RESUMO
Among the key cell cycle regulators, cyclin D1 has been implicated most strongly in oncogenesis. This G1 cyclin is a putative proto-oncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias. We have now raised a series of monoclonal antibodies to human cyclin D1 and analysed its regulation at the protein level in 40 human tumour cell lines. We found that 12 cell lines displayed low or undetectable cyclin D1 protein level, while the remaining lines accumulated the protein to a level comparable to, or moderately higher than, that of four normal diploid non-immortalized cell types. The cell cycle-dependent oscillation and subcellular localization of cyclin D1 were similar in both tumour and normal cells. The protein localized to the nucleus of G1 cells, and it was reduced to immunocytochemically undetectable level in DNA-replicating cells. At the functional level, microinjection and electroporation of anti-D1 antibodies revealed that in most tumour cell lines studied, including those with amplification at the cyclin D1 locus, this cyclin is essential for cell cycle progression in G1. Some tumours, however, seem to have evolved mechanism(s) that enable them to bypass the requirement for functional cyclin D1.
Assuntos
Ciclo Celular , Ciclinas/metabolismo , Proteínas Oncogênicas/metabolismo , Anticorpos Monoclonais , Células Cultivadas , Ciclina D1 , Ciclinas/antagonistas & inibidores , Ciclinas/imunologia , Humanos , Imuno-Histoquímica , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/imunologia , Proto-Oncogene Mas , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
Accumulation of the p53 protein was analysed in 212 human malignant lesions. Immunohistochemical staining with new polyclonal (CM-1) and monoclonal antibodies (BP 53-12 and BP53-24) to p53 on methacarn-fixed paraffin sections showed positive staining in 161 (76%). The positive tumours were found across a wide range of human malignancies including breast, colon, stomach, bladder and testis carcinomas, soft-tissue sarcomas and melanomas. The staining was always confined to the malignant lesion. Immunoprecipitation and quantitative ELISA assays established that the positive staining was associated with accumulation of the protein and that the protein was frequently in a mutant conformation. Accumulation of mutant p53 protein is therefore a common feature of human malignant disease.
Assuntos
Genes p53 , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Anticorpos , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias/patologia , Transfecção , Proteína Supressora de Tumor p53/análiseRESUMO
Reactivity patterns of seven mouse monoclonal antibodies to human keratin 7 were compared using immunoblotting and immunohistochemistry on cultured cells and normal human and animal tissues. Differences in keratin specificities as determined by two-dimensional immunoblots and interspecies cross-reactivity data on 8 mammalian species suggest that at least six nonidentical epitopes of the keratin 7 molecule are recognized by this panel of reagents. Immunohistochemical examination of a panel of various human neoplasms with monoclonal antibodies monospecific for keratins 7, 18 and 19 revealed potential value of keratin subtyping in differential diagnosis of tumors in general and in subclassification of carcinomas in particular.
Assuntos
Anticorpos Monoclonais , Queratinas/imunologia , Neoplasias/diagnóstico , Animais , Neoplasias da Mama/diagnóstico , Neoplasias do Colo/diagnóstico , Reações Cruzadas , Imunofluorescência , Humanos , Proteínas de Filamentos Intermediários/análise , Queratinas/análise , Neoplasias/análise , Coloração e Rotulagem , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/diagnósticoRESUMO
A comparative study with 21 recently raised monoclonal antibodies (3 of which are reported here for the first time) to human keratin polypeptides was performed on a wide range of paraffin-embedded tissues and tumors, aimed at the examination of effects of four different fixatives and protease pretreatment on the immunohistochemical detection of keratins. Our data demonstrated that: (a) formaldehyde-based fixatives modified by acidification and/or addition of methanol gave results superior to those achieved by routinely used formol saline; (b) relatively rare antibodies (4 out of 21) could be identified which gave reliable immunostaining patterns even on routine formalin-fixed material; (c) a proteolytic digestion step preceding the immunostaining was beneficial for the performance of the majority of antibodies in our panel. Additional options which could potentially lead to further improvement of keratin immunohistochemistry in paraffin embedded specimens are also suggested. This work provides the necessary basis for wider application of the anti-keratin antibodies of the C-series in both routine oncopathology and research-oriented retrospective studies.
