DNA amplification and metastasis of the human melanoma cell line MeWo

A positive correlation was found between the presence of amplified DNA in the form of homogeneously staining regions (HSRs), and the formation of spontaneous metastases by the human melanoma cell line MeWo. HSR+ and HSR- MeWo sublines and clones were injected s.c. into BALB/c nude mice. All MeWo lines produced primary tumors that were allowed to grow to a similar size before the animals were sacrificed and examined for the presence of metastatic nodules in the lungs and abdominal cavity. HSR+ lines gave extensive metastases (greater than 100 nodules) in the lungs and/or liver and abdomen in 18 of 19 animals. The HSR- MeWo lines were effectively nonmetastatic, producing metastases in 3 of 20 animals, two of which had only a single metastatic lung nodule each. Evidence for the presence of HSRs in the primary tumors and metastatic nodules was obtained by DNA dot-blot hybridization to a sequence (D15Z1) amplified in the HSRs, flow cytofluorometry for cellular DNA content, and quinacrine staining of metaphase spreads. The HSR+ clones also colonized the lung to a much greater extent than HSR- clones following i.v. injection. In addition, the HSR+ clone had a selective advantage in lung colonization, since i.v. injection of a 50:50 mixture of HSR+ and HSR- clones resulted in extensive metastases populated exclusively by HSR+ cells. The results suggest that DNA sequences amplified in the HSRs of human melanoma MeWo cells may confer enhanced metastatic properties to these cells.     

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.     

Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns

The apathogenic Newcastle disease virus (NDV) strain Ulster has been used successfully as an adjuvant component for active specific immunotherapy of malignant mouse lymphoma, and in nude mice it was shown to be able to lead to retardation of the s.c. growth of xenotransplanted human melanoma cells. In order to improve in vivo effectiveness of virotherapy of human tumors without significantly increasing the risk of unspecific viral replication in host cells, we adapted the virus for growth in a human melanoma line (MeWo M). For this purpose NDV Ulster was mutagenized and a variant was selected which could replicate and reinfect the tumor line. The mutant (NDV 1E 10) performed late lysis on the melanoma line. Replication was found to be at least 100 times more efficient in MeWo M than in 6 of 8 other human tumor cell lines of different tissue origin. In 10 of 11 murine cell lines, NDV 1E 10 did not replicate via multicycles. Chick embryonic fibroblasts were permissive for nonlytic replication. Neither the virulent wild-type NDV Italian nor the avirulent strain NDV Ulster shared these specific replication properties with the new variant. We also established MeWo melanoma sublines with different metastatic capacities and tested them as targets for NDV 1E 10 infection. The MeWo subpopulations exhibited comparatively small differences in permissivity for multicyclic replication, but the more metastatic MeWo Met, like allogeneic melanoma lines, was more resistant to lysis. NDV Italian, in contrast, showed no differences in replication and lysis on any of the tested melanoma lines. Trypsin-activation experiments suggested an incomplete cleavage of mutant envelope glycoprotein F by the permissive cell line and, thus, mechanisms of specific infection and replication not requiring fully activated envelope glycoproteins.     

Relative tumorigenicities of hybrid cells with and without HSR-bearing chromosomes from a human melanoma cell line

Some cell types within the human melanoma cell line MeWo contain homogeneously staining regions (HSRs) consisting of repetitive DNA sequences and ribosomal RNA (rRNA) genes derived from chromosome 15. To further examine the association between enhanced tumorigenicity and the presence of HSR-bearing chromosomes, hybrid cell lines were constructed by fusing X-HSR-containing MeWo cells with ouabain-resistant, HPRT-deficient Chinese hamster ovary cells and culturing in HAT medium containing ouabain. A hybrid containing the X-HSR chromosome and several MeWo chromosomes was more tumorigenic in BALB/c nude mice than derivative cells lacking the X-HSR and human chromosome 18. However, since this enhanced tumorigenicity could be due to sequences on either the X-HSR or chromosome 18, a second series of hybrids was constructed by micro-cell fusion. In this case, the tumorigenicity of hybrid cells containing 2 copies of the X-HSR as the only MeWo chromosome was similar to that of derivative cells lacking these chromosomes. Cytogenetic analysis revealed that the nucleolar organizer regions (NORs) on the HSR were inactive in the hybrid cells. Our data indicate that DNA sequences amplified on MeWo HSRs do not enhance tumorigenicity under experimental conditions in which rRNA genes are not expressed. As the only active NORs in MeWo HSR-containing cells are on the HSRs, we suggest that expression of these amplified rRNA genes is responsible for the selective growth advantage of these cell types in nude mice. Our data also indicate that the enhanced tumorigenicity of MeWo HSR-containing cells is not due to co-amplification of a dominant oncogene.     

