RESUMEN

Tumor-derived vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) plays an important role in neovascularization and the development of tumor stroma. Furthermore, VEGF receptors are over-expressed in the endothelial cells of tumor vasculature and almost non-detectable in the vascular endothelium of adjoining normal tissues. The differential expression of receptor offers a selective advantage for targeting cytotoxic toxin polypeptides. We have prepared a vascular targeting reagent by chemically linking recombinant VEGF to a truncated form of diphtheria toxin. The VEGF-toxin conjugate was selectively toxic to endothelial cell lines and inhibited experimental neovascularization of the chick chorioallantoic membrane. In the present study, we examined the effects of VEGF-toxin conjugate on solid tumor growth. Athymic nude mice with established subcutaneous tumors were treated with daily intraperitoneal injections of the VEGF-toxin conjugate or free toxin. When compared with control animals treated with the toxin polypeptide alone, the conjugate-treated animals displayed a significant inhibition of tumor growth. Histological analysis of tumors from conjugate-treated animals revealed hemorrhagic necrosis consistent with a vascular-mediated injury. In contrast, highly vascularized normal tissues from conjugate-treated animals demonstrated no evidence of hemorrhage or tissue injury. The conjugate was well tolerated without apparent toxicities. Our results illustrate the anti-tumor activity of a VEGF-toxin conjugate selectively targeting the tumor neovasculature.

Asunto(s)

Toxina Diftérica/uso terapéutico , Factores de Crecimiento Endotelial/uso terapéutico , Inmunotoxinas/uso terapéutico , Linfocinas/uso terapéutico , Neoplasias Ováricas/irrigación sanguínea , Animales , Toxina Diftérica/química , Factores de Crecimiento Endotelial/química , Femenino , Hemorragia/inducido químicamente , Hemorragia/patología , Humanos , Inmunotoxinas/química , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Hígado/irrigación sanguínea , Hígado/efectos de los fármacos , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Linfocinas/química , Ratones , Ratones Desnudos , Necrosis , Trasplante de Neoplasias , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

BACKGROUND: Tumor angiogenesis is essential for solid tumor growth. Yet, the importance of any particular factor in neoplastic proliferation is poorly defined. This study examines the clinical significance of increased expression of one of the angiogenic factors, vascular endothelial growth factor (VEGF), in early stage ovarian carcinoma. METHODS: Tumor specimens from 68 patients with International Federation of Gynecology and Obstetrics Stage I and II ovarian carcinoma were evaluated for VEGF expression. Antisense and corresponding sense (control) RNA probes were transcribed from the pCRII construct (Invitrogen, San Diego, CA), which contained human VEGF cDNA. The antisense probe was designed to include a highly conserved region of the VEGF coding sequence and thus detect all known variants. After in situ hybridization, sections were assessed for overexpression of VEGF. RESULTS: Twenty-nine of the tumor samples overexpressed VEGF, whereas 39 specimens did not. In patients whose tumors demonstrated elevated VEGF expression, 25% were without evidence of disease recurrence at last follow-up. In contrast, 75% of the patients whose tumors did not overexpress VEGF were without evidence of disease at last follow-up (P < 0.001). Median disease free survival for the VEGF positive group was 22 months, compared with > 108 months for the VEGF negative group (P < 0.001). When borderline tumors were excluded from the survival analysis, median disease free survival for the VEGF positive group was 18 months, compared with >120 months for the VEGF negative group (P < 0.001). Other possible prognostic variables had minimal impact on survival; these included age, stage, grade, cytology, and tumor size (P > 0.05). Assignment to a high risk group, as defined by the Gynecologic Oncology Group of the National Cancer Institute, was somewhat predictive of a shorter relapse free interval (P = 0.056). In a multivariate analysis, however, only elevated VEGF expression was associated with poorer survival. CONCLUSIONS: In this analysis, patients with early stage ovarian carcinoma with increased VEGF expression had a poorer prognosis. Further study of VEGF may ultimately lead to identification of patients with high risk lesions whose tumor biology portends a worse prognosis and who therefore may benefit from aggressive adjuvant therapy.

