RESUMO
BACKGROUND: Low-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1. METHODS: We transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1. RESULTS: Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB activity and reduced p53 induction. Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression. CONCLUSIONS: The co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions.
Assuntos
Coinfecção/patologia , Infecções por Vírus Epstein-Barr/patologia , Neoplasias Bucais/genética , Infecções por Papillomavirus/patologia , Lesões Pré-Cancerosas/patologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Coinfecção/genética , Coinfecção/virologia , Dano ao DNA , Reparo do DNA , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Feminino , Fibroblastos , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno/genética , Papillomavirus Humano 11/patogenicidade , Papillomavirus Humano 6/metabolismo , Humanos , Camundongos , Mucosa Bucal/patologia , Mucosa Bucal/virologia , Neoplasias Bucais/patologia , Neoplasias Bucais/virologia , Mutação , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/virologia , Cultura Primária de Células , Proteínas da Matriz Viral/metabolismoRESUMO
BACKGROUND: Our aim was to investigate the disease-free survival in patients with tongue squamous cell carcinoma receiving metronomic neoadjuvant chemotherapy with 5-fluorouracil prodrugs (UFT or S-1) plus bleomycin compared with those who had up-front surgery retrospectively. METHODS: In this retrospective study, 108 patients with stages I to II tongue squamous cell carcinoma who had undergone surgery were divided into the "surgery group" or "neoadjuvant chemotherapy group." RESULTS: A total of 41 patients received up-front surgery; 67 received metronomic neoadjuvant chemotherapy with UFT plus bleomycin (39) or S-1 plus bleomycin (28). The rate of disease-free survival was the primary outcome measure. Neoadjuvant 5-fluorouracil prodrugs did not correlate higher with improved disease-free survival than up-front surgery (72 and 54%, respectively; hazard ratio for recurrence or death, 0.54; 95% confidence interval [CI], 0.28 to 1.03; P = 0.06). Patients who received S-1 were more likely than those who received UFT to have pathological complete response (46% vs. 15%; P = 0.007). Neoadjuvant S-1 significantly improved disease-free survival as compared with up-front surgery (79% vs. 54%; hazard ratio, 0.41; 95% CI, 0.15 to 0.98; P = 0.04). However, neoadjuvant UFT did not improve disease-free survival as compared with up-front surgery (67% vs. 54%, respectively; hazard ratio, 0.66; 95% CI, 0.31 to 1.33; P = 0.24). CONCLUSIONS: Neoadjuvant S-1 chemotherapy, as compared with up-front surgery, significantly improved disease-free survival among patients with tongue squamous cell carcinoma. CLINICAL RELEVANCE: A choice of drugs before neoadjuvant metronomic chemotherapy is needed.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/cirurgia , Terapia Neoadjuvante , Neoplasias da Língua/tratamento farmacológico , Neoplasias da Língua/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica , Bleomicina/uso terapêutico , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Fluoruracila/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Pró-Fármacos/uso terapêutico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Although cell therapy using adipose-derived mesenchymal stem cells (AdMSCs) regulates immunity, the degree to which cell quality and function are affected by differences in immunodeficiency of donors is unknown. We used liquid chromatography tandem-mass spectrometry (LC MS/MS) to identify the proteins expressed by mouse AdMSCs (mAsMSCs) isolated from normal (C57BL/6) mice and mice with severe combined immunodeficiency (SCID). The protein expression profiles of each strain were 98%-100% identical, indicating that the expression levels of major proteins potentially associated with the therapeutic effects of mAdMSCs were highly similar. Further, comparable levels of cell surface markers (CD44, CD90.2) were detected using flow cytometry or LC MS/MS. MYH9, ACTN1, CANX, GPI, TPM1, EPRS, ITGB1, ANXA3, CNN2, MAPK1, PSME2, CTPS1, OTUB1, PSMB6, HMGB1, RPS19, SEC61A1, CTNNB1, GLO1, RPL22, PSMA2, SYNCRIP, PRDX3, SAMHD1, TCAF2, MAPK3, RPS24, and MYO1E, which are associated with immunity, were expressed at higher levels by the SCID mAdMSCs compared with the C57BL/6 mAdMSCs. In contrast, ANXA9, PCBP2, LGALS3, PPP1R14B, and PSMA6, which are also associated with immunity, were more highly expressed by C57BL/6 mAdMSCs than SCID mAdMSCs. These findings implicate these two sets of proteins in the pathogenesis and maintenance of immunodeficiency.
