RESUMO
The Center for Basic and Translational Science was formed to address the unique challenges faced by surgeon-scientists. Shortly after its inception, COVID-19 upended research workflows at our institution. We discuss how the collaborative Center for Basic and Translational Science framework was adapted to support laboratories during the pandemic by assisting with ramp-down, promoting mentorship and community building, and maintaining research productivity.
Assuntos
COVID-19/prevenção & controle , Colaboração Intersetorial , Pesquisadores/organização & administração , Cirurgiões/organização & administração , Pesquisa Translacional Biomédica/organização & administração , COVID-19/epidemiologia , Eficiência , Humanos , Mentores , Michigan/epidemiologia , PandemiasRESUMO
BACKGROUND: Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis. METHODS: Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line. RESULTS: Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model. CONCLUSIONS: These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc.
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Adenocarcinoma/patologia , Carcinogênese/patologia , Metástase Neoplásica/patologia , Proteína de Ligação a Fosfatidiletanolamina/genética , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Camundongos , Camundongos Knockout , Metástase Neoplásica/genética , Neoplasias da Próstata/genéticaRESUMO
Glucagon-like peptide-2 (GLP-2) has been shown to be effective in patients with short bowel syndrome (SBS), but it is rapidly inactivated by dipeptidyl peptidase IV (DPP4). We used an orally active DPP4 inhibitor (DPP4-I), MK-0626, to determine the efficacy of this approach to promote adaptation after SBS, determined optimal dosing, and identified further functional actions in a mouse model of SBS. Ten-week-old mice underwent a 50% proximal small bowel resection. Dose optimization was determined over a 3-day post-small bowel resection period. The established optimal dose was given for 7, 30, and 90 days and for 7 days followed by a 23-day washout period. Adaptive response was assessed by morphology, intestinal epithelial cell (IEC) proliferation (proliferating cell nuclear antigen), epithelial barrier function (transepithelial resistance), RT-PCR for intestinal transport proteins and GLP-2 receptor, IGF type 1 receptor, and GLP-2 plasma levels. Glucose-stimulated sodium transport was assessed for intestinal absorptive function. Seven days of DPP4-I treatment facilitated an increase in GLP-2 receptor levels, intestinal growth, and IEC proliferation. Treatment led to differential effects over time, with greater absorptive function at early time points and enhanced proliferation at later time points. Interestingly, adaptation continued in the group treated for 7 days followed by a 23-day washout. DPP4-I enhanced IEC proliferative action up to 90 days postresection, but this action seemed to peak by 30 days, as did GLP-2 plasma levels. Thus DPP4-I treatment may prove to be a viable option for accelerating intestinal adaptation with SBS.
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Inibidores da Dipeptidil Peptidase IV/farmacologia , Síndrome do Intestino Curto/metabolismo , Adaptação Fisiológica , Amilases/sangue , Animais , Glicemia/metabolismo , Proliferação de Células/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Modelos Animais de Doenças , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 2 , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/citologia , Jejuno/efeitos dos fármacos , Jejuno/ultraestrutura , Masculino , Camundongos , Receptor IGF Tipo 1/biossíntese , Receptores de Glucagon/biossíntese , Transportador 1 de Glucose-Sódio/biossíntese , Triazóis/uso terapêutico , Regulação para CimaRESUMO
BACKGROUND: We previously demonstrated angiotensin converting enzymes (ACE) over-expression in a dextran-sodium sulfate colitis model; ACE inhibitor (ACE-I) treatment reduced colitis severity in this model. However, ACE-I has not been tested in more immunologically relevant colitis models. AIM: We hypothesized that ACE-I would decrease disease severity in an IL-10 knockout (-/-) colitis model. METHODS: Colitis was induced by giving 10-week old IL-10-/- mice piroxicam (P.O.) for 14 days. The ACE-I enalaprilat was given transanally at a dose of 6.25 mg/kg for 21 days. Prednisolone (PSL) with or without enalaprilat were used as therapeutic, comparative groups. All groups were compared to a placebo treated group. Outcome measures were clinical course, histology, abundance of pro-inflammatory cytokines/chemokines, and epithelial barrier function. RESULTS: Enalaprilat exhibited better survival (91 %) versus other treatment groups (PSL: 85.7 %, PSL + ACE-I: 71.4 %, placebo: 66.6 %). The ACE-I and PSL + ACE-I groups showed significantly better histological scores versus placebo mice. ACE-I and the PSL groups significantly reduced several pro-inflammatory cytokines versus placebo mice. FITC-dextran permeability was reduced in the ACE-I and PSL + ACE-I groups. Blood pressure was not affected in ACE-I treated mice compared to placebo mice. CONCLUSIONS: ACE-I was effective in reducing severity of colitis in an IL-10-/- model. The addition of prednisolone minimally augmented this effect. The findings suggest that appropriately dosed ACE-I with or without steroids may be a new therapeutic agent for colitis.
