RESUMEN
BACKGROUND: A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination. METHODS: In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions. RESULTS: The L1 loop (aa 112-124), a region within the S7-S8 loop (aa 214-236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53wt and p53mut cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53wt and p53mut cells were in response to sustained increases in p53. CONCLUSION: These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.
Asunto(s)
Azurina/farmacología , Fragmentos de Péptidos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Azurina/química , Azurina/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/química , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Terminal replicative senescence (TRS) is a physiological process associated with terminal differentiation, shortening of the telomere, and lack of proliferative activity. Immortalised and tumour cells have lost their differentiation potential and the ability to develop a senescence phenotype. Recently, others and we [11] have observed that some antitumour agents and radiation induce a senescence-like phenotype (SLP) in human immortalized and tumour cell lines. The main purpose of this study was to identify senescence-like cells (SLC) in mammary tumours of rats and assess whether chemopreventive agents that have been used for the prevention and/or treatment of breast cancer can induce a SLP in tumour cells. Sprague-Dawley rats with N-methyl-N-nitrosourea (MNU)-induced mammary tumours were randomised and treated with tamoxifen, vorozole, 4-(hydroxyphenyl)retinamide (4-HPR), or 9-cis-retinoic acid (9cRA). The SLC in mammary tumours were identified and characterised by: (a) SA-beta-Gal staining method, which has been considered specific for human cells in TRS (b) staining for lipofuscin, which, although not specific, accumulates in the cytoplasm of cells in senescence; (c) lack of 5-Bromodeoxyuridine (BrdU) labelling after continuous (7 days) infusion of BrdU via osmotic pumps; (d) 90 degrees side light scatter (9OLS) as evaluated by flow cytometry; and (e) decreased telomerase activity. We found that in control tumours, SA-beta-Gal-positive cells were rare (below 1.0%) among the tumour cells, stroma fibroblast, myoepithelial and endothelial cells. SA-beta-Gal-positive cells increased significantly in the tumours treated with chemopreventive agents and this was associated with a lack of proliferative activity, increased cell granularity, lipofuscin accumulation, and decreased telomerase activity. Thus, in this study we provide for the first time evidence that cells in replicative senescence are present in mammary tumours of rats and that chemopreventive agents can suppress tumor growth by a novel cellular mechanism, inducing a SLP in the tumor cells.
Asunto(s)
Anticarcinógenos/uso terapéutico , Senescencia Celular , Neoplasias Mamarias Experimentales/prevención & control , Tamoxifeno/uso terapéutico , Animales , Gránulos Citoplasmáticos , Femenino , Citometría de Flujo , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Fenotipo , Ratas , Ratas Sprague-Dawley , Telomerasa/metabolismoRESUMEN
The incidence of melanoma is estimated to be growing at the second fastest rate among all cancers in the United States. The progression of the melanocyte to a malignant melanoma involves various sequential steps: development of benign naevocellular naevus, preneoplastic dysplastic naevus, primary melanoma, and metastatic melanoma. Despite these clearly defined stages, very little is known about the molecular events leading to melanoma progression. We established a human congenital naevus cell line (UISO-CMN-1). UISO-CMN-1 cells were confirmed to have melanocytic origin by S100 immunoreactivity and the presence of melanin granules and melanosomes. UISO-CMN-1 cells, even though they showed structural and numerical abnormalities in karyotype, were non-tumorigenic when transplanted into athymic mice. However, following frequent exposure to ultraviolet C radiation, UISO-CMN-1 cells acquired tumorigenic potential. Transformation of UISO-CMN-1 cells into tumorigenic cells was accompanied by induction of ganglioside-2 expression without any significant changes in cellular ganglioside-3. These transformed and non-transformed UISO-CMN-1 cell lines can serve as excellent research tools for studying the molecular changes associated with melanoma development and progression, and for identifying agents that might prevent development of malignant melanoma.
