RESUMO
OBJECTIVE: Triple-negative breast cancer (TNBC) is an aggressive subtype with a poor prognosis. Minichromosome maintenance genes (MCM2-7) crucial for DNA replication are significant biomarkers for various tumor types; however, their roles in TNBC remain underexplored. MATERIALS AND METHODS: We utilized four TNBC-related GEO databases to examine MCM2-7 gene expression and predict its prognosis in TNBC, performing single-cell analysis and GSEA to discover MCM6's potential function. The Cancer Dependency Map gene effect scores and CCK8 assay were used to assess MCM6's impact on TNBC cell proliferation. The correlations between MCM6 expression, immune infiltrates, and immune cells were also analyzed. WGCNA and LASSO Cox regression built a risk score model predicting TNBC patient survival based on MCM6-related gene expression. RESULTS: MCM2-7 gene expression was higher in TNBC tissues compared to adjacent normal tissues. High MCM6 expression correlated with shorter TNBC patient survival time. GSEA and single-cell analysis revealed a relationship between elevated MCM6 expression and the cell cycle pathway. MCM6 knockdown inhibited TNBC cell proliferation. A risk model featuring MCM6, CDC23, and CCNB1 effectively predicts TNBC patient survival. CONCLUSIONS: MCM6 overexpression in TNBC links to a worse prognosis and reduced cell proliferation upon MCM6 knockdown. We developed a risk score model based on MCM6-related genes predicting TNBC patient prognosis, potentially assisting future treatment strategies.
Assuntos
Componente 6 do Complexo de Manutenção de Minicromossomo , Neoplasias de Mama Triplo Negativas , Humanos , Biomarcadores , Ciclo Celular , Proliferação de Células/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Prognóstico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Objective: To evaluate clinical outcomes of autologous and allogeneic peripheral blood stem cell transplantation (PBSCT) for aggressive peripheral T-cell lymphoma (PTCL). Methods: From June 2007 to June 2017, clinical data of PTCL patients who underwent PBSCT were assessed retrospectively. Results: Among 41 patients, 30 was male, 11 female, and median age was 38(13-57) years old. Seventeen patients with autologous PBSCT (auto-PBSCT) and 24 patients with allogeneic PBSCT (allo-PBSCT) were enrolled in this study. Eight patients (8/17, 47.1%) in auto-PBSCT group were ALK positive anaplastic large cell lymphoma (ALCL), 7 patients (7/24, 29.2%) with NK/T cell lymphoma and 9 patients (9/24, 37.5%) with PTCL-unspecified (PTCL-U) in allo-PBSCT group (P=0.035). There were 58.8% patients (10/17) in complete response (CR) status and 11.8% (2/17) in progression disease (PD) status before transplantation in auto-PBSCT group, and 8.3% (2/24) in CR status and 45.8% (11/24) in PD status before transplantation in allo-PBSCT group (P=0.026). The 2-years cumulative overall survival (OS) were (64.0±10.8)% and (53.5±9.7)% for auto-PBSCT and allo-PBSCT respectively (P=0.543). The 2-years cumulative disease-free survival (DFS) were (57.1±12.4)% and (53.5±10.6)% for auto-PBSCT and allo-PBSCT respectively (P=0.701). In patients with dead outcomes after PBSCT, 83.3% (5/6) of death cause was relapse in auto-PBSCT and 41.7% (5/12) of death cause was relapse in allo-PBSCT. Conclusion: Both auto-PBSCT and allo-PBSCT were effective for PTCL. Allo-PBSCT maybe was better than auto-PBSCT for high-risk PTCL with poor prognosis.