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Inibidores da Enzima Conversora de Angiotensina/farmacologia , Colite/induzido quimicamente , Enalaprilato/farmacologia , Interleucina-10/metabolismo , Animais , Colite/tratamento farmacológico , Colite/patologia , Sulfato de Dextrana/toxicidade , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Accurate prediction of survival from adrenocortical carcinoma (ACC) is difficult and current staging models are unreliable. Central sarcopenia as part of the cachexia syndrome is a marker of frailty and predicts mortality. This study seeks to confirm that psoas muscle density (PMD), lean psoas muscle area (LPMA), lumbar skeletal muscle index (LSMI), and intra-abdominal (IA) or subcutaneous fat (SC) can be used in combination to more accurately predict survival in ACC patients. METHODS: PMD, LPMA, IA, and SC fat were measured on serial CT scans of patients with ACC. Clinical outcome was correlated with quantitative data from patients with ACC and analyzed. A linear regression model was used to describe the relationship between PMD, LPMA, LSMI, IA, and SC fat, time to recurrence, and length of survival according to tumor stage. RESULTS: One hundred twenty-five ACC patients (94 females) were treated from 2005 to 2011. Significant morphometric predictors of survival include PMD, LPMA, and IA fat (p ≤ 0.0001, ≤ 0.0024, <0.0001, respectively) and improve prediction of survival compared to using stage alone. A 100-mm(2) increase in LPMA confers an 8 % lower hazard of death. LSMI does not change significantly between stages (p = 0.3196). CONCLUSION: Decreased PMD, LPMA, and increased IA fat suggest decreased survival in ACC patients and correlate with traditional staging systems. A more precise prediction of survival may be achieved when staging systems and morphometric measures are used in combination. Serial measurements of morphometric data are possible. The rate of change of these variables over time may be more important than the absolute value.
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Neoplasias do Córtex Suprarrenal/mortalidade , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/mortalidade , Carcinoma Adrenocortical/patologia , Gordura Intra-Abdominal/patologia , Sarcopenia/patologia , Caquexia/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Região Lombossacral , Masculino , Músculo Esquelético/patologia , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Músculos Psoas/patologia , Estudos Retrospectivos , Gordura Subcutânea/patologiaRESUMO
BACKGROUND: Bone metastasis occurs frequently in advanced prostate cancer (PCa) patients; however, it is not known why this happens. The epidermal growth factor receptor (EGFR) ligand EGF is available to early stage PCa; whereas, TGF-alpha is available when PCa metastasizes. Since the microenvironment of metastases has been shown to play a role in the survival of the tumor, we examined whether the ligands had effects on cell survival and proliferation in early and late PCa. METHODS: We used LNCaP cells as a model of early stage, non-metastatic PCa and the isogenic C4-2B cells as a model of late stage, metastatic PCa. RESULTS: We found that the proliferation factor MAPK and the survival factor AKT were differentially activated in the presence of different ligands. TGF-alpha induced growth of C4-2B cells and not of the parental LNCaP cells; however, LNCaP cells expressing a constitutively active AKT did proliferate with TGF-alpha. Therefore, AKT appeared to be the TGF-alpha-responsive factor for survival of the late stage PCa cells. LNCaP cells exposed to EGF produced more osteoprotegerin (OPG), an inhibitor of bone remodeling, than C4-2B cells with TGF-alpha, which had increased expression of RANKL, an activator of bone remodeling. In concordance, TGF-alpha-treated C4-2B conditioned medium was able to differentiate an osteoclast precursor line to a greater extent than EGF-treated C4-2B or TGF-alpha-treated LNCaP conditioned media. CONCLUSION: The switch in EGFR ligand availability as PCa progresses affects cell survival and contributes to bone remodeling.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Ósseas/metabolismo , Remodelação Óssea/fisiologia , Receptores ErbB/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Adenocarcinoma/patologia , Neoplasias Ósseas/secundário , Remodelação Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Progressão da Doença , Fator de Crescimento Epidérmico/farmacologia , Humanos , Ligantes , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Estadiamento de Neoplasias , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
PURPOSE: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.
