RESUMO
To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.
Assuntos
Antígenos de Superfície/genética , Carcinoma Hepatocelular/enzimologia , Colangiocarcinoma/enzimologia , Oxigenases de Função Mista/genética , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Western Blotting , Neoplasias da Mama/imunologia , Carcinoma Hepatocelular/genética , Linhagem Celular Transformada , Colangiocarcinoma/genética , Clonagem Molecular , Neoplasias do Colo/imunologia , Biblioteca Gênica , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Recent observations based on immunohistochemistry and RT-PCR demonstrate that early placenta insulin-like peptide (EPIL), encoded by the INSL4 gene, is present in the placenta during gestation. In the present study, we report on the development of a specific immunoassay, entirely based on two monoclonal anti-peptide antibodies (mAbs), and its use for the detection of pro-EPIL forms in biological fluids during pregnancy. One mAb directed against the C-connecting peptide was used to capture pro-EPIL forms and their binding was revealed by a radiolabeled anti-A chain mAb as the indicator. A composite synthetic peptide, encompassing the C- and A-domains, was utilized as the standard. Under these experimental conditions, the assay displays a sensitivity limit of 2 ng/mL. Pro-EPIL molecular forms were detected in both amniotic fluid and maternal serum of pregnant women. At 12 to 16 weeks of pregnancy, the pro-EPIL level was higher in amniotic fluid (246 +/- 50.8 ng/mL) than in maternal serum (5 +/- 2.0 ng/mL). As gestation advanced, so the concentration of pro-EPIL forms decreased in amniotic fluid while its level increased in maternal serum. Interestingly, in amniotic fluid, the pattern of pro-EPIL concentration during pregnancy is very similar to that observed for human chorionic gonadotropin (hCG) and its free subunits. The pattern of serum pro-EPIL concentration is similar to that of the free alpha-subunit. Together with our previous immunohistochemical observations, these results indicate that pro-EPIL is preferentially secreted by cytotrophoblasts in amniotic fluid and that the biosynthesis of hCG subunits and EPIL may be regulated by common pathways. Overall, our observations strongly suggest that EPIL may play a critical physiological role during embryonic and foetal development.
Assuntos
Líquido Amniótico/metabolismo , Substâncias de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas da Gravidez/metabolismo , Gravidez/metabolismo , Adulto , Líquido Amniótico/química , Anticorpos Monoclonais , Gonadotropina Coriônica/metabolismo , Feminino , Humanos , Ensaio Imunorradiométrico , Gravidez/sangue , Proteínas da Gravidez/sangue , Proteínas da Gravidez/imunologia , Valores de Referência , Fatores de TempoRESUMO
Recently, a new member of the insulin gene superfamily termed insulin-like 4 (INSL4) was identified in the human placenta and uterus. The present study investigated whether placenta translates INSL4 mRNA into a putative peptide named early placenta insulin-like (EPIL). Among antibodies elicited against the C chain of pro-EPIL, one antibody (AB7381) was specifically directed against the C chain 59-88 portion, and among those elicited against the A and B chains of EPIL, one antibody (Ab1661) was directed against the A chain 115-139 and the B chain 23-52 portions. Immunohistochemistry based on antibody 7381 to pro-EPIL and antibody 1661 to EPIL demonstrated that the cytotrophoblast from early placenta preferentially expresses the pro-EPIL peptide, whereas the EPIL peptide is expressed by both the cytotrophoblast and the syncytiotrophoblast. At term, the pro-EPIL peptide was detected in villous cytotrophoblast cells, whereas the EPIL peptide was not detected. Moreover, in vitro experiments performed on term placenta showed that the steady state levels of INLS-4 mRNA in the cytotrophoblast are 10 times (one log unit) lower than in the differentiated villous syncytiotrophoblast cells. Taken together, these findings reveal that expression of EPIL peptides in the villous cytotrophoblast is different from that displayed by the syncytiotrophoblast. Finally, these data are the first demonstration that INSL4 mRNA are translated into pro-EPIL and EPIL peptides.
Assuntos
Substâncias de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular , Placenta/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Biossíntese de Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , Sequência de Aminoácidos , Feminino , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Gravidez , Transcrição GênicaRESUMO
To characterize the membrane changes associated with cisplatin resistance, we raised monoclonal antibodies (MAbs) against a cisplatin-resistant subline (OV1/DDP) derived from a human ovarian carcinoma cell line (OV1/p). An MAb, designated OCP02, was selected for its particularly high affinity for the resistant cell line. It bound 3.1-fold higher to OV1/DDP cells than to OV1/p cells and recognized an M(r) 45K antigen. This antigen appeared to be present in several normal and tumorous tissues. Its distribution in normal tissues was mainly detected in tissues involved in secretory processes, suggesting that this antigen could be related to a transport mechanism in normal cells as well as in drug-resistant cells.
Assuntos
Antígenos de Neoplasias/biossíntese , Cisplatino/farmacologia , Neoplasias Ovarianas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Resistência a Medicamentos/imunologia , Feminino , Humanos , Hibridomas/imunologia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Ovarianas/tratamento farmacológico , Células Tumorais Cultivadas/imunologiaRESUMO
In order to characterize the membrane changes related to Vinca alkaloid resistance, we raised monoclonal antibodies (mAbs) against a Vincristine resistant subline (OV1/VCR) derived from a human ovarian adenocarcinoma cell line (OV1/p). Among three monoclonal antibodies selected for a higher binding to OV1/VCR than to OV1/p cells, one designated OVR09, recognized a Mr 92,000 protein. This protein appears to be gradually overexpressed along the drug resistance establishment in vitro, and to decrease slowly in absence of drug. Further, mAb OVR09 showed a much higher binding to the vinblastine resistant epidermoid tumor cell line KbV1 than to its parental counterpart. The Mr 92,000 protein was also detected in various tumor cell lines and in an ovarian carcinoma surgical sample.
Assuntos
Antígenos de Neoplasias/imunologia , Células Tumorais Cultivadas/imunologia , Vincristina/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Carcinoma/imunologia , Membrana Celular/imunologia , Resistência a Medicamentos , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Neoplasias Ovarianas/imunologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A library of monoclonal antibodies (MoAbs) has been produced against a human hepatocellular carcinoma (HCC) cell line designated FOCUS in order to study the antigenic properties of transformed hepatocytes. Several monoclonal antibodies (MoAbs) were initially selected for study since they bound to antigens which were overexpressed in HCC tissues compared with the adjacent uninvolved normal liver counterpart; in addition, these MoAbs revealed low level antigen expression on other normal human tissues. Subsequently, HCC cell lines were metabolically labelled and the antigens further characterized by immunoprecipitation and Western blot analysis. If the MoAb recognized a primary linear epitope on a protein, cloning was performed using a lambda GT11 cDNA expression library prepared from the FOCUS HCC cell line. These studies characterized the HCC associated antigen(s) at the molecular level. This review illustrates the value of such an experimental approach to search for and identify HCC associated antigens and emphasizes the biological properties of novel proteins may be defined and characterized by these techniques. More important, our investigations have described unique proteins that may not only be important in the pathogenesis of HCC but also demonstrates how such antigen-antibody systems may be used to develop strategies for immunotargetting and gene therapy of HCC.