RESUMO
In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P-labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P-induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P-dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.
Assuntos
Endométrio/metabolismo , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/biossíntese , Progesterona/fisiologia , Animais , Northern Blotting , Divisão Celular , Linhagem Celular , Endométrio/irrigação sanguínea , Endométrio/citologia , Estradiol/fisiologia , Estro , Feminino , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Substâncias de Crescimento/genética , Humanos , Hibridização In Situ , Macaca mulatta , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , RNA Mensageiro/biossíntese , Células Estromais/metabolismo , Útero/metabolismoRESUMO
BACKGROUND: The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. METHODS: The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. RESULTS: KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1alpha increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. CONCLUSION: KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1alpha.
Assuntos
Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Cistos Odontogênicos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Adolescente , Adulto , Distribuição de Qui-Quadrado , Cisto Dentígero/metabolismo , Cisto Dentígero/patologia , Feminino , Humanos , Imuno-Histoquímica , Interleucina-1alfa/metabolismo , Masculino , Cistos Odontogênicos/patologia , Estatísticas não Paramétricas , Adulto JovemRESUMO
OBJECTIVES: Cell death is detected in the ducts of labial salivary glands (LSG) of patients with primary Sjögren's syndrome (pSS). However, the counter-mechanism to inhibit the apoptotic process remains unclear. In this study, we studied the ability of epidermal growth factor (EGF) to activate the PI3K-Akt pathway and NF-kB in primary cultured salivary gland epithelial cells (SGEC) of pSS patients. METHODS: SGEC, obtained from 2 female pSS patients, were cultured and used for Hoechst staining and deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay. The frequency of apoptosis, detected by Hoechst staining, was quantified, and statistical significance was determined through unpaired student's t-test. RESULTS: Following twelve hours of stimulation, both PI3K inhibitors and anti-Fas antibody failed to induce apoptosis in primary cultured SGEC. However, the combination of anti-Fas antibody, along with LY294002 or Bay 11-7082, induced apoptosis which was statistically more significant than apoptosis found in the control cells (p < 0.01). Interestingly, the apoptosis induced by anti-Fas antibody along with LY294002 was clearly inhibited by the addition of 10 ng/ml EGF. Furthermore, the results of the TUNEL assay clearly indicated apoptosis through stimulation with anti-Fas antibody and LY294002 or Bay 11-7082. Furthermore, the apoptosis was completely blocked by the addition of EGF. CONCLUSION: Our results suggest that salivary epithelial cells are protected from Fas mediated apoptosis, through cell survival factors including either the PI3K-Akt pathway or NF-kB.
Assuntos
Apoptose/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Glândulas Salivares/citologia , Síndrome de Sjogren/patologia , Receptor fas/fisiologia , Anticorpos/imunologia , Apoptose/fisiologia , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Morfolinas/farmacologia , Nitrilas/farmacologia , Glândulas Salivares/efeitos dos fármacos , Sulfonas/farmacologia , Receptor fas/imunologiaRESUMO
The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6-diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE.
Assuntos
Carcinoma Hepatocelular/terapia , Embolização Terapêutica , Neoplasias Hepáticas/terapia , Adulto , Idoso , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Atrofia , Carcinoma Hepatocelular/enzimologia , Feminino , Frutosedifosfatos/sangue , Frutosefosfatos/sangue , Humanos , Fígado/patologia , Neoplasias Hepáticas/enzimologia , Masculino , Pessoa de Meia-Idade , alfa-Fetoproteínas/análiseRESUMO
Lectin-affinity electrophoretic separation of serum alpha-fetoprotein (AFP) was carried out using AFP Differentiation Kits, which used Lens culinaris agglutinin-A (Kit L) and erythroagglutinating phytohemagglutinin (Kit P). Separated AFP bands were detected with a sensitive antibody-affinity blotting technique and determined quantitatively by densitometry, and the results were expressed as percentages of the intensity of total AFP bands. Sera from 424 patients with acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, and extrahepatic tumors were assayed for proportion of AFP present as Lens culinaris agglutinin-A-reactive AFP (AFP-L3) and erythroagglutinating phytohemagglutinin-reactive AFPs (AFP-P4+P5). From the maximum Youden indices determined, cutoff levels were set at 15% for both AFP-L3 and AFP-P4+P5 to discriminate between patients with chronic hepatitis and liver cirrhosis and patients with hepatocellular carcinoma. AFP-L3 and AFP-P4+P5 showed sensitivities of 55.3 and 61.0% at specificities of 93.9% and 82.3%, respectively. Thirty-eight % of tumors that measured less than 20 mm in diameter were positive for AFP-L3 and AFP-P4+P5. AFP-L3 exceeded the cutoff level of 15% 4.0 +/- 4.9 months before detection of hepatocellular carcinomas by imaging techniques with a sensitivity of 48% and a specificity of 81%. Thus, these tests are useful for the early detection of hepatocellular carcinomas in patients with hepatitis or liver cirrhosis.
