TNFα receptor1 drives hypoxia-promoted invasiveness of human melanoma cells.

AIM: Oxygen deprivation leading to hypoxia represents a common feature of advanced solid tumors, able to control several aspects of tumor progression. Indeed, ability to respond to changes in oxygen partial pressure represents a hallmark of malignant cells. Aim of this study is to disclose new pathway of hypoxia-induced tumor cell invasion. MATERIALS AND METHODS: Hs294T human melanoma cells were grown in a gas mixture containing 0.3% O2 and used to evaluate invasion on Matrigel-coated polycarbonate filters mounted in Boyden's chambers, MMP release and expression of inflammatory receptors and their ligands. RESULTS: We demonstrate that hypoxia promotes the expression of TNFα receptor 1 (TNFαR1) able to drive a higher ability to penetrate Matrigel-coated filters of Hs294T human melanoma cells, an effect does not mediated by hypoxia-inducible factor (HIF)-1α. CONCLUSION: Expression of inflammatory cytokine receptors in hypoxic human melanoma cells might provide a new target for improving strategies against local and distant tumor cell diffusion.

Extracellular acidity as favouring factor of tumor progression and metastatic dissemination.

The bidirectional interactions between tumor cells and the so-called "host reactive stroma" play a critical role in most of the events characterizing tumor progression and distant organ colonization. This review discusses critical components of tumor environment involved in tumor cell dissemination. More specifically, it addresses some of the experimental evidences providing that acidity of tumor environment facilitates local invasiveness and metastasis formation, independently from hypoxia, with which acidity may be associated. Besides, acidity renders tumor cells resistant to radiation therapy and chemotherapeutic drugs. Therefore, this review examines the strategies for raising the low extracellular pH of tumors that might have considerable potential in cancer therapy.

The change in leukotrienes and lipoxins in activated mouse peritoneal macrophages.

The aim of this study was to investigate to what extent the generation of leukotrienes (LTs) and lipoxins (LXs) was affected by the expression of definite levels of macrophage activation. We used a system of murine peritoneal macrophages at different states of activation consisting in resident macrophages and FCS-, thioglycollate- or Corynebacterium parvum-elicited macrophages. The profile of lipoxygenase metabolites in resident macrophages was characterized by the presence of high levels of 12-HETE, followed by 15-HETE, 5-HETE, LTB(4) and 6-trans-LTB(4), 6-trans-12-epi-LTB(4). A comparable pattern was also found in FCS-elicited macrophages which appeared not to be responsive to the challenge with interferon gamma plus LPS, as measured by the generation of NO and tumor necrosis factor alpha. Resident as well as FCS-elicited macrophages also generated appreciable quantities of LXs (A(4) and B(4)). Thioglycollate-elicited macrophages, which expressed a state of 'responsive' macrophages, showed a block of the LT and LX synthesis. This block was also present in C. parvum-elicited macrophages which expressed a fully 'activated' phenotype, reflected by their capacity of releasing NO and tumor necrosis factor alpha even though they were not challenged. These results provide the first evidence that the level of 'responsive' as well as 'activated' macrophages was associated with of a simultaneous block of LTB(4) and LXs.

Lipid characteristics in metastatic cells.

Interest in lipid characteristics of metastatic cells was aroused by the consideration that the various lipid components of cell membranes influence a broad spectrum of cell surface biological functions which are involved in different steps of the metastatic cascade. Correlation between invasive properties and characteristics of cell surface components has been appropriately studied in a limited number of metastatic cell systems isolated by in vivo and in vitro procedures. The major findings of this study have been reported in this review. Among membrane lipid components, glycolipids and phospholipids appeared particularly affected in tumor cells which acquired a metastatic phenotype. In fact, the reduction of complex gangliosides typical of transformed cell lines was even more evident in a highly metastatic variant selected from RSV-transformed murine fibroblasts. The reduction of complex gangliosides, mainly GD1a, particularly affected the adhesion sites of this variant. In a fibrosarcoma line, T3 cells, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line. Moreover, a particularly high level of ether-linked lipids was found in high metastatic variants isolated from murine melanoma and fibrosarcoma lines, as well as in human mammary carcinomas. The findings considered in this review are discussed for their possible relevance to the invasive properties of metastatic cells.

