RESUMO
BACKGROUND: This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. STUDY DESIGN AND METHODS: PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. RESULTS: Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. CONCLUSION: Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.
Assuntos
Eritrócitos/metabolismo , Plasma/metabolismo , Plasma Rico em Plaquetas/metabolismo , Anticoagulantes/farmacologia , Fatores de Coagulação Sanguínea/metabolismo , Gasometria , Preservação de Sangue , Eritrócitos/efeitos dos fármacos , Eritrócitos/efeitos da radiação , Hemoglobinas/análise , Humanos , Tempo de Tromboplastina Parcial , Contagem de Plaquetas , Testes de Função Plaquetária , Plasma Rico em Plaquetas/efeitos dos fármacos , Plasma Rico em Plaquetas/efeitos da radiação , Tempo de Protrombina , Riboflavina/farmacologia , Raios UltravioletaRESUMO
A perfluorocarbon (PFC) investigated for treatment of traumatic brain injury (TBI) delivers oxygen to support brain function, but causes transient thrombocytopenia. TBI can cause acute inflammation with resulting thrombocytopenia; an interaction between the PFC effects and TBI inflammation might exacerbate thrombocytopenia. Therefore, PFC effects on platelet (PLT) function and hemostasis in a lipopolysaccharide (LPS) model of inflammation in the baboon were studied. Animals were randomized to receive saline ±LPS, and ± one of two doses of PFC. PLT count, transmission electron microscopy, and microparticle populations were quantified at baseline (BL) and at 2, 24, 48, 72, and 96 hours; hemostatic parameters for aggregometry and for blood clotting were measured at baseline (BL) and days 3 and 4. Injection of vehicle and LPS caused thrombocytopenia within hours; PFCs caused delayed thrombocytopenia beginning 48 hours post-infusion. LPS+PFC produced a more prolonged PLT decline and decreased clot strength. LPS+PFC increased ADP-stimulated aggregation, but PFC alone did not. Microparticle abundance was greatest in the LPS+PFC groups. LPS+PFC caused diffuse microvascular hemorrhage and death in 2 of 5 baboons in the low dose LPS-PFC group and 2 of 2 in the high dose LPS-PFC group. Necropsy and histology suggested death was caused by shock associated with hemorrhage in multiple organs. Abnormal morphology of platelets and red blood cells were notable for PFC inclusions. In summary, PFC infusion caused clinically significant thrombocytopenia and exacerbated LPS-induced platelet activation. The interaction between these effects resulted in decreased hemostatic capacity, diffuse bleeding, shock and death.
Assuntos
Fluorocarbonos , Inflamação , Animais , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Fluorocarbonos/intoxicação , Hemorragia/induzido quimicamente , Hemostáticos , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Trombocitopenia/induzido quimicamenteRESUMO
Bioluminescent reporter genes are sensitive in situ tools for following disease progression in preclinical models, albeit they are subject to scattering and absorption in deep tissues. We have generated a bicistronic Cre/LoxP reporter mouse line that pairs the expression of firefly luciferase with quantifiable expression of a human placental alkaline phosphatase that is secreted into the serum (SeAP). With the use of this dual-modality bioreporter with a novel, inducible Pax7-CreER line for tracking muscle satellite cells, we demonstrate the longitudinal kinetics of muscle stem cell turnover, accounting for a doubling of the signal from satellite cell and progeny every 3.93 wk in the transition from adolescence to early adulthood. We also show that this dual-modality bioreporter can be incorporated in preclinical cancer models, whereby SeAP activity is reflective of tumor burden. Thus, this dual bioreporter permits both spatial localization and accurate quantification of biological processes in vivo even when the tissue of interest is deep within the animal.