Asunto(s)
Línea Celular , Melanoma/metabolismo , Melanoma/patología , Nevo/congénito , Animales , Línea Celular Transformada , Femenino , Gangliósido G(M2)/biosíntesis , Gangliósido G(M3)/biosíntesis , Humanos , Inmunohistoquímica , Lactante , Cariotipificación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transformación Genética , Rayos UltravioletaRESUMEN
The patterns of periodic acid-Schiff (PAS) staining of extracellular matrix in histological sections of certain melanomas may be predictive of outcome. Recent in vitro and molecular genetic data suggest that the appearance of these patterns in both uveal and cutaneous melanoma is a function of aggressive tumor cells. We studied 96 patients with primary cutaneous melanomas treated at the University of Illinois at Chicago who were monitored for disease-free survival. Survival probabilities were determined by Kaplan-Meier estimates, and prognostic factors were evaluated by multivariate analysis. By univariate analysis, there was a significant decrease in disease-free survival among patients whose tumors contained parallel with cross-linking or network patterns (PXNs; P = 0.0070). Stepwise regression with Cox models that included the combinations of the PAS-positive patterns, tumor thickness, female gender, ulceration, and age yielded a model with thickness and the PAS-positive parallel with cross-linking or networks. Despite the relatively small sample size in this study, the detection of the PAS-positive parallel with cross-linking or networking in cutaneous melanoma was associated with a decrease in disease-free outcome. Additional studies of the prognostic significance of these patterns is warranted on larger data sets.
Asunto(s)
Melanoma/diagnóstico , Melanoma/patología , Reacción del Ácido Peryódico de Schiff , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Regresión , Factores de TiempoRESUMEN
Micropthalmia transcription factor (Mitf) is involved in melanocyte development and differentiation. The current study was undertaken to determine whether there is a relationship between Mitf expression and survival in patients with intermediate-thickness (1.0-4.0 mm) melanoma. Original paraffin blocks or slides of the primary tumor were accessible in 63 such patients. Mitf expression was evaluated by immunocytochemistry and analyzed visually. Slides were graded as follows according to the percentage of cells whose nuclei stained positive for Mitf: (a) 0, 0%; (b) +1, 1-25%; (c) +2, 26-50%; (d) +3, 51-75%; and (e) +4, > 75%. Median follow-up was 50 months. Mean thickness was 2.2 +/- 0.7 mm. Mean overall survival was 171.90 +/- 13.12 months. Mean disease-free survival was 168.53 +/- 13.96 months. Fifty-two melanomas (82.5%) stained positive for Mitf. By univariate analysis, mean overall survival and disease-free survival in patients whose melanomas did not express Mitf were 80.89 +/- 17.98 months (median, 51 months) and 71.36 +/- 19.87 months (median, 40 months), respectively. This compares with 187.90 +/- 13.41 months (median, not reached) and 186.78 +/- 13.84 months (median, not reached), respectively, for patients whose melanomas expressed Mitf (P = 0.0086 and P = 0.0054). These findings persisted in multivariate analysis. In addition, patients with > 50% Mitf expression had significantly fewer nodal metastases after node dissection than patients with < or = 50% Mitf expression (P = 0.04). Our data suggest that Mitf may be a new molecular prognostic marker in patients with intermediate-thickness melanoma.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Pronóstico , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Factores de Transcripción/biosíntesisRESUMEN
Previously, we found that vorozole (Vz), a nonsteroidal aromatase inhibitor, suppresses the development and progression of mammary tumors in rats. Here we evaluated for the first time the expression of cell death-related proteins Bcl-2 and Bax in hyperplastic, premalignant (carcinoma in situ), or malignant (carcinoma) lesions of mammary carcinogenesis; we also assessed whether these proteins are involved in mediating Vz-induced cell death in tumors. We found that Bcl-2 and Bax were equally expressed in epithelial cells of terminal end buds, ducts, and alveoli. However, in myoepithelial cells, the level of Bax expression was much higher than the level of Bcl-2 expression. Bcl-2 and Bax levels in hyperplastic lesions were similar to those of normal mammary epithelial cells but lower in most carcinomas in situ and carcinomas. In animals with established mammary tumors, Vz induced apoptotic cell death, which was primarily associated with a decrease in Bcl-2 and, to a lesser extent, with a decrease in Bax. These data support the hypothesis that Bcl-2 loss is more potent than Bax gain in regulating apoptotic cell death in mammary tumors.
