Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

Newly identified type of beta actin reduces invasiveness of mouse B16-melanoma

Low metastatic parent B16 melanoma and isolated B16-F1 cell lines have a third actin designated as beta m(Ax:previously). beta m actin is scantily or not at all detected in highly metastatic cell lines, such as B16-F10 and BL6. To directly assess the physiological role of beta m in phenotypic changes of B16 melanoma, we transfected expression plasmids of beta m into B16-F10 cells. The actin expressed in the transfectants is located largely in cytoskeletal fractions. The transfectants exhibited a larger number of stress fibers and a lower invasiveness than did the recipient cells. Thus, beta m actin plays an important role in the organization of actin stress fibers, the result being a decrease in invasiveness of B16 melanoma.     

Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines.

Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

The receptor for alpha-melanotropin of mouse and human melanoma cells. Application of a potent alpha-melanotropin photoaffinity label

The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.     

Ultraviolet-A radiation induces changes in cyclin G gene expression in mouse melanoma B16-F1 cells

Background  

We have previously shown that ultraviolet-A (UVA) radiation enhances metastatic lung colonization capacity of B16-F1 melanoma cells. The aim of this study was to examine changes in expression profile of genes in mouse melanoma B16-F1 cells exposed to UVA radiation.     

Effect of PKC412, a selective inhibitor of protein kinase C,on lung metastasis in mice injected with B16 melanoma cells

PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 microM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16-F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 microM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or platelet-aggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent.     

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

Differences in expression of a variant actin between low and high metastatic B16 melanoma

The polypeptides of mouse B16 melanoma lines of defined metastatic potential have been analyzed by two-dimensional electrophoresis. Parent B16 melanoma and two independently isolated B16-F1 lines, which are low metastatic, exhibited a new polypeptide, Ax (pI 5.2; Mr = 43,000), comprising approximately 30% of the total actin, in addition to normal beta- and gamma-actin. The Ax is present in the Triton-insoluble fraction (cytoskeleton and nuclear matrix) as well as in the Triton-soluble fraction at a constant ratio of about 0.5 to beta- plus gamma-actin. The Ax polypeptide has been identified as a variant form of actin by immunostaining with anti-actin antibody and by a comparison of its tryptic patterns with those produced by beta- and gamma-actin polypeptides; the Ax is also identified as a component of microfilaments. On the other hand, the Ax polypeptide disappears or its expression is very low in high metastatic lines, two independently isolated B16-F10s and B16-BL6. By in vitro translation, we have identified the mRNA species that code for Ax in B16-F1, but not in B16-F10.     

The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of ¹²⁵I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.     

Expression of the cell adhesion molecules N-CAM and L1 in B16 melanoma cells

The cell adhesion molecules N-CAM and L1 are important for cell-cell recognition and cell migration and so may be involved in the metastatic process. We have studied the biosynthesis of N-CAM and L1 in the B16 melanoma cell lines B16-F1 and B16-F10 which differ in metastatic capacity. N-CAM was synthesised as two glycosylated polypeptides with Mr of 150,000 and 210,000; L1 was synthesised as one polypeptide with Mr of 215,000. In fetal neurons N-CAM is synthesised as a 135,000 and a 200,000 Mr polypeptide and L1 as a 200,000 Mr polypeptide. Thus, the Mr of N-CAM and L1 in tumour cells appeared to be 10,000-15,000 higher than in the normal cells. L1 was phosphorylated in the tumour cells as in neurons. The tumour cells also phosphorylated the 210,000 Mr N-CAM polypeptide, whereas no phosphorylation of the 150,000 Mr polypeptide was observed. In neuronal cells both the corresponding polypeptides are phosphorylated and thus the biosynthesis of N-CAM in tumour cells seem to differ from that in neuronal cells with regard to phosphorylation. No differences in biosynthesis of N-CAM or L1 were apparent between the two tumour cell lines, B16-F1 and B16-F10.     

Caveolin-1-enhanced motility and focal adhesion turnover require tyrosine-14 but not accumulation to the rear in metastatic cancer cells

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.     

Enhancing CTL responses to melanoma cell vaccines in vivo: synergistic increases obtained using IFNgamma primed and IFNbeta treated B7-1+ B16-F10 melanoma cells

Sequentially treating human melanoma cell lines by priming with interferon-gamma before adding interferon-beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7-1, intercellular adhesion molecule-1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule-based protocol is applied to murine B16-F10 melanoma cells as well as preclinical studies using the B16-F10 model as a poorly immunogenic melanoma. Thus, treating B16-F10 cells or a highly expressing B7-1 transfected subline (B16-F10/B7-1 hi) by priming with interferon-gamma for 24 h before adding interferon-beta for a further 48 h (interferon-gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon-gamma 72/beta 48 treated B16-F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16-F10 cells expressing high levels of B7-1 and treated using the interferon-gamma 72/beta 48 protocol (interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7-1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16-F10 cells with tenfold higher killing than for syngeneic EL-4 lymphoma cells. Killing proceeded via the perforin-mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi vaccine in vivo remained tumour free after challenge with 5 x 105 live B16-F10 cells expressing intermediate B7-1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.     

