Mouse Lewis Lung Carcinoma Cells Research Articles

Gene Therapy for Murine Lung Cancer Using an Adenoviral Vector Expressing lnterleukin-15

It has been proven that interleukin-15 (IL-15) has structural and functional features similar to those of interleukin-2 (IL-2). In this study we examined the anti-tumor effect of IL-15 using an adenoviral vector in a mouse model. We constructed an adenoviral vector expressing the IL-15 gene, and introduced it into a mouse Lewis lung carcinoma (LLC) cell line. Then we inoculated pulmonary tissue of a syngeneic mouse strain (C57BL/6 CR) with the IL-15 gene-expressing LLC cells (LLC/IL-15). At the early phase (Day 14), a large number of NK cells accumulated around the IL-15 expressing tumors. At the late phase (Day 28), a much larger number of NK cells had accumulated around the tumors and quite a few NK cells had invaded the IL-15 expressing tumors. The LLC/IL-15-inoculated group survived significantly longer compared to the control groups, i.e., a group inoculated with LacZ gene-expressing LLC cells or a group inoculated with IL-2 gene-expressing LLC cells. After 60 days, the surviving mice of the LLC/IL-15-inoculated group were re-challenged with an additional inoculation of LLC cells alone. Tumor growth was significantly inhibited in the rechallenged LLC/IL-15 group. Induction of cytotoxic T cells was confirmed by interferon-gamma production by splenocytes. We concluded that the IL-15 expressed in the IL-15 expressing tumor cells enhanced the accumulation of NK cells and activated them, leading to suppression of proliferation of the tumor cells.

Jun oncoproteins do not function as primary transcription factors for the mouse major histocompatibility complex class I H-2 genes in fibroblasts.

There are several reports in the literature focusing on regulation of major histocompatibility complex (MHC) class I genes by transcription factors of the jun family. The methods employed in these reports differed in various respects, and their results are inconsistent. In mouse Lewis lung carcinoma, B16-melanoma and F9-teratocarcinoma cell lines, c-jun was characterized as a transcriptional activator of the murine MHC class I H2-Kb gene, while c-jun was identified as a direct transcriptional repressor of the swine class I PD1 gene, and c-jun stably transfected clones of mouse L-fibroblasts markedly reduced their H-2 class I gene expression. In this study, we attempted to reproduce this last effect by means of transient transfection coupled to Northern hybridization, upon transfecting L-fibroblasts with expression vectors for all jun family members as well as with an array of c-jun-derived dominant negative mutants. No change in H-2 class I expression could be identified. Next, we derived two additional fibroblastic cell lines from the fibrosarcoma of the H2-Kk/v-jun transgenic mouse and transfected them with the two most potent c-jun dominant negative mutants, again without eliciting any change in H-2 class I mRNA level. We conclude that the negative regulation of H-2 class I genes by c-jun in cells of the fibroblastic lineage is not a primary effect.

92. Calcitriol and its analog down-regulates αvβ3 integrin expression and suppresses the tumorigenesis of lewis lung carcinoma (LLC) cell line

Aim Calcitriol (CAL) [1,25-(OH)203], active hormonal form of vitamin D3, reveals antitumor activity both in vitro and in vivo. CAL may activate the β3-integrin subunit gene and promote the plasma membrane appearance of αvβ3 on osteociast precursors and on HL-60 leukemia cells. In this study we evaluated the effect of calcitriol and its new analogue (PRI-2191), synthesized in order to avoid hypercalcemic effect of calcitriol, on αvβ3 integrin-dependent mouse lung cancer invasiveness. Material and Methods The influence of CAL or PRI-2191 on the proliferation, adhesion properties and celi cycle of mouse LLC cells were tested by FACS analysis, adhesion and antiproliferative assays. Monoclonal antibodies and flow cytometry were applied for evaluation of integrins expression. Antitumor activity in vivo was examined in the model of subcutaneously growing tumors in C57BI/6 mice. Results We have shown, that LLC cells express a high level of αvβ3 (but not of αllbβ3) integrin and this expression was signiticantly reduced by the in vitro treatment with CAL or PRI-2191. Both compounds inhibited (in about 60%) LLC cells proliferation after 72 h of in vitro treatment. Moreover, both agents augmented adhesion of LLC cells to collagen (ligand tor allb integrin subunit) and diminished adhesion to fibrinogen (ligand for β3 integrin subunits). Further, in vitro incubation of LLC cells with both agents retarded the growth of subcutaneous tumors in mice. n all experiments, biological activity of PRI-2191 was higher than this of CAL, what is in accordance with our previous experiments, showing higher antiproliterative, lower calcemic activities and lower lethal toxicity of new analog as compared to parental drug. Conclusions Our data demonstrate that new analog of calcitriol, PRI-2191, is a good candidate for an antitumor drug, especially in the treatment of cancers in which the invasive potential is, atleast in part, dependent on expression of β3 integrins.

Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction.

Inflammatory conditions are associated with tumor development. IL-1beta is a multifunctional and proinflammatory cytokine that affects nearly all types of cells. To investigate the role of IL-1beta in tumor growth in vivo, we transduced the retroviral vector coding human IL-1beta gene into mouse Lewis lung carcinoma (LLC) cells and subsequently inoculated the transformant (LLC/IL-1beta) to syngeneic C57BL/6 mice. Tumors derived from LLC/IL-1beta grew faster (240%, day 18, vs null-vector control LLC/neo; p < 0.01) and showed more abundant vasculature (250%, vs LLC/neo; p < 0.05), whereas LLC/IL-1beta cells, LLC/neo cells, and wild-type LLC cells did not show any significant difference in the growth rate in vitro. As compared with LLC/neo cells, LLC/IL-1beta cells secreted 2-fold the amount of vascular endothelial growth factor and >10-fold the amount of macrophage-inflammatory protein-2 (CXCL2), one of whose main functions is angiogenesis. Although LLC/IL-1beta itself did not secrete hepatocyte growth factor (HGF), the tumor derived from LLC/IL-1beta cells also contained a >4-fold higher concentration of HGF, another angiogenic factor. In situ hybridization of HGF mRNA in LLC/IL-1beta tumor sections demonstrated that stromal fibroblasts and infiltrating cells overexpressed HGF mRNA. Moreover, when cultured in the presence of HGF in vitro, LLC/IL-1beta cells secreted even larger amounts of vascular endothelial growth factor and macrophage-inflammatory protein-2. The antiangiogenic agent TNP-470 and anti-CXCR2 Ab inhibited the tumor growth of LLC/IL-1beta cells in vivo. These results indicated that secreting IL-1beta into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide x cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Proteolytic Activation of Tissue-Type Plasminogen Activator by the Culture Media of Mouse Cancer Cells

Human single-chain tissue-type plasminogen activator (tPA) was activated by the culture media of mouse B16 melanoma and Lewis lung carcinoma cells. This activation was due to the proteolytic conversion of single-chain tPA to two-chain tPA. Typical serine proteinase inhibitors, such as diisopropylfluorophosphate and aprotinin, strongly inhibited the proteolytic activation, suggesting that the enyzme responsible for this activation is a serine proteinase. Through a process of gel filtration, the molecular weight of the putative tPA-activating enzyme was estimated to be approximately 35 kDa. Expression of the tPA mRNA was demonstrated by Northern blot analysis both for the melanoma and carcinoma cells. Zymographic experiments showed that the culture medium of the melanoma cells contained active two-chain tPA. These results indicate that a common serine proteinase may be involved in the proteolytic activation of single-chain tPA in these cancer cells.

Cytotoxicity of 2-deoxy-D-glucose and its tetra-acetate ester in tumoral cell lines

Mouse Lewis lung carcinoma cells (3LL-HH), mouse leukemic cells (L1210), mouse melanoma cells (B16F1), rat gliosarcoma cells (9L), rat hybrid insulin-producing cells (BRIN-BD11) and human hepatocellular carcinoma cells (HepG2) were cultured for 8 to 96 h in the absence or presence of 2-deoxy-D-glucose or its tetra-acetate ester (45 mu M to 0.8 mM). The ester was more efficient than the unesterified glucose analog in decreasing the growth of the tumoral cells, as assessed by either the generation of formazan from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide or direct cell counting. At high concentration (0.8 mM), the ester even decreased the cell number below its initial value. Even under these conditions, a partial restoration ol-cell growth was observed, however, when the cells were cultured in a control medium after having been exposed for 8 h to 2-deoxy-D-glucose tetraacetate. These findings indicate that such an ester acts as a powerful cytostatic and cytotoxic agent in several tumoral cell lines.

Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins.

Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e. I, Y and I-C). This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin. It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated. We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes.

Presence of atypical laminin on the surface of mouse Lewis lung carcinoma cells.

We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.

Inhibitory effects of green tea infusion on in vitro invasion and in vivo metastasis of mouse lung carcinoma cells

The peroral administration of green tea infusion reduced the number of lung colonies of mouse Lewis lung carcinoma cells in a spontaneous metastasis system. The experiments with artificially reconstituted basement membrane suggested that this reduction could be understood by the inhibitory effects of the green tea infusion and its constituent catechins on the penetration of the cells through the basement membrane.

Anti-metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent.

We have demonstrated that urinary trypsin inhibitor (UTI) purified from human urine is able to inhibit lung metastasis of mouse Lewis lung carcinoma (3LL) cells in experimental and spontaneous metastasis models. In this study, we have investigated whether UTI in combination with an anti-cancer drug, etoposide, can prevent tumour metastasis and show an enhanced therapeutic effect. Subcutaneous (s.c.) implantation of 3LL cells (1 x 10(6) cells) in the abdominal wall of C57BL/6 female mice resulted in macroscopic lung metastasis within 21 days. Microscopic lung metastasis was established by day 14 after tumour cell inoculation, and surgical treatment alone after this time resulted in no inhibition of lung metastasis. The number of lung tumour colonies in the group of mice which received surgery at day 21 was greater than in mice which had tumours left in situ (P = 0.0017). Surgical treatment on day 7, followed by UTI administration (s.c.) for 7 days, led to a decrease in lung metastasis compared with untreated animals. A significant inhibition of the formation of pulmonary metastasis was obtained with daily s.c. injections of UTI for 7 days immediately after tumour cell inoculation. UTI administration did not affect the primary tumour size at the time of operation. In addition, etoposide treatment alone led to a smaller primary tumours and yielded reduction of the formation of lung metastasis in the group of mice which received surgery at day 14 (P = 0.0026). Even in mice which received surgical treatment on day 14, followed by the combination of UTI (500 micrograms per mouse, days 14, 15, 16, 17, 18, 19 and 20) with etoposide (40 mg kg-1, days 14, 18 and 22), there was significant reduction of the formation of lung metastasis (P = 0.0001). Thus, the combination of an anti-metastatic agent with an anti-cancer drug, etoposide, might provide a therapeutically promising basis for anti-metastatic therapy.

Retrovirus-directed expression of PDGF-B in human lymphocytes and monocytes

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer with an extremely low survival rate. It is characterized by a chronic inflammatory process with intense mast cell infiltrate that is associated with reduced survival. The aim of this study was to test the hypothesis that mast cells have an enhancing effect on NSCLC proliferation. To assess the tumor-promoting potential of mast cells, we used the human alveolar basal adenocarcinoma (A549) and the mouse Lewis lung carcinoma (LLC) cell lines, umbilical cord blood-derived mast cells (CBMC) and the mast cell-deficient mouse Sash model. The proliferation rate of A549/LLC cells was markedly increased by mast cells and histamine. Histamine proliferating activity was mediated via H1, H2 and H4 receptors and caused ERK phosphorylation. LLC induced in Sash mice or in wild-type mice treated with the mast cell stabilizer nedocromil sodium displayed an accelerated growth (number of metastic colonies in the lungs, total lung area and lung/total mice weight ratio). In summary, we have shown a significant effect of mast cells and histamine in enhancing NSCLC/LLCX growth in vitro, while in a mouse LLC model in vivo we have found that mast cells are important negative regulators of cancer development. Therefore our results would indicate a pro-tumorogenic effect of the mast cells in vitro on established lung tumor cell lines, and anti-tumorogenic effect in mice at lung cancer induction. In conclusion, mast cell/anti-histamine targeted therapies should carefully consider this dual effect.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. I. Selection and in vivo properties