Asunto(s)
Carcinoma/metabolismo , Células Epiteliales/metabolismo , Hiperplasia/metabolismo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/metabolismo , Lesiones Precancerosas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , Inhibidores de la Aromatasa , Carcinoma/patología , Femenino , Hiperplasia/patología , Glándulas Mamarias Animales/anatomía & histología , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Animales/patología , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Triazoles/farmacología , Proteína X Asociada a bcl-2RESUMEN
Vorozole (Vz) is a competitive non-steroidal inhibitor of aromatase, which has been used to treat breast cancer in postmenopausal women and in various chemoprevention pre-clinical studies. Recently, we assessed the inhibitory effect of Vz on MNU-induced mammary carcinogenesis (Lubet et al., 1994), as well as on the progression of mammary tumors (Lubet et al., 1998). In this study we evaluated the effects of Vz on tumor growth, serum estradiol, cell proliferation, apoptotic and non-apoptotic cell death to determine whether any of these 'surrogate' markers might reflect the efficacy of various doses of Vz. Vz at doses of 2.5 (Hi), 0.32 (Md), and 0.08 (Lo) mg/kg body weight induced complete (100%), 60%, and 20% regression of mammary tumors, respectively. Vz at Hi and Md doses caused a decrease in serum estradiol within the first two days of treatment, and the estradiol values remained low with additional treatment for 4 and 10 days. When Vz was administered to animals bearing palpable tumors a time and dose-dependent decrease in the proliferating cells (BrdU-L1) was observed. The percentage of apoptotic cells (A1) sharply increased 2 days after initiation of Vz treatment and then decreased followed by an increase in non-apoptotic dead cells. Interestingly even the Lo dose of Vz, which was only moderately effective in suppressing tumor growth, decreased cell proliferation and increased cell death in the peripheral tumor areas at 4 and 10 days after initiation of treatment. The time- and dose-dependent alterations in various cell parameters suggest two different phases of Vz-induced cellular responses: (1) an early phase (2-4 days of treatment) with a sharp increase in apoptotic cells and decrease in proliferating cells, and (2) a later phase (10 days) with disintegration of tumor parenchyma, increase in non-apoptotic dead cells, and decrease in apoptotic cells. The dose-dependent decrease in proliferating cells and increase in apoptotic and non-apoptotic cell death in Vz-treated animals suggest that these biomarkers might be used as potential surrogate endpoints for efficacy in breast cancer chemoprevention and therapy studies with aromatase inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de la Aromatasa , Inhibidores Enzimáticos/farmacología , Neoplasias Mamarias Experimentales/patología , Triazoles/farmacología , Animales , Biomarcadores de Tumor/análisis , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estradiol/sangre , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
Previous studies have shown that terminal end buds (TEBs) in the murine mammary gland have high proliferative activity and demonstrate apoptotic cell death (ACD). Since TEBs are considered the place of origin of most chemically induced mammary carcinomas, we hypothesized that the development of hyperplastic and premalignant (carcinoma in situ, CIS) lesions in TEBs is associated with either a further increase in cell proliferation and/or with a decrease in ACD. To test this hypothesis we used the N-methyl-N-nitorosourea (MNU) carcinogenesis model in rats, where the occurrence of mammary tumors is preceded by hyperplastic and premalignant lesions arising mostly in TEBs, as well as in ducts and alveoli. The percentage of proliferating cells, as evaluated by 5-bromodeoxyuridine labeling (BrdU-LI), was similar in TEBs to those in terminal endbud hyperplasia (TEBH), CIS, and carcinomas (CA), whereas the percentage of apoptotic cells (apoptotic index, AI) was relatively high in TEBs and decreased in TEBH, CIS, and CA. This indicates that neoplastic transformation of mammary epithelial cells in TEBs is not associated with an increase in cell proliferation, but with a decrease in ACD. In addition to TEBH, hyperplastic lesions developed in ductal branching areas (ductal hyperplasia, DH) and alveolar structures (alveolar hyperplasia, AH). However, BrdU-LI in both DH and AH was lower than in TEBH, whereas the AI values were similar, suggesting that TEBH has a higher potential for progression and malignant transformation than DH and AH. In mammary tumors apoptotic cells were rare in the peripheral, proliferative areas, but frequent close to the necrotic areas, suggesting that intratumoral factors may significantly affect ACD. Thus, it appears that dissociation between cell proliferation and apoptosis occurs in the hyperplastic stages of mammary carcinogenesis and that neoplastic transformation of mammary epithelial cells is associated with decreased ACD but not with increased cell proliferation.
