Changes in the expression of B cell surface markers on complement receptor-positive and complement receptor-negative B cells induced by phorbol myristate acetate

The ability of phorbol myristate acetate (PMA) to induce changes in the expression of B cell surface markers on CR- and CR+ B cells from normal mice in an in vitro culture system was examined. The markers studied were CR, sIgM, sIgD, and sIa. CR- B cells acquired the CR after overnight incubation with PMA. A twofold increase in sIa expression on CR- and CR+ B cells was also noted, whereas the staining intensity of sIgM and sIgD decreased on both B cell populations. These changes in the expression of surface markers took place without detectable increases in cell proliferation, cell size, or RNA content. Furthermore, the same effects were observed when CR- and CR+ B cells were prepared from a small B cell population purified by elutriation. It therefore appears that PMA can exert its effect directly on small, resting B cells.     

Dickkopf-1 activates cell death in MDA-MB435 melanoma cells

Dickkopf-1 (DKK-1) is known inhibitor of the canonical Wnt pathway. Recent studies strongly suggested that activation of DKK-1 expression results in inhibition of cell tumorigenicity. Reduced levels of DKK-1 in melanomas were recently shown. However, it is not known if DKK-1 activation in melanoma cells will inhibit cell tumorigenicity. In the present study, we overexpressed DKK-1 in melanoma cell line MDA-MB435. We show that while DKK-1 did not affect cell growth in soft agar, weak but significant inhibition of tumorigenicity in nude mice in vivo was observed. Analysis of resulting tumors revealed activation of cell death. In tumors originating from cells transduced with DKK-1, tumor mass was permeated with areas of necrosis. In tumors, originated from control cells, areas of necrosis were limited to the central region, a common feature of large tumors growing in nude mice. TUNEL assay revealed that in tumors originating from cells transduced with DKK-1 apoptotic cells were detected along the border of necrotic and viable areas of the tumors indicating significant increase in apoptotic process. Thus, our results indicate that activation of DKK-1 in melanoma cells leads to activation of apoptosis in vivo and, thus, is incompatible with tumor growth in nude mice.     

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.     

Calretinin and calretinin-22k increase resistance toward sodium butyrate-induced differentiation in CaCo-2 colon adenocarcinoma cells.

Calretinin (CR) and the alternatively spliced form calretinin-22k (CR-22k) are members of the EF-hand family of Ca(2+)-binding proteins (CaBPs). CR is expressed in more than 60% of poorly differentiated human colon tumors and both isoforms are present in several colon carcinoma cell lines (e.g., WiDr). They are absent in normal enterocytes and in well-differentiated adenocarcinoma cell lines such as CaCo-2. Calretinins are thought to act as Ca(2+) buffers and to be involved in the regulation of Ca(2+)-dependent processes. Down-regulation of calretinins in WiDr cells by antisense oligonucleotides leads to growth inhibition and treatment with sodium butyrate (NaBt, an inducer of differentiation) leads to a blockage of the cell cycle and, in parallel, to down-regulation of CR. It has been proposed that CR and/or CR-22k may be involved in maintaining the undifferentiated phenotype of WiDr cells and contributing to the transformation of enterocytes. Expression levels and distribution of CR-22k were investigated in WiDr cells. CR-22k was down-regulated in NaBt-treated cells and the normally cytoplasmic protein was preferentially localized in the nucleus either as a result of translocation or selective nuclear maintenance, a process more pronounced than in the case of CR. To compare functional differences of calretinins, CR-negative Caco-2 cells were stably transfected with cDNAs encoding CR or CR-22k. Cell growth of CR-transfected cells was increased, an effect that was not observed in CR-22k-transfected ones. The CR-expressing clones were almost completely resistant to treatment with 0.5 mM NaBt, a concentration significantly reducing cell growth in control cells. The same effect was obtained in the CR-22k-expressing clones, although to a lesser extent. This implicates that expression of CR and/or CR-22k in colon tumor cells may contribute to tumorigenesis by blocking differentiation-promoting signals.     

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.     

Suppression of tumor formation in vivo by expression of the JE gene in malignant cells.

The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.     

Melanotransferrin induces human melanoma SK-Mel-28 cell invasion in vivo

The expression of melanotransferrin (MTf), a membrane-bound glycoprotein highly expressed in melanomas, is correlated with tumor vascularization and progression, suggesting a proinvasive function associated with MTf in malignant tumors. To test this hypothesis, we silenced MTf in human melanoma SK-MEL-28 cells using small interfering RNA (siRNA) and examined the plasmin activity and invasiveness of MTf-silenced melanoma. In vitro, the siRNA-mediated MTf knockdown inhibited by 58% the cell surface activation of plasminogen into plasmin. In addition, decreased expression of MTf in melanoma cells reduced cell migration. In vivo, we used a nude mice invasion model in which tissue factor (TF) induces vascular [125I]-fibrin deposition following injection. Using this metastasis model, the invasive potential of MTf-silenced cells into the lungs was reduced by fivefold. Altogether, these findings strongly suggest that MTf overexpression in melanoma cells contributes to tumor progression by stimulating plasmin generation as well as cell migration and invasion.     

Potential for Cripto-1 in defining stem cell-like characteristics in human malignant melanoma

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal co-receptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Spontaneous CD4+ T cell responses against TRAG-3 in patients with melanoma and breast cancers

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-gamma-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRbeta gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-3(34-48) is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.     

