins were not post-translational cleavage products of the larger proteins. Tryptic peptide mapping showed that almost all of the P75 sequences were contained within P105 and almost all of the P105 sequences were contained within P180. The syntheses of all three proteins were inhibited by m7GTP, indicating that they were translated from capped mRNA's. Although a 24S polyadenylylated RNA had been identified as the intracellular mRNA for env precursor polyprotein, no such protein could be translated from the 24S polyadenylylated RNA isolated from the virions. However, translation of a 14S size class of polyadenylylated virion yielded four prominent proteins of about 36,000, 23,000, 21,000, and 20,000 daltons. These proteins were unrelated to murine mammary tumor viral structural proteins, as suggested from tryptic peptide mapping and immunoprecipitation data. They might be the products of an as-yet-unidentified gene located near the 3' terminus of the murine mammary tumor viral genomic RNA.     

Phosphorylation of murine mammary tumor virus precursor polypeptides.

Phosphorylation of the murine mammary tumor virus (MuMTV) structural proteins was studied in an MuMTV-infected epithelial cell line derived from a BALB/cf C3H mouse mammary tumor. Immunoprecipitation of 32P-labeled cell extracts with monospecific anti-p27 serum revealed that phosphorylation occurred at the stage of the core-protein polyprotein precursor prp75. Two forms of phosphorylated prp75 were found: one migrating with an apparent molecular weight of 80,000, and the other with a molecular weight of 76,000. The 80,000-molecular-weight species was found to be the most heavily phosphorylated. In addition, a relatively stable phosphorylated processing intermediate of 34,000 molecular weight was observed as well. Tryptic peptide mapping analysis of the 32P-labeled viral proteins indicated a precursor product relationship between the intracellular phosphorylated, high-molecular-weight peptides and the mature MuMTV phosphoproteins p23 and p27. Phosphopeptide analysis also suggested that phosphorylation of the viral proteins occurred in discrete steps and that the attached phosphate groups were conserved throughout the processing steps.     

Purification and translation of murine mammary tumor virus mRNA's

We have studied the functions of the intracellular RNAs of mouse mammary tumor virus (MMTV) by purification and translation in vitro. Two major size classes of MMTV RNA, 35S and 24S RNA, were isolated from MMTV-infected rat (XC) cells and cultured mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with antisera against gag and env products, and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including three previously reported in extracts of infected cells; 24S RNA directed synthesis of two polypeptides closely related to env proteins from infected cells. Therefore, 35S RNA includes mRNA's for gag and gag-pol, whereas 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.     

Radioimmunolocalization of murine mammary tumor virus proteins in gels

Nycodenz is a new nonionic iodinated gradient medium which readily dissolves in water to give nontoxic, autoclavable solutions. This paper describes the use of diffusion techniques to prepare isotonic Nycodenz gradients with maximum densities up to 1.15 g/ml, which is sufficient to band most types of cells.     

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.