RESUMO
BACKGROUND: Recombinant human basic fibroblast growth factor (rHu-bFGF) is known to stimulate proliferation in some tumor cells and to modulate tumor vascularization. PURPOSE: The purpose of this study was to examine the possible role of this agent in the development of tumors. The study was designed to determine the effects of modulating bFGF activity in vivo in tumor models from cell lines with different responses to bFGF and with different content and receptor levels of bFGF. METHODS: Two tumor cell lines (human DLD-2 colon carcinoma and rat C6 glioma) were characterized for bFGF content and bFGF receptor levels by Western blot analysis in cultured cells and by studies of [125I]rHu-bFGF binding to sections from xenografts grown in nude mice. Tumor cell proliferation was monitored after treatment with rHu-bFGF or the DG2 or DE6 IgG monoclonal antibody to rHu-bFGF in culture and in vivo. RESULTS: C6 cells exhibited 7800 high-affinity receptors for rHu-bFGF per cell (dissociation constant [Kd] = 46 pM), while DLD-2 cells lacked high-affinity receptors. rHu-bFGF stimulated [3H]thymidine uptake by C6 cells, but the addition of DG2 IgG prevented this stimulation; rHu-bFGF had no effect on [3H]thymidine incorporation by DLD-2 cells. C6 cells had higher levels of immunoreactive bFGF than did DLD-2 cells. The xenografts from both cell lines exhibited high-affinity [125I]rHu-bFGF binding that was concentrated on vascular-like structures. rHu-bFGF at a dosage of 0.25 mg/kg given intraperitoneally daily for 18 days caused a twofold increase in DLD-2 tumor weight but had little effect on the growth of C6 xenografts. In contrast, daily intravenous injections of DG2 IgG given to mice had no effect on DLD-2 tumor growth but reduced growth of C6 tumors by approximately 30%--a statistically significant difference. CONCLUSIONS: The addition of exogenous rHu-bFGF or of a neutralizing antibody resulted in significant alterations in tumor growth in vivo, which were specific for tumor type and bFGF characteristics. While some of these effects may be mediated by the bFGF-responsive endothelial cells of the tumor vasculature (DLD-2 colon carcinoma), others may result from inhibition of bFGF-dependent tumor cell proliferation (C6 glioma). IMPLICATIONS: Studies that measure tumor blood flow are necessary to confirm that these effects are mediated by changes in tumor vasculature.
Assuntos
Neoplasias do Colo/patologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glioma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Autorradiografia , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/terapia , Citosol/ultraestrutura , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/farmacocinética , Glioma/terapia , Humanos , Imunoglobulina G , Radioisótopos do Iodo , Camundongos , Camundongos Nus , Testes de Neutralização , Ratos , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Suramin, an anticancer agent in current clinical trials, is a prototype of a pharmacological antagonist of growth factors, including basic fibroblast growth factor (bFGF). Suramin inhibited angiogenesis in the chick chorioallantoic membrane assay in a dose-dependent fashion. Suramin, 200 mg/kg i.v., inhibited rat corneal angiogenesis induced by bFGF-impregnated polymers; addition of heparin stimulated angiogenesis and counteracted the inhibition of suramin. The half-maximal inhibitory concentration (IC50) of suramin was determined for key cellular mechanisms that regulate angiogenesis: (a) low and high affinity cellular binding of bFGF to bovine capillary endothelial (BCE) cells with IC50s, respectively, of 24.3 and 71.5 micrograms/ml; (b) spontaneous migration of bovine pulmonary artery endothelial and normal AG 7680 fetal bovine aortic endothelial cells; bFGF-stimulated migration of BCE and transformed GM 7373 fetal bovine aortic endothelial cells with IC50s of 200-320 micrograms/ml; (c) proliferation of bovine pulmonary artery endothelial cells at > 100 micrograms/ml and of BCE cells at > 250 micrograms/ml; and (d) urokinase-type plasminogen activator activity of GM 7373 endothelial cells stimulated by bFGF with an IC50 of 211 micrograms/ml and of BCE cells stimulated by bFGF at > 100 micrograms/ml, but not plasminogen activator activity induced by phorbol 12-myristate 13-acetate. Suramin inhibited multiple control points of angiogenesis, including those stimulated by bFGF. Because tumor growth is angiogenesis dependent, the clinical efficacy of suramin may relate, in part, to angiosuppression.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/prevenção & controle , Suramina/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Córnea/irrigação sanguínea , Ensaios de Seleção de Medicamentos Antitumorais , Endotélio Vascular/patologia , Indução Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Masculino , Ratos , Ratos WistarAssuntos
Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glioma/terapia , Receptores de Superfície Celular/genética , Animais , Antineoplásicos/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Glioma/genética , Glioma/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoterapia , Cinética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Ensaio de Cápsula Sub-Renal , Timidina/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The amounts of G-actin and F-actin were measured in cultured cells grown under various conditions. The percent of total actin as F-actin in monolayer cultures of asynchronous cells was 72.4% in Chinese hamster ovary (CHO) cells, 57.7% in HeLa cells, 69.8% in V79 cells, and 79.5% in 1080 cells. Actin comprises 2.4-3.1% of the total protein in these cell lines. Treatment of cells with 20 microM cytochalasin B (CB) caused different cytological effects from treatment with 10 microM colchicine, but the effects characteristic of each drug were observed throughout the range of cell lines used. Of the five cell lines treated with CB only the V79 and CHO cells showed a decrease (5-8%) in the level of F-actin. Colchicine treatment of HeLa cells resulted in a 13% increase in the percent F-actin, but similar treatment of CHO cells caused no significant change in F-actin. Therefore, a change in the steady state level of F-actin is not necessary for the observed cell shape change. The F-actin levels in CHO cells treated with 7 mM procaine decreased from 72 to 65% over the first 15 min of exposure, a time during which the cells rounded. After continuous exposure of the cells to procaine for 1 h, the F-actin percentage returned to control levels and the cells, though abnormal in appearance, flattened on the culture dish. The relationship between the level of F-actin and cell density was studied on the culture dish. The relationship between the level of F-actin and cell density was studied in the 10T 1/2 cells, a cell line which demonstrates density-dependent growth regulation. While contact inhibition was accompanied by a decrease in the F-actin percentage (from greater than 95% to about 60%) in one strain of 10T1/2 cells, two other strains of the same cells progressed from log phase growth (highly motile) to late plateau phase (non-motile, contact-inhibited) with a constant level of F-actin (about 60%). A spontaneous transformant of this cell line, which no longer demonstrated contact-inhibited cell growth, also maintained the same constant F-actin (60%). Thus, the maintenance of contact-inhibited growth control does not appear to depend upon the net distribution of actin between the globular and filamentous forms.
