Homologous recombination in variants of the B16 murine melanoma with reference to their metastatic potential

Genomic instability has been accepted as providing a phenotypic variety of malignant cells within a developing tumour. Defects in genetic recombination can often lead to phenotypic differences; therefore, it is possible that metastatic variant cell lines exhibit their particular phenotype as a result of an altered ability to catalyse homologous recombination. We have investigated recombination efficiency in B16 melanoma metastatic variants, using a plasmid, pDR, as a recombination substrate. The plasmid contains two truncated, nontandem but overlapping segments of the neomycin resistance gene (neo 1 and neo 2), separated by the functional gpt gene unit. Only a successful recombination of the two neo segments will generate a functionally intact neomycin gene. Extrachromosomal recombination here was a transient measure of the cells to recombine the neo fragments in an intra- or intermolecular manner. Extrachromosomal recombination frequencies were higher in the high metastasis variants (BL6, ML8) compared with the low metastatic F1 cells. On the other hand, the frequency of chromosomal recombination (after plasmid integration) was higher for the low metastasis (F1) cell line compared with the highly metastatic variants, BL6 and ML8. Since the recombination assay measures only successful recombination events, we have interpreted the observed higher incidence of chromosomal recombination in the low metastatic variant line as indicative of a more stable genome. Similarly, a higher inherent instability in the genome of the high metastasis variants would render these less efficient at producing and maintaining successful recombination events, and this was found to be true by Southern analysis. The results presented show that frequency of recombination may be adduced as evidence for implicating genomic instability in the generation of variant cell populations during metastatic spread. Such an interpretation is also compatible with the Nowell hypothesis for tumour progression. © 1996 Wiley-Liss, Inc.     

The cell surface sulphydryl content of metastatic variants of B16 murine melanoma

The cell surface sulphydryl content of three metastatic variants of the B16 murine melanoma has been determined using isoelectric equilibrium techniques. The F1 variant, which has no ability for natural metastasis, and the F10 variant with moderate metastatic ability appeared to have no detectable surface thiol groups. The variant BL6, which shows a high degree of natural metastasis, possessed surface thiol groups. The variants were found to be heterogeneous in isoelectric distribution. Three subpopulations were identified based on isoelectric criteria. The size of the pI 5.0 subpopulation appeared to increase with metastatic ability. A proportion of this subpopulation, approximately 4% in the F10 and 10% in the BL6, was found to possess surface thiol groups. In the BL6 line, 10-20% of the pI 4.6 subpopulation possessed surface thiol groups. The surface negative charge density of the cell types showed no correlation with their natural metastatic ability.     

DNA repair and repair fidelity in metastatic variants of the B16 murine melanoma

We have examined the concept of genomic instability in relation to the metastatic progression of low (F1) and high metastasis (BL6, ML8) clones of the B16 mouse melanoma, by using a mutation assay, and DNA strand break repair and repair fidelity assays. The frequency of induced ouabain resistant colonies between the variant cell lines was consistent with the difference between their metastatic properties. Survival data for X-irradiation and bleomycin were similar among the 3 cell lines. When X-rays or bleomycin were used to induce strand breakage, no difference was detectable in either the rate or extent of DNA repair using the techniques of alkaline unwinding and alkaline elution for total strand breaks, and neutral elution for double strand breaks. DNA repair fidelity was measured using the PMH16 plasmid. A Kpn I restriction site was used to introduce a break within the gpt gene of the plasmid, prior to transfection. We found that ～ 100% and ～ 65% of the highly metastatic ML8 and BL6 clones, respectively, religated the gene with the required fidelity, compared with only ～ 25% of the low metastasis F1 clones. In summary, the metastatic variants show similar sensitivities to X-irradiation and bleomycin, but a differential response to EMS. This difference is not reflected in any subsequent DNA strand break religation, but the variants do differ in their fidelity of repair. However, although the fidelity of DNA religation is related to metastatic potential, it is not consistent with the mutation frequency data. © 1993 Wiley-Liss, Inc.     