Assuntos
Linfoma de Células T Periférico , Transplante de Células-Tronco de Sangue Periférico , Adolescente , Adulto , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Linfoma de Células T Periférico/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Retrospectivos , Transplante Autólogo , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND AND AIMS: Dyslipidemia predicts higher risk of coronary events and stroke and might be associated with cerebral small vessel disease (SVD). Previous studies linking blood lipids and SVD have yielded inconsistent results, which may be attributable to sex differences in lipids metabolism. The aim of this study was to investigate the relationships between blood lipids and SVD in neurologically healthy men and women. METHODS AND RESULTS: Consecutive 817 people aged 50 years or more were enrolled and underwent magnetic resonance imaging scans to evaluate the periventricular white matter lesions (PVWMLs), deep white matter lesions (DWMLs) and silent brain infarction (SBI). Fasting total cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol, apolipoprotein A-1 (apoA-1) and apolipoprotein B were assessed. Multivariable logistic regression analyses were performed to determine the associations of blood lipids with PVWMLs, DWMLs and SBI. HDL-C (for PVWMLs: OR 0.36, 95% CI 0.19-0.71; for DWMLs: OR 0.35, 95% CI 0.20-0.63) and apoA-1 (for PVWMLs: OR 0.27, 95% CI 0.11-0.66; for DWMLs: OR 0.22, 95% CI 0.10-0.48) were inversely associated with the severity of PVWMLs and DWMLs in women but not in men after adjustment for age, hypertension, diabetes, current smoking, daily drinking, body mass index and uric acid. Additionally, no blood lipids were significantly associated with SBI. CONCLUSIONS: Our findings demonstrate that sex differences may exist in the associations between lipids and SVD. HDL-C and apoA-1 levels were inversely associated with the severity of PVWMLs and DWMLs in women.
Assuntos
Infarto Encefálico/sangue , Doenças de Pequenos Vasos Cerebrais/sangue , Dislipidemias/sangue , Leucoencefalopatias/sangue , Lipídeos/sangue , Idoso , Apolipoproteína A-I/sangue , Biomarcadores/sangue , Infarto Encefálico/diagnóstico por imagem , Infarto Encefálico/etiologia , Doenças de Pequenos Vasos Cerebrais/diagnóstico por imagem , Doenças de Pequenos Vasos Cerebrais/etiologia , HDL-Colesterol/sangue , Estudos Transversais , Dislipidemias/complicações , Dislipidemias/diagnóstico , Feminino , Humanos , Leucoencefalopatias/diagnóstico , Leucoencefalopatias/etiologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Índice de Gravidade de Doença , Fatores SexuaisRESUMO
Objective: To explore whether diabetes mellitus (DM) impairs functions of bone marrow-derived endothelial progenitor cells (BM-EPC) and circulating EPC. Methods: Diabetic model of rabbit was induced by Alloxan injection and the rabbits were then randomly divided into three groups: BM-EPC group, circulating EPC group, and DM group, with six rabbits in each group. Another 6 normal rabbits were enrolled as normal control group as well. 8 weeks later, BM-EPC and circulating EPC from diabetic and healthy rabbits were isolated and cultured. Colony number, proliferation, adhesion and tube formation function were detected. Exogenous diabetic BM-EPC and circulating EPC were analyzed for therapeutic efficacy in acute ischemia model of diabetic rabbits. Left ventricular (LV) function was assessed using Echocardiography. Capillary density and fibrosis area were evaluated by confocal laser scanning microscope (CLSM) and Masson-trichrome staining. The mRNA expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was analyzed using real-time quantitive PCR. Results: Colony number, proliferation, adhesion and tube formation function of diabetic circulating EPC were significantly reduced compared with healthy rabbits. DM impaired tube-forming ability of BM-EPC, but did not influence colony number, proliferation and adhesion function. Compared with circulating EPC and control group, BM-EPC group had fewer fibrosis area (6.98%±0.94% vs 13.03%±2.97% and 15.84%±4.74%, both P=0.001), higher capillary density [(792±87) vs (528±71) and (372±77) vessels/mm(2,) both P<0.001], higher mRNA expression of VEGF (6.25±2.33 vs 2.19±1.01 and 1.55±0.52, both P<0.001) and bFGF (6.38±2.65 vs 1.24±0.76 and 1.18±0.82, both P<0.001), higher left ventricular ejection fraction (LVEF) (61%±4% vs 47%±5% and 50%±10%, both P<0.05). Conclusions: DM not only impaired functions of circulating EPC, but also influenced tube formation function of BM-EPC. Auto transplantation of BM-EPC may rescue the ischemic myocardium by neovascularization and paracrine effect in diabetic rabbits.
