Assuntos
Pareamento Incorreto de Bases/genética , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Reparo do DNA/genética , Mutação , Transativadores , Fatores de Transcrição/fisiologia , Proteína Axina , Linhagem Celular , Proteínas do Citoesqueleto/biossíntese , Análise Mutacional de DNA , Genes Reporter , Humanos , Transdução de Sinais , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transfecção , beta CateninaRESUMO
Fas/Fas Ligand (FasL) interactions play a significant role in peripheral T lymphocyte homeostasis and in certain pathological states characterized by T cell depletion. In this study, we demonstrate that antigen-presenting cells such as monocyte-derived human macrophages (MDM) but not monocyte-derived dendritic cells express basal levels of FasL. HIV infection of MDM increases FasL protein expression independent of posttranslational mechanisms, thus highlighting the virus-induced transcriptional upregulation of FasL. The in vitro relevance of these observations is confirmed in human lymphoid tissue. FasL protein expression is constitutive and restricted to tissue macrophages and not dendritic cells. Moreover, a significant increase in macrophage-associated FasL is observed in lymphoid tissue from HIV (+) individuals (P < 0.001), which is further supported by increased levels of FasL mRNA using in situ hybridization. The degree of FasL protein expression in vivo correlates with the degree of tissue apoptosis (r = 0.761, P < 0. 001), which is significantly increased in tissue from HIV-infected patients (P < 0.001). These results identify human tissue macrophages as a relevant source for FasL expression in vitro and in vivo and highlight the potential role of FasL expression in the immunopathogenesis of HIV infection.
Assuntos
HIV/fisiologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/biossíntese , Apoptose , Células Cultivadas , Células Dendríticas/metabolismo , Proteína Ligante Fas , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Regulação para CimaRESUMO
A novel region of amplification in breast tumors has recently been identified on chromosome 17q22-23. In an effort to identify the oncogenes in the region that are targeted by the amplification process, we determined the structure of the amplicon in breast cancer cell lines and tumors. Physical and transcription maps of the approximately 3.5-Mb region were established and used as the basis for copy number analysis within the region by Southern blot and fluorescence in situ hybridization. Seven specific and independent amplification maxima were identified in breast cancer cell lines and breast tumors. We present correlative amplification and overexpression studies for the FLJ21316 and Hs.6649 genes suggesting a role for these candidates as amplification-dependent oncogenes.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Cromossomos Humanos Par 17/genética , Amplificação de Genes , Sequência de Aminoácidos , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Oncogenes , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Amplification of the 17q23 region occurs frequently in breast tumors. To characterize the structure of 17q23 amplicons and to identify oncogene targets associated with this alteration, we performed a copy number analysis of 87 17q23 localized expressed sequence tags in seven breast cancer cell lines. Three major regions of amplification were detected in the MCF7 and BT474 cell lines. Amplification of at least one of four known genes (PAT1, PS6K, RAD51C, and SIGMA1B) was detected in the cell lines and in 28% of 94 breast tumors. In most cases, these four genes were overexpressed when amplified, but there was a particularly good association between amplification of the SIGMA1B gene and elevated expression in tumors, which suggested a possible role for this gene in tumor progression. Our data show that this region contains at least four independent targets of amplification, which suggests that there is considerable variability in the structure of the 17q23 amplicon.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Cromossomos Humanos Par 17/genética , Proteínas de Saccharomyces cerevisiae , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Amplificação de Genes , Expressão Gênica , Humanos , Proteínas de Ligação a RNA , Rad51 Recombinase , Proteínas Quinases S6 Ribossômicas/biossíntese , Proteínas Quinases S6 Ribossômicas/genética , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/genética , Células Tumorais CultivadasRESUMO
Members of the protein kinase C (PKC) family appear to play important roles in colorectal carcinogenesis. To investigate the potential involvement of PKC isozymes in adenomatous transformation induced by inactivation of the adenomatous polyposis coli (APC) gene product, we examined protein levels and localizations of ten PKC isozymes by immunohistochemistry in normal and adenomatous ileal epithelium of ApcMIN mice. Compared with surrounding normal epithelium, adenomas showed dramatically reduced staining for PKCs a, beta1, and zeta, as well as dysplasia-specific punctate nuclear staining of PKC mu. We conclude that reduced protein expression of PKC alpha, beta1, and zeta, and nuclear localization of PKC mu are markers of, and are perhaps involved in, adenomatous transformation induced by APC inactivation in ApcMIN mice.