Assuntos
Carcinoma Hepatocelular/química , Tumor do Seio Endodérmico/química , Neoplasias Gastrointestinais/química , Hepatite Viral Humana , Cirrose Hepática , Neoplasias Hepáticas/química , alfa-Fetoproteínas/análise , Carcinoma Hepatocelular/sangue , Tumor do Seio Endodérmico/sangue , Feminino , Seguimentos , Neoplasias Gastrointestinais/sangue , Hepatite Viral Humana/sangue , Humanos , Lectinas , Cirrose Hepática/sangue , Neoplasias Hepáticas/sangue , MasculinoRESUMO
Plasma membranes prepared from rat livers inhibited the in vitro growth of various mammalian cells including hepatoma cells in a concentration-dependent manner, showing almost complete arrest of cell growth at 0.1 mg protein/ml. Some of these cells tested, i.e., leukemia (L1210 and P388) and myeloma (P3-NS-1/1-Ag4-1) cells, were labile in the presence of plasma membranes (losing the viability), and CHO (Chinese hamster ovary) cells became round without detaching from the substratum. The culture medium preincubated with liver plasma membranes no longer supported the growth of hepatoma cells (AHI3 and AH66F). However, the 'conditioned' medium supplemented with L-arginine, supported the growth of the cells. Moreover, the addition of L-ornithine to the cultures containing plasma membranes markedly reduced the inhibitory effect of plasma membranes. The plasma membrane preparations were found to possess considerable arginase activity. There results seem to indicate the possible involvement of arginase in the inhibition of cell growth by liver plasma membranes.
Assuntos
Arginase/metabolismo , Fígado/enzimologia , Animais , Arginase/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/enzimologia , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Cinética , Leucemia Experimental/fisiopatologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Masculino , Camundongos , Neoplasias Experimentais/fisiopatologia , Ovário , Ratos , Ratos EndogâmicosRESUMO
Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowman's capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN.
Assuntos
Nefropatias Diabéticas/imunologia , Interleucina-6/análise , Interleucina-6/biossíntese , Rim/imunologia , Adolescente , Adulto , Biópsia , Nitrogênio da Ureia Sanguínea , Divisão Celular , Creatinina/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Feminino , Fibrinogênio/análise , Taxa de Filtração Glomerular , Mesângio Glomerular/imunologia , Mesângio Glomerular/patologia , Hemoglobinas Glicadas/análise , Humanos , Hibridização In Situ , Rim/patologia , Masculino , Pessoa de Meia-Idade , Proteinúria , RNA Mensageiro/análiseRESUMO
Glutathione S-transferase (GST) functions in xenobiotic biotransformation and drug metabolism. Increased expression of GSTpi, an isozyme of GST, has been found in cancer cells resistant to doxorubicin hydrochloride (DOX) or cis-diamminedichloroplatinum (II) (CDDP), and this increase was believed to be correlated with drug resistance of cancer cells. GST is mainly expressed in the cytoplasm; GSTpi in the nucleus has been reported in cancer cells, but the meaning of this result is not known. Here, we studied changes in the amount of nuclear GSTpi after exposure of cancer cells to anticancer drugs, and role of the nuclear GSTpi in drug resistance. We found nuclear GSTpi in cancer cells resistant to DOX, and the amount of nuclear GSTpi was enhanced by treatment of the cancer cells with DOX or CDDP. We also found that a mushroom lectin, an inhibitor of nuclear transport, inhibited the nuclear transfer of GSTpi, suggesting the existence of a specific transport system for the nuclear transfer of GSTpi. Nuclear GSTpi protected DNA against damage by anticancer drugs. These results suggest a possible role of GSTpi in the acquisition of resistance to anticancer drugs by cancer cells.