Growth factors and proto-oncogenes in early mouse embryogenesis and tumorigenesis.

Growth factors and proto-oncogenes play an important role in the regulation of embryonic growth and differentiation as well as in tumorigenesis. Insulin and insulin-like growth factor I (IGF I) are secreted by embryonic tissues during the prepancreatic stage of mouse development. Measureable amounts of these factors were found in 8- to 12-day-old embryos. Embryonic cells derived from 8- to 10-day-old embryos secrete insulin and IGF I in serum-free medium. Relatively high levels of c-myc, c-fos and c-H-ras oncoproteins were also detected in 8- to 12-day-old embryos. Insulin and IGF I, when added to the culture of embryonic cells, stimulate their proliferation. Similar results were obtained in some animal or human tumors. Murine myeloid leukemias and melanoma B 16 secrete a substance immunologically cross reactive with insulin (SICRI) both in vivo and in serum-free media. In culture, the DNA synthesis rate per leukemic or melanoma cell is proportional to cell density and is reduced by antiinsulin serum in case of leukemic cells. Human hemangiosarcoma secrete IGF I, which also plays a role as an autocrine factor. Purified IGF I efficiently induce c-myc and c-fos mRNA, which is among the earliest events following growth factor stimulation, leading to mitosis. These results lead us to the conclusion that IGF I and insulin together with oncoproteins stimulate the growth of embryonic and tumor cells, which is indirect evidence for a paracrine (or autocrine) type of action.     

Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115.

An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgen-induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 micrograms/ml. DNA synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosterone-treated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.     

Induction of antibody-dependent cellular cytotoxicity in vivo by IFN-alpha and its antitumor efficacy against established B16 melanoma liver metastases when combined with specific anti-B16 monoclonal antibody

We have previously shown the ability of different cytokines to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells in vitro. In addition we found that the administration to mice of IL-2-induced cells which mediated ADCC and that these cells were phenotypically similar to the cells induced in vitro. In the present study we tested the ability of various cytokines, including IL-1, TNF, IFN-alpha, and IFN-gamma to induce ADCC in vivo. We found that both IFN-alpha and IFN-gamma induced ADCC in the livers and spleens of C3H/Hen-treated mice and that these cytokines together with TNF enhanced the IL-2-induced ADCC in vivo. In C57BL/6 mice which, as previously shown, exhibit relatively low ADCC activity, IFN-alpha and IFN-gamma increased the IL-2-induced ADCC only when 100,000 U of IL-2 were used for priming. The effect of IFN-alpha on ADCC was dose dependent and was optimal after the administration of 200,000 U of the cytokine given three times a day for 3 days. Similar to the cells induced in vivo by IL-2, the precursors of the cells mediating ADCC were asialo GM1+ whereas the effectors were mainly nonadherent, Thy-1+ cells. IFN-alpha-generated cells mediating ADCC in the liver and spleen and, when combined with IL-2, ADCC was induced in the thymus as well. This effect of IFN-alpha on the induction of ADCC was exploited in an immunotherapy model in which we found that IFN-alpha significantly enhanced the antibody-mediated antitumor effect on established B16 melanoma liver micrometastases. Furthermore, when IL-2 and IFN-alpha administration was combined with the administration of mAb, a significantly reduced number of established 6- to 8-day B16 melanoma liver macrometastases and prolonged survival of tumor-bearing mice were seen. These studies imply that the administration of appropriate cytokine combinations may be a useful adjunct to the administration of mAb for the treatment of cancer in humans.     

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.     

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.     

Activation of AMP-activated protein kinase is involved in vincristine-induced cell apoptosis in B16 melanoma cell

The molecular basis for induction of apoptosis in melanoma cells by vincristine remains unknown. Here we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found for the first time that vincristine induces AMPK activation (AMPKα, Thr 172) and Acetyl-CoA carboxylase (ACC, Ser 79) (a downstream molecular target of AMPK) phosphorylation in cultured melanoma cells in vitro. Reactive oxygen species (ROS) dependent LKB1 activation serves as the upstream signal for AMPK activation. AMPK inhibitor (compound C) or AMPKα siRNA knockdown inhibits vincristine induced B16 melanoma cell apoptosis, while AMPK activator 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) enhances it. AMPK activation is involved in vincristine induced p53 phosphorylation and stabilization, the latter is known to mediate melanoma cell apoptosis. Further, activation of AMPK by vincristine inhibits mTOR Complex 1 (mTORC1) in B16 melanoma cells, which serves as another important mechanism to induce melanoma cell apoptosis. Our study provides new insights into understanding the cellular and molecular mechanisms of vincristine induced cancer cell death/apoptosis. We suggest that combining AMPK activator AICAR with vincristine may have potential to be used as a new therapeutic intervention against melanoma.     

Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor

We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.     

Bronchial epithelial cell matrix production in response to silica and basic fibroblast growth factor

BACKGROUND: Previous studies show that macrophages, lung fibroblasts, and their soluble mediators are responsible for the onset and development of pulmonary fibrosis. This study was conducted to determine whether airway epithelial cells are also directly involved in response to fibrogenic agents and consequently in the pathogenesis of lung fibrosis. To verify the hypothesis, we determined whether silica acts directly on human bronchial epithelial cells by stimulating cytokine and growth factor release and by modifying matrix production. MATERIALS AND METHODS: An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o (16HBE), was used. The expression profile of some proinflammatory interleukins (ILs), such as IL-1alpha, IL-1beta and IL-6 and their modulation by silica, were evaluated by polymerase chain reaction (PCR) analysis. Transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) mRNA levels were tested by Northern blotting in the presence and in the absence of silica. The silica- and/or bFGF-induced effects on matrix components (total proteins, collagen, and fibronectin) were also evaluated using radio-labeled precursors. RESULTS: The results demonstrated 16HBE internalized silica particles. Silica induced a little IL-6 secretion, without affecting IL-1 and TGFbeta isoform production and strongly stimulated bFGF mRNA level and bFGF protein secretion. Silica also induced changes in 16HBE production of total proteins, collagen, and fibronectin production. When added in combination with the growth factor, it strengthened bFGF stimulation of matrix component secretion. CONCLUSIONS: These results support the hypothesis that the changes in matrix components are due to a direct effect of silica on bronchial epithelial cells. Silica-induced over-secretion of bFGF suggests that autocrine and paracrine differentiation loops for bFGF may also be operative and that these mechanisms may be involved in the pathogenesis of pulmonary fibrosis. In the future, cytokine-directed therapeutic strategies might find a place in clinical practice.     

IFN-alpha induces autocrine production of IL-6 in myeloma cell lines.

IL-6 is a major tumor growth factor in human multiple myeloma. Myeloma cell lines, which have the same phenotypic characteristics and Ig gene rearrangements as the original fresh myeloma cells and whose growth is strictly dependent on exogenous IL-6 similar to fresh myeloma cells, have been reproducibly established. We show here that IFN-alpha stimulated the growth of five of six of these human myeloma cell lines by inducing an autocrine production of IL-6 in myeloma cells. Indeed, IFN-alpha induced IL-6 mRNA accumulation and IL-6 production in myeloma cells and the IFN-alpha-induced growth of these cells was inhibited by anti-IL-6 mAb. Moreover, IFN-alpha made possible the rapid emergence of autonomously growing myeloma cell sublines, which produced IL-6 as an autocrine growth factor. As IFN-alpha has a potential therapeutical interest for multiple myeloma, the present study opens up new directions for studying its effects on the myeloma clone in vivo.     

A factor in conditioned medium of rabbit costal chondrocytes inhibits the proliferation of cultured endothelial cells and angiogenesis induced by B16 melanoma: its relation with cartilage-derived anti-tumor factor (CATF)

Serum-free medium conditioned by exposure to rabbit costal chondrocytes in culture inhibited the proliferation and DNA synthesis of bovine pulmonary endothelial cells in culture. The factor in conditioned medium did not inhibit DNA synthesis in B16 melanoma cells, L1210 cells or 3T3 fibroblasts in culture, but it inhibited angiogenesis in the chorioallantoic membrane of chick embryos induced by B16 melanoma and growth of the tumor transplanted onto the membrane. These findings strongly suggest that rabbit costal chondrocytes produce an anti-angiogenesis factor that is similar to cartilage-derived anti-tumor factor (CATF).     