Assuntos
Células-Tronco Adultas/metabolismo , Genes Reporter , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Fosfatase Alcalina/genética , Animais , Sequência de Bases , Primers do DNA/genética , Proteínas Ligadas por GPI , Humanos , Isoenzimas/genética , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição PAX7/genéticaRESUMO
Ept1, Ept2, Ept6, and Ept9 are quantitative trait loci mapped in crosses between the ACI and Copenhagen (COP) rat strains as genetic determinants of responsiveness of the pituitary gland to estrogens. We have developed four congenic rat strains, each of which carries, on the genetic background of the ACI rat strain, alleles from the COP rat strain that span one of these quantitative trait loci. Relative to the female ACI rats, female ACI.COP-Ept1 rats exhibited reduced responsiveness to 17beta-estradiol (E2) in the pituitary gland, as evidenced by quantification of pituitary mass and circulating prolactin, and in the mammary gland, as evidenced by reduced susceptibility to E2-induced mammary cancer. The ACI.COP-Ept2 rat strain exhibited reduced responsiveness to E2 in the pituitary gland but did not differ from the ACI strain in regard to susceptibility to E2-induced mammary cancer. Interestingly, female Ept2 congenic rats exhibited increased responsiveness to E2 in the thymus, as evidenced by enhanced thymic atrophy. The ACI.COP-Ept6 rat strain exhibited increased responsiveness to E2 in the pituitary gland, which was associated with a qualitative phenotype suggestive of enhanced pituitary vascularization. The ACI.COP-Ept9 rat strain exhibited reduced responsiveness to E2 in the anterior pituitary gland, relative to the ACI rat strain. Neither Ept6 nor Ept9 impacted responsiveness to E2 in the mammary gland or thymus. These data indicate that each of these Ept genetic determinants of estrogen action is unique in regard to the tissues in which it exerts its effects and/or the direction of its effect on estrogen responsiveness.
Assuntos
Resistência a Medicamentos/genética , Estrogênios/farmacologia , Hipófise/efeitos dos fármacos , Locos de Características Quantitativas/fisiologia , Animais , Animais Congênicos , Atrofia/genética , Feminino , Marcadores Genéticos/fisiologia , Predisposição Genética para Doença , Incidência , Neoplasias Mamárias Animais/epidemiologia , Neoplasias Mamárias Animais/genética , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Prolactina/sangue , Ratos , Timo/patologiaRESUMO
Exposure to estrogens is associated with an increased risk of breast cancer. Our laboratory has shown that the ACI rat is uniquely susceptible to 17beta-estradiol (E2)-induced mammary cancer. We previously mapped two loci, Emca1 and Emca2 (estrogen-induced mammary cancer), that act independently to determine susceptibility to E2-induced mammary cancer in crosses between the susceptible ACI rat strain and the genetically related, but resistant, Copenhagen (COP) rat strain. In this study, we evaluate susceptibility to E2-induced mammary cancer in a cross between the ACI strain and the unrelated Brown Norway (BN) rat strain. Whereas nearly 100% of the ACI rats developed mammary cancer when treated continuously with E2, BN rats did not develop palpable mammary cancer during the 196-day course of E2 treatment. Susceptibility to E2-induced mammary cancer segregated as a dominant or incompletely dominant trait in a cross between BN females and ACI males. In a population of 251 female (BN x ACI)F(2) rats, we observed evidence for a total of five genetic determinants of susceptibility. Two loci, Emca4 and Emca5, were identified when mammary cancer status at sacrifice was evaluated as the phenotype, and three additional loci, Emca6, Emca7, and Emca8, were identified when mammary cancer number was evaluated as the phenotype. A total of three genetic interactions were identified. These data indicate that susceptibility to E2-induced mammary cancer in the BN x ACI cross behaves as a complex trait controlled by at least five loci and multiple gene-gene interactions.
Assuntos
Cocarcinogênese , Estradiol/farmacologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Animais , Mapeamento Cromossômico , Feminino , Predisposição Genética para Doença , Neoplasias Hipofisárias/genética , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BNRESUMO
Estrogens stimulate proliferation and enhance survival of the prolactin (PRL)-producing lactotroph of the anterior pituitary gland and induce development of PRL-producing pituitary tumors in certain inbred rat strains but not others. The goal of this study was to elucidate the genetic bases of estrogen-induced pituitary tumorigenesis in reciprocal intercrosses between the genetically related ACI and Copenhagen (COP) rat strains. Following 12 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), pituitary mass, an accurate surrogate marker of absolute lactotroph number, was increased 10.6-fold in ACI rats and 4.5-fold in COP rats. Composite interval mapping analyses of the phenotypically defined F(2) progeny from the reciprocal crosses identified six quantitative trait loci (QTL) that determine the pituitary growth response to DES. These loci reside on chromosome 6 [Estrogen-induced pituitary tumor (Ept)1], chromosome 3 (Ept2 and Ept6), chromosome 10 (Ept9), and chromosome 1 (Ept10 and Ept13). Together, these six Ept loci and one additional suggestive locus on chromosome 4 account for an estimated 40% of the phenotypic variance exhibited by the combined F(2) population, while 34% of the phenotypic variance was estimated to result from environmental factors. These data indicate that DES-induced pituitary mass behaves as a quantitative trait and provide information that will facilitate identification of genes that determine the tumorigenic response of the pituitary gland to estrogens.
