Expression of tumor antigen correlated with metastatic potential of Lewis lung carcinoma and B16 melanoma clones in mice

Expression of a tumor-associated antigen, recognized by a monoclonal antibody (MoAb 135-13C) to lung carcinoma cells, has been studied in cloned Lewis lung carcinoma (3LL) and in B16 melanoma (F1 and F10) tumor lines endowed with different metastatic potentials. MoAb 135-13C recognizes a protein complex (tumor-specific Mr 180,000 protein) that appears on the cell surface of several murine lung carcinomas but is not detected on normal cells in culture. Standard metastatic variants of B16 melanoma (F1 and F10) and two variant sublines of 3LL (M1087 and BM21548) together with the parental line of 3LL have been used for these experiments. The two cloned variant lines derived from 3LL have been shown to retain high (M1087) and low (BM21548) metastatic phenotypes during in vivo passaging. We found that all three cell lines of 3LL bind monoclonal antibody specifically, but one cell variant with higher metastatic potential shows a higher capacity to bind MoAb 135-13C than did the other variant. Similarly we found that B16 F10 cells bind higher amounts of MoAb 135-13C than did B16 F1 cells. In addition the analysis of the amounts of MoAb 135-13C bound to the cell surface of several other in vitro and in vivo tumor lines with different metastatic capacity demonstrates that all tumor lines which express high ability to colonize to the lung also express, on the cell surface, higher amounts of tumor-specific Mr 180,000 protein. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms of immunoprecipitates from cell lysates of 3LL and B16 tumor lines demonstrate that MoAb 135-13C specifically precipitated three proteins banding at molecular weights of 204,000, 134,000, and 116,000. We conclude that MoAb 135-13C recognizes a surface protein complex which is present in higher amounts in 3LL and B16 cells which possess higher capacity to metastasize to the lung.     

Long exposure of non-cytotoxic concentrations of methylselenol suppresses the invasive potential of B16F10 melanoma

To assess the inhibitory effects of methylselenol on the invasion of murine B16F10 melanoma cells, we carried out in vivo and in vitro experiments using Se-methylselenocysteine (Se-MSC) and selenomethionine (SeMet), respectively. In an animal experiment, the supplementation of drinking water with Se-MSC (4 ppm Se) led to a significant increase in Se levels in the lung, liver and serum in mice. Mice given a mash diet or water supplemented with Se-MSC (2, 4 and 6 ppm Se in the mash diet, and 2 and 4 ppm Se in the drinking water) displayed an almost completely diminished pulmonary metastasis of B16F10 melanoma cells and an enhanced survival, compared to the control mice which were given a basal diet. Treatment with non-cytotoxic concentrations of SeMet (2.5, 5 and 10 microM plus 0.02 U/ml METase, methioninase) induced a substantial decrease in the expression of integrin alphavbeta3, the FN receptor and adhesion ability to vitronectin (VN) and fibronectin (FN) in B16F10 melanoma cells. Moreover, these compounds suppressed gelatinase activity, invasive ability and wound migration in the culture system. SeMet-METase prevented the conversion of pro-MMP-9 to its active form and decreased pro-MMP-2 activities in a zymogram. The pre-treatment of B16F10 melanoma cells with SeMet-METase led to a decrease in pulmonary metastasis and extended survival in mice injected with tumor cells. Collectively, our results indicate that integrin expression is crucial in promoting the metastatic phenotype in murine B16F10 melanoma cells by supporting specific adhesive and invasive properties, suggesting that Se-MSC effectively reduces the metastasis of B16F10 melanoma cells as a nutritional adjuvant. Methylselenol may also contribute to the suppression of integrin expression.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

Nitric oxide-mediated regulation of hypoxia-induced B16F10 melanoma metastasis

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.     

