ABSTRACT
BACKGROUND: Effective cancer biomarkers for early detection, prognosis, or therapy response prediction are urgently needed in ovarian cancer. Kallikrein-related peptidases, including KLK5, have been reported to play an important role in the course of the disease. PATIENTS AND METHODS: KLK5 antigen content was determined by enzyme-linked immunosorbent assay in ovarian cancer patients' [FIGO (International Federation of Gynecology and Obstetrics) stages I-IV, n = 52] serum as well as ascitic fluid and compared with KLK5 content in serum of patients with benign ovarian tumors (n = 45). RESULTS: KLK5 antigen content was significantly elevated in the serum of ovarian cancer patients compared with the serum of patients with benign ovarian tumors. Forty-two of 52 ovarian cancer serum samples, 42 of 43 benign ovarian tumor serum samples, and all 41 ascitic fluid samples were KLK5 positive. Elevated KLK5 antigen in serum and ascitic fluid of ovarian cancer patients was a prognostic factor for progression-free survival. CONCLUSIONS: Our data support the finding that ovarian cancer patients release significant amounts of KLK5 into serum and ascitic fluid but KLK5 antigen is low in serum of patients with benign ovarian tumors. Increased serum and ascitic fluid KLK5 levels are associated with poor patient outcome, thus underlining the importance of KLK5 as a biomarker for early detection as well as for disease management in ovarian cancer.
Subject(s)
Biomarkers, Tumor/blood , Kallikreins/blood , Ovarian Neoplasms/blood , Ovarian Neoplasms/mortality , Adult , Aged , Ascitic Fluid/enzymology , Disease-Free Survival , Female , Humans , Middle Aged , Neoplasm Staging , Ovarian Neoplasms/pathologyABSTRACT
The aim of this study was to assess the value of TPS and CA 19-9 in a long-term follow-up analysis of 11 patients with chronic pancreatitis (CP) and 15 patients with pancreatic cancer (PC). In all monitored patients with chronic pancreatitis the initial TPS level was below 200 U/L, whereas CA 19-9 was elevated in two of them. In one patient a dramatic increase in the TPS concentration (820 U/L) was measured at the last follow-up visit (after 8.6 months), which led to the detection of PC. In all patients with PC the preoperative TPS level exceeded 200 U/L, whereas CA 19-9 was elevated in only nine patients. After the Kausch-Whipple operation 11 patients showed no evidence of disease and in eight of these patients both TPS and CA 19-9 were within the reference range; however, in three patients liver metastases were detected after 8-24 months from the last tumor marker measurement. In four of the 15 patients both markers were elevated at the end of the follow-up period and distant metastases were clinically confirmed. Our results indicate that in patients with CP and PC undergoing long-term follow-up, TPS reflects the clinical status of patients more accurately than CA 19-9.
Subject(s)
Biomarkers, Tumor/blood , CA-19-9 Antigen/blood , Pancreatic Neoplasms/blood , Pancreatitis/blood , Peptides/blood , Cohort Studies , Follow-Up Studies , Humans , Neoplasm Metastasis , Pancreatic Neoplasms/mortality , Pancreatitis/complications , ROC Curve , Time FactorsABSTRACT
VEGF is an important angiogenic cytokine with a critical role in tumor angiogenesis. VEGF concentrations were measured using an ELISA assay, detecting VEGF165 isoform, in tumor cyst and/or ascitic fluids and in sera of 86 patients with malignant neoplasms and in 53 patients with benign ovarian neoplasms. VEGF levels were significantly elevated in the sera and cyst fluids of carcinoma patients compared with patients who had benign neoplasms. In carcinoma patients, statistically higher VEGF levels were detected in tumor effusions than in corresponding sera. The differences between VEGF values in sera and tumor effusions in relation to histological subtypes of ovarian carcinoma and FIGO stages were statistically insignificant. High VEGF levels in ascitic fluids appeared to be significantly associated with shorter disease-free survival and overall survival In multivariate analysis, besides FIGO stage and age of patients, only serum VEGF concentration was an independent prognostic factor for overall survival. The elevated VEGF levels in sera and tumor effusions of patients with FIGO stages I/II indicated that angiogenesis promoted by VEGF is a continuous process, independent of clinical advancement of the disease.
Subject(s)
Ovarian Neoplasms/metabolism , Vascular Endothelial Growth Factor A/metabolism , Ascitic Fluid/metabolism , Ascitic Fluid/pathology , Cyst Fluid/metabolism , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Humans , Ovarian Neoplasms/blood , Ovarian Neoplasms/pathology , ROC Curve , Survival Rate , Vascular Endothelial Growth Factor A/bloodABSTRACT
BACKGROUND: The analysis of mutual relations between HPV infection and the expression of cancer gene products and proliferative activity in cervical carcinomas and dysplasias. MATERIALS AND METHODS: The expression of p53, c-erbB-2 oncoproteins and proliferative activity (Ki-67) was evaluated immunohistochemically in 41 cervical carcinomas and 29 dysplasias. HPV infection (type 16, 18) was assessed by in situ hybridization technique. RESULTS: HPV-positive carcinomas were found in 68.3% of cases. HPV type 16 infection were detected in 54% and HPV 18 in 39% of carcinomas. Simultaneous appearance of both virus types was shown in 25% of carcinomas. In dysplastic lesions, HPV infection was observed in 62.1% of cases. HPV type 16 was found in 34.5% and HPV 18 in 44.8% of patients. Both virus types were found in 17.2% of dysplasias. HPV infection was more extensive in cervical carcinomas than in dysplasias. Similarly the expression of oncoproteins was more intensive and referred to a higher percentage of cells in carcinomas. No relations between p53, c-erbB-2 overexpression and HPV infection were found. Ki-67 activity was found in a higher percentage of HPV-positive than in HPV-negative, both carcinomas and dysplasias. CONCLUSIONS: HPV infection, especially accompanied by increase of proliferative activity in dysplasias may define the cell subpopulation predisposed to malignant process development. The employment of in situ hybridization technique appears to be useful in detecting the viral infection in cytological smears even with no morphological changes in the cells.
Subject(s)
Ki-67 Antigen/biosynthesis , Papillomaviridae/isolation & purification , Papillomavirus Infections/metabolism , Receptor, ErbB-2/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Tumor Virus Infections/metabolism , Uterine Cervical Dysplasia/metabolism , Uterine Cervical Neoplasms/metabolism , Female , Humans , Ki-67 Antigen/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Receptor, ErbB-2/genetics , Tumor Suppressor Protein p53/genetics , Tumor Virus Infections/pathology , Tumor Virus Infections/virology , Uterine Cervical Dysplasia/classification , Uterine Cervical Dysplasia/pathology , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virologyABSTRACT
BACKGROUND: The value of serum tissue polypeptide specific antigen (TPS) as a complement to CA 19-9 in the detection of pancreatic carcinoma was determined prospectively. TPS and CA 19-9 levels obtained at the time of diagnosis in patients suspected of having chronic pancreatitis or pancreatic carcinoma were evaluated in receiver operating characteristic (ROC) curve analysis. METHODS: Serum TPS and CA 19-9 levels were measured by immunoassays in 122 subjects, 48 with pancreatic carcinoma and 74 with chronic pancreatitis. RESULTS: Elevated levels of CA 19-9 were detected preoperatively in 70% of pancreatic carcinoma patients and in 19% of chronic pancreatitis patients. Elevated levels of TPS were detected in 100% of patients with pancreatic carcinoma and in 22% of patients with chronic pancreatitis. The median levels of TPS and CA 19-9 for pancreatic carcinoma were significantly higher than those for chronic pancreatitis (P < 0.0001). Increasing the upper reference value of TPS allowed for better discrimination between chronic pancreatitis and pancreatic carcinoma. ROC curve analysis showed that the introduction of 200 U/L as a decision criterion for TPS did not reduce its sensitivity but significantly improved its specificity. At a specificity of 98% for TPS, discrimination between pancreatic carcinoma and chronic pancreatitis was found to be 97%. Increasing the upper reference level for CA 19-9 to attain a specificity of 98% decreased its sensitivity from 70% to 33%. CONCLUSIONS: At an elevated cut-off level for TPS (200 U/L), almost complete discrimination between pancreatic carcinoma and chronic pancreatitis was obtained. TPS will be more useful than CA 19-9 in the differential diagnosis of pancreatic carcinoma and chronic pancreatitis.
Subject(s)
Biomarkers, Tumor/analysis , CA-19-9 Antigen/analysis , Carcinoma/diagnosis , Pancreatic Neoplasms/diagnosis , Pancreatitis/diagnosis , Tissue Polypeptide Antigen/analysis , Adult , Aged , Carcinoma/immunology , Chronic Disease , Diagnosis, Differential , Female , Humans , Immunoassay , Male , Middle Aged , Pancreatic Neoplasms/immunology , Pancreatitis/immunology , Prospective Studies , Sensitivity and SpecificityABSTRACT
The presence of p53 autoantibodies and p53 protein overexpression in ovarian carcinoma patients were determined and compared. p53 antibodies were detected in sera samples, cyst and/or ascitic fluids of individual patients by two separate techniques (ELISA assay and immunoblot). p53 protein accumulation was assessed immunohistochemically in tissue sections and corresponding tumor effusion cells. The relations between p53 overexpression, the presence of p53 autoantibodies and histology of tumors, grade of differentiation and clinical stage of the disease were considered. p53 expression was found in 20 of 46 (43.5%) ovarian carcinomas and significant relationship between p53 reactivity in tumor tissue and effusion cells in individual patients was evident. In the subset of carcinomas with detectable p53 accumulation only two cases (one serous, one endometrioid) were associated with the presence of p53 autoantibodies (10%). Among 26 p53- negative carcinomas also two cases (7.6%) were seropositive. The strong correlation between the presence of p53 autoantibodies in the sera and respective cyst or ascitic fluids were revealed with no exception of this coincidence. There was no association between the detection of antibodies against p53 and FIGO stage and tumor grade. Our results clearly indicate that p53 overexpression is not sufficient to elicit p53 humoral response in ovarian carcinoma patients. The presence of p53 autoantibodies in this type of cancer is not a frequent event and their importance as independent prognostic factor seems to be very limited.
Subject(s)
Ascitic Fluid/metabolism , Autoantibodies/metabolism , Ovarian Cysts/metabolism , Ovarian Neoplasms/metabolism , Tumor Suppressor Protein p53/immunology , Autoantibodies/blood , Blotting, Western , Cell Differentiation/physiology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunohistochemistry , Neoplasm Staging , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Tumor Suppressor Protein p53/biosynthesisABSTRACT
Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The present study examines the existence and location of the constitutive isoform eNOS (endothelial NO synthase) accompanying the already substantiated neurogenic NOS (nNOS) in the human corpus cavernosum of men with and without erectile dysfunction. Activities of NOS enzymes were examined in specimens of 11 potent and nine long-term impotent patients by means of light and electron microscopy using NADPH-diaphorase staining and immunohistochemical eNOS-specific, smooth muscle actin-specific and nNOS-specific markers. Cavernosal smooth muscle shows a distinct expression of eNOS. In contrast to the weaker expression of eNOS and nitrinergic innervation found in larger veins, the small intracavernosal helicine arteries express large quantities of eNOS and possess a dense nitrinergic innervation. Long-term impotent patients display a broad heterogeneity in eNOS expression and nitrinergic innervation while no overall correlation between NOS expression and erectile function was observed. The expression of eNOS indicates eNOS as a main source of NO alongside nNOS. The differentiated localization of eNOS supports at least a role of this isoform in vascular regulation.
Subject(s)
Muscle, Smooth, Vascular/enzymology , Nitric Oxide Synthase/metabolism , Penile Erection/physiology , Penis/enzymology , Erectile Dysfunction/physiopathology , Humans , Immunohistochemistry , In Vitro Techniques , Male , Muscle Relaxation/physiology , Muscle, Smooth, Vascular/physiology , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type III , Penis/blood supply , Penis/physiologyABSTRACT
BACKGROUND: The association between p53 and c-erbB-2 overexpression relation to ER status in ductal breast carcinoma is still unclear. Our aim was investigate the prognostic importance of the overexpression of c-erbB-2, p-53 factor, and ER status in stage II of human ductal breast cancer. MATERIALS AND METHODS: Th. expression of c-erbB-2 and p53 oncoproteins was evaluated by immunoperoxidase technique (PAP) in 62 cases of ductal breast carcinoma. The relationship between these cell growth regulatory factors was estimated and compared with the presence estrogen receptor (ER), tumor grading, tumor size, lymph node involvement, age patients and number of relapses up to the second year after surgery. RESULTS: c-erbB-2 overexpression was found in 44% and p53 in 45% of carcinomas. ER level was usually inversely proportional to the presence of studied molecular markers. Stratifying patients on the basis of c-erbB-2, p53 and ER status revealed that the combination c-erbB-2 and p53 overexpression accompanied by undetectable ER, identified the population of poorly differentiated tumors and patients with a high incidence of axillary lymph node metastases and shorter relapse time. On the other hand, undetectable values of molecular markers were associated with a low grade of tumors and a lack of lymph nodes involvement. CONCLUSIONS: Estimation of c-erbB-2, p53 and ER status seems to be a powerful tool to discriminate between different phenotypes of breast carcinoma. c-erbB-2 and p53 oncoproteins have been recognized as independent molecular markers of aggressive tumor behaviour.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Receptor, ErbB-2/metabolism , Tumor Suppressor Protein p53/metabolism , Adult , Aged , Female , Humans , Immunohistochemistry , Middle Aged , Neoplasm Recurrence, Local , Prognosis , Prospective Studies , Receptors, Estrogen/metabolismABSTRACT
The serum markers TPS and CA 125 were determined in serum, cyst fluid and ascites in ovarian carcinoma patients and in patients with benign ovarian neoplasms. The levels of TPS and CA 125 were significantly higher in malignant and benign tumor cysts and ascitic fluids than in corresponding patients sera (p < 0.005 for TPS, p < 0.0001 for CA 125). The concentrations of cyst fluid TPS and CA 125 were also usually higher in cancer patients than in patients with benign ovarian neoplasms. TPS and CA 125 were elevated in a higher proportion of ovarian cancer patients sera than in benign ovarian patients' sera (p < 0.001 for both markers). Serum preoperative TPS and CA 125 levels were significantly higher in patients with advanced disease (FIGO stage III/IV) than in patients with early stage disease (FIGO stage I/II, p = 0.002, p < 0.05 respectively). When histology was considered, small differences in the marker signals were noted for TPS and CA 125 with the exception of mucinous neoplasms, where TPS showed a markedly higher signal. These results suggest that the combined use of TPS and CA 125 could be of additive value for the identification of epithelial ovarian neoplasms. Postsurgical TPS sera levels achieved the normal values faster than CA 125 suggesting that determination of TPS concentration before and 3 days after surgery seems to be valuable for the evaluation of radical surgery.
Subject(s)
Biomarkers, Tumor/analysis , CA-125 Antigen/analysis , Carcinoma/pathology , Ovarian Cysts/pathology , Ovarian Neoplasms/pathology , Peptides/analysis , Adenocarcinoma/blood , Adenocarcinoma/pathology , Ascites/blood , Ascites/pathology , Biomarkers, Tumor/blood , CA-125 Antigen/blood , Carcinoma/blood , Carcinoma/surgery , Carcinoma, Endometrioid/blood , Carcinoma, Endometrioid/pathology , Cystadenoma/blood , Cystadenoma/pathology , Female , Humans , Neoplasm Staging , Ovarian Cysts/blood , Ovarian Cysts/surgery , Ovarian Neoplasms/blood , Ovarian Neoplasms/surgery , Peptides/bloodABSTRACT
Frequencies of HPV type 16 and 18, evaluated by in situ hybridization technique in uterine cervix carcinomas of the IIIrd stage of clinical advancement, were 54% and 36.5% respectively. Presence of p 21 protein was detected in 85.3% cases of the cancers and showed no relation to HPV infection.
Subject(s)
Biomarkers, Tumor/analysis , Cyclins/analysis , Papillomaviridae/isolation & purification , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Uterine Cervical Neoplasms/chemistry , Uterine Cervical Neoplasms/virology , Cyclin-Dependent Kinase Inhibitor p21 , Female , Humans , Neoplasm Staging , Papillomaviridae/classification , Papillomavirus Infections/complications , Tumor Virus Infections/complicationsABSTRACT
The expression of Ki-67 proliferation antigen and its relation to p53 protein was assessed immunohistochemically in malignant and benign ovarian neoplasms, considering the stage of disease and histology of tumors. The comparison of p53 and Ki-67 in tissue sections and respective cyst and/or ascitic fluid cells was also performed. Significant heterogeneity of staining was observed. However, the relationship between p53 and Ki-67 activity in tissue sections and loose cells in individual patients was evident. Moreover, the presence of Ki-67 antigen was closely correlated with p53 protein. It was observed the trend for serous carcinoma to have a higher Ki-67 and p53 positivity versus endometrioid and mucinous carcinomas. However, it was not statistically significant. Both growth fraction as measured by Ki-67 staining and p53 content were significantly higher in stages III and IV compared to stages I and II of ovarian carcinomas (P<.05, and P<.01, respectively). In benign ovarian neoplasms, no p53 reactivity was observed and Ki-67 staining was very low. Our results showed that p53 is not a feature of benign epithelial ovarian tumors and indicate that increased proliferative activity of cells seems to involve immunohistochemically detectable alterations in p53 gene contributing to the evolution of ovarian carcinoma.
Subject(s)
Carcinoma/pathology , Neoplasm Proteins/analysis , Nuclear Proteins/analysis , Ovarian Neoplasms/pathology , Tumor Suppressor Protein p53/analysis , Adenocarcinoma, Mucinous/chemistry , Adenocarcinoma, Mucinous/diagnosis , Adenocarcinoma, Mucinous/pathology , Ascitic Fluid/chemistry , Carcinoma/chemistry , Carcinoma/diagnosis , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/pathology , Cystadenoma/chemistry , Cystadenoma/diagnosis , Cystadenoma/pathology , Female , Humans , Immunoenzyme Techniques , Ki-67 Antigen , Neoplasm Proteins/biosynthesis , Neoplasm Staging , Nuclear Proteins/biosynthesis , Ovarian Cysts/chemistry , Ovarian Cysts/diagnosis , Ovarian Cysts/pathology , Ovarian Neoplasms/chemistry , Ovarian Neoplasms/diagnosis , Paraffin Embedding , Tumor Suppressor Protein p53/biosynthesisABSTRACT
Infection with HPV type 16 was demonstrated in 54% of the cases and infection with HPV type 18 in 36.5% of the cases of uterine cervix carcinoma. Both types of viruses were present in 24.3% of the patients. P53 protein accumulation in cell nuclei was observed in 12.2% of the cases of uterine cervix carcinomas and the cases were mostly HPV negative while the cytoplasmic expression of p53 protein was present in 39% of the cases and was frequently accompanied by HPV infection. Presence of p21 protein was detected in 85.3% of the cases, independently of HPV infection or expression of p53 protein.
Subject(s)
Oncogene Protein p21(ras)/metabolism , Papillomaviridae/isolation & purification , Papillomavirus Infections/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Virus Infections/metabolism , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/virology , Female , Humans , Immunoenzyme Techniques , Papillomavirus Infections/pathology , Tumor Virus Infections/pathology , Uterine Cervical Neoplasms/pathologyABSTRACT
Serum levels of tissue polypeptide antigen specific (TPS), a cytokeratin 18 marker, were determined and compared with serum levels of carcinoembryonic antigen (CEA) in 45 patients with colon adenocarcinoma and in 34 patients with benign diseases (adenomatous polyps and ulcerative colitis) at the time of diagnosis. In colon carcinoma patients 58% had an elevated TPS level (cut-off 100 U/l) and 53% had an elevated CEA level (cut-off 3.0 ng/ml). The sensitivity of the cytokeratin marker TPS was related to the stage of the disease. Significant correlation was observed between TPS and Dukes stages in colon cancer patients and the highest TPS values were achieved in Dukes stage D. The combined use of the two markers increased the sensitivity to 82% compared with the use of only one. Simultaneous raise of both serum markers TPS and CEA was observed in 36% of cases. In the majority of the patients with adenomatous polyps and ulcerative colitis the serum TPS and CEA levels were below the upper reference limit. However the initial high levels in some patients could be considered as a prognostic indicator for identifying a group of patients with increased risk of cancer development. No significant correlation was observed between serum TPS and CEA concentrations in individual patients with benign diseases.
ABSTRACT
The aim of this study was to establish whether different subsets of ovarian neoplasms express a restricted isotype of carcinoembryonic antigen (CEA) which can be detected in solid tumors and detached cells. Sixty-one cases of mucinous, serous, endometrioid, and Krukenberg tumors were studied by immunohistochemistry using two monoclonal antibodies (MAbs), commercial anti-CEA and D14 with a higher specificity for colorectal adenocarcinomas. The results with both antibodies showed a considerable degree of heterogeneity between cases of nonserous tumors, with a more restrictive pattern observed with the D14 MAb. The proportion of immunostained cells was comparable in tumors and fluids.
Subject(s)
Carcinoembryonic Antigen/immunology , Ovarian Neoplasms/immunology , Adenocarcinoma/immunology , Adenocarcinoma, Mucinous/immunology , Antibodies, Monoclonal , Antibody Specificity , Ascitic Fluid/immunology , Cystadenocarcinoma/immunology , Cystadenoma/immunology , Endometriosis/immunology , Epitopes , Female , Humans , Immunohistochemistry , Krukenberg Tumor/immunologyABSTRACT
The dimeric and monomeric forms of NCA-50 from normal lung showed differences in binding to nitrocellulose in an immunoblotting procedure. The NCA dimer showed weaker binding, it passed through nitrocellulose and was more easily washed out from the blot. The NCA monomer was bound strongly and was detected with higher sensitivity following immunoblotting.
Subject(s)
Antigens, Differentiation, Myelomonocytic/isolation & purification , Antigens, Neoplasm , Blotting, Western/methods , Cell Adhesion Molecules , Membrane Glycoproteins/isolation & purification , Antibodies, Monoclonal , Antigens, Differentiation, Myelomonocytic/chemistry , Autoradiography , Carcinoembryonic Antigen/isolation & purification , Collodion , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Humans , Lung/immunology , Membrane Glycoproteins/chemistryABSTRACT
CEA and NCA show an increased mobility in SDS-polyacrylamide gel electrophoresis if the samples are preheated in 1% SDS for 5 min at 100 degrees C. Now we found that the slow-to-fast form conversion of CEA and NCA also occurs under milder conditions and is reversible at low SDS concentration. At 100 degrees C the conversion was completed within few minutes in 0.03% SDS, while at lower temperatures it required higher SDS concentration and longer time of incubation. After the antigens were heated at 100 degrees C at 0.05% or lower SDS concentration, they showed slow reassociation at room temperature. Under conditions insufficient for the complete slow-to-fast or fast-to-slow form conversion two CEA or NCA bands were present at various proportions. No material with an intermediate mobility was detected for CEA, while a weak transient intermediate band (located closer to the fast band) was visible in some gels showing an incomplete dissociation or association of NCA. The ratio of mol. wts. of slow and fast band was approx. 2:1 for NCA and 1.5:1 for CEA. Despite some deviations from the expected mol. wt. of the presumably undissociated CEA, the results obtained supplied a support for the conclusion that the two electrophoretic forms of CEA and NCA represent the dimer-monomer interconversion.