Assuntos
Estrogênios/farmacologia , Hipófise/efeitos dos fármacos , Neoplasias Hipofisárias/induzido quimicamente , Neoplasias Hipofisárias/genética , Animais , Biomarcadores , Mapeamento Cromossômico , Cruzamentos Genéticos , Epistasia Genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Marcadores Genéticos , Genótipo , Masculino , Modelos Genéticos , Modelos Estatísticos , Tamanho do Órgão , Fenótipo , Hipófise/patologia , Prolactina/metabolismo , Locos de Características Quantitativas , Ratos , Sensibilidade e Especificidade , Fatores Sexuais , Especificidade da EspécieRESUMO
Hormonal, genetic, and environmental factors play major roles in the complex etiology of breast cancer. When treated continuously with 17beta-estradiol (E2), the ACI rat exhibits a genetically conferred propensity to develop mammary cancer. The susceptibility of the ACI rat to E2-induced mammary cancer appears to segregate as an incompletely dominant trait in crosses to the resistant Copenhagen (COP) strain. In both (ACI x COP)F(2) and (COP x ACI)F(2) populations, we find strong evidence for a major genetic determinant of susceptibility to E2-induced mammary cancer on distal rat chromosome 5. Our data are most consistent with a model in which the ACI allele of this locus, termed Emca1 (estrogen-induced mammary cancer 1), acts in an incompletely dominant manner to increase both tumor incidence and tumor multiplicity as well as to reduce tumor latency in these populations. We also find evidence suggestive of a second locus, Emca2, on chromosome 18 in the (ACI x COP)F(2) population. The ACI allele of Emca2 acts in a dominant manner to increase incidence and decrease latency. Together, Emca1 and Emca2 act independently to modify susceptibility to E2-induced mammary cancer.
Assuntos
Mapeamento Cromossômico , Estradiol/farmacologia , Predisposição Genética para Doença , Neoplasias Mamárias Experimentais/genética , Animais , Feminino , Ligação Genética , Marcadores Genéticos , Neoplasias Mamárias Experimentais/induzido quimicamente , Ratos , Ratos Endogâmicos ACIRESUMO
BACKGROUND: Many military and civilian centers have shifted to a damage-control resuscitation approach, focused on providing oxygen-carrying capacity while simultaneously mitigating coagulopathy with a balanced ratio of platelets and plasma to red blood cells. It is unclear to what degree this strategy is used during burn or soft tissue excision. Here, we characterized blood product transfusion during burn and soft tissue surgery and reviewed the published literature regarding intraoperative coagulation changes. We hypothesized that blood product resuscitation during burn and soft tissue excision is not hemostatic and would be insufficient to address hemorrhage-induced coagulopathy. METHODS: Consented adult patients were enrolled into an institutional review board-approved prospective observational study. Number, component type, volume, and age of the blood products transfused were recorded during burn excision/grafting or soft tissue debridement. Component bags (packed red blood cells, fresh frozen plasma, platelets, and cryoprecipitate) were collected, and the remaining sample was harvested from the bag and tubing. Aliquots of 1/1,000th the original volume of each blood product were obtained and combined, producing an amalgam sample containing the same ratio of product transfused. Platelet count, rotational thromboelastometry, and impedance aggregometry were measured. Significance was set at p < 0.05. RESULTS: Amalgamated transfusate samples produced abnormally weak clots (p ≤ 0.001) particularly if they did not contain platelets. Clot strength (48.8 [2.6] mm; reference range, 49-71 mm) for platelet-containing amalgams was below the lower limit of the reference range despite platelet-red blood cell ratios greater than 1:1. Platelet aggregation was abnormally low; transfused platelets were functionally inferior to native platelets. CONCLUSION: Our study and focused review demonstrate that further work is needed to fully understand the needs of patients undergoing tissue excision. The three studies reviewed and the results of our observational work suggest that coagulopathy and thrombocytopenia may contribute to intraoperative hemorrhage. Blood product resuscitation during burn and soft tissue excision is not hemostatic. LEVEL OF EVIDENCE: Epidemiologic study, level V.