Assuntos
Anticorpos Monoclonais , Endopeptidases , Queratinas/análise , Proteínas de Neoplasias/análise , Fixação de Tecidos/métodos , Adenocarcinoma/química , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/biossíntese , Eletroforese em Gel de Poliacrilamida , Secções Congeladas , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Inclusão em Parafina , Células Tumorais CultivadasRESUMO
A comparative study of six mouse monoclonal antibodies against human 45 kDa keratin polypeptide (keratin No. 18) was undertaken using three experimental approaches: immunohistochemistry on normal human tissues, examination of interspecies cross-reactivity and identification of the target polypeptides in 1-D and 2-D immunoblots. The data suggest that at least five different antigenic sites of keratin 18 are recognized by this panel of reagents. The C-04 epitope is keratin 18-specific and widely conserved among mammalian species, while the antibodies DA7 and DC10 also react specifically with the 45 kDa keratin but stain simple epithelia of human origin only. Two antibodies, C-11 and C-66, decorate simple as well as stratified epithelia in human and in all seven animal species tested, but their respective target epitopes are shared by different groups of keratin polypeptides, which indicates their non-identity. In contrast to keratin specificity of the five above mentioned antibodies, the C-08 antibody cross-reacts with a 70 kDa nuclear lamina protein found in human and bovine tissues. The results of the present study provide the necessary basis for future applications of these antibodies in both routine immunodiagnostic work and as probes to study the biology of epithelial cells in general and the significance of keratin intermediate filaments in particular.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Queratinas/imunologia , Animais , Especificidade de Anticorpos , Bovinos , Células Cultivadas , Citoesqueleto/imunologia , Epitélio/imunologia , Imunofluorescência , Humanos , Hibridomas/imunologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
In an attempt to raise a set of monoclonal antibody (MAb) probes for the study of biology and diagnostic value of human keratin No. 8, a series of hybridomas was prepared and their characteristics investigated by immunohistochemistry and immunoblotting. The polypeptide specificities of individual MAbs ranged from monospecificity for keratin No. 8 (MAbs C-15, C-23, C-36, C-43, C-47, C-51, and C-61) up to the "pan-keratin" MAbs C-11 and C-66 recognizing a broad range of keratins. The target epitopes of the remaining 4 MAbs (C-10, C-22, C-50, C-69) appear to be shared by various pairs of keratin polypeptides. The immunohistochemical investigation of tissues and/or cultured cells from 10 animal species revealed considerable variability of the interspecies crossreactivity of individual MAbs. The target epitopes of 5 MAbs appear to be phyllogenetically conserved from man to Xenopus, 6 of 12 MAbs tested show reactivity with several mammalian species (but not chicken or Xenopus) and the MAb C-15 reacts with human and sheep keratin only. The data obtained demonstrate that at least 9, and possibly as many as 12, nonidentical epitopes of the human keratin 8 polypeptide can be distinguished by this set of antibodies. The results of the present study suggest that our MAbs represent a significant contribution to the list of reagents applicable in both research of intermediate filament (IF) biology and diagnostic histopathology.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Queratinas/imunologia , Filogenia , Animais , Linhagem Celular Transformada/imunologia , Reações Cruzadas , Cães , Epitélio/imunologia , Humanos , Filamentos Intermediários/imunologia , Camundongos , Especificidade de Órgãos/imunologiaRESUMO
The results of the present study of the p53 antioncogene in a broad panel of human cell lines (n = 32) and biopsy specimens (n = 435) from both normal and tumour tissues can be summarized as follows: 1. Cells in primary cultures from normal tissues express very low levels of the p53 protein while strong nuclear accumulation of p53 can be seen in all SV 40 transformed human cell lines and the vast majority of tumour--derived cell lines studied. 2. Similarly, in human tissues strong nuclear p53 expression is found in high proportion of malignancies of various histogenesis, in contrast to benign lesions, nonmalignant tissues surrounding malignant tumours and normal tissues in which the p53 protein levels remain below the limits detectable by common immunohistochemical methods. 3. Sequence analysis of p53 mRNA amplified by polymerase chain reaction (PCR) revealed point mutations in the central region of the p53 gene in several cancer cell lines. Furthermore, very good correlation was found between the presence of such mutations and accumulation of the mutated p53 protein. This study confirms and extends our current view of p53 as the gene most frequently altered in human cancer and suggests that simple immunohistochemical methods can be used to screen for aberrations of this antioncogene.