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages.

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parnum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors.

Diminution of the development of experimental metastases produced by murine metastatic lines in essential fatty acid-deficient host mice.

In a previous study we found that the capacity for spontaneous metastases of tumors developed after subcutaneous transplantation of RSV-transformed Balb/c 3T3 cells was reduced in essential fatty acids (EFA)-deficient host animals. In the present study, we have extended our investigation by considering the requirement of EFA for the formation of lung colonies obtained by i.v. injection of two metastatic murine cell lines of different origin: (1) T3 cells, a highly metastatic cell line isolated from a fibrosarcoma, and (2) the F10 variant of B16 melanoma (B16-F10 cells). We found that EFA deficiency reduces the lung colonization of both T3 cells and B16-F10 cells without affecting the retention of tumor cells in the lung. NK cells did not seem to be involved in the diminution of lung colonization in EFA-deficient animals. Furthermore, by examining histologically the lung parenchyma at successive intervals after tumor cell injection, we found that, in comparison with control mice, EFA-deficient animals had fewer lung colonies and a prevalence of smaller microcolonies during the entire period of observation. This led us to conclude that the diminution in development of tumor colonies in the lungs of EFA-deficient host animals was related to a reduced growth rate of tumor cells at this site.

Enhancement of lung-colonizing potential of murine tumor cell lines co-cultivated with activated macrophages.

In order to explore the influence of activated macrophages on tumor cells, we cultured a series of weakly metastatic clones isolated from the murine T3 fibrosarcoma line (T3 clones) and the B16-F10 melanoma cells on feeder layers of C. parvum- or thioglycollate-elicited macrophages, or 'resident' (unstimulated) macrophages. Co-cultivation with C. parvum-elicited macrophages, but not with resident macrophages, induced an increase of the lung-colonizing potential of T3 clones and B16-F10 cells. An enhancement of lung-colonizing potential was also found in tumor cells grown in media conditioned by C. parvum-elicited macrophages. Thioglycollate-elicited macrophages also generated a pro-clonogenic activity which was however effective only on T3 clones but not on B16-F10 cells. The increase in the lung-colonizing potential of tumor cells stimulated by activated macrophages was retained to some degree after subcultivation in tissue culture medium or transplantation into syngeneic animals. In conclusion, our data support the notion that macrophages at different states of activation may enhance lung colonization of tumor cells by inducing a partially stable change of phenotype.

Induction by human interferon and murine h-2l(d) gene of the surface expression of endogenous hla class-I free heavy-chains in hct human colon-carcinoma cells, deficient in Beta-2-microglobulin.

The colon cancer cell line HCT fails to express HLA class I antigens on the cell surface as a consequence of two independent mutations occurring in both copies of the beta2-microglobulin (beta2-mu) gene. Restoration of HLA class I antigen expression in HCT cells transfected with the wild-type human beta2-mu gene is accompanied by the expression of beta2-mu-free HLA class I heavy chains on the cell surface. HCT cells expressing a transfected H-2L(d) gene (HCT-L(d) transfectants) exhibited high levels of H-2L(d) heavy chains on the cell surface, following treatment with human interferon a (IFN-alpha). IFN-treated HCT-L(d) transfectants also expressed high levels of endogenous beta2-g-free HLA class I heavy chains on the cell surface. Incubation of HCT-L(d) transfectants at 26-degrees-C for 24 hours is associated with a striking increase in the surface expression of H-2L(d) heavy chains (comparable to that observed at 37-degrees-C in the presence of IFN), without a concomitant increase in the surface expression of beta2-mu-free HLA class I heavy chains, suggesting that IFN and low temperature act by different mechanisms. These results also indicate that human IFN and H-2L(d) heavy chain can act synergistically in inducing the surface expression of endogenous beta2-mu-free HLA class I heavy chains by HCT cells. The combination of 26-degrees-C incubation and IFN treatment results in the surface expression of beta2-mu-free HLA class I heavy chains in HCT-neo transfectants, suggesting that IFN plays a crucial role in allowing the surface expression of endogenous HLA class I heavy chains in beta2-mu-negative HCT cells.