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Núcleo Celular/enzimologia , DNA de Neoplasias/efeitos dos fármacos , Doxorrubicina/farmacologia , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Western Blotting , Camptotecina/farmacologia , Núcleo Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Cisplatino/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Dano ao DNA , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glutationa S-Transferase pi , Glutationa Transferase/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Irinotecano , Isoenzimas/efeitos dos fármacos , Lectinas/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The present study was undertaken to define the relationship between histological grade (Gleason grade) and prostate-specific antigen (PSA) mRNA expression and to evaluate the level of PSA mRNA expression as a possible prognostic marker for untreated prostate cancers. The primary grade areas of 104 prostatic biopsy specimens were analyzed for the expression of PSA mRNA and its protein by nonradioactive in situ hybridization and immunohistochemistry, respectively. A multivariate survival analysis was performed to examine the correlation between PSA mRNA expression and several clinicopathological parameters, e.g., the immunostaining level of PSA protein in biopsy specimens. The percentage of specimens positive for PSA mRNA increased significantly with advanced histological grade. Image analysis of the signal intensity for PSA mRNA showed a significant correlation between the signal intensity in both primary and secondary grade areas of each specimen and the histological grade (P < 0.0001). Only 26.0% of specimens positive for PSA protein were also positive for PSA mRNA (and vice versa, 6.7%). Other tumors were either positive for both (66.3%) or negative for both (1.0%). When the Cox's proportional hazards regression model was used to analyze cancer-specific survival, untreated patients with higher levels of PSA mRNA expression in the higher grade (representing higher grade of either primary or secondary grade) area of tumors were at high risk for cancer-related death (P = 0.017). Furthermore, in cancer-specific survival curves based on PSA mRNA expression status, patients with high levels of PSA mRNA expression in the higher grade area of tumors had a significantly poorer prognosis (P = 0.001), compared with those with tumors expressing low levels of PSA mRNA. Our results suggested that analysis of PSA mRNA expression in specific areas in biopsy specimens of patients with untreated prostate cancer may provide a good assessment of prognosis of prostate cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno Prostático Específico/biossíntese , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , DNA de Neoplasias/metabolismo , Dimerização , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Timina/metabolismoRESUMO
We investigated apoptosis in tumor-infiltrating lymphocytes (TILs) obtained from 41 colorectal carcinomas by in situ nick translation (ISNT). When the ISNT labeling index (LI) was determined as the number of positive nuclei per 1000 nuclei of TIL in tissue sections, the median LI was 12.0 (range, 2-30). The ISNT LI of colorectal carcinoma with lymph node metastasis was higher than that of colorectal carcinoma without metastasis. The cases with a high LI of 212.0 had a significantly poorer prognosis than those with a low LI. We also confirmed immunohistochemically that a part of the TILs expressed Fas using the sections adjacent to what contained abundant ISNT-positive TILs. Moreover, Fas ligand (FasL) expression was detected on the cell surface as well as the cytoplasm of colorectal cancer cells in 61% of cases. Apoptosis in TILs was consistently seen more frequently in FasL-positive cases than in FasL-negative ones. These findings indicate that the FasL expressed in colorectal carcinoma cells may kill the Fas-positive immune effective TILs by means of a Fas-FasL system termed Fas counterattack. This tumor immune evasion induced by FasL may therefore affect the malignant potential of human colorectal carcinoma.
Assuntos
Apoptose/fisiologia , Neoplasias Colorretais/imunologia , Linfócitos do Interstício Tumoral/citologia , Glicoproteínas de Membrana/biossíntese , Receptor fas/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Fragmentação do DNA , Proteína Ligante Fas , Feminino , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Prognóstico , Coloração e Rotulagem/métodos , Receptor fas/imunologiaRESUMO
We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoylphorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.