Asunto(s)
Apoptosis , Carcinoma in Situ/patología , Carcinoma Ductal de Mama/patología , Transformación Celular Neoplásica/patología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Animales , Carcinógenos/toxicidad , Carcinoma in Situ/inducido químicamente , Carcinoma Ductal de Mama/inducido químicamente , División Celular , Transformación Celular Neoplásica/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Hiperplasia , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/inducido químicamente , Metilnitrosourea/toxicidad , Necrosis , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Ratas , Ratas Sprague-DawleyRESUMEN
In most previous chemoprevention studies on inhibition of mammary carcinogenesis, the formation of palpable tumors has been used as an end-point. Little is known about whether chemopreventive agents may similarly or selectively suppress hyperplastic and premalignant stages of the neoplastic process. In this study, we evaluated the effect of 4-(hydroxyphenyl)retinamide (4-HPR) on the development and progression of hyperplastic lesions and carcinoma in situ (CIS) in the N-methyl-N-nitrosourea (MNU) mammary carcinogenesis model in rats. 4-HPR was used as the chemopreventive agent because of its proven inhibitory effect on both the early and late phases of mammary carcinogenesis. Treatment with 4-HPR (2.0 mM/kg diet), beginning 2 days after MNU administration and administered continuously for 10 weeks, suppressed all mammary gland lesions (hyperplasia, CIS and invasive carcinoma) in 35% of animals. In the remaining 65%, 4-HPR allowed the development of hyperplastic lesions, alone or combined with CIS, and/or invasive carcinomas (CA). 4-HPR also increased by 2-fold the ratio between CIS and CA (0.75 per animal in control versus 1.5 in 4-HPR-treated animals), suggesting that it may also suppress the transition of CIS into CA. 4-HPR, when administered beginning 4 weeks after MNU administration [when hyperplastic and premalignant (CIS) lesions are present in the mammary gland], inhibited the frequency of terminal end bud hyperplasia (TEBH) and CA but did not significantly suppress ductal hyperplasia, ductal alveolar hyperplasia, alveolar hyperplasia and CIS. In these animals, 4-HPR induced partial disintegration of mostly peripheral areas of lesions, including carcinomas. Taken together, our data indicate that 4-HPR selectively suppresses the development and progression of hyperplastic lesions and CIS in TEBs. Furthermore, it appears that, in addition to mammary carcinomas, TEBH and CIS could also be used as end-point biomarkers in breast cancer chemoprevention studies.
Asunto(s)
Anticarcinógenos/uso terapéutico , Carcinoma in Situ/prevención & control , Fenretinida/uso terapéutico , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/prevención & control , Lesiones Precancerosas/prevención & control , Animales , Carcinógenos , Carcinoma in Situ/patología , Progresión de la Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Hiperplasia/inducido químicamente , Hiperplasia/patología , Hiperplasia/prevención & control , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Metilnitrosourea , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Ratas , Ratas Sprague-DawleyRESUMEN
The detection of telomerase activity has been proposed as a biomarker of breast cancer development and progression. In this study, we used cell proliferation and telomerase in MNU (N-methyl-N-nitrosourea)-induced mammary carcinomas as targets for assessing the response of tumor cells to 4-(hydroxyphenyl)retinamide (4-HPR), a known inhibitor of mammary carcinogenesis in animal models and premenopausal women. In mammary tumors of rats treated for 1, 2, 4 or 6 weeks with 4-HPR, we observed that telomerase activity decreased progressively with the extension of 4-HPR administration. A marked reduction in telomerase activity was already observed by 2 weeks after treatment and the lowest level was found at 6 weeks after initiation of 4-HPR treatment. The changes in telomerase activity were preceded and accompanied by a significant decrease in the percentage of proliferating cells as evaluated by 5-bromodeoxyuridine (BrdU)-labeling. However, when the values of telomerase activity in the individual tumors were compared with the percentage of proliferating cells, no significant correlation was found. These data suggest that the decreased telomerase activity in the animals treated with 4-HPR is not a simple consequence of the changes in cell proliferation, but a more complex phenomenon involving different cellular mechanisms and pathways. The time-dependent and consistent decrease of telomerase activity in the tumors treated with 4-HPR suggests that, in addition to the percentage of proliferating cells, telomerase activity could also be used as an endpoint in breast cancer chemotherapy studies.