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Apoptose/efeitos da radiação , Proteínas de Neoplasias/deficiência , Proteína de Ligação a Fosfatidiletanolamina/deficiência , Neoplasias da Próstata/radioterapia , Tolerância a Radiação/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Indução Enzimática/efeitos da radiação , Masculino , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteína de Ligação a Fosfatidiletanolamina/biossíntese , Proteína de Ligação a Fosfatidiletanolamina/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/enzimologia , Imunodeficiência Combinada SeveraRESUMO
Although oncogenes and their transformation mechanisms have been known for 30 years, we are just now using our understanding of protein function to abrogate the activity of these genes to block cancer growth. The advent of specific small-molecule inhibitors has been a tremendous step in the fight against cancer and their main targets are the cellular counterparts of viral oncogenes. The best-known example of a molecular therapeutic is Gleevec (imatinib). In the early 1990s, IFN-alpha treatment produced a sustained cytologic response in approximately 33% of chronic myelogenous leukemia patients. Today, with Gleevec targeting the kinase activity of the proto-oncogene abl, the hematologic response rate in chronic myelogenous leukemia patients is 95% with 89% progression-free survival at 18 months. There are still drawbacks to the new therapies, such as drug resistance after a period of treatment, but the drawbacks are being studied experimentally. New drugs and combination therapies are being designed that will bypass the resistance mechanisms.
Assuntos
Oncogenes/fisiologia , Animais , Humanos , Neoplasias/patologia , Proto-Oncogene MasRESUMO
Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of alpha(5)beta(1) with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of alpha(5)beta(1) and alpha(4)beta(1) fibronectin receptors in regulating matrix metalloproteinase (MMP)-1-dependent invasion by human breast cancer and mammary epithelial cells. We found that a peptide consisting of fibronectin PHSRN sequence, Ac-PHSRN-NH(2), induces alpha(5)beta(1)-mediated invasion of basement membranes in vitro by human breast cancer and mammary epithelial cells. PHSRN-induced invasion requires interstitial collagenase MMP-1 activity and is suppressed by an equimolar concentration of a peptide consisting of the LDV sequence of the fibronectin connecting segment, Ac-LHGPEILDVPST-NH(2), in mammary epithelial cells, but not in breast cancer cells. This sequence interacts with alpha(4)beta(1), an integrin that is often down-regulated in breast cancer cells. Immunoblotting shows that the PHSRN peptide stimulates MMP-1 production by serum-free human breast cancer and mammary epithelial cells and that the LDV peptide represses PHSRN-stimulated MMP-1 production only in mammary epithelial cells. Furthermore, PHSRN stimulates MMP-1 activity in breast cancer cells and mammary epithelial cells with a time course that closely parallels invasion induction. Thus, down-regulation of surface alpha(4)beta(1) during oncogenic transformation may be crucial for establishment of the alpha(5)beta(1)-induced, MMP-1-dependent invasive phenotype of breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Sequência de Aminoácidos , Membrana Basal/metabolismo , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Metaloproteinase 1 da Matriz/biossíntese , Dados de Sequência Molecular , Invasividade Neoplásica , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologiaRESUMO
To better understand the mechanisms of transformation by the oncogene HER-2, we transduced the human mammary epithelial (HME) cell line MCF-10A with HER-2 and developed a cell line that appeared to moderately overexpress HER-2. These MCF-10HER-2 cells were unable to grow in the absence of epidermal growth factor (EGF). However, coexpression of HER-2 with the HPV-16 oncoproteins E6 and E7 resulted in EGF-independent cells that expressed very high levels of constitutively activated HER-2. Interestingly, coexpression of E7 with HER-2 resulted in cells that were EGF-independent for growth but did not express HER-2 to high levels, and coexpression of E6 with HER-2 resulted in cells expressing higher levels of HER-2, which were still dependent on EGF for growth and survival. The MCF-10HER-2E7 and HER-2/E6E7 cells exhibited constitutive activation of a form of epidermal growth factor receptor (EGFR) that had a faster electrophoretic mobility than EGFR activated by exogenous growth factors. Exposure of cells with EGFR activation to ZD1839 (Iressa), at concentrations specific for EGFR, had little or no influence on proliferation of cells with amplified HER-2 but little or no EGFR. These results indicate that HER-2, E6, and E7 cooperate with endogenous EGFR to yield fully transformed cells.
Assuntos
Transformação Celular Neoplásica , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/biossíntese , Receptor ErbB-2/biossíntese , Proteínas Repressoras/biossíntese , Neoplasias da Mama , Linhagem Celular Transformada , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Glândulas Mamárias Humanas/metabolismo , Proteínas E7 de PapillomavirusRESUMO
We previously demonstrated that erbB-2-overexpressing human mammary epithelial (HME) cells exhibit several transformed phenotypes including growth factor independence, anchorage-independent growth, motility, and invasiveness. Because phosphatidylinositol 3'-kinase (PI3K) is a major target of erbB-2 activation, we tested the contribution that PI3K and its downstream signaling pathways make to these phenotypes. Utilizing a constitutively active form of PI3K, p110CAAX, we show that PI3K can mediate most phenotypes observed in erbB-2-overexpressing cells. To identify pathways leading from PI3K to specific phenotypes, we expressed constitutively active AKT or PTEN in erbB-2-overexpressing cells or in HME cells. HME cells expressing constitutively active AKT were growth factor independent, anchorage independent and motile, but not invasive. PTEN expression blocked erbB-2-mediated invasion but none of the other phenotypes. Rottlerin blocked invasion induced by p110CAAX and erbB-2, suggesting that protein kinase C delta (PKC-delta) is the downstream effector of PI3K responsible for the invasive capacity of the cells. Consistent with these observations, phospho-AKT remained detectable in erbB-2 cells treated with LY294002 or expressing exogenous PTEN, but was abolished by treatment with the p38MAP kinase inhibitor SB202190. Thus, both PI3K-dependent and p38MAP kinase-dependent pathways lead to activation of AKT, and activation of PKC-delta, via PI3K, mediates invasion.
Assuntos
Transformação Celular Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Quimiotaxia , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Fagocitose , Piridinas/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN: Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS: Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION: EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.