Assuntos
Adenoma/enzimologia , Regulação Neoplásica da Expressão Gênica , Genes APC/genética , Neoplasias do Íleo/enzimologia , Proteína Quinase C/metabolismo , Adenoma/genética , Adenoma/patologia , Polipose Adenomatosa do Colo/enzimologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Núcleo Celular/enzimologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias do Íleo/genética , Neoplasias do Íleo/patologia , Íleo/citologia , Íleo/enzimologia , Íleo/patologia , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína Quinase C/química , Proteína Quinase C/genéticaRESUMO
Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colon cancer and in a subset of patients with sporadic colorectal cancer (CRC). In sporadic CRC, three tumor phenotypes have been defined: microsatellite stable (MSS), low-frequency MSI, and high-frequency MSI (MSI-H). Although defective mismatch repair, consisting primarily of alterations in hMSH2 and hMLH1, is believed to be responsible for the MSI phenotype in the majority of patients with hereditary nonpolyposis colon cancer, the genetic defect responsible for this phenotype in sporadic CRC has yet to be clearly delineated. Somatic or germ-line alterations in these two genes have been identified in only a minority of these cases. Analysis of the protein expression patterns of hMSH2 and hMLH1 in unselected CRC, however, suggests that alterations in hMLH1 may account for a majority of the MSI-H cases. In an effort to explore the underlying molecular basis for these findings, we have examined the methylation status of the presumptive hMLHI promoter region in 31 tumors that vary in regard to their MSI status (MSI-H or MSS), their hMLH1 protein expression (MLH- or MLH+), and their gene mutation (Mut+ or Mut-) status. Hypermethylation of the hMLH1 promoter occurred in all 13 MSI-H/ MLH- tumors that did not have a detectable mutation within the hMLH1 gene. Of those MSI-H tumors containing germ-line or somatic alterations in hMLH1 (n = 7, including 3 frameshift, 1 nonsense, 2 missense mutations, and 1 tumor containing multiple mutations: missense, splice-site alteration, and a frameshift), four had a normal methylation pattern, whereas three others demonstrated hypermethylation of the hMLH1 promoter region. Two of these cases had a missense alteration, the other a frameshift alteration. The single MSI-H/Mut+ tumor that had normal hMLH1 and hMSH2 expression, as well as 9 of the 10 MSS cases, lacked methylation of the hMLH1 promoter. Hypermethylation of the hMSH2 promoter was not observed for any of the cases. These results suggest that hypermethylation of the hMLH1 promoter may be the principal mechanism of gene inactivation in sporadic CRC characterized by widespread MSI.
Assuntos
Neoplasias do Colo/genética , Metilação de DNA , Proteínas de Ligação a DNA , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Neoplasias do Colo/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Mutação em Linhagem Germinativa , Humanos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genéticaRESUMO
Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colorectal cancer and in a large number of sporadic tumors. To further characterize the type of alterations at these loci and their frequency of involvement in colon cancer, we studied DNA extracted from paraffin-embedded tissue from 508 patients using 11 microsatellites localized to chromosomes 5, 8, 15, 17, and 18. Overall, MSI at each locus varied in character and frequency and was observed with at least one marker in 191 cases (37.6%). Based on the number of markers displaying instability per tumor, three groups of patients were defined: those with <30% of the markers showing instability (MSI-L,, n = 109, 21.5%); those with > or = 30% (MSI-H, n = 82, 16.1%); and those showing no instability (MSS, n = 317, 62.4%). These groups were tested for correlations with a number of clinical and pathological parameters, including age, sex, stage, ploidy status, and site of tumor. Comparing across the three groups and verified by pair-wise comparisons, the MSI-H group was associated with tumor site (proximal colon, P = 0.001), sex (females, P = 0.005), stage (Dukes' B, P = 0.01), and ploidy status (diploid, P = 0.03). No significant differences were noted between the MSI-L and MSS group for any of the parameters tested. An additional 188 consecutive surgical colorectal cancer cases were examined for the presence of MSI and for the immunohistochemical expression of hMLH1 and hMSH2 proteins. Of this group, 129 (68.6%) were classified as MSS, 17 (9.0%) as MSI-L, and 42 (22.3%) as MSI-H. None of the MSS and none of the MSI-L tumors had altered expression of either hMLH1 or hMSH2. However, the majority of MSI-H (40 of 42, 95%) cases demonstrated absence of staining for these proteins. The most frequently altered protein was hMLH1, occurring in 95% of the tumors with altered expression. Cumulatively, these data suggest that the tumor phenotype MSI-H is distinct from tumor phenotypes MSI-L and MSS, with no apparent differences between MSI-L and MSS. Furthermore, altered hMLH1 protein expression appears to be responsible for the mutator phenotype in the vast majority of MSI-H tumors.