Enhanced tumorigenicity, melanogenesis, and metastases of a human malignant melanoma after subdermal implantation in nude mice

Transplantation of human tumors into the organ or tissue of their origin (orthotopic transplantation) in nude mice can result in significant enhancement of tumor growth and metastases, compared with sc (ectopic) transplantation. Because melanocytes are normally found in the epidermal-dermal junction, intradermal inoculation of melanoma cells might be expected to improve their potential for malignant growth as xenografts. The purpose of our study was to examine this possibility. We found that because mouse epidermis and dermis are so thin, it was not possible to inject a bolus of tumor cells intradermally; instead the cells were actually deposited in the most superficial layer of the subcutis (i.e., subdermally). We evaluated the behavior of cells from a human melanoma cell line after sc or subdermal inoculation into National Institutes of Health Swiss athymic nude mice. The cells used were from (1) the predominantly amelanotic human malignant melanoma cell line MeWo, originally established from a melanotic lymph node metastasis, and (2) two MeWo variants resistant to wheat germ agglutinin (WGAr), which were selected for altered malignant capacities. Whereas 5 X 10(5) MeWo cells were required to achieve 100% tumor take with sc injection, only 2 x 10(4) cells were required with subdermal inoculation. Subdermal injection of the MeWo cells resulted in the development of highly melanotic and nonencapsulated primary tumors, which grew quickly into the dermis and epidermis and metastasized at high frequency to draining lymph nodes. In contrast, the tumors that developed after sc injection were found in the deepest layer of the subcutis and were predominantly amelanotic and encapsulated; they rarely metastasized to lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for selection of homogeneously staining regions in a human melanoma cell line

Cytogenetic analysis of tumor cells from a human malignant melanoma was performed on both the primary tumor colony-forming cells and a cell line (HA-A) established subsequently from the clonogenic population. Chromosome-banding analysis demonstrated identical karyotypic alterations in both the tumor colony-forming cells and the HA-A cell line, documenting their origin from a common precursor. The most distinctive chromosome alterations shared between tumor colony-forming cells and the HA-A cell line were double-minute bodies (DMs) and a homogeneously staining region (HSR) on chromosome 7 at band p22. This represents the first observation of DMs or HSRs in cells from a human malignant melanoma. The frequency of HSR-bearing cells observed in the original tumor was less than 1%, while DM-containing cells were present in greater than 90% of all cells examined. In contrast, serial chromosome harvests at early passage of the HA-A cell line revealed positive selection for HSR-bearing cells with concomitant loss of DM-containing cells after increasing time in vitro. Following the ninth serial passage in vitro, HSRs were observed in 100% of cells with DMs no longer observed in the HA-A cell line. The finding of an HSR-bearing marker in the original tumor sample supports the view that HSRs are not an artifact of in vitro monolayer growth. However, our results demonstrate that the frequency of HSR-bearing cells within established cell lines may reflect in vitro selection and therefore not accurately reflect the frequency of this population in vivo.     

Enhanced natural killer sensitivity with concomitant clonal selection for cells bearing homogeneously staining regions in the human melanoma cell line MeWo upon induction of differentiation with theophylline

Culture of the human melanoma cell line MeWo in the presence of 1 mM theophylline was associated with an increase in susceptibility to natural killer (NK)-mediated cytolysis. The phenomenon was detected as early as 72 hours after initiation of theophylline treatment, reaching maximum values at 3-4 weeks and remaining stable for longer than 3 months of testing, provided the cells were maintained in the presence of theophylline. The alteration in target sensitivity was selective for NK-mediated cytolysis, since other mechanisms of cell-mediated cytolysis, including antibody-dependent cell-mediated cytotoxicity and monocyte-mediated and lectin-induced cytolysis, were comparable between untreated and treated cells. The enhanced susceptibility of theophylline-treated cultures to NK lysis, as compared to NK lysis susceptibility of untreated MeWo cells, was not significantly changed by pretreatment of effector lymphocytes with interferon. Evidence for differentiation in theophylline-treated cultures was obtained. In addition, however, cytofluorometric and karyologic analysis revealed the existence of two subpopulations of differing ploidy in the MeWo line. The hypodiploid, NK-sensitive subpopulation, bearing homogeneously staining regions on two chromosomes, could be selected by growth in theophylline. Therefore, selection of subpopulations in heterogeneous tumor cell lines by chemical inducers suggests an alternative and novel mechanism for enhancement of NK sensitivity.     