New variants of B16 mouse melanoma: differentiation and metastatic properties

A system of tumor transplantation has been developed to select metastatic variants of B16 in mutants of the C57BL/6J black strain of mice. The effects of transplantation into nonagouti a/a and mutant recipients on the production of melanin and on the metastatic potential of tumors were investigated. Transplantation of the pigmented B16 melanoma from a nonagouti black a/a host to a yellow mutant Ay/a recipient resulted in an achromic and metastatic variant melanoma, designated YB16. The amelanotic phenotype occurred consistently after more than ten passages through yellow mice and simultaneously with an increase in the incidence of pulmonary metastases. When YB16 was transplanted back to the nonagouti black a/a host, a second variant, MB16, characterized by its variable pigmentation, was obtained. Pigmented and/or entirely achromic tumors were observed. MB16 was dramatically more metastatic than B16 and YB16 when injected s.c. or i.v. Metastases in the lungs were pigmented and/or achromic. The properties of tumor cells derived from artificially induced metastases were investigated after s.c. and i.v. injections. Whereas the metastatic cells expressed a potent ability to generate metastases when injected s.c., no differences in the incidence of metastases, as compared to the metastatic potential of cells of parental origin, were observed after i.v. injection. In the MB16 variant, there appeared to be an inverse relationship between differentiation (production of melanins) and malignancy. Our results demonstrate that differentiation and metastatic behaviour are dependent on specific mutations in the host environment which generate a pool of tumor cells from which highly metastatic variants can be selected.     

Cryotherapy of metastatic B16 melanoma. Consequences of the immunogenic potential

A significant inhibition of B16 melanoma growth and prolonged survival of mice, immunized with cryolysat at 10 M omega of B16 melanoma cells, are obtained in an immunoprophylactic assays. These results show that the freezing maintains tumor rejection activity of tumor antigen. Immunization with frozen cells or their crude butanol extract induce only an inhibition of tumor growth without a prolongation of survival.     

Differentiation of new metastatic variants of B16 melanoma under different culture conditions.

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow Ay/a, albino c/c, and C+/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5-S-CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C+/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.     

Immunogenicity and tumor protective activity of B16 melanoma vaccines

The immunogenicity and tumor-protective activity of different vaccines were examined and compared with murine B16 melanoma. All vaccines were prepared from material shed into culture medium by B16 melanoma cells. Vaccine I was generated by concentrating the shed material. Vaccine II was partially purified by precipitating the shed material with 50% ammonium sulfate followed by sephadex G-200 column chromatography. Vaccine III was concentrated shed material that was treated with 0.5% NP-40 and then ultracentrifuged to remove transplantation antigens. Mice were immunized to equal protein concentrations of vaccines weekly for 5 weeks or to control buffer. Antibody, cellular, and tumor-protective immunity to melanoma was measured in all mice 2 weeks following the last immunization. All three vaccine preparations were immunogenic. Vaccine preparation I appeared to be the most immunogenic and the one that most consistently augmented tumor-protective immunity. Augmentation in tumor-protective immunity correlated better with increase in cellular than in humoral immunity to melanoma.     

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.     

Correlation between post-replication repair and metastatic potential in B16 mouse melanoma cell lines

The rate of postreplication repair of the B16-F1 and the B16-F10 variant clones was compared to the parent B16CL4 mouse melanoma cells in an attempt to correlate the postreplication repair efficiency with the metastatic potential of these melanoma cells. The rate of postreplication repair of the B16-F10 subline was 47% higher than that of the parent B16CL4 mouse melanoma cells and 20% higher than that of the B16-F1 cells. This higher rate of postreplication repair in the B16-F10 cells correlates with its higher metastatic potential. It was also of interest to notice that the rate of postreplication repair of the B16-F1 and the B16-F10 cells are comparable to their rate of replicon joining in non-irradiated cells, in contrast to the parent B16CL4 cells whose rate of post-replication repair was significantly lower than its rate of replicon joining.     