Assuntos
Genes p53/genética , Neoplasias/genética , Humanos , Imuno-Histoquímica , Mutação , Células Tumorais CultivadasRESUMO
Recent evidence from molecular biology studies of the cell cycle machinery suggests that, apart from oncogenes and tumor suppressor genes, the genes encoding the key cell cycle regulatory proteins could serve as additional targets for oncogenic mutations involved in the multistep process of carcinogenesis. In an attempt to identify such potential cancer-associated aberrations of the cell cycle regulators, the expression of cdc2 and cdk2 kinases, as well as cyclins A, B1 and D1, was analyzed by immunoblotting in a panel of more than 40 human cancer cell lines derived from 17 different tumor types. The expression of cdc2, cdk2, cyclin B1 and cyclin A polypeptides was detectable in all lines examined, and moderate variation in protein level does not provide evidence for any obvious abnormalities in the cancer cell lines studied. The application of a series of novel monoclonal antibodies (Mab) to human cdc2 revealed the existence of an intriguing protein, designated p37, immunologically and structurally related to cdc2, which is strongly and selectively expressed in about 50% of the cancer cell lines. In contrast to cyclin A, which has also been implicated in tumorigenesis, we found pronounced variation in abundance of the cyclin D1 protein. Our data suggest that dysregulation of cyclin D1 (a candidate bcl-1, PRAD1 oncogene) can be involved in the pathogenesis of some additional tumor types (e.g., sarcomas and neuroblastomas) besides those reported for amplification and/or mRNA overexpression of this oncogene.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/genética , Quinases Ciclina-Dependentes , Neoplasias/genética , Proteínas Serina-Treonina Quinases , Animais , Proteína Quinase CDC2/genética , Quinase 2 Dependente de Ciclina , Ciclinas/genética , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Quinases/genética , Células Tumorais CultivadasRESUMO
Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8-10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.
Assuntos
Proteína Glial Fibrilar Ácida/genética , Medula Espinal/metabolismo , Vimentina/genética , Anticorpos Monoclonais , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Expressão Gênica , Idade Gestacional , Proteína Glial Fibrilar Ácida/imunologia , Humanos , Microscopia de Fluorescência , Gravidez , Medula Espinal/embriologia , Coloração e Rotulagem , Vimentina/imunologiaRESUMO
Alterations of the p53 tumour suppressor gene are considered critical events in multistage carcinogenesis of a wide range of human cancers. In an attempt to elucidate the role of various p53 mutations in tumorigenesis and to investigate their relationship to the p53 protein accumulation and subcellular localization, we have raised a new series of 21 mouse monoclonal antibodies (MAbs) to human recombinant p53. The new MAbs (designated the Bp53 series) appear to recognize mainly denaturation-resistant epitopes in immunoblotting and the majority of them are suitable for immunostaining of p53 in cultured cells and frozen sections. Furthermore, at least three MAbs (Bp53-11, Bp53-12, and Bp53-28) proved to be reliable reagents for immunohistochemistry on paraffin-embedded specimens. The immunohistochemical analysis of paraffin sections from 118 human tumours of various histogeneses with Bp53-11 and Bp53-12 showed nuclear accumulation of the p53 protein in variable proportion of tumour cells in 76 cases (64 per cent). The influence of three parameters of tissue processing (type of fixative, period of fixation, and duration of autolysis) on p53 protein detection was also investigated. The results of this study provide the necessary basis for wider application of these novel MAbs as tools in both routine histopathology and functional analyses of the p53 oncoprotein.
Assuntos
Proteína Supressora de Tumor p53/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Técnicas de Preparação Histocitológica , Humanos , Imuno-Histoquímica , Camundongos , Neoplasias/química , Proteínas Recombinantes/imunologia , Proteína Supressora de Tumor p53/análiseRESUMO
A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.