Asunto(s)

Carcinoma/patología , Factores de Crecimiento Endotelial/análisis , Linfocinas/análisis , Neovascularización Patológica/patología , Neoplasias Ováricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Elementos sin Sentido (Genética) , Permeabilidad Capilar , Supervivencia sin Enfermedad , Femenino , Humanos , Modelos Logísticos , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Sondas ARN , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Vascular endothelial growth factor (VEGF) expression and microvessel density were studied in cases of advanced epithelial ovarian carcinoma to evaluate their usefulness as prognostic variables. Tumor samples from 18 patients with advanced stage serous epithelial ovarian cancer were evaluated for VEGF expression by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Immunohistochemical study of corresponding archival tissues with an antibody to von Willebrand factor (vWF; FVIII-RA) was used for tumor microvessel count determinations. The correlation of VEGF expression and mean microvessel counts was determined by an unpaired t-test. Survival analysis for known prognostic factors and VEGF expression was performed. Survival distributions were calculated by the product limit of Kaplan and Meier and significant differences between distributions were analyzed with a log rank test. From the RT-PCR analysis of tumor VEGF expression, 12 samples were found to be strongly positive, whereas six samples had low/negative VEGF expression. The median survival was 60 months for the VEGF-low/negative group and 28 months for the VEGF-positive group (P = 0.058). Other prognostic variables had minimal impact on survival, i.e. age < 65 years (P = 0.873), FIGO stage (P = 0.06), grade (P = 0.236) and debulking status (P = 0.842). Fourteen of 18 tumor specimens were suitable for microvessel counting. The mean microvessel counts of the VEGF-positive group and the VEGF-negative group were 27/hpf and 35/hpf, respectively (P = 0.16). In this preliminary analysis, high VEGF expression in epithelial ovarian carcinomas was associated with poor overall survival. Further study will be necessary to elucidate the lack of association of VEGF expression and tumor microvessel counts.

Asunto(s)

Cistadenocarcinoma Seroso/química , Factores de Crecimiento Endotelial/análisis , Linfocinas/análisis , Neoplasias Ováricas/química , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia Celular/fisiología , Cistadenocarcinoma Seroso/irrigación sanguínea , Cistadenocarcinoma Seroso/mortalidad , Cistadenocarcinoma Seroso/patología , Epitelio/química , Epitelio/patología , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Microcirculación/fisiología , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa/métodos , Pronóstico , Tasa de Supervivencia , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [gamma32P]ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Asunto(s)

Toxinas Bacterianas , Oligopéptidos/genética , Proteínas Recombinantes/aislamiento & purificación , Superantígenos , Adenosina Trifosfato/metabolismo , División Celular/efectos de los fármacos , Clonación Molecular , Secuencia de Consenso/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacología , Electroforesis en Gel de Poliacrilamida , Factores de Crecimiento Endotelial/metabolismo , Enterotoxinas/metabolismo , Enterotoxinas/farmacología , Expresión Génica/genética , Vectores Genéticos/genética , Linfocinas/metabolismo , Peso Molecular , Oligopéptidos/metabolismo , Isótopos de Fósforo , Fosforilación , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ricina/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Levaduras/genética

RESUMEN

OBJECTIVE: Our purpose was to test the ability of an antiserum to vascular permeability factor/vascular endothelial growth factor to inhibit postoperative adhesion formation in a murine model. STUDY DESIGN: After a standardized peritoneal injury, 28 Balb/c mice were randomized and treated intraperitoneally with either vascular permeability factor antiserum (n = 14) or preimmune serum (n = 14) at the time of abdominal closure. Mice were killed on postoperative day 14, and the development of intraabdominal adhesions was determined. Adhesion scoring was based on an overall assessment of the extent, location, and type of adhesions present. Statistical analyses were performed with the Mann-Whitney and Fisher's exact tests. RESULTS: The mice treated with the vascular permeability factor antiserum had significantly lower adhesion scores than did the control group (mean +/- SD 1.5 +/- 0.9, median 1.0, vs control 2.5 +/- 0.7, median 3.0). When the groups were analyzed for the presence of grade 2 or 3 adhesions, the group treated with vascular permeability factor antiserum had a significantly lower incidence of advanced adhesions (38%, vs control 92%). CONCLUSION: This study demonstrates that the intraperitoneal administration of a neutralizing antiserum to vascular permeability factor/vascular endothelial growth factor limits postoperative adhesion formation. These observations, to the best of our knowledge, are the first to suggest a role for vascular permeability factor in the pathogenesis of adhesion formation.