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Reparo do DNA , Feminino , Heterozigoto , Humanos , Imuno-Histoquímica , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Ploidias , Reação em Cadeia da PolimeraseRESUMO
Two microsatellite instability (MSI) phenotypes have been described in colorectal cancer (CRC): MSI-H (instability at >30% of the loci examined) and MSI-L (MSI at 1-30% of the loci examined). The MSI-H phenotype, observed in both hereditary nonpolyposis colon cancer-associated CRC and approximately 15% of sporadic CRC, generally results from mutational or epigenetic inactivation of the DNA mismatch repair (MMR) genes hMSH2 or hMLH1. The genetic basis for the MSI-L phenotype, however, is unknown. Several other proteins, including hMSH3 and hMSH6, also participate in DNA MMR. Inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair single-base mispairs and small insertion-deletion loops but not large insertion-deletion loops. This suggests that hMSH6 mutations are more likely to be associated with a MSI-L phenotype than a MSI-H phenotype in CRC. To explore this possibility, we screened tumors from 41 patients with MSI-L CRC for hMSH6 mutations with conformation-sensitive gel electrophoresis (CSGE) and for hMSH6 protein expression by immunohistochemistry. Alterations found with CSGE were confirmed by DNA sequencing of normal and tumor tissue. One somatic (Asp389Asn) and 15 germ-line changes were found. Of the 15 germ-line changes, 9 were found in an intron (none involving splice junctions), and 6 were found in an exon (Gly39Glu, Leu395Val, and 4 silent alterations). Immunohistochemical staining for hMSH6 performed on 34 of the 41 tumors revealed strong nuclear hMSH6 expression in all of the cases. Overall, our results suggest that hMSH6 mutations do not play a major role in the development of sporadic CRC with a MSI-L phenotype.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Mutação/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Saúde da Família , Feminino , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético/genéticaRESUMO
To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA , Repetições de Microssatélites , Mutação , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Humanos , Imuno-Histoquímica , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/análise , Proteínas Nucleares , Proteínas Proto-Oncogênicas/análiseRESUMO
Electrophoretic analysis of plasminogen activators from pig heart, human uterus, human plasma and human melanoma cells was performed in SDS-polyacrylamide gradient slab gels containing plasminogen and casein. Direct visualization of activator activity bands in polyacrylamide gels was achieved after removal of SDS, incubation in buffer, and staining with Coomassie brilliant blue. Tissue activator extracted from pig hearts displayed a molecular weight of 72000 and migrated similarly to activator secreted by human melanoma cells and to one activator component present in extracts of human uterus. Immunoadsorption experiments with melanoma cell activator antiserum indicated that these 72-kDa activators are all related immunologically. Human uterus also contained a second activator component with a molecular weight 55000, which migrated similarly to a higher molecular weight component of urokinase and cross-reacted with urokinase antiserum. We conclude that the 72-kDa uterine activator component represents a tissue activator and the 55-kDa component represents a urokinase-like activator. A euglobulin solution from venous occlusion plasma displayed multiple bands of plasmin activity in the Mr range 85000-96000. Two activator components were also present, one of Mr 72000 and another of Mr 62000. The 72-kDa euglobulin activator was adsorbed by MCA antiserum, and we conclude that this component represents vascular activator. The 62000 activator also had weak plasminogen-independent caseinolytic activity and was not affected by either melanoma cell activator or urokinase antisera. Conclusions concerning its identity cannot be made at this time.