Assuntos
Células Progenitoras Endoteliais , Aloxano , Animais , Medula Óssea , Diabetes Mellitus Experimental , Neovascularização Fisiológica , Coelhos , Fator A de Crescimento do Endotélio VascularRESUMO
Objective: To explore the efficacy of laparoscopic surgery in treatment of advanced mid-low rectal cancer following a long-term neoadjuvant chemoradiotherapy. Methods: Clinicopathologic and perioperative data were collected retrospectively from 74 patients with advanced mid-low rectal cancer, who received both neoadjuvant chemoradiotherapy and resections between January 2010 and January 2013 at Xinjiang tumor hospital. Routine follow-up was conducted. The safety and long-term survival of 36 patients who underwent laparoscopic resection were compared with those of 38 patients who received conventional resection. Results: The laparoscopic group had less amount of blood loss during surgery (50 ml vs 100 ml, P<0.05). The time needed for recovery of gastrointestinal function in the laparoscopic group was significantly shorter than that in the open surgery group (2.0 d vs 3.0 d, P<0.05). The rate of postoperative complication was 19.4% and 42.1% (P<0.05), respectively. In terms of the range of radical surgery and the numbers of dissected lymph nodes (8 and 10, P>0.05), no significant difference were found in the two groups. The operation duration and hospital stay in the laparoscopic group was longer than that in the open surgery group (240.0 min vs 231.5 min , P>0.05) (22.0 d vs 21.5 d , P>0.05), but no significant difference was found between the two groups. There were no significant difference in the incidence of 3 disease-free survival rate (53.0% vs 43.8%, P>0.05) and overall survival rate (70.0% vs 62.9%, P>0.05) between two groups. Conclusion: Laparoscopic surgery is a safe and feasible option for advanced mid-low rectal cancer patients who undergone the neoadjuvant chemoradiotherapy because of the similar rate of radical resection and satisfied long-term outcomes, which will have a better prospect in the future.
Assuntos
Quimiorradioterapia , Terapia Neoadjuvante , Neoplasias Retais , Intervalo Livre de Doença , Humanos , Laparoscopia , Tempo de Internação , Excisão de Linfonodo , Complicações Pós-Operatórias , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
We investigated the associations between Beclin-1 and microRNA-30a (miR-30a) expression and the severity and treatment response in colorectal cancer (CRC). Our sample size consisted of 139 CRC patients who were treated with surgery alone. Immunohistochemistry was used to investigate the expression and prognostic significance of Beclin-1 in CRC, while the weak expression of Beclin-1 in normal tissue was used as the basis for assessing tumors (control group). Real-time reverse transcription-polymerase chain reaction quantified miR-30a levels. The expression levels of Beclin-1 and miR-30a were associated with clinical variables and prognoses. Beclin-1 was expressed more highly in CRC tissues than in controls. This expression was related to gender (P = 0.023), histological grade (P = 0.006), M stage (P = 0.004), tumor node metastasis stage (P = 0.020), vascular invasion, and nodal involvement. Patients with higher Beclin-1 expression levels had higher survival rates (P = 0.08) than patients with lower Beclin-1 expression levels. Beclin-1 was a prognostic indicator (P < 0.05) in a multivariate analysis. Beclin-1 was overexpressed in CRC tissues and was correlated with lower levels of miR-30a (P < 0.05, r = -0. 4189). In conclusion, Beclin-1 was a good prognostic indicator in CRC and was correlated with survival rate. Beclin-1 is important in the growth and metastasis of CRC. Apoptosis in CRC might be due to the increased autophagy induced by decreased levels of miR-30a.