Aberrant Expression of Basic Fibroblast Growth Factor in NIH-3T3 Cells Alters Drug Resistance and Gene Amplification Potential

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.     

Autocrine FGF signaling is required for vascular smooth muscle cell survival in vitro

Expression of both basic fibroblast growth factor (bFGF) and FGF receptors (FGFR) by vascular smooth muscle cells suggests that autocrine FGF signaling mechanisms may have important functions. Inhibition of smooth muscle cell bFGF expression provokes apoptosis, suggesting that endogenous bFGF generates an anti-apoptotic signal. The purpose of this study was to determine whether the survival function of endogenous bFGF requires signaling through FGFR. A recombinant adenovirus encoding a truncated murine FGFR-1 lacking the kinase domain (DN-FGFR) efficiently expressed the transgene in cultured rat aortic smooth muscle cells. The truncated receptor acted in a dominant negative fashion to effectively prevent receptor-mediated signaling, assessed by phosphorylation of p42/p44 MAP kinase. Expression of DN-FGFR provoked apoptosis of SMC in a dose-dependent fashion that was insensitive to recombinant bFGF but could be rescued by platelet derived growth factor or epidermal growth factor. Heterologous growth factor rescue was inhibited by PD98059, an inhibitor of MEK (MAP kinase-kinase). These data demonstrate that inhibition of FGF receptor activation results in apoptosis and suggest that an intact autocrine FGF signaling loop is required for vascular smooth muscle cell survival in vitro. These findings also implicate the Ras/Raf/MEK /MAP kinase cascade in generating or sustaining the survival signal. The functional significance of an autocrine FGF signaling loop in non-transformed cells has important implications for cardiovascular development, remodeling and disease. J. Cell. Physiol. 177:58–67, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and secretion by mouse 3T3 cells of an inhibitor of cell growth (mIGFBP-3): correlation with cell proliferation.

Our results show that stimulation by serum of dense cultures of 3T3 cells rapidly induced increased synthesis of a growth inhibitor (mIGFBP-3) capable of binding IGF. mIGFBP-3 was secreted by stimulated cells immediately after its synthesis, and accumulated in the medium. Accumulation of mIGFBP-3 in the medium increased, as a function of growth factor (bFGF, PDGF, insulin) concentrations and time. bFGF was the best stimulatory factor for both DNA synthesis and accumulation of mIGFBP-3 in the first 24 h of incubation. DNA synthesis was arrested after 48 h of incubation with bFGF when accumulation of mIGFBP-3 was maximal. Since we showed that mIGFBP-3 is able to inhibit bFGF stimulation of DNA synthesis in mouse fibroblasts, it is possible that the accumulation of mIGFBP-3 induces a feedback regulation of cell growth.     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

bFGF反义寡核苷酸增强鼠黑色素瘤B16细胞化疗药物敏感性研究

目的:研究bFGF反义硫代寡核苷酸增强肿瘤细胞对化疗药物敏感性作用。方法:设计、合成bFGF寡核苷酸,用聚乙烯亚胺(polyemyleneimine,PEI)介导bFGF反义硫代寡核苷酸转染入黑色素瘤B16细胞,MTT法检测bFGF反义硫代寡核苷酸及其与化疗药物联合处理后的细胞增殖率;半定量RT-PCR测定bFGF反义硫代寡核苷酸转染后细胞中bFGF mRNA水平;流式细胞仪分析bFGF反义硫代寡核苷酸诱导的细胞凋亡。结果:bFGF反义硫代寡核苷酸对B16细胞增殖的抑制率为64.8%,且呈剂量依赖效应。B16细胞中bFGF mRNA被bFGF反义硫代寡核苷酸显著降低,为对照细胞的57.9%,且bFGF反义硫代寡核苷酸诱导B16细胞凋亡,凋亡率为41.8%。bFGF反义硫代寡核苷酸转染能显著增强B16细胞对阿霉素、5-氟脲嘧啶及顺铂的敏感性,非特异性硫代寡核苷酸不影响阿霉素、5-氟脲嘧啶及顺铂抑制B16细胞增殖。结论:bFGF反义硫代寡核苷酸显著增强B16细胞的化疗敏感性,表明其可协同化疗药物用于治疗肿瘤。