Assuntos
Caseínas , Ativadores de Plasminogênio/análise , Plasminogênio , Animais , Fenômenos Químicos , Química , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas de Imunoadsorção , Especificidade da Espécie , SuínosRESUMO
BACKGROUND: Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)-approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression. MATERIALS AND METHODS: Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization. RESULTS: Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79% (38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17% (seven of 41) of 2+ cases had increased gene copy. CONCLUSION: Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Genes erbB-2/genética , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Reações Falso-Positivas , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Padrões de Referência , TrastuzumabRESUMO
PURPOSE: To evaluate the prognostic significance of p53 expression in epithelial ovarian cancer, including a subset of stage I patients, and to look for correlations between p53 expression and other disease parameters, including stage, grade, age, histologic subtype, second-look results, ploidy, and percent S phase. PATIENTS AND METHODS: We analyzed p53 expression in 284 patients with epithelial ovarian cancer using immunohistochemical techniques in paraffin-embedded specimens. There were 36 patients with stage I disease, 20 with stage II disease, 186 with stage III disease, and 42 with stage IV disease. RESULTS: p53 immunoreactivity was present in 177 cases (62%). p53 expression was associated with grade 3 to 4 disease (P = .003). The following factors were associated with a decrease in overall survival in a univarate analysis: stage III or IV disease (P = .0001), grade 3 or 4 disease (P = .0001), age above the median (P = .0002), and p53 reactivity (P = .04). In a multivariate analysis, stage, grade, and age retained independent prognostic significance. In the subset of 36 stage I patients, p53 positively approached statistical significance (P = .10) as a negative prognostic factor in a univariate analysis. CONCLUSION: Abnormalities of p53 expression occur commonly in epithelial ovarian cancer. Although associated with decreased survival in a univariate analysis, this biologic marker did not retain independent prognostic significance in a multivariate analysis. p53 expression should be studied in a larger cohort of early-stage patients, where accurate prognostic information is needed to direct therapy.
Assuntos
Carcinoma/química , Neoplasias Ovarianas/química , Proteína Supressora de Tumor p53/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/mortalidade , Carcinoma/patologia , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Estatística como Assunto , Análise de SobrevidaRESUMO
Amyloid deposits that characteristically form in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus (NIDDM) and in insulinomas are both derived from islet amyloid polypeptide (IAPP). Evidence from previous studies has suggested that deposition of IAPP-derived amyloid is related to inherent amyloidogenic sequences present within normal human IAPP, together with an increased production and local concentration of IAPP. However, whether the aggregation of IAPP to form amyloid fibrils is primarily an intra- or extracellular event is not clear. To address this question, we studied 20 human insulinomas by light and electron microscopy. By light microscopy, amyloid deposits were demonstrated in 13 of 20 (65%) human insulinomas. Furthermore, evaluation of Congo red-stained tumor sections showed small, globular or irregular, congophilic amyloid deposits within the cytoplasm of many tumor cells in 10 of 13 (77%) amyloid-containing insulinomas. Dense, punctate areas of IAPP immunoreactivity within tumor cells corresponded with the congophilic intracellular deposits. Ubiquitin immunoreactivity also was observed as punctate intracellular labeling and within large extracellular amyloid deposits. Among the 10 insulinomas available for electron microscopic evaluation, pathological IAPP-immunoreactive (immunogold) deposits were found in 3 of 5 insulinomas in which amyloid was demonstrated by light microscopy and in none of 5 tumors found negative for amyloid by light microscopy. Morphology of IAPP-immunoreactive deposits varied from those with the classical distinct 7- to 10-nm diameter nonbranching fibrils to those with distinct but faint fibrillarity to those without discernable fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Amiloide/análise , Insulinoma/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Criança , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Insulinoma/ultraestrutura , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Masculino , Microscopia Eletrônica , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Neoplasias Pancreáticas/ultraestruturaRESUMO
DNA mismatch repair genes have been reported to play a role in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC). Mutations of DNA mismatch repair genes have accounted for 90% of HNPCC-related colon and endometrial tumors. These mutations have been associated with microsatellite instability (MIN). Because endometrial cancer (EC) is the most common extracolonic malignancy associated with HNPCC, we hypothesized that similar molecular alterations may occur in sporadic endometrial tumors exhibiting MIN. Mutational analysis of the MSH2 and MLH1 genes was undertaken in sporadic EC that demonstrate MIN to determine the role of these genes in the pathogenesis of sporadic ECs. Established microsatellite markers were used to determine the incidence of MIN from 28 patients with sporadic EC. MIN was observed in 32% (9 of 28) of the tumor specimens analyzed. Mutational analysis of MSH2 and MLH1 genes was performed by immunohistochemical analysis and direct sequencing of tumor specimens that exhibited MIN. All 28 tumor specimens exhibited strong nuclear staining with both MSH2 and MLH1 antibodies, suggesting the absence of mutations. Sequencing of all exons of both the MSH2 and MLH1 genes in the nine MIN-positive tumor specimens demonstrated no mutations. We conclude that the MSH2 and MLH1 genes do not play a role in the pathogenesis of sporadic endometrial cancer.