Assuntos
Proteína Beclina-1/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , MicroRNAs/genética , Apoptose , Autofagia , Proteína Beclina-1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de SobrevidaRESUMO
As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.
Assuntos
Animais , Humanos , Mamíferos/fisiologia , Feromônios/fisiologia , Comportamento Animal/fisiologia , Comportamento/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Mucosa Olfatória/fisiologia , Condutos Olfatórios/anatomia & histologia , Condutos Olfatórios/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Feromônios Humano/fisiologia , Olfato/fisiologiaRESUMO
As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.
Assuntos
Cobre/metabolismo , Metalotioneína/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Cobre/análise , Formazans , Células HeLa , Humanos , Metalotioneína/análise , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sais de Tetrazólio , Fatores de TempoRESUMO
BACKGROUND: Little is known about the occurrence of transfusion-related acute lung injury(TRALI) in Chinese paediatric patients. As such, a retrospective review of medical records from January 2008 to December 2011 was undertaken. OBJECTIVE: To determine the incidence of TRALI and its risk factors in children (age <14 years). STUDY DESIGN AND METHODS: All medical records of Sheng Jing Hospital from January 2008 to December 2011 were reviewed retrospectively using the hospital's record system. Paediatric surgical patients who had been diagnosed clinically with acute lung injury were included. Transfusion data were collected, together with risk factors such as sepsis and aspiration. RESULTS: In total, 1495 patients were involved in the study. Thirty-five cases were analysed further as they had acute lung injury, pulmonary oedema and respiratory distress. TRALI was confirmed in two of these cases. The average duration of transfusion was found to be significantly longer in patients with TRALI compared with controls, and the percentage of female donors was significantly higher for patients with TRALI. CONCLUSION: The incidence of TRALI was found to be lower than reported previously, but TRALI is under-recognised, under-reported and undertreated.
Assuntos
Lesão Pulmonar Aguda/epidemiologia , Procedimentos Cirúrgicos Operatórios , Reação Transfusional , Lesão Pulmonar Aguda/etiologia , Adolescente , Criança , Pré-Escolar , Feminino , Registros de Saúde Pessoal , Humanos , Incidência , Lactente , Masculino , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised. METHODS: Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-ß (TGF-ß) signalling derived from CAF-CM, which effect on breast cancer cells. RESULTS: The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-ß1 than TGF-ß2 and TGF-ß3, and activated the TGF-ß/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-ß1 signalling. CONCLUSION: Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-ß1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.
Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , Comunicação Parácrina/genética , Fator de Crescimento Transformador beta/metabolismo , Actinas/biossíntese , Actinas/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Quimiocina CXCL12/biossíntese , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Músculo Liso/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
OBJECTIVE: Aim of the study was to further evaluate the role of caspase 8 and death receptors (DR) in the TRAIL-induced apoptosis of neuroblastoma (NB) cell lines. METHODS: Caspase 8 mRNA expression was monitored by RT-PCR. Caspase 8, DR5 and DR4 protein expression were monitored by Western blot analysis. The effects of IFNγ, TRAIL, IFNγ+TRAIL, caspase 8 inhibitor+TRAIL and IFNγ+chemotherapeutic agents+TRAIL on the growth and apoptosis of NB cells were detected with MTT and flow cytometry. The relative caspase 8 activity was measured with colorimetric assay. RESULTS: Caspase 8 expression was induced by IFNγ in the NB cell line SKNDZ. TRAIL alone did not induce apoptosis compared with controls but a combination of IFNγ+TRAIL and IFNγ+chemotherapeutic agents+TRAIL significantly induced cell apoptosis in SY5Y cells. Etoposide and doxorubicin induced DR5 but not DR4 in NB cell lines. SKNDZ cells expressing caspase 8 after treatment with IFNγ were still resistant to TRAIL but sensitive to TRAIL after the induction of DR5. CONCLUSIONS: Sensitization of NB cells to TRAIL may be mediated by the upregulation of caspase 8 with IFNγ and DR5 with chemotherapeutic agents. This suggests that caspase 8 and death receptors play a very important role in TRAIL-induced apoptosis of NB cells and a combination of IFNγ, TRAIL and chemotherapeutic agents may be a new and interesting anticancer treatment strategy for NB.