Expression of a Mr 41,000 glycoprotein associated with thrombin-independent platelet aggregation in high metastatic variants of murine B16 melanoma

In the previous study, we generated a monoclonal antibody, 8F11, against NL-17, a high metastatic clone derived from a metastatic variant of murine colon adenocarcinoma 26. 8F11 inhibited platelet aggregation induced by NL-17 and recognized a Mr 44,000 membrane protein as antigen. In the present study, the reactivity of 8F11 to murine B16 melanoma and its metastatic variants was examined, and the antigen recognized by 8F11 on the cell surface was characterized. 8F11 was found to strongly react with 3 metastatic variants of B16 melanoma. In contrast, only slight reactivity was observed with parent B16 melanoma. The reactivity of the antibody to these cells was in the order B16F10 greater than B16BL-6 greater than B16F1 much greater than B16. Western blot analysis showed a Mr 41,000 protein as the antigen recognized by 8F11 on the cell surface of B16F10 cells. The Mr 41,000 antigen appeared to be a glycoprotein that bound to wheat germ agglutinin as has been observed for the Mr 44,000 antigen of NL-17. To elucidate the functional role of the Mr 41,000 antigen in B16 melanoma, platelet aggregation induced by B16 and B16F10 was compared. B16 was reported to stimulate platelet aggregation by the generation of thrombin, whereas B16F10 was found to activate platelet by at least 2 mechanisms: one dependent on thrombin and the other independent on thrombin. The activity of B16 and its metastatic variants to induce platelet aggregation in the presence of MD805, a synthetic antagonist of thrombin, well correlated with the reactivity of 8F11 to these cells. 8F11 blocked platelet activation by B16F10 under conditions preventing thrombin activity such as enzymatic formation of lysolecithin through the treatment of the cell surface with phospholipase A2 or in the presence of MD805. These data indicate that Mr 41,000 glycoprotein recognized by 8F11 on metastatic variants of B16 melanoma is involved in the thrombin-independent platelet aggregation. A positive correlation was observed between the levels of Mr 41,000 glycoprotein expression of B16 and its metastatic variants and their pulmonary metastasis after i.v. injection, suggesting Mr 41,000 glycoprotein, as well as other factors reported previously, may play an important role in the hematogenous spread of B16 melanoma.     

Preferential organ attachment and invasion in vitro by B16 melanoma cells selected for differing metastatic colonization and invasive properties

Murine B16 melanoma sublines have been sequentially selected in vivo for low (B16-F1) or high (B16-F10) lung, high ovary (B16-O10) or high brain (B16-B15b) colonization, and in vitro for enhanced tissue invasion (B16-BL6). These B16 sublines were tested in vitro in a syngeneic organ adhesion/invasion assay to determine differences in tumor and/or host tissue properties that might account for preferential metastasis to certain sites. Tissues used were murine C57BL/6 lung, ovary and heart. In 8 independent experiments high lung-colonizing B16-F10 cells bound to and infiltrated into lung tissue better than ovary or heart tissue, while high ovary-colonizing B16-O10 cells attached to and invaded into ovary tissue at higher rates than lung or heart tissue. Only highly tissue-invasive B16-BL6 cells were able to invade heart tissue within 18 h in the experiments. The results suggest that organ metastatic colonization preferences by malignant cells may be determined, in part, by differences in the abilities of metastatic tumor cells to attach to and invade target tissue.     

Antigenic differences among B16 melanoma variants selected for their differing abilities to metastasize: a possible mechanism for effective adjuvant immunotherapy

Most C57BL/6J mice will develop pulmonary metastases four to six weeks after excision of B16 melanoma isografts and begin to die within 40 days. When applied in an adjuvant setting after isograft excision, specific B16 immune RNA (I-RNA) therapy prevents pulmonary metastases and prolongs survival in 50% of treated animals. Since increased in vitro cell-mediated cytotoxicity (CMC) against B16 targets has been demonstrated in animals whose survival is improved by B16 I-RNA therapy, we have proposed that the treatment functioned, at least in part, by specifically altering host CMC in vivo. In this study, C57BL/6J splenocytes were specifically sensitized in vitro by B16 I-RNA exposure and examined for cytotoxic effect on variants of B16 melanoma selected for their differing metastatic potential in vivo. F10 tumor targets (explanted from a B16 melanoma vairant producing frequent pulmonary metastases in vivo) were consistently more sensitive to specific in vitro cytotoxic effect than F1 target cells (from a B16 melanoma variant with low metastatic potential in vivo). Lung metastases (F1 mets) were explanted after intravenous (IV) injection of F1 and used as target cells in the in vitro CMC assays. F1 mets demonstrated greater cytotoxic effect than F1 targets after exposure to B16 I-RNA-treated syngeneic splenocytes. C57BL/6J mice were sacrificed at weekly intervals following adjuvant in vivo B16 I-RNA therapy (after B16 isograft excision), and their splenocytes were shown to be consistently more cytotoxic in vitro to B16 and F10 than to F1 target cells. Splenocytes harvested from mice treated with I-RNA specific to antigenically distinct Lewis lung carcinoma (3LL) after 3LL isograft excision had no cytotoxic effect on any of the B16 variants. When nonsensitized C57BL/6J splenocytes were examined for cytotoxic effect in natural killer (NK) assays or in NK inhibition assays, differences in cytotoxic sensitivity of B16, F10, F1, and F1 mets could not be demonstrated. We conclude, therefore, that specifically sensitized effector cells could distinguish antigenic differences among B16 variants selected for differing in vivo metastatic potiential. These differences were tumor specific and could be demonstrated by cytotoxic splenocytes after either in vivo B16 I-RNA treatment or in vitro B16 I-RNA exposure. The relationship of these antigenic differences to in vivo metastatic potential and to the effectiveness of adjuvant B16 I-RNA therapy is discussed.     