Assuntos
Reparo do DNA/genética , Neoplasias do Endométrio/genética , Repetições de Microssatélites , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias do Endométrio/complicações , Feminino , Humanos , Proteína 1 Homóloga a MutL , Mutação , Proteínas de Neoplasias/genética , Proteínas NuclearesRESUMO
We studied the prognostic value of angiogenesis grading and microvessel density estimation in newly diagnosed multiple myeloma. Seventy-five patients with newly diagnosed myeloma, treated on Eastern Cooperative Oncology Protocol E9486 and Intergroup study 0141 (S9321) at the Mayo Clinic, were studied. Bone marrow microvessels were examined using immunohistochemical staining for von Willebrand factor. Determination of microvessel density and angiogenesis grading was done in a blinded manner. There was a strong correlation between microvessel density and the plasma cell labeling index, rho 0.42, P < 0.001. Angiogenesis grade was also significantly associated with the plasma cell labeling index. Fifteen % of patients with low-grade angiogenesis had a high labeling index (>1%). In contrast, 47% of patients with intermediate or high-grade angiogenesis had high labeling indices (P = 0.02). Overall survival was significantly different among those with high-, intermediate-, and low-grade angiogenesis, with median times of 2, 4, and 4.4 years, respectively (P = 0.02). Similarly, patients with microvessel density >50/x400 field had poorer survival compared with those with 50 or fewer microvessels/field, median survival 2.6 versus 5.1 years, respectively (P = 0.004). There was a strong association between angiogenesis grade and microvessel density (P < 0.001). We conclude that bone marrow angiogenesis is a predictor of poor survival in newly diagnosed myeloma. Angiogenesis is correlated with the plasma cell labeling index but not the bone marrow plasma cell percentage. A simple visual grading of angiogenesis is an efficient alternative to microvessel density estimation.
Assuntos
Medula Óssea/irrigação sanguínea , Mieloma Múltiplo/irrigação sanguínea , Neovascularização Patológica/patologia , Medula Óssea/patologia , Ensaios Clínicos como Assunto , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Neovascularização Patológica/metabolismo , Plasmócitos/metabolismo , Plasmócitos/patologia , Prognóstico , Análise de Sobrevida , Resultado do Tratamento , Fator de von Willebrand/metabolismoRESUMO
The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.
Assuntos
Compostos de Dansil , Mastócitos/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Glicerol/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Leucemia de Mastócitos/enzimologia , Leucemia de Mastócitos/patologia , Leucemia de Mastócitos/fisiopatologia , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Mastocitose/enzimologia , Mastocitose/patologia , Mastocitose/fisiopatologia , Cloreto de Mercúrio/farmacologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Peptídeo Hidrolases/análise , Pele/citologiaRESUMO
To gain insights into 1,25-dihydroxyvitamin D3 receptor (VDR) function during fetal bone development, we examined fetal rat tissues from gestational days 13-21 for the presence and distribution of VDR using immunohistochemistry. Prior to ossification, VDR epitopes were observed in the mesenchyme condensing to form skeletal tissues, on day 13 in the developing vertebral column and limbs, and on day 17 of gestation in developing calvaria. Immunostaining for VDR was seen in proliferating and hypertrophic chondrocytes and in osteoblasts of limb buds and the vertebral column by day 17 of gestation. In calvaria, VDR epitopes were observed in osteoblasts by gestational day 19. VDR immunostaining was also evident in the skin of fetal limbs at all gestational ages examined. We show for the first time that the VDR appears very early in the developing fetal rat skeleton, suggesting that the VDR, in concert with its ligand, 1,25-dihydroxyvitamin D3, may play a role in the differentiation of mesenchymal precursors into bone tissue.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Osso e Ossos/embriologia , Extremidades/embriologia , Feminino , Idade Gestacional , Imuno-Histoquímica , Gravidez , Ratos , Crânio/embriologia , Crânio/metabolismo , Coluna Vertebral/embriologia , Coluna Vertebral/metabolismoRESUMO
PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Osteoblastos/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Retículo Endoplasmático/metabolismo , Feminino , Complexo de Golgi/metabolismo , Lâmina de Crescimento/metabolismo , Humanos , Hipofosfatemia Familiar/genética , Hipofosfatemia Familiar/fisiopatologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Endopeptidase Neutra Reguladora de Fosfato PHEX , Proteínas/genética , Proteínas/imunologia , Pele/embriologia , Pele/metabolismo , Crânio/embriologia , Crânio/metabolismoRESUMO
Cell proliferation requires calmodulin, a protein that regulates calcium-dependent enzymes involved in signal transduction pathways in eukaryotic cells. Calmodulin-like protein (CLP) is found in certain epithelial cell types, including normal breast epithelium, and, although it closely resembles calmodulin in amino acid sequence, CLP interacts with different proteins than does calmodulin. The observation that CLP mRNA expression is dramatically reduced in transformed breast epithelial cells led to two hypotheses: (1) CLP helps to maintain the differentiated state in epithelial cells; and (2) downregulation of CLP accompanies malignant transformation of breast epithelial cells. The objective of this study was to determine if the expression of CLP in human breast cancer specimens is reduced in comparison to its expression in normal breast tissue. Eighty human breast cancer biopsy specimens were analyzed immunohistochemically for CLP expression by using a polyclonal rabbit antihuman CLP antibody. CLP expression was reduced in 79% to 88% of the invasive ductal carcinoma and lobular carcinoma specimens and in a similar fraction of the ductal carcinoma in-situ specimens, compared with normal breast specimens. None of the breast cancer specimens showed an increase in CLP expression. These findings support the hypotheses that CLP behaves as a functional tumor suppressor protein and is downregulated early in breast cancer progression.
Assuntos
Neoplasias da Mama/química , Calmodulina/análise , Animais , Calmodulina/genética , Calmodulina/fisiologia , Regulação para Baixo , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , CoelhosRESUMO
Membrane fractions from fully luteinized rat ovaries contained proteolytic enzymes that were solubilized and reversibly inhibited by the anionic detergent sodium dodecyl sulfate. Electrophoretic analysis in substrate-containing sodium dodecyl sulfate-polyacrylamide slab gels revealed the presence of numerous protease bands in the mol wt range of 26,000 to more than 200,000. When casein served as protease substrate in the gels, enzyme bands of Mr 26,000, 28,000, 30,000, and 90,000 were produced by crude (2,000 X g pellet) ovarian membranes. Gels containing casein and plasminogen also revealed the presence of two plasminogen activators of Mr 63,000-65,000 and 42,000. Subcellular fractionation of luteinized ovaries by centrifugation in sucrose density gradients indicated that the Mr 90,000 protease and the two plasminogen activators were uniformly distributed among microvillous membranes, basolateral membranes (BLM), and the mitochondrial-lysosomal fraction (MLF). The Mr 26,000, 28,000, and 30,000 proteases were enriched in BLM and MLF. Analysis of crude membranes in slab gels which contained gelatin as the protease substrate revealed the presence of two additional enzymes of Mr 52,000 and more than 200,000. The Mr greater than 200,000 protease was present in microvillous membranes, BLM, and MLF. The Mr 52,000 protease was found exclusively in BLM. This enzyme was not consistently demonstrable in crude membranes but could be generated upon incubation of membranes at 30 C. This finding indicated that Mr 52,000 protease can exist in an inactive and/or zymogen form. The Mr 90,000 protease was inhibited by tosyl-lysine chloromethyl ketone and dansyl-glutamyl-glycylarginine chloromethyl ketone. The gelatinase activity of the Mr 52,000 protease was blocked by tosyl lysine chloromethyl ketone, dansyl-glutamyl-glycylarginine chloromethyl ketone and tosylamide-2-phenyl chloromethyl ketone. The activity of the Mr 26,000, 28,000, and 30,000 proteases was not affected by any of the above mentioned inhibitors. These findings demonstrate that the proteolytic potential of ovarian membranes is not limited to plasminogen activators. Numerous plasminogen-independent proteases are also present, and these may play a role in ovulation, luteolysis, and mediation of hormonal stimulation.