Assuntos
Tecido Adiposo/citologia , Biomarcadores , Expressão Gênica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Diferenciação Celular , Separação Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Medicina RegenerativaRESUMO
Human adipose-derived mesenchymal stem cells (hADSCs) are representative cell sources for cell therapy. Classically, Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) has been used as culture medium for hADSCs. A chemically defined medium (CDM) containing no heterologous animal components has recently been used to produce therapeutic hADSCs. However, how the culture environment using a medium without FBS affects the protein expression of hADSC is unclear. We subjected hADSCs cultured in CDM and DMEM (10% FBS) to a protein expression analysis by tandem mass spectrometry liquid chromatography and noted 98.2% agreement in the proteins expressed by the CDM and DMEM groups. We classified 761 proteins expressed in both groups by their function in a gene ontology analysis. Thirty-one groups of proteins were classified as growth-related proteins in the CDM and DMEM groups, 16 were classified as antioxidant activity-related, 147 were classified as immune system process-related, 557 were involved in biological regulation, 493 were classified as metabolic process-related, and 407 were classified as related to stimulus responses. These results show that the trend in the expression of major proteins related to the therapeutic effect of hADSCs correlated strongly in both groups.
Assuntos
Cromatografia Líquida/métodos , Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Tecido Adiposo/citologia , Animais , Bovinos , Técnicas de Cultura de Células , Células Cultivadas , Análise por Conglomerados , Meios de Cultura/química , Meios de Cultura/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteoma/classificação , Soro/químicaRESUMO
Adipose-derived mesenchymal stem cells (ADSCs) have become a common cell source for cell transplantation therapy. Clinical studies have used ADSCs to develop treatments for tissue fibrosis, such as liver cirrhosis and pulmonary fibroma. The need to examine and compare basic research data using clinical research data derived from mice and humans is expected to increase in the future. Here, to better characterize the cells, the protein components expressed by human ADSCs used for treatment, and mouse ADSCs used for research, were comprehensively analyzed by liquid chromatography with tandem mass spectrometry. We found that 92% (401 type proteins) of the proteins expressed by ADSCs in humans and mice were consistent. When classified by the protein functions in a gene ontology analysis, the items that differed by >5% between human and mouse ADSCs were "biological adhesion, locomotion" in biological processes, "plasma membrane" in cellular components, and "antioxidant activity, molecular transducer activity" in molecular functions. Most of the listed proteins were sensitive to cell isolation processes. These results show that the proteins expressed by human and murine ADSCs showed a high degree of correlation.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/metabolismo , Proteoma , Proteômica , Animais , Biomarcadores , Diferenciação Celular , Células Cultivadas , Cromatografia Líquida , Biologia Computacional , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Pessoa de Meia-Idade , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
Human angiosarcoma is a rare malignant vascular tumor associated with extremely poor clinical outcome and generally arising in skin of the head and neck region. However, little is known about the molecular pathogeneses and useful immunohistochemical markers of angiosarcoma. To investigate the mechanisms of angiosarcoma progression, we collected 85 cases of human angiosarcoma specimens with clinical records and analyzed ISO-HAS-B patient-derived angiosarcoma cells. As control subjects, 54 cases of hemangioma and 34 of pyogenic granuloma were collected. Remarkably, consistent with our recent observations regarding the involvement of survivin expression following Hippo pathway inactivation in the neoplastic proliferation of murine hemangioendothelioma cells and human infantile hemangioma, nuclear survivin expression was observed in all cases of angiosarcoma but not in hemangiomas and pyogenic granulomas, and the Hippo pathway was inactivated in 90.3% of yes-associated protein (YAP) -positive angiosarcoma cases. However, survivin expression modes and YAP localization (Hippo pathway activation modes) were not correlated with survival. In addition, we confirmed that survivin small interference RNA (siRNA) transfection and YM155, an anti-survivin drug, elicited decreased nuclear survivin expression and cell proliferation in ISO-HAS-B cells which expressed survivin consistently. Conclusively, these findings support the importance of survivin as a good marker and critical regulator of cellular proliferation for human angiosarcoma and YM155 as a potential therapeutic agent.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Biomarcadores Tumorais/genética , Hemangiossarcoma/genética , Proteínas Inibidoras de Apoptose/genética , Fosfoproteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemangiossarcoma/patologia , Via de Sinalização Hippo , Humanos , Imidazóis , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Naftoquinonas , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Survivina , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