Reexpression of the major histocompatibility complex (MHC) class-I antigen h-2k(b) by m1 (b16-f10) murine melanoma-cells.

We monitored the expression of MHC class I antigens in a clonal isolate (M1) of B16-F10 murine melanoma cells, under various condition of growth in vitro. We found that, compared to exponentially growing cultures, cells from confluent M1 cultures expressed higher levels of H-2D(b) and H-2K(b) surface antigens. Expression of MHC class I antigens could be enhanced further by incubation of M1 cells in serum-free medium for 24-48 hours. Northern blot analyses indicated that the up-regulation of MHC class I antigen expression was associated with increased levels of H-2K(b) and H-2D(b) mRNAs, without a concomitant decrease in c-myc expression. Analyses of the cell cycle distribution of M1 cells stained for MHC class I antigens failed to show any differential segregation of H-2D(b)- or H-2K(b)-positive cells in any phase of the cell cycle. Enhanced MHC class I antigen expression appeared not to be due to the release into the medium of known cytokines like IFN-alpha, -beta, -gamma, TNF-alpha, IL-6 (IFN-beta(2)), and IL-1 by the M1 cells either at confluence, or after growth in serum-free medium. Taken together, our experimental results indicate that expression of MHC class I genes by M1 cells can be greatly enhanced by manipulating culture conditions; these observations are particularly important for the re-expression of the H-2K(b) gene, which was assumed to be phenotypically silent in B16 cells. These data also suggest that re-expression of H-2K(b) antigen in M1 cells at confluence may be related to cell-cell contact, consistent with the concept that H-2K(b) molecules can play a nonimmune role in cellular communication and growth control.

Organ-specific metastases in melanoma: experimental animal models.

Metastases from certain primary tumors frequently exhibit specific organ preference. Animal models have been developed to induce in a reproducible fashion the formation of organ-specific metastases by malignant melanoma cells. Some of these models rely on the use of immunodeficient mice, which can support the growth of murine as well as human malignant melanomas. Moreover, immunodeficient mice, because of their diminished ability to mount an effective immune response, allow the expression of malignant properties (e.g., preferential colonization of certain organs), which are intrinsic to transplanted melanoma cells. This review discusses the relevant factors (and limitations) of some of the animal models used to study the in vivo properties of melanoma cells.

Modulation by MHC class I antigens of the biology of melanoma cells. Non-immunological mechanisms.

Many human as well as experimental tumours, including melanoma, express reduced levels of major histocompatibility complex (MHC) Class I antigens. Decreased MHC Class I antigen expression may be selected during neoplastic progression because it allows tumour cells to escape killing by cytotoxic T lymphocytes. Furthermore, the regulatory role of MHC Class I antigens in the proliferation of T cells suggests that abnormalities in MHC Class I antigen expression may play a role in the disordered proliferation of malignant cells and in their metastatic potential by non-immunological mechanisms. This paper reviews some of the available evidence supporting the concept of non-immune functions of MHC Class I antigens in the biology of malignant cells, with emphasis on experimental models for metastatic melanoma.

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line.