Spontaneous sister chromatid exchange in metastatic variants of the murine B16 melanoma and human astrocytomas in culture

Three metastatic variants, BL6 (high metastasis), F1 (nonmalignant) and F10 (intermediate malignancy) of the B16 murine melanoma, and a pulmonary metastatic line BL6-ML8 of the BL6 primary tumour have been examined for spontaneous sister chromatid exchange (SCE). Two human astrocytoma cell lines were also examined. SCE was encountered in 29 and 13% of second division metaphases of BL6 and F10. In contrast, only 3% of second division metaphases of the F1 showed SCE. In BL6-ML8, 40% of the metaphases showed SCE. Approximately 2-4% of the human astrocytoma second division cells showed SCE. The variant lines were karyotypically heterogeneous. The pattern of cell distribution according to chromosome number showed an overall similar profile in the melanoma variants. However, the metastatic BL6-ML lines showed a marked shift to a hypertriploid state. SCEs occurred with higher frequency in this hypertriploid subpopulation of BL6 and F10 cells than in F1. SCE incidence in the hypertriploid subpopulation was twofold higher in the metastatic line than in the primary BL6 line. The number of SCEs per chromosome was twice as high in F10, BL6 and BL6-ML8 as in the F1 cells. This hypertriploid subpopulation showed a marked increase of SCEs on exposure to mitomycin C and ethyl methane sulphonate, indicating their mutability. It is suggested that the parallelism between SCE and metastatic potential may be relevant in the context of the generation of the metastatic phenotype.     

Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase sense and antisense genes.

Murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for the prepro form of urokinase in an SV40 expression vector (plasmid pSV2-uPA), and cells expressing high amounts of the human urokinase gene product were selected for by an enzyme-linked immunosorbent assay specific for human high molecular weight urokinase. Southern analysis showed one of the cell lines (clone 7) had incorporated 150 copies of the pSV2-uPA plasmid into its genomic DNA. The human urokinase synthesized by the pSV2-uPA-transfected murine B16 cells was found to be glycosylated and did not bind to the murine cell surface urokinase receptor sites. In an in vivo assay that measures metastasis from a primary tumor (spontaneous metastatic assay), clone 7 cells showed an increased ability to metastasize (12 of 12 mice showed metastatic tumors), while control cells showed a lower ability to metastasize (only 2 of 11 mice showed metastatic tumors). In a second in vivo assay, which measures only the steps of the metastatic migration process during which tumor cells extravasate from the blood and then grow into pulmonary tumors (lung colonization assay), a significant multifold increase in the ability to form lung tumors was shown by the high human urokinase-secreting B16-F1 cells. In B16-F10 cells incorporating an antisense sequence to preprourokinase (plasmid pSV1-ASuPA-265) and secreting significantly decreased amounts of murine urokinase, a corresponding significant decrease in lung colonization was observed. These results provide direct experimental support for a role of secreted (non-surface-bound) urokinase in the colonization steps of the metastatic process. Furthermore, the data indicate that the higher lung colonization ability of the B16-F10 line than of the B16-F1 line is primarily based on the quantitative differences in their abilities to produce urokinase.     