Assuntos
Junções Aderentes/metabolismo , Nutrição Enteral , Mucosa Intestinal/fisiopatologia , Intestino Delgado/fisiopatologia , Nutrição Parenteral/efeitos adversos , Junções Íntimas/metabolismo , Adolescente , Biomarcadores/metabolismo , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Modelos Lineares , Masculino , Permeabilidade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Toll-Like/metabolismo , Adulto JovemRESUMO
We have previously shown that human breast cancer cells that overexpress erbB-2 are growth factor-independent. In order to test the contribution of erbB-2 to this and other transformed phenotypes without the genetic instability of cancer cells, erbB-2 was overexpressed in human mammary epithelial (HME) cells. ErbB-2-overexpressing HME cells exhibit several transformed phenotypes including cell surface alpha(4) integrin downregulation and invasiveness. We formulated a model for invasiveness that depends on a cell's ability to downregulate alpha(4) integrin. As small G-proteins play a role in cytoskeleton remodeling and as this is a likely route for alpha(4) integrin trafficking, we investigated the role of small G-proteins and their downstream signals in mediating alpha(4) integrin downregulation and invasiveness using Rac 1. Dominant-negative Rac 1 blocked erbB-2-mediated invasion and reversed erbB-2-mediated alpha(4) integrin downregulation. In addition, constitutively active Rac 1 induced alpha(4) integrin downregulation and invasiveness. In erbB-2-overexpressing and in constitutively active Rac 1-expressing cells, a p38MAP kinase (p38MAPK) inhibitor blocked invasiveness and reversed alpha(4) integrin downregulation. These data suggest a model in which erbB-2 signaling activates Rac 1, which, in turn, activates p38MAPK, leading to the downregulation of alpha(4) integrin. These data strengthen the model where loss of alpha(4) integrin at the cell surface, leading to reduced alpha(4) integrin binding to plasma fibronectin, plays a role in erbB-2-mediated invasiveness.
Assuntos
Regulação para Baixo , Integrina alfa4/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptor ErbB-2/metabolismo , Actinas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular , Transformação Celular Neoplásica , Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Fibronectinas/sangue , Citometria de Fluxo , Genes Dominantes , Humanos , Imidazóis/farmacologia , Modelos Biológicos , Invasividade Neoplásica , Fenótipo , Transporte Proteico , Piridinas/farmacologia , Retroviridae/genética , Transdução de Sinais , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Novel strategies are needed to address the problem of patients with short bowel syndrome. We previously demonstrated a three-fold lengthening of pig bowel after 2 weeks of applied distractive forces, but we have not elucidated the mechanisms facilitating this growth. We used a mouse model of distraction-induced enterogenesis. High molecular weight polyethylene glycol (PEG) osmotically stretched an isolated small bowel segment (PEG-stretch). Significant increases in villus height and crypt depth and in intestinal epithelial cell length and numbers suggested epithelial remodeling in addition to proliferation during enterogenesis. LC-MS/MS analysis showed a two-fold upregulation of α-actinin-1 and -4. We also demonstrated that p-focal adhesion kinase (FAK), FAK, α-actinin, and Rac1 were significantly upregulated and that F-actin was relocalized in PEG-stretch versus controls. Blockade of the phosphotidyl inositol 3' kinase pathway failed to influence the increase in proliferation or decline in apoptosis after stretch, suggesting alternative signaling pathways are used, including MEK and P38MAPK, which were both upregulated during enterogenesis. Our data suggests that several known mechanotransduction pathways drive distraction-induced enterogenesis.
Assuntos
Intestino Delgado/crescimento & desenvolvimento , Intestino Delgado/patologia , Mecanotransdução Celular , Síndrome do Intestino Curto/metabolismo , Síndrome do Intestino Curto/patologia , Actinina/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Espectrometria de Massas , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sus scrofa , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Replacement of a diseased organ with an autologously derived tissue is an ideal therapy for some medical problems. However, it is difficult to recreate many adult human tissues in vitro due to the functionally necessary architecture of most organs and the lack of understanding of methods to direct the development of the organ of interest. The parathyroid gland is ideal for in vitro organ development because this gland is relatively simple, is transplantable, and is commonly affected by a surgical complication rather than an autoimmune disease. We have investigated thymus as a source of autologous endoderm and parathyroid-like precursor cells. Human thymus cells were treated with a differentiation protocol we developed with human embryonic stem cells (The Bingham Protocol) that utilizes timed exposures to Activin A and soluble Sonic hedgehog (Shh). We incrementally changed the protocol to optimize the differentiation of the thymus cells into parathyroid-like cells. The final protocol used 50 ng/mL Activin A and 100 ng/mL Shh over 13 weeks. The differentiated cells expressed the parathyroid markers parathyroid hormone (PTH), calcium sensing receptor, chemokine receptor type-4 (CXCR4), and chorian-specific transcription factor (GCM2) as measured by reverse transcription-polymerase chain reaction and PTH enzyme-linked immunosorbent assay. Cultured thymus cells without Activin A or Shh exposure did not secrete PTH nor express similar markers. The differentiated cells released PTH, which was suppressed in response to increased calcium concentration. The chemically differentiated cells did not form tumors in immune-compromised mice. Our protocol recreated cells with markers of parathyroid tissue that responded as parathyroid cells to physiologic stimuli. This approach is a further step toward a strategy to restore parathyroid function using autologous cells that were directed to differentiate by nongenetic in vitro manipulation.