Altered methionine metabolism in metastatic variants of a human melanoma cell line

In order to identify the biochemical defect(s) responsible for the reduced levels of DNA 5-methylcytosine (5-mCyt) found within highly metastatic (in athymic "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo, the ability of these cells to grow in culture medium devoid of exogenous methionine but containing either homocysteine (Hcy) or 5'-deoxy-5'-methylthioadenosine (MeSAdo) was determined. In contrast to the parental MeWo tumor line, many (but not all) of these malignant variants were completely unable to proliferate in methionine-free homocysteine-containing medium. Many of these malignant variants also exhibited a reduced ability to proliferate in methionine-free MeSAdo-containing medium. Cell lines established from "artificial" metastases of MeWo or its cloned sublines, exhibited no consistent reduction in their ability to grow in methionine-free medium containing either Hcy or MeSAdo. These observations suggest that alterations in S-adenosylmethionine(AdoMet)-dependent transmethylation reactions may contribute to "progression" of the MeWo tumor from a relatively benign to a highly autonomous and malignant state.     

Correlation of growth morphology and clonability with malignancy of WEHI-7 T-cell lymphoma sublines

This study examined the development of subclones of different proliferative capacity and malignancy in the WEHI-7 tumor. The mouse T-cell lymphoma WEHI-7 can be cloned in agar with a cloning efficiency of 40 to 50%. On the basis of growth morphology, two types of colonies were distinguished. Most colonies were compact, but a few, no more than 5%, were diffuse. Sublines of the two colony types were established. The cloning efficiency in agar was 40 to 50% for the compact and 15 to 25% for the diffuse sublines. The recloning efficiency did not change for compact colonies. In contrast, the cloning efficiency of diffuse colonies decreased with repeated reclonings. The mice died within 24 to 34 days of i.v. injection of 10(4) WEHI-7 cells. The same number of cells of the individual sublines administered to mice resulted in improved survival. Injection of compact subline cells resulted in death in 24 to 48 days, while diffuse subline cells resulted in death in 30 to 75 days. The sublines were indistinguishable by light and electron microscopy. Both subline types were negative for terminal deoxynucleotidyl transferase and positive for Thy 1.2. Most cells from the two types of sublines were Lyt-1 and Lyt-2 positive. The doubling time was 10 hr for compact and 14 hr for diffuse sublines. Colony morphology was conserved after passage in vivo and after more than 10 transfers in vitro in liquid and agar media. In conclusion, the different growth morphology in vitro distinguished the subclones of different malignancy in the WEHI-7 tumor cell line.     

Analysis of gene expression profiles in melanoma cells with acquired resistance against antineoplastic drugs

Resistance to various antineoplastic agents is common in the clinical management of malignant melanoma. The biological mechanisms conferring these different drug-resistant phenotypes are still unclear. To identify potential factors mediating drug resistance to melanoma cells, the mRNA expression profiles of the parental drug-sensitive human melanoma cell line MeWo and four derived drug-resistant sublines with acquired resistance against four commonly used drugs for melanoma treatment (cisplatin, etoposide, fotemustine and vindesine) were analysed. We investigated cDNA arrays with 43,000 cDNA clones ( approximately 30,000 unique genes) to study the expression patterns of these cell lines. We were able to simultaneously extract new candidate genes associated with drug resistance in malignant melanoma and to correlate the present findings with previously described resistance-associated genes. Using hierarchical clustering and analysing the overlap of genes with altered expression, we detected similarities between the expression signatures related to cisplatin and fotemustine resistance. The resistance against vindesine required a minimal set of changes in gene expression relative to the parental MeWo cell line. Our study provides new data that may be used to obtain further insight into the resistance characteristics of malignant melanoma.     

Combined effect of proteasome and calpain inhibition on cisplatin-resistant human melanoma cells

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.     