Assuntos
Actinas/análise , Inibição de Contato , Animais , Divisão Celular , Células Cultivadas , Colchicina/farmacologia , Cricetinae , Cricetulus , Citocalasina B/farmacologia , Dimetil Sulfóxido/farmacologia , Feminino , Células HeLa , Humanos , Masculino , Ovário , Procaína/farmacologiaRESUMO
Basic fibroblast growth factor (bFGF) and other members of the FGF family share several biological properties that have the potential to mediate neoplastic cell growth. To test the hypothesis that bFGF may play a role in human ovarian cancer cell growth, three ovarian cancer cell lines, A90, A121(P), and A121(A), were investigated for their ability to respond to bFGF as a mitogen, to express endogenous bFGF protein or message for FGF proteins, and to exhibit FGF receptor or its message. Addition of bFGF to cultures of all three cell lines maintained in chemically defined media resulted in a statistically significant increase in cell number. Cell extracts from A90, A121(P), and A121(A) contained an immunoreactive protein that comigrated with hr-bFGF by Western blot analysis. Several bands of higher molecular weight were also noted. Immunohistochemical staining for bFGF demonstrated a cytoplasmic distribution of bFGF in the three cell lines. Both high- and low-affinity binding sites for human recombinant bFGF (hr-bFGF) were expressed by all three lines. High-affinity sites varied from 2700 sites per cell (Kd = 29 pM) to 13,500 sites per cell (Kd = 71 pM). All three cell lines were screened for mRNA expression for seven FGF proteins and four FGF receptors. In all three lines, mRNA for FGF2 (bFGF) was detected by PCR analysis, and in two lines, mRNA for FGF1 (aFGF) and FGF5 were also found. The FGFR1 receptor subtype (flg) was common to all of the cell lines. Finally, suramin inhibited proliferation of A90 and A121 (P and A) with IC50's of 60 and 210 micrograms/ml, respectively. This is consistent with the A90 cell line having higher levels of endogenous bFGF and flg and therefore being more responsive to suramin inhibition than the A121 cell line. The results indicate that these ovarian cancer cell lines can produce bFGF as well as other members of the FGF family of genes and have the ability to respond to bFGF.
Assuntos
Fator 2 de Crescimento de Fibroblastos/análise , Neoplasias Ovarianas/química , Receptores de Fatores de Crescimento de Fibroblastos/análise , Sequência de Bases , Western Blotting , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Proteínas Filagrinas , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Suramina/farmacologia , Células Tumorais CultivadasRESUMO
Basic fibroblast growth factor (bFGF) is a potent mitogen and angiogenic factor. bFGF is expressed by a variety of solid human tumors and has been implicated as an autocrine regulator of tumor growth. Different solid tumor lines including glioma, colon carcinoma and melanoma were examined for intracellular immunoreactive bFGF, high- and low-affinity bFGF receptors and mitogenic response to bFGF when grown in chemically defined medium. All tumor lines contained significant levels of bFGF. In addition, all tumor lines contained subsets of five forms of immunoreactive bFGF, as well as 0.68-20 x 10(6) low affinity bFGF binding sites (Kd = 15-300 nM). Most, but not all lines exhibited high affinity bFGF receptors (Kd = 25-40 pM). Glioma cell lines were distinguished by expressing the highest levels of bFGF protein as well as the most high-affinity receptors for bFGF. Furthermore, glioma cell lines were the only tumor type mitogenically responsive to bFGF. These results indicate that glioma cells express high levels of this potent mitogen and angiogenic factor relative to human colon carcinoma and melanoma cells. The expression of bFGF and bFGF receptors by glioma cells may be related to abnormal growth and neoplastic progression in these tumors.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Glioma/fisiopatologia , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/fisiopatologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Melanoma/fisiopatologia , Ratos , Receptores de Superfície Celular/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos , Células Tumorais CultivadasRESUMO
OBJECTIVE: To demonstrate expression of transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. METHODS: Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGF beta and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGF beta bioactivity was determined by Mv1Lu assay. RESULTS: Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGF beta and bFGF. Western blot analysis identified a single TGF beta band (MW approximately 25 kd) in each of the cell lysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW approximately 18.0 kd) in normal synovial cell lysates and 4 bFGF bands (MW approximately 18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell lysates. RA and normal synovial cells expressed mRNA transcripts encoding TGF beta 1 but not TGF beta 2, and FGF-2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb 1D11.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 micrograms/ml) had no effect on cell growth. CONCLUSION: RA and normal synovial cells cultured in serum-free medium express TGF beta 1 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGF beta 1 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown.