ABSTRACT
In order to clarify the complete cytomorphology of cytopathic changes as a consequence of human papillomavirus (HPV) infection, we performed three-dimensional (3D) reconstruction from confocal fluorescent images. After confirming 22 HPV types using a DNA chip, we performed 3D confocal image restoration in human uterine cervical swab samples and corresponding tumor tissues. On restoration of 3D confocal images, the multinucleated feature of koilocytes was revealed to be multilobation of a single nucleus, as opposed to true multinucleation.
Subject(s)
Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Cell Nucleus/metabolism , Cell Nucleus/pathology , Female , Genotype , Humans , Imaging, Three-Dimensional , Microscopy, Confocal , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/pathology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/ultrastructure , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/ultrastructureABSTRACT
AIMS: To clarify the fine structure of koilocytes and correlate this with genetic aberration of the G2 checkpoint. METHODS: Three dimensional reconstruction from confocal fluorescent images, together with functional assays for key molecules of the G2 checkpoint-cdc2 and cyclin B1-was performed in human uterine cervical samples. After confirming 22 human papillomavirus (HPV) types using a DNA chip from 30 cervical swabs, previously confirmed as 15 cervical low grade and 15 high grade intraepithelial lesions, the activity of molecules involved in the G2 checkpoint was evaluated using western blotting for cyclin B1, cdc2, and phospho-cdc2 (Y15 and T161), a nuclear extraction fractional assay, and a reverse transcription polymerase chain reaction assay. In addition, three dimensional confocal image restoration was performed on confirmed cervical intraepithelial neoplasia tissue samples. RESULTS: T161 phospho-cdc2 and cyclin B1 expression was higher in HPV infected cervical lesions than in normal samples. Immunofluorescence, revealed that cyclin B1 was present predominantly in the nuclei of HPV infected cells, confirming the results of the nuclear fractional assay. On restoration of three dimensional confocal images, the multinucleation of koilocytes was revealed to be multilobation of a single nucleus, rather than true multinucleation. This multilobation appeared to be associated with chromosomal instability and aberration of the G2 checkpoint. CONCLUSIONS: The multiple nuclei of koilocytes are in fact multilobation of a single nucleus, and this phenomenon is associated with upregulation of gene products related to the G2 checkpoint.
Subject(s)
Epithelial Cells/pathology , G2 Phase/genetics , Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/virology , Blotting, Western/methods , CDC2 Protein Kinase/metabolism , Cell Nucleus/metabolism , Cell Nucleus/pathology , Chromosome Aberrations , Cyclin B/metabolism , Cyclin B1 , DNA, Viral/analysis , Female , Genotype , Humans , Imaging, Three-Dimensional , Microscopy, Confocal , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/pathology , Reverse Transcriptase Polymerase Chain Reaction/methods , Up-Regulation , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/ultrastructure , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/ultrastructureABSTRACT
Angiogenesis is a series of processes that include endothelial proliferation, migration and tube formation. Vascular endothelial growth factor (VEGF) is regarded as a potent mediator of angiogenesis, vascular permeability and tumor cell growth in renal cell carcinoma. This study was designed to evaluate the expression of VEGF and the microvessel count (MVC) and to determine their prediction efficacies for prognosis in renal cell carcinoma. The relationship between the expression of VEGF and MVC were evaluated immunohistochemically in 50 patients with renal cell carcinoma who received a radical nephrectomy at Wonju Christian Hospital between 1989 and 1997. Microvessels were identified by immunostaining endothelial cells for CD-31 antigen. The mean follow-up was 96 months (3 - 133 months). Overall 5-year survival rate was 71.5%. VEGF was expressed in the tumor cell cytoplasm. Of the 50 tumors, 23 (46%) were weak to strongly positive for VEGF but 27 (54%) were unreactive. The respective 5-year survival rates for patients with positive and negative expressions of VEGF were 70% and 73% (p > 0.05). The overall mean MVC was 13.4 in a 400x field. Mean MVCs were significantly higher in VEGF-positive tumors (17.6 +/- 12.1) than in VEGF-negative tumors (9.9 +/- 5.4), and the MVCs of the high vascular density group and the low vascular density groups were significantly different. The 5-year survival rates of patients with high vascular density and low vascular density were 59% and 86%. The median survival period for patients with MVCs higher than or equal to 10 vessels/field was 85 months, whereas for those with MVCs lower than 10 vessels/field the median survival time was 102 months. These results suggest that MVC may be a better prognostic factor in renal cell carcinoma than the expression of VEGF.
Subject(s)
Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/metabolism , Endothelial Growth Factors/metabolism , Kidney Neoplasms/blood supply , Kidney Neoplasms/metabolism , Lymphokines/metabolism , Neovascularization, Pathologic/pathology , Adult , Aged , Female , Humans , Male , Middle Aged , Prognosis , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
We explored a biological role of SET as it relates to cell proliferation and differentiation. Immunohistochemical staining demonstrated that the expression of SET was ubiquitous and diffuse over the whole embryo on gestational day 15. At a later stage of development, SET was expressed at relatively lower levels and localized to specific tissues and cells. On embryonic day 19, specific SET immunoreactivity was found in the epithelium of skin, respiratory tract, intestine, and retina as well as in muscle and cartilage. In these cells SET was stained mostly in the nucleus, which was supported indirectly by nuclear transport of enhanced green fluorescence protein-SET fusion proteins in ECV304 endothelial cells. Set mRNA expression was further confirmed in various cultured cells, including NIH 3T3 cells, L6 myoblast cells, human umbilical vein endothelial cells, and ECV304 cells. Using F9 teratocarcinoma cell lines, which were stimulated to differentiate into the two different cell lineages of parietal and visceral endoderm, we have further examined the role of SET. The expression of set mRNA and SET protein was diminished about three-fold in both differentiated endoderm cells compared to the undifferentiated F9 cells. However, when F9 cells were subjected to serum starvation, reduction of set mRNA abundance also took place at a similar level to that observed in response to differentiation. Consistent with this, quiescent L6 myoblast showed a marked downregulation of set mRNA compared to proliferating cells. These results suggest that SET is involved mainly in the regulation of cell proliferation rather than differentiation during embryonic development.
Subject(s)
Cell Division/genetics , Proteins/genetics , RNA, Messenger/genetics , 3T3 Cells , Animals , Base Sequence , Chromosomal Proteins, Non-Histone , DNA Primers , DNA-Binding Proteins , Embryonic and Fetal Development/genetics , Gene Expression Regulation, Developmental , Histone Chaperones , Humans , Immunohistochemistry , Mice , Proteins/metabolism , Transcription Factors , Tumor Cells, CulturedABSTRACT
ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Integrins/biosynthesis , Neovascularization, Physiologic , Apoptosis/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Division/drug effects , Cell Line , Cell Movement/drug effects , Culture Media, Serum-Free/pharmacology , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Endothelium, Vascular/chemistry , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , Immunohistochemistry , Integrin alpha3beta1 , Integrins/analysis , Transfection , ras Proteins/genetics , ras Proteins/physiologyABSTRACT
We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.
Subject(s)
Down-Regulation , Endothelium/metabolism , Integrins/metabolism , Oncogene Protein p21(ras)/metabolism , Antibodies/pharmacology , Cell Line, Transformed , Cell Movement/drug effects , Chromones/pharmacology , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Endothelium/cytology , Endothelium/drug effects , Endothelium/enzymology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Flow Cytometry , Genes, Dominant/genetics , Humans , Imidazoles/pharmacology , Integrin alpha3beta1 , Integrins/immunology , Morpholines/pharmacology , Oncogene Protein p21(ras)/antagonists & inhibitors , Oncogene Protein p21(ras)/genetics , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Pyridines/pharmacology , Signal Transduction/drug effects , Transfection , Up-Regulation/drug effectsABSTRACT
Angiogenin is a potent angiogenic factor secreted by cultured tumor cells and is found in various normal organs and tissues. The ovary is one of the adult organs in which angiogenesis normally occurs during the female reproductive cycle. In this study, we examined whether angiogenin protein is localized and if angiogenin mRNA is expressed in the normal bovine ovary by immunohistochemistry using polyclonal rabbit anti-bovine angiogenin IgG and by in situ hybridization using bovine angiogenin probe, respectively. The localization and mRNA expression of angiogenin in the ovarian follicle and in the corpus luteum were different in their developmental stages. The intensities of immunoreactivities and angiogenin transcripts in the follicle increased from the primordial to the tertiary (or Graafian) follicle. The early corpus luteum contained strong immunoreactivities and mRNA expression of angiogenin but these intensities weakened during regression. The results suggest that angiogenin is involved in morphological changes and angiogenesis in the ovary.
Subject(s)
Neovascularization, Physiologic , Ovary/blood supply , Ovary/growth & development , Proteins/metabolism , Ribonuclease, Pancreatic , Animals , Cattle , Corpus Luteum/cytology , Corpus Luteum/growth & development , Corpus Luteum/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Factor VIII/analysis , Female , Gene Expression Regulation, Developmental , Granulosa Cells/metabolism , Immune Sera , Immunohistochemistry , In Situ Hybridization , Luteal Cells/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Oocytes , Ovarian Follicle/cytology , Ovarian Follicle/growth & development , Ovarian Follicle/metabolism , Ovary/cytology , Ovary/metabolism , Proteins/genetics , RNA, Messenger/metabolismABSTRACT
Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100-180 ng/mL) and milk (4-8 micrograms/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis.
Subject(s)
Angiogenesis Inducing Agents/analysis , Proteins/analysis , Ribonuclease, Pancreatic , Angiogenesis Inducing Agents/immunology , Angiogenesis Inducing Agents/metabolism , Animals , Blotting, Western , Cattle , Enzyme-Linked Immunosorbent Assay , Gallbladder/chemistry , Immune Sera/biosynthesis , Immunohistochemistry , Liver/chemistry , Mammary Glands, Animal/chemistry , Proteins/immunology , Proteins/metabolismABSTRACT
The presence of immunoreactive atrial natriuretic peptide (irANP) in rabbit, pig and human gallbladders was investigated using radioimmunoassay and immunohistochemistry. Serial dilution curves of gallbladder tissue and bile juice extracts were paralleled to the standard curve of atriopeptin III. Gel filtration profiles of gallbladder tissue extracts showed a major peak corresponding to rat pro-ANP. The amounts of irANP in rabbit, pig and human gallbladders were 30.0 +/- 12.3 pg/mg (n = 7), 7.0 +/- 2.0 fg/mg (n = 7) and 17.7 +/- 2.0 fg/mg wet tissue (n = 8), respectively. Bile juice was also shown to contain irANP but with small molecular mass. The amounts of irANP in the rabbit, pig and human bile juice were 25.0 +/- 2.0 (n = 7) and 0.50 +/- 0.02 (n = 7), and 1.3 +/- 0.1 pg/ml (n = 8), respectively. The immunohistochemical staining of the rabbit gallbladder tissue revealed the presence of irANP in the luminal epithelium and smooth muscle layer. The amount of irANP was higher in the luminal epithelium than in the rest of the gallbladder tissue from rabbits (0.30 +/- 0.06 vs. 0.01 +/- 0.01 pg/microgram protein, P < 0.01). These findings suggest that ANP may be synthesized and stored in the gallbladder, and may have a role in the regulation of fluid balance and cystic motility.
Subject(s)
Atrial Natriuretic Factor/analysis , Bile/chemistry , Gallbladder/chemistry , Animals , Chromatography, Gel , Humans , Immunohistochemistry , Molecular Weight , Rabbits , Radioimmunoassay , SwineABSTRACT
1. The presence and partial characterization of immunoreactive ANP (irANP) in eggs were investigated using high performance liquid chromatography (HPLC), immunohistochemistry and Northern-blot hybridization in mammalian and nonmammalian vertebrates. 2. Serial dilution curves of egg extracts from frogs and freshwater teleostean fishes (silver crusian carp and loach) were parallel to the standard curve of atriopeptin III. Rat oocytes also contained irANP (25-30 pg/egg). 3. The HPLC profile of irANP showed two main peaks corresponding to low and high molecular weight of irANP. 4. ANP mRNA was detected in the fish eggs. With immunohistochemical analysis of the rat ovarian follicles, this ANP-like material was localized mainly at the oocytes. 5. We, therefore, suggest that the vertebrate oocytes synthesize ANP and that the presence of ANP in the oocytes may be linked with the cell differentiation.
Subject(s)
Atrial Natriuretic Factor/analysis , Oocytes/chemistry , Animals , Blotting, Northern , Chromatography, High Pressure Liquid , Cypriniformes , Female , Goldfish , Immunoenzyme Techniques , Radioimmunoassay , Ranidae , Rats , Rats, Sprague-DawleyABSTRACT
Atrial natriuretic peptide (ANP) has been reported to be locally synthesized in the ovary although its physiological roles are still unknown. To define the origin of ovarian ANP, we demonstrated the presence and release of immunoreactive (ir) ANP in pig granulosa cells and characterized its biochemical properties. Serial dilution curves made with the extracts of pig granulosa cells, their perfusates and follicular fluid were paralleled to the standard curve of ANP. The amount of irANP in the granulosa cell was 2 fg/cell. The total amount of irANP in granulosa cells significantly correlated with the levels of irANP in follicular fluid. Additionally, the total content of irANP in the follicle negatively correlated with the follicular size. On reverse phase HPLC, the major form of irANP in granulosa cells and follicular fluid was high molecular weight but that in perfusate was low molecular weight. In Northern blot analysis, ANP mRNA was detected in the pig granulosa cells. Immunohistochemistry showed ANP prohormone location in granulosa cells of rat ovary. These data strongly suggest that the granulosa cells synthesize and secrete ANP.