Assuntos
Antineoplásicos/farmacologia , Caspase 8/biossíntese , Interferon gama/farmacologia , Neuroblastoma/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Humanos , RNA Mensageiro , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Regulação para CimaRESUMO
The object of this work was to measure the levels of monocyte-derived dendritic cell precursors (pDC1) and plasmacytoid dendritic cell precursors (pDC2) in peripheral blood mononuclear cells (PBMC) of patients with thymomatous MG and to assess the ratio of pDC1/pDC2 in these patients. Three-colour monoclonal antibody labelling technology was used to detect the cell counts and ratio of pDC1 and pDC2 in PBMCs of 18 patients with myasthenia gravis (MG), nine thymomatous patients without MG, and 15 normal controls. The number of pDC and pDC subsets in peripheral blood of patients with thymomatous generalized MG was significantly lower than that in thymomatous controls before and after the treatment. After the treatment, patients with both generalized MG and ocular MG had significantly lower number of pDC compared with thymomatous controls. We found no significant differences in pDC1/pDC2 ratio among groups. Before the extended thymomatous treatment, the number of pDC in patients with generalized MG was significantly lower than that in patients with ocular MG (P < 0.05). The counts of peripheral blood pDC and pDC subsets of patients with thymomatous MG were significantly lower 1 week after extended thymectomy. The counts of pDC and pDC subsets decreased in generalized thymomatous MG, and the patients with generalized MG had lower pDC counts than the patients with ocular MG before the treatment. Treatment resulted in decreased counts of pDC and pDC subsets in thymomatous MG. We suggest that the level of peripheral blood pDC can be used as a marker to define the progress of the disease.
Assuntos
Células Dendríticas/imunologia , Subpopulações de Linfócitos/imunologia , Miastenia Gravis/imunologia , Fatores Etários , Idoso , Contagem de Células Sanguíneas , Estudos de Coortes , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Miastenia Gravis/cirurgia , Estatísticas não Paramétricas , TimectomiaRESUMO
The transcription factor nuclear factor (NF)-kappaB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-kappaB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-kappaB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-kappaB1 (p50) knockout mice were used to study the role of NF-kappaB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-kappaB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-kappaB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-alpha and nerve growth factor-alpha genes at elevated levels, with in situ hybridization localizing some of the TNF-alpha expression to epithelial cells. TNF-alpha is NF-kappaB regulated, and its upregulation in NF-kappaB1 knockouts may result from an alleviation of p50-p50 repression. NF-kappaB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.
Assuntos
Colo/anatomia & histologia , Células Epiteliais/fisiologia , Mucosa Intestinal/fisiologia , NF-kappa B/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that can influence cell proliferation, apoptosis, and inflammatory responses. Since these events can contribute to carcinogenesis, we examined the expression of NF-kappaB inhibitory proteins (IkappaBs) in normal and transformed colonic epithelial cells. Immunohistochemical analysis of the mouse colon revealed a high level of IkappaBbeta expression in epithelial cells relative to the rest of the tissue, whereas IkappaBalpha was found primarily in cells of the lamina propria. Mouse colon tumors showed a similar cell-specific staining pattern. Immunoblot analysis of IkappaBbeta from mouse colonocytes and the human HT-29 colon cancer cell line indicated that most of the IkappaBbeta in these cells was similar to the C-terminal-truncated IkappaBbeta2 isoform. Cell fractionation studies were consistent with IkappaBbeta being a major regulator of p65-p50 NF-kappaB complexes in HT-29 cells. Interestingly, two larger proteins specifically recognized by IkappaBbeta antibodies (p106 and p112) were found in HT-29 cells and in colon tissue of carcinogen-exposed mice. The p106 and p112 proteins bound to NF-kappaB, and their levels changed during the transient interleukin-1beta activation of NF-kappaB in HT-29 cells. Evidence was obtained indicating that p106 and p112 are stably ubiquitinated forms of IkappaBbeta. We propose that deficiencies in the proteasomal degradation of IkappaBbeta lead to p106 and p112 accumulation, which in turn alter NF-kappaB regulation in colon cancer cells.