The availability of lectin-resistant cell lines with altered carbohydrate moieties in cell surface glycoproteins and glycolipids has greatly facilitated study of the involvement of cellular glycoconjugates in tumor growth and metastasis. We present here a new animal model for metastasis study based on mouse Lewis lung carcinoma LL2 in vitro cell line. From this line, five lectin-resistant variant sublines were selected with the following lectins: wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR). The correlation of the lectin resistance with their in vitro and in vivo growth properties, and especially lung colonizing ability, were investigated. Three WGAR variants with well-preserved tumorigenicity revealed reduced metastatic ability, both spontaneous, after subcutaneous (s.c.) administration and experimental, after intravenous (i.v.) administration. The RCA IIR variant also possessed reduced spontaneous and experimental metastatic ability, but exhibited higher growth rate of local s.c. tumors. The AAAR variant possessed reduced spontaneous metastatic ability but its ability to colonize the lungs after i.v. administration was five-fold higher than that of the parent LL2 line, whereas its tumorigenicity remained unchanged. The relative differences among WGAR variants and parent LL2 line, concerning their experimental metastatic ability, remained similar in cyclophosphamide-modified mice to those in normal recipients.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.

Glycosphingolipids in lectin-resistant variants of mouse Lewis lung carcinoma cells.

Neutral glycolipids and gangliosides from murine Lewis lung carcinoma cell line LL2 and its lectin-resistant variants, differing in metastatic properties, were studied by fast-atom-bombardment mass spectrometry (FAB-MS), exoglycosidase treatment and an immunostaining procedure. The neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, asialo GM2, globoside and a glycolipid with a preliminary structure of Hex-Hexl-4HexNAc-Hex-Hex-Cer. The major gangliosides were GM3, GM2, GM1 and GD1a. No qualitative differences in glycosphingolipid expression were found between the metastatic cell lines (LL2 and LL2AAA) and the weakly metastatic variants (LL25, LL28, LL230 and LL2RCA II). Some quantitative differences were observed between the cell lines, e.g., in the level of ganglioside-bound sialic acid, which was not apparently correlated with the metastatic capacities.

Evidence for a warfarin-sensitive serum factor that participates in factor X activation by Lewis lung tumor cells.

Mouse Lewis lung (LL) carcinoma cells possess a factor X activator (procoagulant) that is inhibited in vivo by warfarin treatment or diet-induced vitamin K deficiency. This inhibition suggests that vitamin-K-dependent proteins are involved in LL cell activation of factor X. A LL primary tumor clone (LL13) was isolated which contained a warfarin-sensitive vitamin-K cycle of metabolism and expressed factor X procoagulant activity. LL13 cells exposed to media containing warfarin or deficient in vitamin K grew as well as cells in normal media, and activated factor X to similar extents. In contrast, administration of warfarin to mice bearing LL13 cells inhibited factor X procoagulant activity as well as the vitamin K cycle of metabolism in the primary tumors. In relation to LL13 cells grown in media containing fetal bovine serum, those incubated for 20 hr in media containing mouse serum or the sera from LL13-bearing mice exhibited 9- to 10-times higher levels of factor X procoagulant activity. However, LL13 cells exposed to media containing the sera of warfarin-treated LL-13-bearing mice or to barium-sulfate-adsorbed normal mouse serum activated factor X much less efficiently. Collectively, these data suggest that inhibition of vitamin K function in LL cells does not affect the extent of factor X activation and thus the intrinsic factor X procoagulant is not a vitamin-K-dependent protein. They further suggest that both a warfarin-sensitive (vitamin-K-dependent) protein present in normal mouse serum and a LL13 cell component participate in factor X procoagulant activity.

Co-operation between metastatic tumor cells and macrophages in the degradation of basement membrane (type IV) collagen

The co-culture of mouse peritoneal macrophages and Lewis lung carcinoma cells induces the relase of a metal-dependent type IV collagen-degrading proteinase which is not produced in detactable amounts by either cell type cultivated alone. Conditioned media of the co-cultures degrade both pepsin-extracted type IV collagen from human placenta and mouse type IV procollagen. Thus macrophages can interact with tumor cells to degrade basement membrane type IV collagen: this might be of importance to allow cancer invasion and metastasis.

Immunobiological characteristics of lectin-resistant variants of mouse lewis lung carcinoma cells : D. Duś, C. Radzikowski, H. Debray 1, W. Budzyński and A. Apolski. Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland; 1Laboratory of Biochemistry, University of Lille, France

Immunobiological characteristics of lectin-resistant variants of mouse lewis lung carcinoma cells : D. Duś, C. Radzikowski, H. Debray 1, W. Budzyński and A. Apolski. Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland; 1Laboratory of Biochemistry, University of Lille, France