Assuntos
Anticorpos Monoclonais/imunologia , Queratinas/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular , Eletroforese em Gel Bidimensional , Epitélio/imunologia , Imunofluorescência , Humanos , Immunoblotting , Queratinas/análise , Camundongos , Neoplasias/química , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
An immunohistochemical study of the expression of the secretory component (SC) in human mammary gland epithelium at various stages of differentiation, as well as in benign and malignant breast tumours, was undertaken using three mouse monoclonal antibodies. Antibody RICEO-SC-05 (SC-05), raised against a partially purified preparation of human SC, and reacting with a reduction-resistant epitope present in both free and polymeric immunoglobulin-bound SC, was compared in immunoperoxidase and immunofluorescence studies on a diverse range of normal tissues, to 2 reference anti-SC antibodies (LICR-LONLC28 and RICEO-MFG-12). All three antibodies reacted with secretory epithelia only, consistent with known patterns of expression of SC in tissues, although there was an unexpected reaction by all anti-SC antibodies with some Hassal's corpuscles of the thymus. Staining patterns seen in the normal resting, pregnant, lactating and regressing (after weaning) breast provide evidence for differentiation-associated changes in the production of SC, and support the concept of terminal ductal lobular units (TDLUs) as functional compartments of the mammary gland. SC was detected in all but one benign breast lesion (n = 53) as compared to only 24% positive cases with heterogeneous expression of SC found among 176 primary and metastatic breast carcinomas examined. In a series of 40 primary breast carcinomas and their corresponding lymph node metastases, a good overall correlation was found between the expression of SC in the matched specimens; aside from 3 heterogeneously SC-positive carcinomas whose metastatic counterparts were SC-negative. Our results demonstrate a potential application for monoclonal antibodies to SC in the study of human mammary gland differentiation, but suggest that the value of an assay for SC in the diagnosis of breast carcinomas is questionable due to the generally low expression of SC by either primary or metastatic breast lesions.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/diagnóstico , Mama/metabolismo , Colostro/metabolismo , Glicoproteínas de Membrana/metabolismo , Western Blotting , Mama/patologia , Neoplasias da Mama/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-HistoquímicaRESUMO
An immunohistochemical study with four monoclonal antibodies to human beta-casein was carried out to examine the expression of this milk protein in a wide range of normal tissues, in 127 breast tumours and in a heterogeneous panel of 42 malignancies of other histogenesis. The only normal tissue stained positively by the antibodies was the mammary gland in late pregnancy, during lactation and in the post-lactational regression period. None of the tumours of non-mammary origin showed any staining. Furthermore, only two of 40 benign breast lesions and one anaplastic primary carcinoma with its metastasis (among 87 breast carcinomas) showed any reactivity. The immunohistochemical results were supported by immunoblotting data and suggested beta-casein expression has no role to play as a marker in the diagnosis or monitoring of human breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Caseínas/biossíntese , Anticorpos Monoclonais , Mama/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Lactação/metabolismo , Gravidez/metabolismoRESUMO
An extensive series of histological sections reflecting the various states of normal breast tissue, and a range of benign and malignant lesions, were examined for the expression of the p53 protein using a panel of anti-p53 antibodies. In 2 separate series the results of using frozen or methacarn-fixed, paraffin-embedded sections were compared. Strong positive staining for p53 was detected in over 50% of the malignant lesions when frozen sections were used. This number fell to just over 20% when methacarn-fixed sections were examined. In neither series was any p53 staining seen in normal breast or in the benign lesions. Studies by Western blotting on breast cell lines confirmed that this histological signal is due to a pronounced over-expression of the p53 protein. Earlier studies show that this over-expression is associated with mutation of the p53 gene. Mutation of the p53 gene with over-expression of the mutant protein is therefore one of the most frequent specific genetic changes in malignant breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Genes Supressores de Tumor/genética , Proteína Supressora de Tumor p53/metabolismo , Anticorpos Monoclonais , Mama/anatomia & histologia , Mama/fisiologia , Doenças Mamárias/genética , Doenças Mamárias/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Células Cultivadas , Epitélio/metabolismo , Feminino , Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , Imuno-Histoquímica , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Mama/citologia , Transformação Celular Viral , Leite Humano/citologia , Animais , Southern Blotting , Divisão Celular , Linhagem Celular , DNA Viral/genética , Células Epiteliais , Imunofluorescência , Vetores Genéticos , Humanos , Técnicas In Vitro , Queratinas/metabolismo , Camundongos , Camundongos Nus , Proteína Oncogênica p21(ras)/genética , Retroviridae/genética , Vírus 40 dos Símios/genéticaRESUMO
Expression of the p53 oncoprotein was examined in a wide range of primary human testicular germ-cell tumours using a new mouse monoclonal antibody (MAb) BP53-11 raised and characterized in this study, in parallel with a polyclonal rabbit antiserum CM-1. Immunohistochemistry on paraffin sections showed positive nuclear reaction in at least a fraction of malignant cells in 90 (84%) out of 107 cases studied. Aberrant accumulation of the p53 protein was found among testicular tumours of all major histological types, although generally a higher percentage of positive cases and a higher proportion of p53 over-expressing nuclei within individual lesions was observed in embryonal carcinomas when compared with seminomas. The typical heterogeneous staining pattern characteristic of histological specimens was also found in a cultured cell line derived from a human embryonal carcinoma. In contrast to immunohistochemically undetectable levels in normal testes and morphologically normal tissue areas in the tumour-bearing testes, the accumulation of the p53 protein was clearly identified in a high proportion (59% of cases) of the pre-invasive lesions with positive atypical intratubular germ cells often found in the tissue adjacent to invasive tumours. Altered expression of the p53 protein is therefore a unifying feature of the majority of invasive male germ-cell tumours and the change resulting in high levels of p53 appears to be a relatively early step in the human testicular cancer pathogenesis.