RESUMO
The main objectives were to determine the modulating effects of all-trans retinoic acid on the number, size and multiplicity of aberrant crypt foci as well as the in vivo expression of the genes c-myc and c-fos. These foci, which are hypothesized to be the pre-malignant lesions of colon cancer, were induced in Sprague-Dawley rats with a single injection of azoxymethane. Rats were fed either a control diet (AIN-76) or the control diet to which had been added 75 mg/kg or 150 mg/kg all-trans retinoic acid. Within 4 weeks, we observed that the diets containing all-trans retinoic acid reduced the total number and multiplicity of aberrant crypt foci in the colon. However, all-trans retinoic acid increased the size of the lesions that persisted, possibly due to a greater proportion of lesions with dilated crypts. In situ hybridization and immunohistochemistry were performed on the colons for the in vivo analysis of gene expression in these lesions. The expression of myc-specific mRNA and protein in aberrant crypt foci significantly decreased with both levels of all-trans retinoic acid. In contrast, fos-specific mRNA and protein in aberrant crypt foci significantly increased when 150 mg/kg all-trans retinoic acid was added to the diet. The most important findings of this investigation are that intervention with all-trans retinoic acid in the pre-malignant stage of colon carcinogenesis is effective in decreasing the number and growth of aberrant crypt foci and altering the expression of the c-myc and c-fos genes.
Assuntos
Colo/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Genes fos/efeitos dos fármacos , Genes myc/efeitos dos fármacos , Lesões Pré-Cancerosas/prevenção & controle , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Tretinoína/farmacologia , Animais , Azoximetano , Peso Corporal/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/prevenção & controle , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-myc/análise , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Mutation of the p53 gene is the most common genetic change accompanying the sequential development of colon cancer, but it has not been studied in the early stages of colon cancer particularly at the single and multiple crypt levels. The expression of the mutant p53 gene product in aberrant crypt foci and in adenocarcinomas induced by azoxymethane was investigated immunohistochemically, using the rat model system. Aberrant crypt foci, which may be the premalignant lesions of colon cancer, are one of the earliest recognizable lesions evident in the stepwise development of colon carcinogenesis. Immunohistochemistry was performed with three mouse monoclonal antibodies to p53 proteins. These antibodies included PAB1620 specific for the wild-type p53 protein, PAB240 specific for the mutant p53 protein and PAB421 specific for both the wild-type and mutant p53 proteins. Positive reactivity with PAB240 and PAB421 was observed in 27 of 65 (42%) aberrant crypt foci and in six of eight (75%) adenocarcinomas. No reactivity with these antibodies was present in normal adjacent crypts and in colons from untreated animals. Immunohistochemical staining with PAB240 and PAB421 was present mainly in the cytoplasm and occasionally in the nucleus of cells. This is consistent with the known preferential location of the mutant p53 protein. In adenocarcinomas, uniform staining was present throughout the tissue. Reactivity with PAB1620 in premalignant and malignant tissue was not observed. The results indicate that a p53 gene mutation occurs in aberrant crypt foci as an early genetic event in colon carcinogenesis.