Inhibition of melanoma brain metastasis by targeting melanotransferrin at the cell surface

Brain metastases are a common feature of malignant melanoma and are associated with poor prognosis. Melanotransferrin (MTf), one of several antigens associated with the surface of melanoma cells, has been demonstrated to promote cell invasion. In this study, we investigated the role of membrane‐bound MTf in several of the steps leading to the development of melanoma brain metastasis. Our results indicated that MTf‐positive cells were detected in the brains of nude mice injected intravenously with human melanoma SK‐Mel 28 cells. Moreover, administration of a single dose of a monoclonal antibody (L235) directed against human MTf significantly reduced the development of human melanoma brain metastases in nude mice. The ability of melanoma cells to cross the blood–brain barrier (BBB) in vitro is correlated with their MTf expression levels at the cell surface. Overall, our results indicated that membrane‐bound MTf is a key element in melanoma cell transmigration across the BBB and subsequent brain metastasis. Thus, these data suggest MTf as an attractive target and demonstrate the therapeutic potential of an anti‐MTf mAb for preventing metastatic melanoma.     

Extrathymic T cell maturation. Phenotypic analysis of T cell subsets in nude mice as a function of age.

T cell maturation in an extrathymic environment has been studied using as a model the congenitally athymic nude mouse. Phenotypic analyses as a function of age were conducted on lymphocytes obtained from the spleens and lymph nodes of nude mice through use of mAb recognizing T cell surface markers and multiparameter flow cytometry. The data show that nude mice accumulate increasing numbers of lymphocytes bearing Thy-1, CD3, CD4, and CD8 with age characterized by a progression from heterogeneous dim to more homogeneous bright expression. In contrast, the expression of heat-stable Ag (HSA), a marker of immature thymocytes, decreases with age. By analogy to intrathymic maturation, spleens and lymph nodes in nude mice contain T cells defined as immature, transitional, and mature based on the expression of these markers. Although the proportion of CD4+ and CD8+ T cells associated with bright CD3 expression increases with age, at no age are significant numbers of CD4+8+ cells observed, in contrast to intrathymic T cell maturation. In addition to the frequently observed inversion in the ratio of CD4 to CD8, the CD8 T cell subpopulation in older nude mice contains mainly mature cells (CD8+, CD3+, HSA-) whereas only 50% of CD4+ T cells express the mature (CD4+, CD3+, HSA-) phenotype. At any age, the spectrum of phenotypes observed indicates that lymph nodes contain more mature T cells than spleen, suggesting a role for environmental Ag in driving extrathymic maturation, a process occurring most efficiently among CD8+ T cells. Because extrathymic maturation mirrors some but not all aspects of the intrathymic pathway, we propose that the nude mouse may be a useful model for further dissecting those interactions crucial to establishing the T cell repertoire in euthymic individuals as well as elucidating the contribution of extrathymically derived T cells to the peripheral immune system.     

Characteristics of complement receptor-bearing cells in the spleens of tumor-bearing mice.

In the course of mammary tumor development, a population of nylon nonadherent cells with CR appears in the spleens of tumor-bearing mice although none are ever detected in normal mice. These cells apparently arise in response to immunologic stimulation. In a series of studies we have further characterized subsets of T cells (CR+ and CR-) with regard to their responses to mitogens in the lymphocyte transformation assay. Nylon column nonadherent cells from the spleens of tumor-bearing mice were rosetted in a complement receptor assay using EAC rosetting, and CR+ cells were separated from CR- by centrifugation in a discontinuous Ficoll gradient. CR+ T cells responded strongly to PHA and Con A and in addition responded to LPS, an activity not usually associated with conventional T cells. In contrast, CR- T cells from tumor-burdened mice responded to PHA but failed to respond to Con A or LPS.     

Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro

Overexpression of the small heat shock protein Hsp27 has been shown by us to inhibit the in vitro proliferation rate and to delay tumor development of a human melanoma cell line (A375) in nude mice. We hypothesized that Hsp27 may influence the neoplastic phenotype. In the present study Hsp27 transfectants from this cell line were analyzed for various cellular aspects associated with the metastatic process. We found that Hsp27-overexpressing clones exhibited an altered cellular morphology as compared with control transfected cells. The Hsp27-positive cells tended to develop an epithelial-like phenotype growing in clusters and were characterized by a loss of transcytoplasmic stressfibers. In parallel, Hsp27-expressing cells lost the ability to form colonies in soft agar. The invasive potential was studied in vitro by the use of a reconstituted extracellular matrix-coated filter (Matrigel). Compared with controls, Hsp27-overexpressing cells showed decreased cell invasiveness through Matrigel. A correlation between invasion and activation of matrix metalloproteinases (MMPs) has been shown in several cell models. Secretion of MMPs (MMP-2 and MMP-9) was studied by gelatin-substrate zymogram analysis, as well as by a sensitive gelatinase activity assay. The Hsp27-transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 as compared with the control transfected cells. Integrins are adhesion receptors and function in cell invasion by mediating cell movement on matrix molecules and by regulating the expression of MMPs. Both fluorescence-activated cell sorter analysis and immunofluorescence analysis revealed a loss of alpha(v)beta3 integrin in Hsp27-transfected cell colonies. Our results demonstrate that Hsp27 overexpression has a profound impact on several parameters regulating the invasive and metastatic potential of melanoma cells in vitro.     

Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units

The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice. The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation. To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia. Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects. The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors. Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice. The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice. The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0. The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously. Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice. Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary. The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.