Susceptibility of chemoresistant murine and human tumor cells to lysis by interleukin 2-activated lymphocytes

The sensitivity of three different human and murine doxorubicin (Dx)-sensitive or -resistant pairs of tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes was studied. In two pairs of these sublines (LoVo human colon carcinoma and B16 mouse melanoma sublines), resistance to Dx was induced in vitro, while in the third pair (9229 human metastatic melanoma clones), Dx resistance was spontaneously present in clone 9229.24. Dx-resistant cells were efficiently lysed by rIL2-activated lymphocytes in a short-term 51Cr release assay; in some experiments a trend toward higher lysis of Dx-resistant cells was present. We then tested the tumor cell growth-inhibitory activity of rIL2-activated lymphocytes in the human tumor clonogenic assay after lymphocyte-tumor coculture. Complete inhibition of tumor cell growth was obtained with five of six sublines or clones (both Dx sensitive and resistant) after 3 to 6 days of coculture at effector lymphocyte/target tumor cell ratios of 5 to 50/1; a maximum 99% inhibition was observed with the melanoma clone 9229.4 even after coculture for 6 days at an effector lymphocyte/target tumor cell ratio of 50/1. By using lower effector lymphocyte/target tumor cell coculture ratios (1, 5, 25/1), it was shown that all the three Dx-resistant cell types were significantly more affected by activated lymphocytes than their Dx-sensitive counterparts. The LoVo/DX subline was also more lysed than its Dx-sensitive counterpart LoVo/H subline by an antitumor monoclonal antibody in a complement-mediated cytotoxicity assay, despite the fact that both sublines expressed a similar amount of antigen on the cell surface. These data indicate that Dx-resistant cancer cells are more susceptible to the lysis by rIL2-activated lymphocytes than their Dx-sensitive counterparts and that a complete inhibition of their clonogenic potential can be obtained in vitro.     

Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.     

Reduced levels of DNA 5-methylcytosine in metastatic variants of the human melanoma cell line MeWo

The total levels of DNA 5-methylcytosine were determined in a series of related highly metastatic cell lines which had been isolated from the poorly metastatic human melanoma tumor line MeWo. The procedure used to quantitate DNA 5-methylcytosine involved labeling of DNA with [6-3H]uridine, hydrolysis with formic acid, and separation of cytosine and 5-methylcytosine bases by high-performance liquid chromatography. The DNA 5-methylcytosine content of the parental MeWo tumor was 3.07 +/- 0.16%. Metastatic variants of MeWo which had been isolated using a combination of in vitro and in vivo selection procedures had DNA 5-methylcytosine levels between 2.45 +/- 0.15% and 2.75 +/- 0.09%. Metastatic variants which were isolated using a single step in vivo selection procedure exhibited DNA 5-methylcytosine levels of 1.92 +/- 0.02% to 2.68 +/- 0.09%. No consistent decreases in DNA 5-methylcytosine content were found in "artificial" metastases arising in athymic "nude" mice following the i.v. inoculation of MeWo or its cloned sublines. These observations provide further support for the hypothesis that alterations in the cytosine methylation of DNA may play an important role in the generation of tumor cell heterogeneity and the progression of tumors from relatively benign to highly malignant states.     

Coamplification of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in methotrexate-resistant human leukemia cell lines

Methotrexate (MTX)-resistant K562 human myelocytic leukemia sublines with 20- and 200-fold amplified dihydrofolate reductase (DHFR) genes localized to homogeneously staining regions (HSRs) on the long arms of chromosomes 5, 6, and 19 were used to examine whether other genes mapping close to the DHFR genes were coamplified. The gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, located on chromosome 5q13.3-14, was coamplified 4-14-fold, corresponding to the levels of resistance exhibited by these cells. Similar observations were made with a MTX-resistant subline of the promyelocytic leukemia cell line, HL-60R, with 200 gene copies of DHFR. These observations indicate a tight linkage of DHFR and HMG-CoA genes on chromosome 5q.     

MTA1基因表达与人骨肉瘤细胞浸润和转移的关系

Objective： To compare the expression level of metastasis associated-1 （MTA1） in the higher and lower metastasis sublines of human osteosarcoma cells （MG63）, and to investigate the relationship between the expression level of MTA1-EGFP and in vitro invasion and metastasis of human osteosarcoma cells. Methods： The expression level of MTA1 in two sublines of MG63 cells was detected by semi-quantitative RT-PCR, and cell invasion assay and cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines. The lower metastasis line of MG-63 cells were transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine. The changes of the MTA1-EGFP expression and in vitro invasion potential were measured after transfection. Results： M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentials of low metastasis MG63 cell line were increased after MTA1 gene transfection. Conclusion： There may be a relationship between MTA1 and invasive potentials of human osteosarcoma cells, and MTA1 may play a role in the molecular mechanism of tumor metastases and be a potential target for gene therapy of osteosarcoma. Further studies of MTA1 in human ostersarcoma cell metastasis are needed.     