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride-or forskolin-stimulated activity in a murine fibroblast cell line, i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition of cis-9-16:1, cis-9-18:1/cis-9-18:1 or cis-9-18:1/cis-9,12-18:2 sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, while cis-11-18:1/cis-11-18:1 GPC was not effective and only a partial recovery was observed with 16:0/16:0, 16:0/cis-9-18:1 and trans-9-18:1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of two cis double bonds, each located in delta 9 position of the PC acyl chains. The limited effect of cis-9-16:1/cis-9-18:1 GPC and cis-9-18:1/cis-9-16:1 GPC suggests that an equal length of the terminal hydrocarbon chains extending beyond the delta 9 double bonds is also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective loss of human leukocyte class I allospecificities and staining of melanoma cells by monoclonal antibodies recognizing monomorphic determinants of class I human leukocyte antigens.

Immunoperoxidase staining of frozen sections from surgically removed melanoma lesions showed that anti-human leukocyte antigen (HLA)-A2, A28 monoclonal antibody (mAb) stained keratinocytes, but did not stain melanoma cells in 21% of the 14 primary and 44% of the 9 metastatic lesions tested. The loss of HLA-A2 and/or A28 allospecificities did not affect the staining patterns with mAb recognizing monomorphic determinants of HLA Class I antigens, in terms of percentage of stained melanoma cells and intensity of staining. This finding is not likely to reflect the sensitivity of the immunoperoxidase technique, since cytofluorographic analysis detected no significant difference in the staining pattern by mAb to monomorphic determinants of HLA Class I antigens between a melanoma cell line and an autologous transfectant that had acquired HLA-A2 antigens following gene transfer. The results of the present study imply that the frequency of abnormalities in HLA Class I antigen expression by melanoma cells is higher than that described in the literature, since selective losses of HLA Class I allospecificities are not detected by staining of melanoma cells with mAb to monomorphic determinants of HLA Class I antigens. The latter reagents have been used in most of the published studies to characterize the expression of HLA Class I antigens in melanoma lesions. Furthermore, the present results provide a mechanism for the unexpected resistance to cytotoxic T-cell-mediated lysis and the unexpected poor clinical course of the disease in some patients despite a high expression of HLA Class I antigens as measured by staining of melanoma cells with mAb to monomorphic determinants.

Radiation-enhanced expression of major histocompatibility complex class I antigen H-2Db in B16 melanoma cells.

Exposure of eukaryotic cells to ionizing radiation induces several cellular responses including DNA repair, arrest of DNA synthesis, and increased synthesis of specific cellular proteins. We derived from the murine melanoma cell line B16-F10 a clonal isolate (M1) that was exposed to a total dose of 5000 cGy in 25 fractions, according to a protocol that reflects the standard for current radiotherapeutic regimens. We measured, by flow cytometry of fluorescence-stained cells, the surface expression of the two major histocompatibility complex class I antigens H-2Db and H-2Kb in irradiated M1 cells and untreated M1 controls. We found that after 2000 cGy, expression of H-2Db antigen was enhanced in irradiated cells versus controls. Radiation-induced expression of H-2Db antigen appeared to be selective, since no up-regulation of the H-2Kb antigen was detectable, and persisted for at least 5 weeks following the last irradiation. Enhanced H-2Db antigen expression correlated with increased steady-state levels of H-2Db mRNA in irradiated cells. These results are consistent with the notion that enhanced expression of major histocompatibility complex class I antigens is part of a long-lasting stress response elicited in cells by radiation.

Expression of a transfected H-2Kb gene in B16 cells correlates with suppression of liver metastases in triple immunodeficient mice.