Assuntos
Apoptose/fisiologia , Leucócitos Mononucleares/fisiologia , Osteoblastos/fisiologia , Receptor fas/biossíntese , Antígenos de Superfície/biossíntese , Linhagem Celular , Células Cultivadas , Proteína Ligante Fas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Imunoglobulina M/farmacologia , Ionomicina , Ionóforos , Leucócitos Mononucleares/imunologia , Glicoproteínas de Membrana/biossíntese , Osteoblastos/imunologia , Acetato de Tetradecanoilforbol , Receptor fas/imunologiaRESUMO
To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Terapia Genética/métodos , Proteínas de Choque Térmico/genética , Regiões Promotoras Genéticas , Animais , Antivirais/farmacologia , Apoptose , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Feminino , Ganciclovir/farmacologia , Temperatura Alta , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lipossomos/metabolismo , Neoplasias Mamárias Animais/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Plasmídeos/metabolismo , Retroviridae/metabolismo , Temperatura , Timidina Quinase/metabolismo , Fatores de Tempo , Distribuição Tecidual , Transdução Genética , Transfecção , Células Tumorais Cultivadas , Regulação para Cima , Receptor fas/metabolismoRESUMO
Previous immunocytochemical studies indicate that receptor regulation varies in different uterine cell types. In primates, progesterone (P) suppresses estrogen receptor (ER) in glandular epithelial cells in the functionalis, but fails to suppress ER in the glandular epithelial (GE) cells of the basalis. P also fails to suppress ER in the perivascular stromal and smooth muscle cells of the spiral arteries in the functionalis. We used nonradioactive in situ hybridization to determine whether similar cell type differences occur at the ER mRNA level. We used digoxigenin-labeled oligodeoxynucleotides (oligo-DNAs; 45-mer) as probes and detected the hybrids immunocytochemically with horseradish peroxidase-labeled antidigoxigenin antibody. This technique can discriminate between positive and negative cells in closely packed histological associations. In spayed monkeys, most of the GE cells as well as endometrial stromal cells were positive for ER mRNA, while all vascular smooth muscle, endothelium, and perivascular stromal cells were negative. Estradiol treatment for 14 days markedly increased ER mRNA staining in the GE cells, most stromal cells, and the vascular smooth muscle and perivascular stromal cells of spiral arteries in the functionalis. However, in the basalis, these components of the spiral arteries were negative as were the small basal arteries of the basalis. In most positive cells, ER mRNA was not homogeneously distributed in the cytoplasm, but, rather, was concentrated in their perinuclear regions. The GE cells in the basalis had especially intense concentrations of perinuclear signal at their apical poles. After sequential estradiol plus P treatment, the signal was greatly reduced in the GE cells of the functionalis, but not in the GE cells of the basalis or in the vascular smooth muscle or perivascular stromal cells of the spiral arteries of the functionalis. In myometrium, ER mRNA was localized to the perinuclear region of smooth muscle cells, but the staining intensity was not dramatically affected by hormonal manipulation. Unexpectedly, we observed clusters of stromal cells characterized by extremely high positive signals for ER mRNA ("hot cells") at the endometrial/myometrial border and deeper in the connective tissue of the myometrium, although such cells did not express high levels of ER protein. In general, however, the cellular distribution of ER mRNA and its hormonal regulation paralleled those of ER protein.
Assuntos
Digoxigenina , Hibridização In Situ , Sondas de Oligonucleotídeos , RNA Mensageiro/análise , Receptores de Estrogênio/genética , Útero/química , Animais , Sequência de Bases , Endométrio/química , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Estradiol/farmacologia , Feminino , Macaca mulatta , Dados de Sequência Molecular , Miométrio/química , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Oligonucleotídeos Antissenso , Progesterona/farmacologia , Distribuição Tecidual , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.