Assuntos
Neoplasias do Colo/química , Genes Supressores de Tumor , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Animais , Azoximetano , Núcleo Celular/química , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Citoplasma/química , Imuno-Histoquímica , Masculino , Mutação , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/análiseRESUMO
The main objective of the present investigation was to understand the molecular events involved in the genesis of aberrant crypt foci. Aberrant crypt foci were induced in Sprague-Dawley rats with a single injection of azoxymethane. Aberrant crypts have been identified topographically in the colon and are hypothesized to represent preneoplastic lesions. In order to understand the molecular events involved in the early stages of colon cancer, PCR-amplified DNA from aberrant crypts was hybridized with oligonucleotide probes specific for the detection of point mutations in codon 12 of K-ras. The mutation identified was a G to A transition resulting in the substitution of the amino acid aspartic acid (asp) for glycine (gly). This mutation was present in 6/19 (32%) of aberrant crypts examined. The identical mutation was also identified in adenomacarcinoma tissue while no mutation could be detected in normal intestinal mucosa. For further confirmation of these results, the presence of the mutated ras protein (rasAsp-12) was detected in aberrant crypts by immunohistochemistry. This investigation provides the first identification of a ras point mutation in aberrant crypt foci.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Genes ras , Mutação , Lesões Pré-Cancerosas/genética , Adenocarcinoma/induzido quimicamente , Animais , Azoximetano , Sequência de Bases , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , DNA de Cadeia Simples , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The expression of the ras oncogene in aberrant crypt foci was studied by both in situ hybridization and immunohistochemical approaches. Aberrant crypt foci are hypothesized to represent the earliest identifiable microscopic lesions of colon cancer in rodent colons. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg s.c.) once. Twelve weeks later, aberrant crypt foci were identified topographically, microdissected and processed for histology. In situ hybridization with an antisense oligomer of c-ras demonstrated increased expression of ras-specific RNA in aberrant crypts compared to normal crypts. A low amount of non-specific hybridization was obtained with the corresponding sense oligomer. The percentage of cells with grains (labeling index) was calculated in early and advanced aberrant crypt foci. This index was also calculated in normal appearing crypts. The labeling indices for the early and advanced aberrant crypt foci were significantly greater than that of normal crypts (18.0 and 25.0 versus 11.9). In the same tissue specimens, immunohistochemical staining for ras p21 with the monoclonal antibody (Y13-259) revealed strong staining intensity in early aberrant crypts (15/22) and advanced aberrant crypts (22/30) compared to normal crypts (3/50). The immunohistochemical results demonstrate the presence of elevated levels of ras p21 in the same tissue as increased levels of ras-specific message. This investigation provides the earliest demonstration of increased expression of the ras oncogene in precursor lesions of colon cancer possessing dysplastic features.
Assuntos
Colo/patologia , Neoplasias do Colo/genética , Expressão Gênica/genética , Genes ras/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro/genética , Animais , Colo/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/patologia , Masculino , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Aberrant crypt foci represent the earliest detectable lesions of colon cancer. The expression of c-fos in these aberrant crypts was determined by in situ hybridization and immunohistochemistry. These lesions were induced in the colonic epithelium with azoxymethane using the rat model system. Expression of c-fos was markedly increased in the aberrant colonic crypts. Increases of approximately 60 and approximately 70% in the proportion of epithelial cells labelled were observed in the early and advanced aberrant crypts respectively. This was found to be statistically significant (P less than 0.001). Consistent with cell proliferation patterns in the colonic crypts, the epithelial cells of the lower crypt had greater levels of c-fos mRNA than those of the upper part of the crypts. In addition, immunohistochemical staining with c-fos polyclonal antibodies demonstrated increased levels of c-fos protein in aberrant crypts. This combined approach using in situ hybridization and immunohistochemistry has shown that increased c-fos expression at the RNA level in these lesions is associated with increased amounts of c-fos protein. Since c-fos has been implicated in the process of cell proliferation and differentiation, modifications in its expression may be significant to understanding the mechanisms whereby preneoplastic lesions transform to neoplastic lesions in colon cancer.
Assuntos
Neoplasias do Colo/genética , Expressão Gênica , Genes fos , Lesões Pré-Cancerosas/genética , Animais , Feminino , Imuno-Histoquímica , Hibridização de Ácido Nucleico , Proteínas Proto-Oncogênicas c-fos/análise , RNA Mensageiro/análise , Ratos , Ratos EndogâmicosRESUMO
A new retinoic acid-related receptor has been identified. It is called the retinoid X receptor and is distinct from the retinoic acid receptors, alpha, beta and gamma. It represents an "orphan" receptor because its function and ligand are unknown. What is known is that it is structurally related to the steroid receptor genes. The ligand to which the retinoic acid X receptor binds has recently been determined to be either retinoic acid itself or another metabolically derived molecule. It is speculated that retinoic acid could serve as a precursor that is metabolized to a more active form as is the case for vitamin D and estrogen (24). The elucidation of an alternative pathway in retinoid metabolism will increase our understanding of the scope of retinoid action.