Assuntos
Transdiferenciação Celular , Engenharia Genética , Glândulas Paratireoides/citologia , Timo/citologia , Biomarcadores/metabolismo , Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Criança , Endoderma/citologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Glândulas Paratireoides/efeitos dos fármacos , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/metabolismoRESUMO
BACKGROUND: Muscle weakness and wasting are known manifestations of hypercortisolism (HC). Central sarcopenia is a marker of frailty and predicts mortality. The hypothesis of this study is that central sarcopenia can be used as a marker of disease severity and frailty in patients with HC. METHODS: Psoas muscle area and psoas muscle density (PMD) were measured at specific points on CT scans of patients with HC using a defined protocol. We compared 24-hour urine cortisol (24HUC) levels by time point to each CT scan. A linear regression model was used to describe the relationship between 24HUC and morphometric variables. A comparison with matched non-HC patients was performed. RESULTS: We identified 45 patients (34 female). The median age was 46 years (range, 14-80); the median 24HUC was 211 mg/dL (range, 9.5-39,500); the median PMD was 50.1 24HUC (range, 20-72). An inverse correlation (-0.29) between 24HUC levels and PMD was noted (P = .045). Intra-abdominal fat was also significantly and positively correlated with 24HUC: 27 non-HC patients matched for age, sex, and body-mass index showed higher (58 vs 51) PMD (P = .0127) compared to those with HC. CONCLUSION: PMD and intra-abdominal fat are significantly related to 24HUC levels. Morphometric measures of the overall burden of HC may allow more precise assessment of disease severity.
Assuntos
Síndrome de Cushing/complicações , Hidrocortisona/urina , Músculos Psoas/diagnóstico por imagem , Sarcopenia/classificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/urina , Estudos de Casos e Controles , Síndrome de Cushing/urina , Feminino , Humanos , Gordura Intra-Abdominal/diagnóstico por imagem , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Músculos Psoas/patologia , Estudos Retrospectivos , Sarcopenia/diagnóstico , Sarcopenia/etiologia , Índice de Gravidade de Doença , Gordura Subcutânea Abdominal/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
BACKGROUND: Hypoparathyroidism is the most frequent permanent complication of thyroid surgery. Our hypothesis is that human precursor cells in culture can be differentiated into parathyroid cells and used to reconstitute function. Human embryonic stem cells (hESCs) are a stable model to study differentiation into parathyroid-like cells. In prior work, the BG01-hESC line was stimulated to form parathyroid-like cells. This cell line is no longer available, however, and additional studies were needed to confirm and extend prior observations. METHODS: Increasing concentrations of fetal bovine serum and timed exposure to Activin A were used to differentiate H1-hESC into parathyroid-like cells. The potential benefit of Sonic hedgehog exposure on parathyroid-like cell development also was evaluated by serial alterations of culture conditions. Calcium-sensing receptor (CaSR), GCM2, and PTH expression (RT-PCR) and PTH protein secretion (ELISA) were used as markers of differentiated cells. RESULTS: We successfully modified our prior protocol to generate cells that express CaSR, GCM2, and PTH RNA from undifferentiated H1-hESC. The cells also secreted PTH. CONCLUSION: We replicated parathyroid differentiation using H1-hESC cells. Our data advance the project toward in vitro differentiation of precursor cells isolated from individual patients for autotransplantation.