Asunto(s)
Antineoplásicos/farmacología , Fenretinida/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Telomerasa/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Femenino , Fenretinida/uso terapéutico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratas , Ratas Sprague-Dawley , Telomerasa/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: The limitations of morphologic criteria alone in determining the prognosis for a patient with a particular intermediate-thickness primary melanoma have prompted efforts to identify other markers. METHODS: In this study, the authors analyzed expression of p53, beta1 integrin, and beta3 integrin in primary tumors from 111 patients with intermediate-thickness malignant melanoma. RESULTS: Eighty-nine (80%) had detectable p53 protein, 58 (52%) expressed beta1 integrin, and 71 (64%) expressed beta3 integrin. Patients with beta3 positive melanomas were more likely to die of their disease (32 of 71 patients, 45%) than those with beta3 negative tumors (3 of 40 patients, 8%) (P < 0.0001). The number of involved lymph nodes, Clark's level, beta1 integrin expression, thickness, and mitotic rate also had prognostic significance. beta3 integrin was associated with subsequent lung metastases and beta1 integrin with lymph node involvement. CONCLUSIONS: Integrin expression, along with histopathologic criteria, is a prognostic marker for intermediate-thickness malignant melanoma and may indicate the site of subsequent metastasis. These observations may have clinical utility and suggest areas for future investigation.
Asunto(s)
Antígenos CD/análisis , Biomarcadores de Tumor/análisis , Integrina beta1/análisis , Melanoma/química , Glicoproteínas de Membrana Plaquetaria/análisis , Neoplasias Cutáneas/química , Proteína p53 Supresora de Tumor/análisis , Humanos , Integrina beta3 , Melanoma/diagnóstico , Melanoma/secundario , Metástasis de la Neoplasia , Pronóstico , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patología , Análisis de SupervivenciaRESUMEN
Metastatic melanoma, compared with other cancers, appears to be unusual because of its low frequency of p53 mutations and prevalence of wild-type p53 protein in advanced malignancy. Here, we examined the effects of wild-type and mutated p53 (143 Val-Ala) on tumorigenic and metastatic potential of two human melanoma cell lines. The cell line UISO-MEL-4 contains wild-type p53 and is tumorigenic, whereas UISO-MEL-6 lacks p53 and produces lung and liver metastasis upon s.c. injection into athymic mice. Our study showed that UISO-MEL-4 stably transfected with wild-type p53 cDNA driven by cytomegalovirus promoter-enhancer sequences expressed high levels of p53 and p21 and formed s.c. tumours in vivo. Mutated p53 (143 Val-Ala) expression, on the other hand, inhibited tumour growth in 50% of cases and produced significantly slower growing non-metastatic tumours. Reduced tumour growth involved necrotic as well as apoptotic cell death. Inhibition of tumour growth was abrogated by the addition of Matrigel (15 mg ml(-1)). With UISO-MEL-6 cells, stably transfected with mutant p53, tumour growth was delayed and metastasis was inhibited. In soft agar colony formation assay, both wild-type and mutant p53 transfectants reduced anchorage-independent colony formation in vitro. These data suggest that mutated (143 Val-Ala) p53, which retains DNA binding and some of the transactivation functions of the wild-type p53 protein, suppresses tumorigenic and metastatic potentials of human melanoma cell lines in vivo.
Asunto(s)
Melanoma/patología , Melanoma/secundario , Mutación , Proteína p53 Supresora de Tumor/fisiología , Animales , Materiales Biocompatibles/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Colágeno/farmacología , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Combinación de Medicamentos , Expresión Génica , Genes p53 , Humanos , Laminina/farmacología , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Pulmonares/secundario , Melanoma/genética , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteoglicanos/farmacología , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Células Tumorales Cultivadas/patología , Adulto , Animales , Western Blotting , Neoplasias de la Mama/química , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/secundario , División Celular/efectos de los fármacos , Colágeno , Combinación de Medicamentos , Estradiol/farmacología , Femenino , Humanos , Laminina , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/análisis , Trasplante de Neoplasias/métodos , Proteoglicanos , Trasplante Heterólogo , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/trasplanteRESUMEN
Normal human breast epithelial cells (HBL100) are immortalized by endogenous SV40 genome and are not tumorigenic in nude mice if injected in the first 50 passages in culture. This cell line also depends on the expression of basic fibroblast growth factor (bFGF) for its viability in culture. The present study was designed to transform these cells with chemical carcinogens to establish malignant HBL100 cell lines in order to evaluate the eventual modulation in the expression of bFGF. In the first experiment, HBL100 cells were treated with 2 mu g/ml of 7, 12, dimethylbenz(a)anthracene (DMBA) for two 24 h periods. In a second experiment, HBL100 cells were transformed by exposing them to 20 mu g/ml of N-methyl-N-nitrosourea (MNU) twice a day for two days. Unlike the HBL100 parental cell line, the two newly derived cell lines (named respectively HBLTDMBA and HBLTMNU) were tumorigenic when injected in nude mice. The expression of bFGF, as measured by immunocytochemistry and Western blot analysis, was higher in the DMBA-transformed cell line than in the parental HBL100 and MNU-transformed lines. These results differentiate between transformation selectivity by two different carcinogens. The direct-acting carcinogen MNU, which induces point mutation, does not require enhanced expression of bFGF, whereas bFGF may be necessary for early events leading towards transformation by carcinogens requiring metabolism for their action, such as DMBA.