Highly pigmented human melanoma variant which metastasizes widely in nude mice, including to skin and brain

The properties of a highly malignant human melanoma variant cell line which metastasizes in nude mice in a tissue-specific pattern are described. The variant, called 70-W, was isolated from the MeWo malignant melanoma by exposure of the latter to stepwise increasing concentrations of the toxic lectin, wheat germ agglutinin. After nine cycles of treatment a population of wheat germ agglutinin-resistant cells was obtained that manifested a 4-fold resistance to wheat germ agglutinin, a property which was found to be stable in culture for over 6 months in the absence of the lectin. Intravenous inoculation of 70-W cells into 4-6-week-old nude mice revealed remarkable differences in metastatic (organ colonization) behavior. Whereas the parent MeWo cells gave rise only to lung metastases, most of which were amelanotic, injection of the 70-W cells resulted in multiple skin (s.c.) and brain and, to a lesser extent, bone marrow, ovarian, mesenteric (gut-associated), muscle, and abdominal metastases all of which were highly melanotic. This is the first report of brain metastases of a human tumor in nude mice. They were found to be bilateral and confined to the deeper layers of the cerebral cortex. The unique malignant behavior of 70-W cells in nude mice should facilitate studies of host and tumor cell factors involved in human melanoma metastasis, melanogenesis, and development of new treatment strategies for disseminated human malignant melanoma.     

Analysis of the Cell Membrane Proteolytic Enzymes of the B16, Fl, F10, and BL6 Melanoma and Their Role in Target Cell Destruction

The tumor-induced red blood cell (RBC) cytolysis assay has been used to demonstrate that three B16 melanoma sublines, the Fl, F10, and BL6, cause the cytolysis of normal red blood cells in vitro. RBC cytolysis was inhibited for all three sublines by metalloprotease inhibitors. Cell membrane preparations have been prepared for all three sublines and tumor cell membrane-induced RBC cytolysis was also shown to be inhibited by metalloprotease inhibitors. The FIO and BL6 sublines were shown to have cell membrane-bound proteases. The BL6 subline has a cell membrane enriched in an enzyme with a trypsin-like arginine specificity. The trypsin-like protease may have a metal dependence. The BL6 subline has a collagenolytic cell membrane enzymes and a chymotrypsin-like cell membrane enzyme. B16 cell membrane enzymes may be responsible for RBC cytolysis in vitro in a process requiring divalent cations.     

Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production

The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis-associated cellular properties of human and mouse melanoma cells. Treatment of early-passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density-dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time- and dose-dependent induction of heparanase, a heparan-sulfate-specific endo-β-D-glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain-colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species-specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain-colonizing murine B16-B15b variant (B16-B15b > B16-BL6, B16-F10, B16-F1). NGF also increased the invasive capacity of the human 70W and murine B16-B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low- and high-affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm-derived melanoma cells.     

Brain surface invasion and metastasis of murine malignant melanoma variants

Mouse B16 melanoma sublines were selected sequentially for their abilities to colonize brain meninges and leptomeninges of C5713L/6 mice. After 14 selections subline 1316-B14b was established that formed significantly more brain tumor colonies than the parental B16 line. Examination of brains at various times after intravenous or intra-arterial injection of B16 cells by electron microscopy revealed that B14b melanoma cells lodged in small brain blood vessels, proliferated and invaded through vessel walls into brain parenchyma and also along small blood vessels at perivascular sites. Invasion into brain parenchyma was characterized by extension of melanoma cell filopodia resulting in fragmentation and sometimes enfulgment of glial and neural cells.Analysis of cell surface proteins of B16 melanoma sublines revealed increased exposure of a M_r 90 000 glycoprotein on the high brain-colonizing cells. Antibodies against the M_r 90 000 glycoprotein reacted with a variety of human melanoma cell lines and with some fetal and adult tissues, indicating that this melanoma-associated component is not species-, tumor- or tissue-specific. The glycoprotein could be a cell surface receptor important in the survival and growth properties of melanoma cells in brain microenvironments.