An intracellular form of cathepsin B contributes to invasiveness in cancer

Cathepsin B is a lysosomal cysteine proteinase whose expression and trafficking are frequently altered in cancer, and plasma membrane and secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of cathepsin B in several tumor cell lines and measured their capacity to invade through a reconstituted extracellular (Matrigel) matrix. Transient expression of human cathepsin B in a poorly metastatic B16F1 murine melanoma variant produced a 3-5-fold increase in cathepsin B activity and a comparable increase in invasiveness. Stable antisense cathepsin B-expressing clones of the highly metastatic human melanoma A375M and prostate carcinoma PC3M cell lines produced 40-50% less cathepsin B than control cells and were proportionately less invasive. In contrast, manipulating cathepsin B levels had no effect on cell migration across an uncoated membrane. The anionic cathepsin B inhibitor (L-3-trans-propylcarbamoyloxirane-2-carbony)-L-isoleucyl-L-proline (CA-074), at a concentration of 1 microM, caused a nearly quantitative inhibition of extracellular cathepsin B but had no effect on Matrigel invasion. In contrast, the equally potent but less selective inhibitor, trans-epoxysuccinyl-L-leucylamino(4-guanidino)butane (E-64) inhibited invasion by 75%. Surprisingly, at a concentration of 10 microM, CA-074 slowly permeated the cells, causing an 80-95% inhibition of intracellular cathepsin B after 12 h, the duration of the invasion assay. The membrane-permeant cathepsin B inhibitor, CA-074 methyl ester, and the higher concentration of CA-074 that inhibited intracellular cathepsin B both significantly reduced Matrigel invasion. Collectively, these results identify an intracellular role for cathepsin B in matrix degradation. They also indicate that caution should be exercised in assuming that CA-074 is unable to enter cells when it is used to inhibit biological processes of long duration.     

Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: role of the cell surface heparan sulfate glycosaminoglycans

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.     

Anti-metastatic Effects on B16F10 Melanoma Cells of Extracts and Two Prenylated Xanthones Isolated from Maclura amboinensis Bl. Roots

Inhibitory effects of Maclura amboinenesis Bl, one plant used traditionally for the treatment of cancers, onmetastatic potential of highly metastatic B16F10 melanoma cells were investigated in vitro. Cell proliferationwas assessed using the MTT colorimetric assay. Details of metastatic capabilities including invasion, migrationand adhesion of B16F10 melanoma cells were examined by Boyden Chamber invasion and migration, scratchmotility and cell attachment assays, respectively. The results demonstrated that n-hexane and chloroformextracts exhibited potent anti-proliferative effects (p<0.01), whereas the methanol and aqueous extracts hadless pronounced effects after 24 h exposure. Bioactivity-guided chromatographic fractionation of both activen-hexane and chloroform extracts led to the isolation of two main prenylated xanthones and characterizationas macluraxanthone and gerontoxanthone-I, respectively, their structures being identified by comparison withthe spectral data. Interestingly, both exhibited potent effective effects. At non-toxic effective doses, n-hexaneand chloroform extracts (10 and 30 μg/ml) as well as macluraxanthone and gerontoxanthone-I (3 and 10μM) significantly inhibited B16F10 cell invasion, to a greater extent than 10 μM doxorubicin, while reducingmigration of cancer cells without cellular cytotoxicity. Moreover, exposure of B16F10 melanoma cells to highconcentrations of chloroform (30 μg/ml) and geratoxanthone-I (20 μM) for 24 h resulted in delayed adhesionand retarded colonization. As insights into mechanisms of action, typical morphological changes of apoptoticcells e.g. membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic bodies and loss ofadhesion as well as cell cycle arrest in the G1 phase with increase of sub-G1 cell proportions, detected by Hoechst33342 staining and flow cytometry were observed, suggesting DNA damage and subsequent apoptotic cell death.Taken together, our findings indicate for the first time that active n-hexane and chloroform extracts as well asmacluraxanthone and gerontoxanthone-I isolated from Maclura amboinensis Bl. roots affect multistep of cancermetastasis processes including proliferation, adhesion, invasion and migration, possibly through induction ofapoptosis of highly metastatic B16F10 melanoma cells. Based on these data, M. amboinensis Bl. represents apotential candidate novel chemopreventive and/or chemotherapeutic agent. Additionally, they also support itsethno-medicinal usage for cancer prevention and/or chemotherapy.     