RESUMEN
BACKGROUND: Monoclonal Antibody (MAb) 29-13 reacts with the human sarcoma-associated antigen (SAA) p200. We report here engineering and primary characterization of a single chain antibody (scFV2913). MATERIALS AND METHODS: The scFV2913 recombinant gene, consisting of VH-linker-VK, was constructed with RT-PCR. This gene was cloned and expressed in E. coli. The renatured scFV2913 was used in the immunostaining study. RESULTS: Consistent with its parent MAb 29-13, purified and renatured scFV2913 showed affinity and specificity to the SAA p200 according to the immuno-histochemical staining study of 99 specimens of human sarcomas and other tissues. CONCLUSIONS: Due to its retained specificity and affinity, scFV2913 may be useful in immunodiagnosis and immunotherapy for sarcoma patients.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos/genética , Antígenos de Neoplasias/inmunología , Proteínas Recombinantes , Sarcoma/inmunología , Secuencia de Aminoácidos , Anticuerpos/inmunología , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Clonación Molecular , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Anticuerpos de Cadena ÚnicaRESUMEN
BACKGROUND: Experimental evidence suggests that integrins are key regulators of the development of melanoma metastases, influencing both the likelihood and site of metastases. Whereas effective treatment of cutaneous melanoma remains surgical, elective lymph node dissection (ELND) is controversial. The present study was designed to investigate the relationship between integrin expression by a given primary melanoma and occult regional lymph node metastases. MATERIALS AND METHODS: We studied beta 1 integrin expression, by quantitative immunohistochemistry using an image analyzer, in the primary melanomas of 90 ELND patients. RESULTS: beta 1 integrin was expressed in > or = 10% of the primary tumor in 92% of cases eith lymph node involvement versus 9% of node negative cases (p < 0.001). CONCLUSIONS: Our data demonstrate that quantitative immunohistochemistry for beta 1 integrin expression in primary melanomas can identify patients likely to have occult lymph node metastases. This suggests that beta 1 integrins play a role in the lymphatic dissemination of cutaneous melanoma.
Asunto(s)
Biomarcadores de Tumor/análisis , Integrina beta1/análisis , Melanoma/química , Neoplasias Cutáneas/química , Femenino , Humanos , Escisión del Ganglio Linfático , Metástasis Linfática , Masculino , Melanoma/secundarioRESUMEN
Previous studies have suggested that differential expression and/or activation of integrins facilitates metastatic progression in murine and human melanoma. While recent data show that the integrin alphavbeta3 is involved in tumor angiogenesis and that tumor growth may be abrogated by alphavbeta3 inhibitors in vitro, the clinical significance of beta3 integrin expression in human malignant melanoma is not known. To assess the prognostic value of beta3 integrin expression, we examined primary cutaneous melanomas from 160 patients followed for a mean of 98 months or until death. We quantified the percentage of tumor area stained with beta3 integrin Ab CD-61 using an image analyzer. beta3 integrin expression was detected in 107/160 primary melanomas (69%). beta3-integrin-positive (beta3+) tumors were thicker (mean 2.98 +/- 0.3 mm) than beta3-integrin-negative (beta3-) melanomas (mean 1.64 +/- 0.2 mm) (P = 0.002). Patients with beta3+ melanomas were more likely to relapse (57/107, 53%) and to die from disease (45/107, 42%) than those with beta3- tumors (6/53, 11%; and 4/53, 8%, respectively) (P < 0.001). Overall survival was greater for beta3- than for beta3+ patients (mean 102 +/- 9 vs 69 +/- 6 months) (P = 0.001). These data show that beta3 integrin expression in primary cutaneous melanoma predicts subsequent metastatic progression. Further study of beta3 integrins in the development of melanoma metastases may yield new therapeutic strategies, as well as prognostic information, for the treatment of this cancer.