Effect of <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> on TIMP-1 secretion from mouse melanoma cell

Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.     

Interaction of tumor and surrounding tissue of mice inoculated B16 melanoma variants in terms of enzyme activity

1. The interactions of B16-F1 and B16-F10 tumors with their surrounding tissues in terms of enzyme activities such as cathepsin B, hemoglobin(Hb)-hydrolase, acid phosphatase, beta-glucuronidase and plasminogen activator were investigated when said tumors proliferated locally and at secondary sites throughout the host's circulatory system. 2. In the case of B16-F1 and B16-F10 tumor cells proliferating under the skin, statistical differences were not detected between the enzyme activities of the skin surrounding the tumors and control skin, nor between B16-F1 and B16-F10 tumors, except for beta-glucuronidase. 3. In the case of B16-F1 and B16-F10 tumor cells metastasizing to lung, statistical differences were detected between numerous enzyme activities of the lung tissues surrounding the tumors and control lung tissue, and also between B16-F1 and B16-F10 tumors. 4. The activities of cathepsin B and acid phosphatase of lung tissue surrounding B16-F1 tumor were lower than those of the control lung. 5. beta-Glucuronidase activity of lung tissue surrounding B16-F10 tumor was higher than that of the control lung. 6. The activities of cathepsin B, Hb-hydrolase and beta-glucuronidase of the B16-F10 tumor were higher than those of the B16-F1 tumor. 7. Results indicate that metastasized B16 melanoma tumor cells interact with surrounding lung tissues, and that cathepsin B, Hb-hydrolase and beta-glucuronidase might play important roles in the metastasis of the malignant tumor.     

Secretion of metastasis related gangliosides by mouse B16-melanoma in circulation in vivo and in culture media in vitro

Mouse B16LuF1 melanoma cells of lower metastatic potential to lung were treated in vitro with same concentration (50 microM) of gangliosides prepared from plasma of mice bearing lung metastasis of B16LuF5, B16LuF9 or B16LuF10 melanoma cell lines of increasing metastatic potential to lung (LuF1 < LuF5 < LuF9 < LuF10) and injected to normal mice through tail vein. The number of metastatic tumor nodules formed in lung increased gradually in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment. Similarly, mouse B16LuF1 melanoma cells treated in vitro with 50 microM concentration of gangliosides prepared from spent culture media of B16LuF5, B16LuF9 or B16LuF10 cells cultured in vitro were injected to normal mice through tail vein. The number of metastatic tumor nodules formed in lung increased gradually in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment. The results indicated that metastasis-associated gangliosides present in plasma and culture media of B16-melanoma of increasing metastatic potential to lung enhanced metastatic potential of B16LuF1 cells. The increasing concentration of metastasis-associated gangliosides present in plasma and in culture media of B16-melanoma of increasing metastatic potential possibly determined increase in metastatic potential of B16LuF1-melanoma cells.     

Inhibition by tyroserleutide (YSL) on the invasion and adhesion of the mouse melanoma cell

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 microg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells.     

Cuprous oxide nanoparticles inhibit the growth and metastasis of melanoma by targeting mitochondria

Metal and its oxide nanoparticles show ideal pharmacological activity, especially in anti-tumor therapy. Our previous study demonstrated that cuprous oxide nanoparticles (CONPs) selectively induce apoptosis of tumor cells in vitro. To explore the anti-tumor properties of CONPs in vivo, we used the particles to treat mouse subcutaneous melanoma and metastatic lung tumors, based on B16-F10 mouse melanoma cells, by intratumoral and systemic injections, respectively. The results showed that CONPs significantly reduced the growth of melanoma, inhibited the metastasis of B16-F10 cells and increased the survival rate of tumor-bearing mice. Importantly, the results also indicated that CONPs were rapidly cleared from the organs and that these particles exhibited little systemic toxicity. Furthermore, we observed that CONPs targeted the mitochondria, which resulted in the release of cytochrome C from the mitochondria and the activation of caspase-3 and caspase-9 after the CONPs entered the cells. In conclusion, CONPs can induce the apoptosis of cancer cells through a mitochondrion-mediated apoptosis pathway, which raises the possibility that CONPs could be used to cure melanoma and other cancers.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16–010 cells grew in circular in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melainin pigment (B16-010 < B16-05 < B16-01 ? B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.     

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.