Asunto(s)

Abdomen/cirugía , Anticuerpos/inmunología , Factores de Crecimiento Endotelial/inmunología , Linfocinas/inmunología , Complicaciones Posoperatorias/prevención & control , Animales , Femenino , Inmunización Pasiva , Incidencia , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Adherencias Tisulares/epidemiología , Adherencias Tisulares/patología , Adherencias Tisulares/prevención & control , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Inhibition of tumor neovascularization has profound effects on the growth of solid tumors. An endothelial cell-specific cytotoxic conjugate was prepared by chemically linking recombinant vascular endothelial growth factor (VEGF165) and a truncated diphtheria toxin molecule (DT385). The treatment of subconfluent cultures of human umbilical vein endothelial cells and human microvascular endothelial cells with the VEGF165-DT385 conjugate resulted in a selective, dose-dependent inhibition of growth. Parallel experiments with either the free toxin or a mixture of VEGF and the toxin polypeptide did not affect proliferation (DNA synthesis) of these cells. The selective cytotoxicity correlated with the appropriate receptor expression (KDR/flk-1 positive) on the target cells. VEGF-toxin conjugate inhibited the growth of a murine hemangioma-derived endothelial cell line (Py-4-1), which was positive for flk-1 expression. Under similar conditions, the conjugate did not affect the proliferation of a receptor-negative ovarian cancer cell line in vitro. In an in vivo model of angiogenesis, the VEGF165-DT385 conjugate blocked basic fibroblast growth factor-induced neovascularization of the chick chorioallantoic membrane. These studies demonstrate the successful targeting of a cytotoxic polypeptide to proliferating vascular endothelial cells (normal and tumorigenic) and the potential utility of such conjugates in blocking tumor neovascularization.

Asunto(s)

Toxina Diftérica/farmacología , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Hemangioma/irrigación sanguínea , Inmunotoxinas/farmacología , Linfocinas/farmacología , Neovascularización Patológica/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras , Receptores de Factores de Crecimiento , Animales , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , ADN/efectos de los fármacos , Toxina Diftérica/administración & dosificación , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Factores de Crecimiento Endotelial/administración & dosificación , Endotelio Vascular/química , Endotelio Vascular/citología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hemangioma/química , Humanos , Inmunotoxinas/administración & dosificación , Linfocinas/administración & dosificación , Ratones , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/química , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Cordón Umbilical/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Somatostatin and other neuropeptides are known to modulate the proliferative capacity of immune cells. In the present study, we investigated the expression of Somatostatin receptor (SSTR) subtypes on rat thymocytes. RT-PCR analysis of fresh thymocytes showed significant levels of transcripts for the SSTR2 whereas transcripts for the SSTR1 and SSTR3 were not detectable. Interestingly, when the thymocytes were activated with low concentration of Phytohemagglutinin and interleukin 1, the transcript for SSTR1 was markedly increased. Lymphokine induced activation of thymocytes selectively upregulated the SSTR1 since, transcripts for SSTR2 remained the same after activation and SSTR3 was not detectable. PCR amplified fragment of SSTR1 from the activated thymocytes showed identical sequence to the rat brain receptor. The physiological significance of the increase of SSTR1 mRNA in thymocytes after activation remains to be elucidated but it may be possible that these two different subsets of receptors (SSTR1 and SSTR2) are involved in the modulation of thymocyte proliferation and differentiation.

Asunto(s)

Regulación de la Expresión Génica , Activación de Linfocitos , Receptores de Somatostatina/genética , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Interleucina-1/farmacología , Masculino , Datos de Secuencia Molecular , Fitohemaglutininas/farmacología , ARN Mensajero/metabolismo , Ratas , Receptores de Somatostatina/clasificación , Linfocitos T/inmunología , Transcripción Genética/genética , Regulación hacia Arriba

RESUMEN

Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) is emerging as an important growth factor in a variety of tumor types. As a potent endothelial cell mitogen and vascular permeabilizing agent, VEGF/VPF has the unique functional capacity to mediate the component events of solid tumor neovascularization and ascites tumor growth. In the present series of investigations, our experimental hypothesis was that VEGF/VPF is a critical mediator of ovarian carcinoma-associated ascites formation and solid tumor growth. Athymic nude mice xenotransplanted with human ovarian carcinoma cell lines received either a preimmune rabbit serum or VEGF/VPF antiserum. Compared with the control group receiving the preimmune serum, the antiserum-treated animals displayed a 10- and 12-fold reduction in ascites accumulation and solid tumor growth, respectively. The administration of a neutralizing antiserum to VEGF/VPF conferred a modest survival advantage to animals harboring intraperitoneal tumors. These data demonstrate the significance of VEGF/VPF in the pathogenesis of ovarian carcinoma and suggest that interventions targeting this growth factor and/or its receptor may be of therapeutic value in the management of ovarian cancer.