Lowered superoxide dismutase in highly metastatic B16 melanoma cells

Superoxide dismutases (SOD), which are enzymes scavenging the superoxide radical, were studied in two variant lines of the B16 melanoma: B16F1 with low metastatic potential and B16F10 with high metastatic potential. SOD activity was measured by a method utilizing reduction in the chemiluminescence of luminol. Using cell free extracts it was shown that the highly metastatic B16F10 cell line has a SOD activity lower (20.70 +/- 3.07) units/mg protein, n = 8, than that of the less metastatic B16F1 cell line (81.38 +/- 6.78) units/mg protein, n = 8. Acrylamide gel electrophoresis suggested that Mn-SOD activity is higher in B16F1 cells.     

Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells

The human tissue inhibitor of metalloproteinases (TIMP) is a glycoprotein with a molecular weight of 28,000. It appears to be ubiquitous in human mesoderm tissues and has previously been shown to be identical to the collagenase inhibitor isolated from human skin fibroblasts. TIMP inhibits type I- and IV-specific collagenases and other neutral metalloendoproteinases that may be responsible for the degradation of extracellular matrix in tumor cell metastasis. In this work we have utilized recombinant human TIMP (rTIMP) obtained by expression of its cDNA gene (Carmichael et al., Proc. Natl. Acad. Sci. USA, 83:2407, 1986). The rTIMP is shown to have similar inhibition properties as natural TIMP against human skin fibroblast collagenase. In an in vitro amnion invasion assay system, rTIMP inhibited the invasion of B16-F10 murine melanoma cells through the human amniotic membrane at an identical concentration to that reported previously for natural TIMP. The mechanism by which rTIMP inhibits amniotic membrane invasion was compared to the mechanism by which the fibronectin receptor binding peptide RGDS and the aminin receptor binding peptide YIGSR inhibit amnion invasion. RGDS and YIGSR inhibited strong binding of the tumor cells to the amniotic membrane. In contrast rTIMP did not inhibit the cell adhesion step in amnion invasion, but actually increased the number of tumor cells that were tightly bound to the amnion. Thus rTIMP appears to inhibit a later step in the amnion invasion process, following B16-F10 cell adhesion. C57BL/6 mice treated with i.p. injections of rTIMP every 12 h for 6.5 days showed a significant inhibition of metastatic lung colonization by B16-F10 murine melanoma cells. While the rTIMP inhibited the number of metastatic lung tumors formed, it had no significant effect on the size of the lung tumors. Furthermore, tumors grown s.c. in mice receiving 12-h i.p. injections of rTIMP for 6.5 days, as in the in vivo colonization assay, showed no difference in size from controls. Thus the anticolonization effect of rTIMP appears not be due to an effect on tumor growth, but on the invasion step itself. The inhibition of lung colonization in C57BL/6 mice by rTIMP is one of the first examples showing an antimetastatic effect of a selective metalloproteinase inhibitor in a mammalian animal model, and supports an essential role for metalloproteinase(s) in the extravasation and invasion of tumor cells during lung colonization by blood-borne tumor cells.     

Altered expression of a third actin accompanying malignant progression in mouse B16 melanoma cells

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two-dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found AX actin (Mr = 43,000, pI = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high-metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony-forming ability following iv administration, increased with increase in the F number. The half life of AX actin was much the same as that of beta- and gamma-actin and the different expressions of AX actin between the low- (F = 1) and high-metastatic (F = 10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of AX actin. The AX actin was incorporated into the cytoskeletal fraction with the same efficiency as beta- and gamma-actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in AX expression. The actin stress fibers, observed staining with rhodamine-conjugated phalloidin, were organized better in a low-metastatic cell line (F = 1) than in a high-metastatic one (F = 10). These results suggest to us that depression of AX actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.     