Assuntos
Tubas Uterinas/química , Imuno-Histoquímica/métodos , Micro-Ondas , Receptores de Estrogênio/análise , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Cílios/ultraestrutura , Criopreservação , Endotélio Vascular/química , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Antagonistas de Estrogênios/farmacologia , Estrogênios/sangue , Etinilestradiol/análogos & derivados , Etinilestradiol/farmacologia , Feminino , Secções Congeladas , Macaca mulatta , Microscopia de Contraste de Fase , Progesterona/sangue , TrítioRESUMO
The physiological effects of estrogen on the pituitary, including cellular proliferation and regulation of hormone synthesis, are mediated by the nuclear estrogen receptor (ER). The purpose of this study was to determine ontogenetic expression of two types of ERs (ERalpha and ERbeta) in the pituitary using specific antibodies, monoclonal antibody (1D5) for ERalpha and polyclonal antibody generated against ERbeta. First, we confirmed the detection of 66- and 55-kDa bands for ERalpha and ERbeta, respectively, in the rat pituitary extract by Western blotting. Then immunostaining with these antibodies was performed using fetal and adult Wistar rat tissues, combined with PRL or LHbeta immunohistochemistry. Intense ERbeta signal was detected throughout the pituitary from day 12 of gestation. However, staining for ERalpha only became detectable from day 17 of gestation. In contrast with the fetal period, nuclei stained for ERalpha were widely distributed in the anterior lobe in the adult rat, whereas ERbeta-positive cells were restricted in the anterior lobe. LHbeta, but not PRL, was colocalized in ERbeta-positive cells. Our results indicated that the major population of ER subtypes in the rat pituitary gland has changed around the day of birth and that the expression of ERbeta may be involved in the differentiation of pituitary cell function to synthesize a specific hormone.
Assuntos
Envelhecimento/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Hipófise/metabolismo , Receptores de Estrogênio/genética , Animais , Anticorpos Monoclonais , Western Blotting , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Idade Gestacional , Imuno-Histoquímica , Hormônio Luteinizante/análise , Masculino , Ovário/metabolismo , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Adeno-Hipófise/embriologia , Adeno-Hipófise/crescimento & desenvolvimento , Adeno-Hipófise/metabolismo , Prolactina/análise , Próstata/metabolismo , Ratos , Ratos Wistar , Receptores de Estrogênio/análiseRESUMO
Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.
Assuntos
Apoptose/fisiologia , Atresia Folicular/fisiologia , Células da Granulosa/fisiologia , Receptor fas/fisiologia , Animais , Western Blotting , Técnicas de Cocultura , Corantes , DNA/análise , Feminino , Gonadotropinas Equinas/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Oócitos/fisiologia , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
Apoptosis, a physiological process of cell death, may modulate the mass of the thyroid gland. We investigated the role of apoptosis and the possible involvement of Fas/Fas ligand (FasL) system in apoptosis during goiter formation and involution in a rat model of goiter. Rats were fed a low iodine diet and a goitrogen, 6-propyl-2-thiouracil, to induce goiter. Rats with goiter were then fed a high iodine diet to study the phase of involution. We examined the presence of apoptosis by electron microscopy (EM) and terminal deoxy-UTP nick end labeling (TUNEL). We also investigated the association between Fas and FasL expression and thyrocyte apoptosis using immunohistochemistry and Western blotting. To evaluate the proliferation of thyrocytes, proliferating cell nuclear antigen was examined immunohistochemically. The number of apoptotic cells increased during goiter formation and the early stage of involution, which were also associated with increased number of Fas-positive thyrocytes, and some of these cells contained TUNEL-positive nuclei. However, the expression of FasL was almost constant throughout the experiment. Proliferating cell nuclear antigen/TUNEL ratio markedly increased during goiter formation but decreased particularly during the late stage of goiter involution. Our results indicate that apoptosis of thyrocytes is a main factor of cell loss during goiter formation and involution and suggest that the Fas/FasL system is involved in the induction of apoptosis of these cells. Moreover, the delicate balance between apoptosis and cell proliferation may play an important role in the control of thyroid gland mass.
Assuntos
Apoptose/fisiologia , Bócio/patologia , Glândula Tireoide/patologia , Receptor fas/fisiologia , Animais , Western Blotting , Dieta , Bócio/induzido quimicamente , Bócio/fisiopatologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Tamanho do Órgão , Antígeno Nuclear de Célula em Proliferação/análise , Propiltiouracila , Ratos , Ratos Wistar , Glândula Tireoide/fisiopatologia , Tiroxina/sangue , Tri-Iodotironina/sangue , Receptor fas/análiseRESUMO
Two antibodies were prepared for use in a sandwich enzyme immunoassay of human IgG. Completely purified guinea pig anti-human IgG was labelled with beta-D-galactosidase (EC 3.2.1.23), using a heterobifunctional cross-linker named GMBS. Partially purified anti-human IgG was immobilized on a new solid support: Amino-Dylark balls. Optimal conditions for immobilizing the antibody, using glutaraldehyde as the coupling reagent, were studied in detail. With the enzyme-labelled antibody and the solid-phase anti-human IgG, a sandwich enzyme immunoassay of human IgG with a lower limit of detection at 10.5 pM (0.3 ng/tube) was developed. A comparative study of the EIA method and a laser nephelometric method showed a good correlation. The specificity of the assay was excellent: all 4 types of IgG tested showed the maximum 0.0001%; human IgA, IgM and albumin possessed the maximum 0.54% in their cross-reactivity values with human IgG.