In vivo experiments performed with NIH (nu/nu, bg/bg, xid/xid) triple immunodeficient (TD) mice revealed the striking ability of i.v. injected B16-F1 and B16-F10 murine melanoma cells to colonize not only the lungs but also the liver of TD mice. Subsequently, B16 melanoma cell cultures, which express very low levels of H-2Kb antigen, were cotransfected with plasmids pRSVneo, containing the neomycin resistance gene, and 6-2B1pMT, expressing the H-2Kb complentary DNA under the control of the metallothionein enhancer-promoter. Several neomycin-resistant clones were analyzed for H-2Kb and H-2Db expression by RNase protection and flow cytometry assays. All parental lines and transfected clones expressed normal levels of H-2Db mRNA, while only some of the transfected clones expressed easily detectable levels of H-2Kb mRNA. Moreover, in these clones H-2Kb expression could be enhanced in the presence of Zn2+, indicating that the metallothionein enhancer was functioning properly. Parental cells and transfected clones were injected i.v. in TD mice to assess the possible involvement of H-2Kb antigen in regulating the metastatic potential of B16 melanoma cells. We observed a remarkable correlation between expression of H-2Kb antigen and suppression of liver-specific metastases in TD mice. Identical results were obtained when we gave TD mice injections of mixed populations of transfectants expressing H-2Kb antigen, obtained by fluorescence-activated cell sorting. These experiments allowed us to rule out the possibility that the observed changes in metastatic potential were due to clonal variability among individual transfected clones. Taken together, the results of our in vivo studies with immunodeficient mice support the notion that specific major histocompatibility complex Class I molecules modulate the metastatic potential of malignant cells also by mechanisms which are independent of their well-established role in antigen presentation.

Reduction of the metastatic potential of a RSV-transformed fibroblastic line (B77-AA6 cells) upon transplantation in essential fatty acid-deficient mice.

Metastatic dissemination has been shown to be influenced by dietary lipids. In particular, diets enriched with linoleic acid have been consistently found to be effective in enhancing the metastatic potential of murine mammary tumors. This study explored whether an essential fatty acid (EFA)-deficient host can affect the capacity of a tumor to metastasize to the lung. Tumors were developed by subcutaneously injecting high metastatic B77-AA6 cells (a subclone isolated from the Balb/c 3T3 cells transformed by the B77 strain of RSV) into syngeneic mice which had been fed either a control or an EFA-deficient diet for 9 weeks. EFA deficiency did not modify the incidence, latency and growth rate of primary tumors developed from B77-AA6 cells, while it markedly reduced their capacity to reproduce lung macro- and micrometastases. These findings demonstrate that physiological levels of EFA are important for a tumor to metastasize in secondary organs.

Lipid characteristics of RSV-transformed Balb/c 3T3 cell lines with different spontaneous metastatic potentials.

To determine whether a metastatic phenotype may be correlated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18:1) and a decrease of arachidonic (20:4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18:1 and decrease of 20:4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells. Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.

Eradication of Mycoplasma contamination from cell lines of different origin by the 5-bromouracil-fluorochrome procedure.

Because mycoplasmas contaminate irreplaceable, established cell lines, several laboratories have been looking for effective procedures to eradicate the infection. This report presents the experience of our laboratory in using the 5-bromouracil-fluorochrome method developed by Marcus et al. which turned out to be effective in eliminating Mycoplasma orale, M. hyorhinis and M. hominis from transformed cell lines of different origin. The metastatic potential of a highly metastatic murine fibroblastic cell line infected with M. orale was restored to its original level after the contamination was eliminated.

Lipid composition of cultured B16 melanoma cell variants with different lung-colonizing potential.

Lipid components influence several cell surface properties that are critical in different stages of the metastatic process. In this study, we examined whether the different lung-colonizing potential of B16-F1 and B16-F10 melanoma cells could be related to a characteristic lipid profile. The lipid analyses, carried out on the same cell cultures used for the assay of lung-colonizing potential, revealed characteristics in the lipid composition of both B16-F1 and B16-F10 melanoma cells that are common to other systems of malignant cells: a high level of 18:1 associated with low proportions of polyunsaturated fatty acids in phospholipids, accumulation of ether-linked lipids and absence of complex gangliosides. The two B16 melanoma variants differed significantly only with respect to ether-linked lipids, due to a higher level of alkyl-PC in B16-F10 than in B16-F1.