抑制整合素α9基因的表达对黑色素瘤细胞B16F1的生长和肺转移的影响

目的 观察shRNA干扰整合素α9（ITGA9）的表达对黑色素瘤细胞B16F1的生长和肺转移的影响。方法 用RNA干扰技术下调B16F1中ITGA9的表达,建立小鼠皮下成瘤和肺转移模型,观察肿瘤生长情况,计数肺转移灶数量。结果 ITGA9-shRNA转染组的肿瘤生长速度减慢（P＜0.05）,实验终点,该组肿瘤平均体积与scramble-shRNA组相比下降36%;肺转移灶数量显著减少（P＜0.05）。结论 下调ITGA9的表达可抑制黑色素瘤细胞B16F1在小鼠体内的生长和肺转移。ITGA9可能成为黑色素瘤的治疗靶点。     

High levels of Met-72 antigen expression: correlation with metastatic activity of B16 melanoma tumor cell variants

In the present study, both poorly and highly metastatic clones derived from the C57BL/6 mouse B16 melanoma were used with cyclophosphamide in an attempt to elicit host antibody responses against cell surface markers expressed on highly metastatic tumor variants. The immunizations, performed in both syngeneic and xenogeneic combinations in Lewis rats, resulted in the production of 3 mouse and 2 rat monoclonal antibodies (MoAb) that preferentially react with highly metastatic clones derived from the B16 melanoma. These MoAb all immunoprecipitated a 72,000-dalton, cell surface-expressed glycoprotein, referred to as Met-72. In this study, 2 of the mouse anti-Met-72 MoAb were examined in detail for a) tumor specificity, b) reactivity against normal mouse tissue by in vivo absorption, and c) their ability to discriminate highly metastatic clones derived from the B16 melanoma.     

Type-1 transforming growth factor-beta differentially modulates tumoricidal activity of murine peritoneal macrophages against metastatic variants of the B16 murine melanoma

Transforming growth factor-beta 1 (TGF-beta 1) renders mouse peritoneal macrophages tumoricidal against metastatic variants of the B16 mouse melanoma in vitro. Both direct cytotoxicity and indirect cytotoxicity were observed. A subthreshold concentration (10 U/ml) of recombinant murine interferon-gamma (rMuIFN-gamma) enhanced the direct tumoricidal activity of TGF-beta 1-activated macrophages from 29% to 88% but did not change their indirect tumoricidal profile. Data obtained from macrophages preincubated with either TGF-beta 1 or rMuIFN-gamma showed that TGF-b1 can initiate tumoricidal activity better than rMuIFN-gamma. These effects were plasma-membrane mediated because targeting macrophages with liposomal TGF-beta 1 was ineffective. The order of tumoricidal susceptibility of the B16 melanoma lines to activated macrophages was B16F1 > B16F10 > B16BL6, in inverse order of metastatic potential.     

Altered Expression of a Third Actin Accompanying Malignant Progression in Mouse B16 Melanoma Cells

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two-dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found A^x actin (Mr = 43,000, pl = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high-metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony-forming ability following iv administration, increased with increase in the F number. The half life of A^x actin was much the same as that of β-and γ-actin and the different expressions of A^x actin between the low- (F=l) and high-metastatic (F=10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of A^x actin. The A^x actin was incorporated into the cytoskeletal fraction with the same efficiency as β-and γ-actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in A^x expression. The actin stress fibers, observed staining with rhodamine-conjugated phalloidin, were organized better in a low-metastatic cell line (F=l) than in a high-metastatic one (F = 10). These results suggest to us that depression of A^x actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.     

A new in vitro assay for quantitating tumor cell invasion

The attachment to and penetration of basement membranes by tumor cells is required to complete the metastatic cascade which culminates in the establishment of secondary tumor foci. Therefore, basement membranes are critical barriers to the passage of disseminating tumor cells. We have developed a simple, in vitro model using matrigel-coated transwell chambers (Costar) for use in a tumor cell invasion assay. Two variants of the K1735 UV-induced murine melanoma cell line were assayed for their invasive capabilities and compared with their ability to colonize the lung in an experimental metastasis assay. The K1735-M2 cells, which are highly metastatic in vivo, invaded through basement membrane matrigel at a significantly higher rate than the low metastatic cells, K1735-16, in a 72-hour assay. As a negative control, normal murine fibroblasts were incapable of penetrating the barrier. Tumor cell invasion in vitro correlated with lung colonization in vivo. Therefore, this model may provide a valuable tool to study the mechanisms involved in the pathogenesis of tumor cell invasion during hematogenous dissemination.