OBJECTIVE: To identify useful cytological findings for detecting premalignant lesions in postmenopausal women, cervicovaginal smear samples were analyzed and compared between women with or without premalignant lesions based on endocrine indices and presence of parakeratosis (PK). METHODS: The cervicovaginal smear samples of postmenopausal women with premalignant lesions (n = 94) and those who were without (n = 344), who were diagnosed between 2012 and 2014 were retrieved and analyzed. Women cytologically diagnosed with malignancy or those with suspicion of malignancy were excluded from this study. Cytological endocrine indices, such as the maturation index (MI) and eosinophilic index (EI) and the prevalence of PK were compared between the groups and analyzed using the 2 × 2 χ2 test. The association of endocrine indices combined with the presence of PK and histological findings was also evaluated. RESULTS: Postmenopausal women with premalignant lesions had higher endocrine indices (EI of ≥11%; 65% vs. 43%, P < 0.01, f = 0.18) and a higher prevalence of PK positivity (PK ≥ 1; 46% vs. 7%, P < 0.01, f = 0.44) than those without lesions. Further analysis indicated that the combination of high EI and the presence of PK in postmenopausal women with cytological premalignant cases was highly associated with histological squamous intraepithelial lesions (SIL) (86% in women with premalignant lesions vs. 53% in those without; P = 0.01, f = 0.34). CONCLUSION: Our research demonstrated that high EI and PK positivity were correlated with SIL in postmenopausal women. These cytological findings could provide potential diagnostic clues for detecting dysplasia.
Assuntos
Paraceratose , Lesões Pré-Cancerosas , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Feminino , Humanos , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/epidemiologia , Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal , Teste de Papanicolaou , Pós-Menopausa , Lesões Pré-Cancerosas/epidemiologiaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) Tax affects cellular genomic stability and senescence. As yet, the mechanism(s) for these events caused by Tax is incompletely understood. Here, we show that Tax expression in primary human cells induces reactive oxygen species (ROS), which elicits DNA damage and the expression of senescence marker. Treatment with a ROS scavenger or knockdown of Tax expression by small interfering RNA (siRNA) abrogated Tax-induced DNA damage and the expression of senescence marker. Our data suggest that ROS induction explains Tax-induced cellular DNA damage and cellular senescence.
Assuntos
Dano ao DNA , Produtos do Gene tax/fisiologia , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Sulfotransferases/biossíntese , Células Cultivadas , Senescência Celular , Técnicas de Silenciamento de Genes , Produtos do Gene tax/antagonistas & inibidores , Instabilidade Genômica , HumanosRESUMO
The insulin promoter is regulated by ubiquitous as well as pancreatic ß-cell-specific transcription factors. In the insulin promoter, GG2-GG1/A2-C1 (bases - 149 to - 116 in the human insulin promoter) play important roles in regulating ß-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2-GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2-GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR-Cas9 technology. The mice with homozygous mutations in the GG2-GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2-GG1/A2 element in mice is important for Ins transcription in vivo.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Regiões Promotoras Genéticas , Animais , Sistemas CRISPR-Cas , Feminino , Teste de Tolerância a Glucose , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Pâncreas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Well-differentiated head and neck squamous cell carcinoma (HNSCC), accounts for approximately 10% of all HNSCCs and, while these cases are associated with good prognosis after surgery, these are resistant to chemotherapy. Here we designed a retrospective study to evaluate the effects of histological differentiation on tongue squamous cell carcinoma (TSCC) patients undergoing surgery or metronomic neoadjuvant chemotherapy. The metronomic neoadjuvant chemotherapy significantly improved overall survival of patients with poorly or moderately differentiated tumour, but not those with well-differentiated tumour. Analysis of the Cancer Genome Atlas (TCGA) showed that FAT1 mutations were significantly enriched in more differentiated HNSCC while ASPM mutations were significantly enriched among the poorly differentiated HNSCC. Interestingly, Wnt/ß-catenin pathway was activated in well-differentiated HNSCC. Active ß-catenin is translocated to the nucleus in the well-differentiated oral squamous cell carcinoma cell lines. Wnt inhibitor, Wnt974, were synergistic with methotrexate in killing well-differentiated oral squamous cell carcinoma (OSCC) cell lines. TCGA data analyses reveal a signature in patients with well-differentiated HNSCC who have no benefits from metronomic neoadjuvant chemotherapy, suggesting that there might be novel nosology and therapeutic candidates for improving HNSCC patient survival. Well-differentiated OSCC is synergistically killed by combination chemotherapy with Wnt inhibitor, making it promising therapeutic candidates.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Neoplasias da Língua/tratamento farmacológico , Administração Metronômica , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Terapia Neoadjuvante , Pirazinas/administração & dosagem , Piridinas/administração & dosagem , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Taxa de Sobrevida , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Adulto JovemRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T-cell leukemia (ATL). The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. ATL is a highly virulent cancer that is resistant to chemotherapeutic treatments. To understand this disease better, it is important to comprehend how HTLV-1 promotes cellular growth and survival. Tax activation of NF-kappaB is important for the proliferation and transformation of virus-infected cells. We show here that prolyl isomerase Pin1 is over expressed in HTLV-1 cell lines; Pin1 binds Tax and regulates Tax-induced NF-kappaB activation.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , NF-kappa B/metabolismo , Peptidilprolil Isomerase/metabolismo , Linhagem Celular , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Ligação ProteicaRESUMO
Genome editing technologies such as CRISPR-Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR-Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs ß cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, ß cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR-Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo.