Drug sensitivity of heat-resistant mouse B16 melanoma variants

Induction of transient thermotolerance by heat or other cytotoxic stressors has been reported to confer a moderate degree of drug resistance to tumor cells in vitro. In this study, a genetically stable, heat-resistant mouse B16 melanoma variant (W-H75) was tested for its sensitivity to various cytotoxic and antiproliferative agents. The heat-resistant W-H75 cells displayed a moderate two- to threefold resistance to doxorubicin, VP-16, VM-26, colchicine, cis-dichlorodiammineplatinum(II), HgCl2, and CdCl2. Marginal resistance to 4'(9-acridinylamino)methanesulfon-m-anisidide vinblastine, 1,3-bis(2-chloroethyl)-1-nitro-sourea, and NaAsO2 was observed, while no difference in sensitivity to the anticancer drugs, actinomycin D and camptothecin, was observed. Although W-H75 cells were generally more resistant than the parental cells to most of the agents that were tested, they were collaterally sensitive to the antimetabolites methotrexate and 6-mercaptopurine. Resistance of the W-H75 cells to epipodophyllotoxins and anthracyclines was not due to differences in steady-state drug accumulation. For the epipodophyllotoxin VP-16, resistance may be related to a relative decrease in the number of drug-induced DNA strand breaks in W-H75 cells. However, no difference in DNA strand breakage was observed between W-H75 and parental cells which were treated with doxorubicin, suggesting that resistance to this drug occurred by a different mechanism. The possible involvement of glutathione and glutathione S-transferase in resistance was also investigated. The glutathione content in W-H75 cells was 35% higher than that in the parental line. However, glutathione S-transferase activity appeared to be identical in both cell lines. Two other heat-resistant B16 melanoma variants, B-H103 and R-H92, were also tested for sensitivity to doxorubicin and VP-16. In contrast to the W-H75 cells, these two heat-resistant variants were hypersensitive to doxorubicin. The B-H103 cells were also hypersensitive to VP-16. This study suggests that selection for cellular resistance to heat may result in cells that have an altered sensitivity to drugs.     

Isolation and visualization of Met-72-positive, metastatic variants present in B16 melanoma tumor masses

Metastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000-dalton glycoprotein (Met-72) on their cell surface (Kimura AK, Xiang J: J Nat Can Inst 76:1247-1253, 1986). Monoclonal antibodies (MoAb) directed against the Met-72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met-72-positive metastatic variants within tumor masses. Biotinylated anti-Met-72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)-streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met-72-positive cells were then cloned and reanalyzed after several weeks of in vitro expansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met-72 staining were positioned perivascularly and at the invading front of B16 melanoma tumors.     

Reconstitution of the Gs protein from B16 melanoma clones of high and low experimental metastatic potential into S49 cyc-membranes

The ability of a series of B16 melanoma clones to form experimental lung metastases in syngeneic mice has been shown to correlate positively with adenylate cyclase activity. (Sheppard et al, Int. J. Cancer 37 (1986) 713-722). To begin to identify the components of the adenylate cyclase complex that account for enhanced enzyme activity in highly metastatic tumor populations, cholate extracts containing the GTP-binding protein GS from B16 melanoma clones of different metastatic capacities were reconstituted with membranes prepared from S49 cyc-, a variant lymphoma cell line that lacks GS function. The results revealed that extracts from a highly metastatic B16 clone (F10-C23) reconstituted significantly greater adenylate cyclase activities in S49 cyc- membranes than parallel preparations from a B16 clone (F1-C29) of low metastatic capacity. The data suggest that aberrations in GS function may contribute to the heightened responsiveness of adenylate cyclase observed in B16 melanoma clones of increased metastatic potential.     

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.     

Actin in B16 melanoma cells of differing metastatic potential : Effects of trypsin and serum

Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.     