RESUMEN

VEGF is a potent mitogen for vascular endothelial cells in vitro and acts as an angiogenic factor in vivo. VEGF121 differs from the other isoforms in that it lacks the heparin-binding domain. To study the potential differences in biological functions of the VEGF isoforms, we cloned and expressed VEGF121 in a yeast expression system. VEGF121 was secreted from the yeast cells as a homodimer with a molecular weight of 34-36 kDa. By taking advantage of the consecutive histidine residues present at position 11 and 12 in VEGF, a novel method of purification using Nickel affinity chromatography was developed. Since all the isoforms of VEGF have an identical amino terminal end, this method can be used to purify not only VEGF121 but also the other forms of VEGF. The level of expression achieved using this system was as high as 40 mg/L. The recombinant protein was biologically active in stimulating the in vitro proliferation of vascular endothelial cells and positively reacted to an antiserum made against recombinant VEGF165.

Asunto(s)

Factores de Crecimiento Endotelial/aislamiento & purificación , Linfocinas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía de Afinidad , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Linfocinas/biosíntesis , Linfocinas/farmacología , Sustancias Macromoleculares , Peso Molecular , Níquel , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae , Transfección , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine tag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [gamma-32P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The 32P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [32P] phosphorylation.

Asunto(s)

Ricina/metabolismo , Secuencia de Aminoácidos , Cromatografía de Afinidad , Histidina/química , Hidrólisis , Datos de Secuencia Molecular , Oligopéptidos/química , Radioisótopos de Fósforo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ricina/genética , Ricina/aislamiento & purificación

RESUMEN

Ricin is a toxic glycoprotein made of two polypeptide chains (A and B) linked by a disulfide bond. Ricin binds to cells by the B chain and is then internalized. The interchain disulfide bond is believed to be reduced in endosomes, and the A chain is then subsequently translocated to cytoplasm where it inactivates ribosomes. To understand the role of the disulfide bond in ricin toxicity, we prepared two types of ricin molecules. First, cysteine 259 of the A chain was mutated to an alanine residue. The mutant A chain was then reassociated with the native B chain to determine whether ricin is biologically active in the absence of an interchain disulfide bond. Reassociated mutant ricin showed a 40-fold reduction in biological activity. Binding studies using a hydrophobic fluorescence probe indicated that the associated complex was stable only at neutral pH and became highly unstable at a lower pH characteristic of the endosomal milieu. In the second construct, the interchain disulfide bond was replaced with a non-reducible bond by chemical derivatization. Interestingly, the non-reducible ricin molecule was equally cytotoxic as native ricin. These results show: (i) that the interchain disulfide bond is necessary to hold the A chain and the B chain together at endosomal pH, and (ii) that intact ricin may be transported to the cytoplasm where proteolysis or hydrolysis may occur to release the biologically active moiety.

Asunto(s)

Antineoplásicos/farmacología , Reactivos de Enlaces Cruzados , Disulfuros/química , Ricina/química , Ricina/farmacología , Alanina , Antineoplásicos/química , Secuencia de Bases , Muerte Celular , Estabilidad de Medicamentos , Femenino , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis , Neoplasias Ováricas , Proteínas Recombinantes/farmacología , Ricina/genética , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Células Tumorales Cultivadas

RESUMEN

Vascular endothelial growth factor (VEGF) is a glycoprotein consisting of two identical polypeptide chains linked by a disulfide bond. The unique biological activities of VEGF include its potent mitogenic and permeability inducing properties specific for the vascular endothelium. VEGF is implicated in tumor angiogenesis, wound healing, and the stimulation of collateral vessel formation at the site of arterial occlusion. Therefore, in order to produce large quantities of biologically active VEGF, a splice variant (VEGF165) was cloned and expressed in a yeast expression system. The coding region of VEGF165 was isolated from U937 cells by RT-PCR, sequenced and then cloned into the yeast expression vector pHILS1. VEGF165 was secreted into the medium as a dimer. Recombinant VEGF reacted to antibodies raised against the N-terminal and C-terminal synthetic polypeptides of human VEGF. As much as 35-40 mg/L of purified VEGF could be obtained from the yeast expression system. The recombinant protein was biologically active in inducing vascular endothelial cell proliferation in vitro and permeability changes in vivo.