Assuntos
Técnicas Imunoenzimáticas , Imunoglobulina G/análise , Técnicas de Imunoadsorção , Animais , Especificidade de Anticorpos , Reações Cruzadas , Cobaias/imunologia , Cavalos , Humanos , Camundongos , Coelhos , Suínos , beta-GalactosidaseRESUMO
The lung is one of the primary targets of acute graft-versus-host disease (GVHD), which is the principal complication that occurs after allogeneic intestinal transplantation. The purpose of this study is to investigate the involvement of Fas/Fas ligand system in pulmonary injury after rat semi-allogeneic intestinal transplantation. The lungs were serially harvested from LEW x BN F1(LBNF1) recipients of either LEW heterotopic intestinal allografts or LBNF1 isografts, on days 1, 3, 5, 9, and 13 posttransplant. In light microscopy, pulmonary injury became apparent on day 13 in the allogeneic combination, showing a thickening of the alveolar septa. The incidence of apoptosis, examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling, was observed to increase steadily in the alveolar cells accompanied by a progression of GVHD. In an immunohistochemical study, Fas was constitutively expressed in the lung, although Fas ligand was expressed most extensively on day 9. The immunoreactivity of both Fas and Fas ligand were observed in alveolar cells, in addition to leukocytes. An analysis by reverse transcription polymerase chain reaction also revealed that the expression of Fas mRNA was constitutive without any significant change, although that of Fas ligand mRNA increased substantially and peaked on day 9, which was significant compared to the isogeneic combination. In conclusion, transcriptionally up-regulated Fas ligand and increased number of apoptosis suggests that the Fas system may play a role in the pathophysiology of GVHD-induced pulmonary injury.
Assuntos
Doença Enxerto-Hospedeiro/complicações , Doença Enxerto-Hospedeiro/imunologia , Intestino Delgado/transplante , Pneumopatias/etiologia , Pneumopatias/imunologia , Glicoproteínas de Membrana/biossíntese , Receptor fas/biossíntese , Animais , Especificidade de Anticorpos , Apoptose/imunologia , Proteína Ligante Fas , Immunoblotting , Pneumopatias/patologia , Masculino , Glicoproteínas de Membrana/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Receptor fas/imunologiaRESUMO
Regulation of gene transcription requires an interaction between specific segments of nuclear DNA and specific proteins. We describe a method to localize the specific DNA-binding proteins using haptenized double-stranded (ds) DNAs. To demonstrate this method, an oligodeoxynucleotide (oligo-DNA) with a consensus base sequence of cyclic adenosine monophosphate-responsive element (CRE) (TGACGTCA) with three TTA repeats at the 5' end was synthesized. Since the CRE sequence is palindromic, the oligo-DNA was allowed to self-anneal and form ds DNA with three TTA repeats at both ends. The CRE ds-oligo-DNA was irradiated with UV light to form haptenic thymine-thymine (T-T) dimers. The haptenized CRE ds-oligo-DNA reacted by Southwestern analysis with a distinct set of proteins, previously identified as CRE-binding proteins, ranging from 40-90 KD. When the haptenized CRE ds-oligo-DNA reacted with frozen sections fixed with 4% paraformaldehyde in PBS (pH 7.4) followed by enzyme immunohistochemical localization of the T-T dimers, nuclei of intestinal epithelial cells and brain cells were heavily stained. Nuclear staining was blocked when the sections were reacted with the haptenized CRE ds-oligo-DNA in the presence of an excess amount of non-haptenized CRE ds-oligo-DNA. This method, henceforth referred as Southwestern histochemistry, should be a useful tool to localize proteins that bind to a specific DNA sequence and regulate the transcriptional activity of genes.