Assuntos
Linhagem Celular Transformada , Colo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Animais , Células Epiteliais/metabolismo , Células HT29 , Humanos , CamundongosRESUMO
Alterations in transforming growth factor beta1 (TGF-beta1) and its type II receptor (TbetaR-II) have been implicated in the pathogenesis of a variety of human cancers and animal tumor models. We postulated that TGF-beta1 and TbetaR-II alterations may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen, azoxymethane (AOM). In the present study, normal colon tissues and AOM-induced colon tumors from SWR/J mice were analyzed for mutational changes in the TbetaR-II gene, and the expression and localization of TGF-beta1 and TbetaR-II were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemisty. Direct DNA sequencing of the coding sequence of the TbetaR-II gene revealed no mutational changes in tumors induced by AOM when compared with the sequence from normal colon tissue. However, TGF-beta1 and TbetaR-II mRNA levels in tumor samples were increased 1.8-fold (p<0.01) and 1.3-fold (p<0.01), respectively, when compared with control mouse colon tissue. The results of immunohistochemical analysis of TGF-beta1 and TbetaR-II were correlated with mRNA expression data. An increase in staining intensity of both TGF-beta and TbetaR-II were observed in colon tumors. These findings suggest that alterations in the expression of TGF-beta1 and TbetaR-II may be involved in the pathogenesis of colon tumors induced by AOM in mice.
Assuntos
Adenocarcinoma/metabolismo , Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Substituição de Aminoácidos , Animais , Códon/genética , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Masculino , Camundongos , Proteínas de Neoplasias/genética , Mutação Puntual , Proteínas Serina-Treonina Quinases , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
Alterations in the p16(INK4a) gene have been implicated in the pathogenesis of different human cancers and animal tumors. We postulated that alterations in the p16(INK4a) gene may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen azoxymethane (AOM). In the present study, p16(INK4a) deletion status and its expression were examined in an AOM-induced mouse colon tumor model. Polymerase chain reaction-based deletion analysis of p16(INK4a) exon 2 showed no deletions in the colon tumors. The expression and localization of p16(INK4a) and its gene product were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses, respectively. The p16(INK4a) mRNA levels were low, and in some cases undetectable, in control colon tissue. However, colon tumors exhibited an eightfold increase in p16(INK4a) mRNA level when compared with control colon tissue (P < 0.01). Whereas control colon epithelium was uniformly negative for p16(INK4a) immunoreactivity, p16(INK4a)-immunoreactive cells were markedly increased in preneoplastic lesions and adenomas isolated from AOM-treated mice. To further examine the p16(INK4a) regulatory pathway, the retinoblastoma tumor-suppressor protein (Rb) was also examined immunohistochemically in these tissues. A heterogeneous Rb immunostaining was observed in preneoplastic lesions and adenomas. Immunohistochemical analysis also showed a reciprocal relationship between p16(INK4a) and Rb protein expression. These findings suggest that alterations in the p16(INK4a)/Rb pathway may play an important role in AOM-induced mouse colon tumorigenesis. Mol. Carcinog. 28:139-147, 2000.
Assuntos
Adenocarcinoma/genética , Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias do Colo/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes p16/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/induzido quimicamente , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Azoximetano/farmacologia , Carcinógenos/farmacologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Genes do Retinoblastoma/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína do Retinoblastoma/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.