Subject(s)
Antigens, Neoplasm , Carcinoembryonic Antigen/isolation & purification , Cell Adhesion Molecules , Glycoproteins/isolation & purification , Antigens/chemistry , Antigens/isolation & purification , Carcinoembryonic Antigen/chemistry , Electrophoresis, Polyacrylamide Gel , Glycoproteins/chemistry , Hot Temperature , Humans , Molecular Weight , Protein Conformation , Sodium Dodecyl SulfateABSTRACT
CEA and NCA, which behave as dimeric molecules in SDS-polyacrylamide gel electrophoresis, were submitted to gel filtration on the Superose 12HR column in phosphate-buffered saline (PBS) pH 7.0 or in 6 M guanidine hydrochloride. The elution volumes of these antigens were compared with those of protein markers. The reference proteins showed almost linear relation between log Mr and elution volume, but they were eluted distinctly faster in guanidine that in PBS. The elution volumes of NCA corresponded to Mr approx. 110,000 in PBS and 50,000 in guanidine. These values were accordant with those obtained by other methods and suggested that guanidine dissociates NCA into subunits. The apparent molecular weights of CEA (migrating as 180 kDa molecule in SDS-electrophoresis) estimated by gel filtration on Superose were approx. 700,000 in PBS and 160,000 in guanidine. These results also suggested the dissociation of CEA by guanidine, but due to anomalous elution of CEA from the Superose column they are less convincing than in the case of NCA.
Subject(s)
Antigens, Neoplasm , Antigens/isolation & purification , Carcinoembryonic Antigen/isolation & purification , Cell Adhesion Molecules , Glycoproteins/isolation & purification , Chromatography, Gel , Guanidine , Guanidines/pharmacology , Humans , In Vitro Techniques , Molecular Weight , Protein Conformation/drug effects , SepharoseABSTRACT
The results of SDS-polyacrylamide gel electrophoresis and cross-linking experiments indicated that carcinoembryonic antigen is a dimer composed of two identical or closely similar noncovalently bound subunits, dissociating on heating in the presence of SDS. The dissociation is reversible upon the detergent removal.
Subject(s)
Adenocarcinoma/immunology , Carcinoembryonic Antigen/isolation & purification , Colonic Neoplasms/immunology , Liver Neoplasms/secondary , Electrophoresis, Polyacrylamide Gel , Humans , Liver Neoplasms/immunology , Macromolecular Substances , Molecular WeightABSTRACT
NCA was purified from normal human lung (L-NCA) and from liver metastases of colon adenocarcinoma (T-NCA), L-NCA and T-NCA had different carbohydrate compositions, but showed an immunological identity in double immunodiffusion with the use of anti-CEA and anti-L-NCA sera. In SDS-polyacrylamide gel electrophoresis L-NCA and T-NCA showed similar molecular weights of about 110,000 and 120,000, respectively. However, if the samples were heated for 3 min at 100 degrees C in the presence of 1% SDS before electrophoresis, their apparent mol. wt decreased to about 50,000, suggesting the dissociation of NCA molecules into subunits. The dissociation of NCA was independent of the presence of mercaptoethanol and did not destroy the ability of NCA to precipitate with anti-CEA and anti-NCA sera. Besides NCA (and CEA in tumor tissue), the presence of a lower molecular weight cross-reacting antigen was observed in lung and tumour. Double immunodiffusion showed that this additional antigen was not a dissociated form of NCA.