Assuntos
Proteínas de Transporte/fisiologia , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , DNA/metabolismo , Humanos , Receptores do Ácido Retinoico , Retinoides/metabolismo , Distribuição TecidualRESUMO
Four cases of variant Philadelphia (Ph1) translocations were found in 72 patients (5.5%) with Ph1-positive chronic myeloid leukemia (CML). One previously unreported case was a simple variant translocation, namely, 46,XY,t(11;17)(q13;p13),t(17;22)(q25;q22); 46,XY,t(1;21)(q32;q11),t(11;17)(q13;p13), t(17;22)(q25;q11). Complex variant translocations were observed in three cases, namely, 46,XY,t(5;9;22)(q31;q34;q11),46,XX,t(8;9;22) (q22;q34;q11) and 46,XX,t(9;15;22) (q34;q15;q11). The chromosomal breakpoints in the cases of variant Ph1 translocations were the following: 1q32, 5q31, 8q22, 11q13, 15q15, 17p13, 17q25 and 21q11. Eight of the eight (100%) breakpoints were located in Giemsa-negative bands. Furthermore, seven of the eight (87%) variant Ph1 breakpoints correspond to the breakpoints present in consistent cancer arrangements. Three of the eight (38%) correspond to fragile sites and four of the eight (50%) correspond to oncogenes.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Cromossomo Filadélfia , Variação Genética , Humanos , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Oncogenes/genética , Translocação Genética/genéticaRESUMO
Transposition of the abl gene was demonstrated in seven Philadelphia (Ph'-) negative patients with either chronic myeloid leukaemia (CML) or acute lymphocytic leukaemia (ALL) by chromosomal in situ hybridization. In six out of seven CML patients and one out of two ALL patients, a significant accumulation of abl hybridization grains was localized to chromosome 22. Hybridization with both abl and sis resulted in the consistent formation of double hybridization events on chromosome 22. Transposition of abl was not apparent in one patient with Ph'-negative CML and in one patient with Ph'-negative ALL. The data suggest that transposition of abl to chromosome 22 is a feature of a particular subgroup of Ph'-negative leukaemias.
Assuntos
Elementos de DNA Transponíveis , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Mapeamento Cromossômico , Cromossomos/análise , Cromossomos/efeitos dos fármacos , Cromossomos/ultraestrutura , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Humanos , Cariotipagem , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/patologia , Masculino , Fator de Crescimento Derivado de Plaquetas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-abl , Proteínas Proto-Oncogênicas c-sisRESUMO
New information is revealed concerning the frequency of expression and distribution of aphidicolin-induced fragile sites in eight leukaemic patients, namely, four chronic myeloid leukaemic patients (CML), three acute lymphocytic leukaemic (ALL) patients, and one acute myeloid leukaemic (AML) patient. The cytogenetic data demonstrate a statistically significant (p less than 10(-6] increase in the frequency of aphidicolin-induced fragile sites in seven of the eight leukaemic patients compared with healthy age-matched and sex-matched controls. The chromosomal band locations of the aphidicolin-induced fragile sites from 400 metaphase spreads of these leukaemic patients reveal a nonrandom distribution in the karyotype. Some aphidicolin-induced fragile sites in these leukaemic patients were located at chromosome bands known to be induced specifically by folic acid, distamycin A, bromodeoxyuridine or azacytidine. The cross-induction of fragile sites in the leukaemic patients may be indicative of shared molecular homology in the sequence composition of nonrandom chromosomal DNA.