Assuntos
Diabetes Mellitus Experimental/genética , Insulina/genética , Mutação , Regiões Promotoras Genéticas , Animais , Sistemas CRISPR-Cas , Diabetes Mellitus Experimental/patologia , Feminino , Vetores Genéticos , Insulina/metabolismo , Células Secretoras de Insulina , Masculino , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Oncoprotein E6 of high-risk human papillomavirus (HPV) plays a critical role in inducing cell immortalization and malignancy. E6 downregulates caspase-dependent pathway through the degradation of p53. However, the effect of HPV E6 on other pathways is still under investigation. In the present study, we found that HPV E6 directly binds to all three forms (precursor, mature, and apoptotic) of apoptosis-inducing factor (AIF) and co-localizes with apoptotic AIF. This binding induced MG132-sensitive reduction of AIF expression in the presence of E6 derived from HPV16 (16E6), a cancer-causing type of HPV. Conversely, E6 derived from a non-cancer-causing type of HPV, HPV6 (6E6), did not reduce the levels of AIF despite its interaction with AIF. Flow cytometric analysis revealed that 16E6, but not 6E6, suppressed apoptotic AIF-induced chromatin degradation (an indicator of caspase-independent apoptosis) and staurosporine (STS, a protein kinase inhibitor)-induced apoptosis. AIF knockdown reduced STS-induced apoptosis in both of 16E6-expressing and 6E6-expressing cells; however, the reduction in 16E6-expressing cells was lower than that in 6E6-expressing cells. These findings indicate that 16E6, but not 6E6, blocks AIF-mediated apoptosis, and that AIF may represent a novel therapeutic target for HPV-induced cervical cancer.
Assuntos
Fator de Indução de Apoptose/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Apoptose , Cromatina/metabolismo , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Adipose-derived mesenchymal stem cells (MSC-ATs) are representative cell sources for cell therapy. However, how cell stress resulting from passage influences the MSC-AT protein expression has been unclear. In this study, a protein expression analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using mouse primary cultured cells (P0) and cells passaged three times (P3) as samples. A total of 256 proteins were classified as cellular process-related proteins, while 179 were classified as metabolic process-related proteins in P0. These were considered to be adaptive responses of the cells to an in vitro environment. However, seven proteins of growth were identified (Csf1, App, Adam15, Alcam, Tbl1xr1, Ninj1, and Sbds) in P0. In addition, four proteins of antioxidant activity were also identified (Srxn1, Txndc17, Fam213b, and Apoe) in P0. We identified 1139 proteins expressed in both P0 and P3 cells that had their expression decreased to 69.4% in P3 cells compared with P0 cells, but 1139 proteins are very likely proteins that are derived from MSC-AT. The function of MSC-ATs was maintained after three passages. However, the LC-MS/MS analysis data showed that the protein expression was degraded after three passages. MSC-ATs retained about 70% of their protein expression ability in P3 cells.
RESUMO
We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100-120 (passages 15-20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the OCT4 and NANOG promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells.
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Kaposi's sarcoma-associated herpesvirus (KSHV) causes both AIDS-related Kaposi's sarcoma (KS) and classic KS, but their clinical presentations are different, and respective mechanisms remain to be elucidated. The KSHV K1 gene is reportedly involved in tumorigenesis through the immunoreceptor tyrosine-based activation motif (ITAM). Since we found the sequence variations in the K1 gene of KSHV isolated from AIDS-related KS and classic KS, we hypothesized that the transformation activity of the K1 gene contributes to the different clinical presentations. To evaluate our hypothesis, we compared the transformation activities of the K1 gene between AIDS-related KS and classic KS. We also analyzed ITAM activities and the downstream AKT and NF-κB. We found that the transformation activity of AIDS-related K1 was greater than that of classic K1, and that AIDS-related K1 induced higher ITAM activity than classic K1, causing more potent Akt and NF-κB activities. K1 downregulation by siRNA in AIDS-related K1 expressing cells induced a loss of transformation properties and decreased both Akt and NF-κB activities, suggesting a correlation between the transformation activity of K1 and ITAM signaling. Our study indicates that the increased transformation activity of AIDS-related K1 is associated with its clinical aggressiveness, whereas the weak transformation activity of classic type K1 is associated with a mild clinical presentation and spontaneous regression. The mechanism of spontaneous regression of classic KS may provide new therapeutic strategy to cancer.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno/genética , Sarcoma de Kaposi/genética , Neoplasias Cutâneas/genética , Proteínas Virais/genética , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/patologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HeLa , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/patogenicidade , Humanos , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Remissão Espontânea , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Índice de Gravidade de Doença , Transdução de Sinais , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Transformação Genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Alkylating reagent chemotherapy for human cancers is not curative, and relapse occurs due to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD cells after chemotherapy, a phenomenon referred to as intrinsic resistance, depends on reactive oxygen species. Well-differentiated regions of the tumor are intrinsically resistant to chemotherapy. Receptor tyrosine kinase erythropoietin-producing human hepatocellular receptor A4 (EphA4) protein is highly expressed in the well-differentiated tumor-derived cervical cancer cell line Caski, but not in poorly differentiated tumor-derived cervical cancer cell lines such as HeLa or SiHa. Here, we report that reactive oxygen species produced by cisplatin exposure induce tyrosine phosphorylation of EphA4. After observing that EphA4 is activated by cisplatin, we rationalized a combination chemotherapy that induces well-differentiated cervical cancer death. Pharmacological inhibition of EphA4 increased cisplatin-induced cell death in Caski cells. Moreover, we observed increased expression levels of the senescence marker cyclin-dependent kinase inhibitor 2A (p16) in the absence of EphA4 kinase function after stimulation of Caski cells with cisplatin exposure. Mechanistically, cisplatin induces chemotherapy resistance of Caski cells by upregulating Lyn, a Src family kinase (SFK) that interacts with EphA4, through a pathway involving reactive oxygen species. Thus, the reactive oxygen species-SFK-EphA4 axis presents new potential drug targets for chemotherapy resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Terapia de Alvo Molecular , Receptor EphA4/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Interleucinas/metabolismo , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Neoplasias do Colo do Útero/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
Preservation of adipose tissue before the isolation of cells is one of the most important steps in maintaining the cell viability of adipose tissue-derived mesenchymal stem cells (ADSCs) for clinical use. Hank's balanced salt solution (HBSS) is one of the main ADSC preservation solutions used clinically. However, this step is known to lead to decreased cell viability. The University of Wisconsin (UW) solution is recognized by transplant physicians as an excellent organ preservation solution. We aimed to investigate the effectiveness of UW solution in preservation of the viability of ADSCs. We collected adipose tissue from the inguinal fat pad of mice and compared preservation in UW solution and HBSS overnight by measuring cell viability after isolation. We found that the number of viable cells harvested per gram of adipose tissue mass was higher in UW solution- than HBSS-preserved tissue.
RESUMO
Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem cells, several issues related to the use of iPS cells in clinical applications remain unresolved, including the issue of teratoma formation. We previously reported that the induction of induced tissue-specific stem (iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by the transient overexpression of reprogramming factors, combined with tissue-specific selection and the generation of iTS cells, could have important implications for the clinical application of stem cells. At the same time, we also generated "induced fibroblast-like (iF) cells" that were capable of self-renewal, which had a similar morphology to fibroblast cells. In this study, we evaluated iF cells. iF cells are unlikely to show adipogenic/osteogenic differentiation. Moreover, iF cells have the ability to form tumors and behave similarly to pancreatic cancer cells. The technology used in the generation of iPS/iTS cells is also associated with the risk of generating cancer-like cells.
RESUMO
Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.