Asunto(s)

Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Permeabilidad Capilar/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Clonación Molecular , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/aislamiento & purificación , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Azul de Evans/metabolismo , Vectores Genéticos/genética , Cobayas , Humanos , Linfocinas/biosíntesis , Linfocinas/química , Linfocinas/aislamiento & purificación , Linfocinas/farmacología , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sefarosa/análogos & derivados , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

GnRH agonists have been found to be clinically useful in several hormone-sensitive conditions, including cancer. However, there is controversy regarding a direct action of these agents on the pathologic tissue of a given disease process, in particular, tumors. In this study, we examined the effects of D-Trp(6) -LHRH, a potent GnRH agonist, on the expression of an increasingly important angiogenesis factor, VPF (vascular permeability factor)/VEGF (vascular endothelial growth factor). Ovarian carcinoma cell lines exposed to D-Trp(6) -LHRH in culture demonstrated a reversible inhibition of VPF mRNA expression and a parallel decrease in the endothelium-specific mitogenic activity of the conditioned media from these treated cultures. This study reports a novel activity of a GnRH agonist and provides a starting point to investigate the in vivo anti-angiogenic properties of GnRH peptide analogs.

RESUMEN

Epithelial ovarian cancer is an aggressive malignancy with a generally poor outcome. To improve survival, novel therapeutic strategies for this disease are needed and require elucidation of the biological events that underlie transformation and tumor growth. Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a homodimeric glycoprotein that acts on vascular endothelium as a potent permeability-inducing agent and mitogen. The present study demonstrates for the first time the constitutive gene expression of VPF in normal and neoplastic human ovaries. Abundant levels of VPF have been identified by an immunoassay in the ascites of patients with epithelial ovarian cancer (K-T. Yeo et al., Cancer Res., 53: 2912-2918, 1993). We have identified the malignant epithelium as one source of VPF in the ascites. Reverse transcription-polymerase chain reaction has demonstrated the expression of the two secreted isoforms, VPF121 and VPF165, in normal and neoplastic ovaries. Western blotting and an endothelial cell proliferation assay confirmed secretion of a biologically active product. VPF may be an important mediator of ascites formation and tumor metastasis observed in neoplastic conditions of the ovary.

Asunto(s)

Factores de Crecimiento Endotelial/análisis , Linfocinas/análisis , Neoplasias Ováricas/química , Ovario/química , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Western Blotting , Factores de Crecimiento Endotelial/genética , Femenino , Humanos , Linfocinas/genética , Linfoma de Células B Grandes Difuso , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Neoplásico/análisis , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Ascitic fluid from human ovarian cancer patients often contains a large number of leukocytes along with tumor cells. Some of the recent evidence suggests that the ascitic fluid contains factors capable of inducing the growth of ovarian cancer cells in vitro and in vivo. While these factors have not yet been completely characterized, growth factors secreted by the tumor cells could influence the tumor growth by paracrine and autocrine mechanisms. Earlier, we reported that ovarian epithelial cancer cells produce macrophage colony-stimulating factor. It appears that these tumor cells produce more than one cytokine. Identifying the various products secreted by the tumor cells would provide valuable information needed to understand the biology of ovarian cancer. In the present study, evidence is provided for the first time that five different human ovarian epithelial tumor cell lines and tumor cells isolated from the ascitic fluid of four cancer patients express interleukin (IL) 1 alpha and beta genes constitutively. Production of the lymphokine was determined by analyzing the cellular RNA for IL-1-related transcripts and by immunological assays. Ovarian cancer cells also secrete another pleiotropic cytokine, IL-6, constitutively. In many systems, IL-1 induces the expression of the IL-6 gene. To determine whether the basal levels of IL-6 production are dependent on the endogenous IL-1, neutralization studies were carried out. Addition of antibodies to IL-1 did not decrease the levels of IL-6 secreted by the cancer cell lines. These results suggest that multiple cytokines are produced by ovarian cancer cells and that the endogenous IL-1 may not be directly involved in the regulation of IL-6 gene expression in these cells.

Asunto(s)

Interleucina-1/genética , Neoplasias Ováricas/genética , ARN Mensajero/análisis , Líquido Ascítico/química , Femenino , Humanos , Interleucina-1/metabolismo , Interleucina-1/farmacología , Interleucina-6/metabolismo , Neoplasias Ováricas/metabolismo , Células Tumorales Cultivadas

Asunto(s)

Inmunotoxinas , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Humanos