Assuntos
Hipoparatireoidismo/cirurgia , Hipoparatireoidismo/terapia , Glândulas Paratireoides/citologia , Transplante de Células-Tronco/métodos , Animais , Bovinos , Técnicas de Cultura de Células , Diferenciação Celular , Fibroblastos/citologia , Humanos , Camundongos , Transplante Autólogo/métodosRESUMO
Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.
Assuntos
Ativinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Glândulas Paratireoides/citologia , Animais , Antígenos de Diferenciação/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Glândulas Paratireoides/metabolismo , Fatores de TempoRESUMO
Men with androgen-independent prostate cancer (AIPC) frequently have bone metastasis. The effects of chemotherapy on markers of bone metabolism have not been well characterized. We conducted a prospective study of patients with AIPC randomized in the first cycle to receive either docetaxel/estramustine or zoledronic acid, a bisphosphonate, to inhibit osteoclastic activity. Here we report the effects of therapy on markers of bone metabolism in these patients following the first cycle of therapy. Serum levels of several indices of bone remodeling were evaluated using commercial enzyme-linked immunosorbent assays. Changes in markers of bone metabolism were compared in patients receiving initial chemotherapy versus bisphosphonate. There was no significant difference in median change in any of the measured bone markers in patients given zoledronic acid when compared to chemotherapy. When comparing responders to nonresponders, overall interleukin-6 (IL-6) decreased by 35% in prostate-specific antigen responders; whereas, IL-6 levels increased by 76% in nonresponders (p = 0.03). Elevated IL-6 levels and reductions in IL-6 levels early in treatment may reflect ultimate clinical response to docetaxel-based regimens.
Assuntos
Antineoplásicos/uso terapêutico , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Interleucina-6/sangue , Neoplasias da Próstata/tratamento farmacológico , Taxoides/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Biomarcadores/sangue , Biomarcadores/metabolismo , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/metabolismo , Difosfonatos/administração & dosagem , Difosfonatos/uso terapêutico , Docetaxel , Estramustina/administração & dosagem , Estramustina/uso terapêutico , Humanos , Imidazóis/administração & dosagem , Imidazóis/uso terapêutico , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , Taxoides/administração & dosagem , Ácido ZoledrônicoRESUMO
BACKGROUND: HER-2 is an epidermal growth factor receptor (EGFR) family receptor tyrosine kinase that is overexpressed in about 30% of human breast cancers correlating with a poor prognosis. Previous work in our laboratory has found that HER-2 overexpression plays a role in growth factor independence, anchorage independence, motility, and invasion of naturally occurring basement membranes. We also found that AKT was activated by p38MAPK in these cells, but this activation did not play a role in invasion. Since AKT has been shown in other systems to be a survival factor, we hypothesized that HER-2 mediated activation of AKT is necessary for growth factor independence. METHODS: Human mammary epithelial cells transduced to overexpress HER-2, HER-2, PTEN, and Myr-AKT and the primary breast cancer cell lines SUM-149 and SUM-225 were used to dissect the signaling pathways leading to growth factor independence and anchorage-independent growth in HER-2 overexpressing cells. RESULTS: We found that, in the absence of EGF, p38MAPK-activated AKT is necessary for HER-2 overexpressing cells to survive and to form colonies in soft agar. We show that EGF works as a survival signal in the absence of p38MAPK-mediated activation of AKT. We also show that human mammary epithelial cells expressing a constitutively active AKT do not require EGF for growth or colony formation in soft agar. CONCLUSIONS: The data presented here indicate that AKT activation can compensate for EGF-mediated cell survival signals leading to growth factor independence and anchorage-independent growth.