Assuntos
Azoximetano , Carcinógenos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Animais , Suscetibilidade a Doenças , Resistência a Medicamentos , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos AKR , Especificidade da EspécieRESUMO
A differential susceptibility phenotype to the organotropic colon carcinogen azoxymethane (AOM) has been described in mice. The following studies were undertaken to test the hypothesis that intraspecific susceptibility can be accounted for by the specific complement of genetic alterations acquired by precancerous colon lesions referred to as aberrant crypt foci (ACF). As an initial approach to this question, mutations in codons 12 and 13 of the Ki-ras proto-oncogene were assessed in ACF, normal-appearing AOM-treated colonic epithelium, and tumors from A/J and SWR/J (susceptible) as well as AKR/J (resistant) mice. Four-week-old male mice were injected intraperitonealy, with AOM once a week for a total of 6 wk and killed 4 and 24 wk after the last injection. DNA was isolated from microdissected tissue, and polymerase chain reaction (PCR)-amplified products of Ki-ras exon 1 (codons 12 and 13) were directly sequenced from microdissected tissues. At 4 wk after AOM exposure, there was no significant difference in the frequency of Ki-ras activation (20-33%) between the three strains. Ki-ras mRNA expression was also evaluated by reverse transcription (RT)-PCR analysis and was comparably reduced (40-50%) in all three strains at the 4 wk time point. However, Ki-ras expression returned to normal by 24 wk after treatment. Finally, to gain further insight into the molecular pathogenesis underlying this experimental tumor model, analysis of the adenomatous polyposis coli (APC) protein within the colonic epithelium was undertaken by using an immunohistochemical approach. Although the APC protein was lost to a varying extent in tumors from A/J and SWR/J mice, the full-length form of the protein was still present in precancerous ACF isolated from each of the three strains, regardless of the degree of dysplasia of the lesion. A further molecular genetic analyses of ACF will be required to gain a more complete understanding of the molecular basis of tumor susceptibility phenotype in this murine model.
Assuntos
Azoximetano/farmacologia , Colo/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes ras , Animais , Sequência de Bases , Colo/metabolismo , Primers do DNA , Éxons , Imuno-Histoquímica , Masculino , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The murine non-pancreatic secretory phospholipase A(2) (sPLA(2)) has been proposed as a tumor modifier of multiple intestinal neoplasia (Min). A genetic polymorphism in the mouse gene that causes a disruption in exon 3 results in loss of functional protein. Mouse strains with a disrupted sPLA(2) gene are susceptible to the Min phenotype and develop numerous intestinal polyps, whereas mice with normal sPLA(2) develop only a limited number of polyps. The following study was undertaken to test the hypothesis that sPLA(2) plays an equivalent role in murine susceptibility to the colon carcinogen azoxymethane (AOM). sPLA(2) status was confirmed by sequencing in mice that are highly susceptible (A/J), susceptible (SWR/J) and resistant (AKR/J) to AOM-induced tumorigenesis. Constitutive expression of sPLA(2) mRNA was compared in small intestine and colon of untreated mice using semi-quantitative RT-PCR. Whereas mRNA expression was nearly absent in A/J mice, AKR/J mice exhibited extensive expression throughout the intestine. Despite the wild-type sPLA(2) gene, colonic mRNA expression in SWR/J mice was significantly lower relative to AKR/J. Immunohistochemical analysis of sPLA(2) protein confirmed the mRNA data. The effect of AOM on colonic sPLA(2) expression was also examined. Twenty-four weeks after the last of six weekly injections of AOM (10 mg/kg i.p.), RT-PCR analysis of distal colons revealed a significant increase in mRNA in normal-appearing epithelium and tumor tissue from AOM-treated mice relative to controls. However, there was no corresponding increase in protein expression in A/J mice. The absence of sPLA(2) expression within control colons of tumor-susceptible A/J mice together with low expression in SWR/J colons is consistent with its potential role as an intestinal tumor modifier, but the carcinogen-induced increase in expression raises doubts as to the significance of sPLA(2) in inhibiting carcinogenesis.