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Antígenos CD/biosíntesis , Melanoma/patología , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Neoplasias Cutáneas/patología , Antígenos CD/análisis , Supervivencia sin Enfermedad , Humanos , Inmunohistoquímica , Integrina beta3 , Melanoma/inmunología , Melanoma/mortalidad , Análisis Multivariante , Metástasis de la Neoplasia , Glicoproteínas de Membrana Plaquetaria/análisis , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Tasa de Supervivencia , Factores de TiempoRESUMEN
BACKGROUND: Elective lymph node dissection for malignant melanoma is still controversial. Experimental studies suggest that differential expression, activation, or both of beta1 integrins facilitate melanoma metastases. However, the clinical significance of beta1 integrin expression in human melanoma is unclear. METHODS: We examined primary cutaneous melanomas from 76 patients undergoing elective lymph mode dissection. We quantified the percentage of tumor area stained by beta1 integrin antibody with an image analyzer. RESULTS: beta1 integrin was expressed in all 23 primary tumors from patients with pathologically positive lymph nodes (LNs) but in only 14 (26%) of 53 cases with pathologically negative nodes (p < 0.001). No patients with beta1 integrin-negative tumors had LN involvement, whereas 23 (62%) of 37 patients with beta1 integrin-positive tumors had LN metastases (p < 0.001). Furthermore, 21 (91%) of 23 cases with LN metastases but only 4 (8%) of 53 cases without had beta1 integrin staining of 10% or more of tumor area (p < 0.001). CONCLUSIONS: Our study is the first to show a correlation between expression of a molecular marker in the primary cutaneous melanoma and likelihood of regional LN metastases. beta1 immunostaining of 10% or more of tumor area reliably predicts patients most likely to harbor occult LN metastases and likely to benefit from ELND.
Asunto(s)
Biomarcadores de Tumor/análisis , Integrina beta1/análisis , Metástasis Linfática , Melanoma/patología , Proteínas de Neoplasias/análisis , Neoplasias Cutáneas/patología , Adulto , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Procesamiento de Imagen Asistido por Computador , Integrina beta1/biosíntesis , Integrina beta1/genética , Tablas de Vida , Escisión del Ganglio Linfático , Metástasis Linfática/diagnóstico , Masculino , Melanoma/química , Melanoma/mortalidad , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Neoplasias Cutáneas/química , Análisis de SupervivenciaRESUMEN
We established and characterized three human breast carcinoma cell lines (two with metastatic potential) by in vivo-in vitro propagation of tumor tissues. Tissues were processed in culture as i) direct explants (n = 185); ii) cell suspensions obtained after enzymic digestion (n = 29); or iii) explants of xenografts established in vivo (n = 18). Tissues processed as explants or cell suspensions generated no viable cell lines. However, three cell lines (MAXF-401, MAXF-583 and OHSTMAM-4) were established from xenografts. All three showed characteristic features of human breast tumor tissue. All were tumorigenic in mice and MAXF-401 and MAXF-583 showed frequent distant metastasis following s.c. transplantation. All three overexpressed EGFR, p53 and c-erbB2 proteins.
RESUMEN
BACKGROUND: Few studies exist that describe Merkel cell carcinoma (MCC) growth characteristics in vitro, in vivo, or both. OBJECTIVE: Our purpose was to evaluate the pathologic features of MCC implanted into athymic mice and to determine cytogenetic abnormalities in the established cell line. METHODS: Tumor tissues from a patient with MCC were grown in culture. Cultured cells were karyotyped and inoculated subcutaneously into athymic mice. Nude mouse tumors were re-implanted into other athymic mice. Tissues from the primary skin tumor and the nude mouse tumor were processed for light and electron microscopy and immunocytochemistry. RESULTS: The cell line showed a doubling time of 64.8 hours. Xenografts of 4 x 10(6) cells produced tumors in athymic mice with a doubling time of 16.1 days. The nude mouse tumors showed pathologic features similar to those of the primary skin tumor. Cytogenetic studies showed a t(1;17) (p36;q21) translocation in 100% of the cells. CONCLUSION: MCC implanted into athymic mice retained the pathologic features of the primary skin tumor and behaved aggressively. The t(1;17) (p36;q21) translocation may be a marker of an aggressive phenotype.