Characterization of Mg2+-ATPase activity in isolated B16 murine melanoma melanosomes

In view of the accumulation of H₂O₂ in the myocardium due to ischemia-reperfusion and changes in -adrenoceptor mechanisms in the ischemic-reperfused heart, we investigated the effects of H₂O₂ on the -adrenoceptor, G-protein and adenylyl cyclase complex. Rat hearts were perfused with 1 mM H₂O₂ for 10 min before isolating membranes for measuring the biochemical activities. The stimulation of adenylyl cyclase by different concentrations of isoproterenol was depressed upon perfusing hearts with H₂O₂. Both the affinity and density of ₁-adrenoceptors as well as the density of the ₂-adrenoceptors were decreased whereas the affinity of ₂-adrenoceptors was increased by H₂O₂ perfusion. Competition curves did not reveal any effect of H₂O₂ on the proportion of coupled receptors in the high affinity state. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities were depressed by perfusing the heart with H₂O₂. Catalase alone or in combination with mannitol was able to significantly decrease the magnitude of alterations due to H₂O₂. The positive inotropic effect of 1 M isoproterenol was markedly attenuated upon perfusing hearts with 200-500 M H₂O₂ for 10 min. These results suggest that H₂O₂ may depress the ₁-adrenoceptor, G_s-proteins and catalytic subunit of the adenylyl cyclase enzyme and thus may play an important role in attenuating the -adrenoceptor linked signal transduction due to ischemia-reperfusion injury.     

Role of transglutaminase 2 in quercetin-induced differentiation of B16-F10 murine melanoma cells

Flavonoids belong to the class of plant polyphenolic compounds with over 6,000 individual structures known. These phytochemicals have attracted the interest of the scientists, because they possess a remarkable spectrum of biological activities, such as antiallergic, antiinflammatory, antioxidant, antimutagenic and anticarcinogenic. In this work, we compared the anticancer potential of two flavonoids, quercetin and pelargonidin, on highly metastatic B16-F10 melanoma murine cells. We have evaluated different parameters related to cell proliferation and differentiation, such as cell number, toxicity, intracellular content of polyamines and transglutaminase (TG, EC 2.3.2.13) activity. The higher inhibition of tumor cell growth, with respect to control, was obtained with quercetin cell treatment, i.e. 32% reduction after 48 h and 39% reduction after 72 h of incubation (P < 0.001). In parallel, quercetin-treated cells showed a similar decrease in polyamine content. TG activity was fourfold increased, with respect to control, after 48 h and twofold increased after 72 h (P < 0.001). Pelargonidin treatment did not show significant antiproliferative effects and any increase in TG activity. Proteomic approach was used to investigate changes in protein expression profiles in tumor cells following quercetin treatment. Changes in expression of 60 proteins were detected, i.e. 8 proteins were down-regulated, 35 up-regulated, 11 “de novo” synthetized proteins and 6 suppressed proteins were present in treated cells. A 80 kDa spot, identified as TG type 2 by Western blot analysis, presented a fourfold increase in intensity, confirming the key role played by TG in the induction of cancer cell differentiation.     

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.     

MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells

MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.     

Substratum acidification by murine B16F10 melanoma cultures.

Murine B16F10 melanoma cells, adherent to thin films of crosslinked, fluorescein-labeled collagen I, and covered by a thin layer of 0.7% agarose, exhibit a decrease in fluorescence emission in the substratum region immediately beneath adherent cells. The relative diminution in fluorescence intensity is dependent on excitation wavelength and is observed following excitation at 490 nm, but is not observed following excitation at 452 nm. The decrease in fluorescence emission is not due to quenching or concentration effects and is attributed to the decrease in extracellular pH in the substratum region. Fluorescence measurements of (I490/I452) within these substratum regions, correlate with an average extracellular pH of 6.4 +/- 0.2 which drops to pH less than 5 after 5 h. It is suggested that this region is sufficiently acidic to activate secreted or cell-surface acid proteinase enzymes and that the activity of these enzymes may be important in invasiveness by this cell-line.