Assuntos
Queimaduras/cirurgia , Lesões dos Tecidos Moles/cirurgia , Adulto , Transfusão de Componentes Sanguíneos , Perda Sanguínea Cirúrgica , Transfusão de Sangue , Queimaduras/fisiopatologia , Humanos , Período Intraoperatório , Agregação Plaquetária/fisiologia , Ressuscitação , Lesões dos Tecidos Moles/fisiopatologia , TromboelastografiaRESUMO
The IGF-II/mannose 6-phosphate (Man-6-P) receptor (IGF2R) binds IGF-II and Man-6-P-bearing ligands at distinct binding sites. Analysis of IGF2R expression and function suggested that decreased IGF2R expression could partly account for the increased growth of lymph node carcinoma of the prostate (LNCaP) human prostate cancer cells observed with increasing passage in culture. However, LNCaP cells that expressed a Myc-tagged IGF2R (IGF2RMyc) proliferated more rapidly than control cells transfected with the empty vector. LNCaP cells expressing a mutant IGF2R incompetent to bind IGF-II (IGF2RMyc I/T) proliferated more rapidly than both vector-transfected cells and cells expressing the IGF2RMyc. In contrast, forced expression of IGF2RMyc in PC-3 human prostate cancer cells resulted in decreased proliferation, compared with control cells. As in LNCaP cells, PC-3 cells expressing IGF2RMyc I/T proliferated more rapidly than vector-transfected cells. The subcellular distribution and ability to internalize cell-surface IGF-II of IGF2RMyc were indistinguishable from endogenous IGF2R in PC-3 cells. These data suggest that the IGF-II- and Man-6-P-binding functions of the IGF2R have opposing activities, with respect to growth of prostate cancer cells. The magnitude of each activity in a given cell type seems to determine whether the net effect of the IGF2R on cell growth is inhibitory or stimulatory.
Assuntos
Fator de Crescimento Insulin-Like II/metabolismo , Manosefosfatos/metabolismo , Neoplasias da Próstata/patologia , Receptor IGF Tipo 2/fisiologia , Divisão Celular , Endocitose , Expressão Gênica , Vetores Genéticos , Humanos , Immunoblotting , Radioisótopos do Iodo , Masculino , Receptor IGF Tipo 2/genética , Transfecção , Células Tumorais CultivadasRESUMO
HT-29 are colonic carcinoma cells that follow a unique pattern of differentiation dependent on the medium's carbohydrate source. This study compared levels of insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 when HT-29 cells were grown with standard glucose-containing medium versus galactose-containing (glucose-free) medium. Serum-free media conditioned for 24h were collected at low density, pre-confluence, confluence, and 48-h post-confluence. Ligand blotting of the conditioned galactose medium demonstrated low IGFBP-4 levels until the cells approached confluence, when the levels increased significantly. In standard medium, IGFBP-4 levels increased with increasing cell numbers except for a transient decrease at confluence. Radioimmunoassay showed little change in IGF-II concentrations, although HT-29 cells grown with galactose had lower IGF-II concentrations. HT-29 cells treated with retinoic acid had dose-dependent increases in IGFBP-4 and reduced IGF-II expression. These studies suggest that HT-29 cell differentiation correlates with an increase in IGFBP-4 levels.
Assuntos
Enterócitos/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Metabolismo dos Carboidratos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like II/metabolismo , Metaloendopeptidases/metabolismo , Proteína Plasmática A Associada à Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tretinoína/farmacologiaRESUMO
When treated with 17ß-estradiol, female ACI rats (Rattus norvegicus) rapidly develop mammary cancers that share multiple phenotypes with luminal breast cancers. Seven distinct quantitative trait loci that harbor genetic determinants of susceptibility to 17ß-estradiol-induced mammary cancer have been mapped in reciprocal intercrosses between susceptible ACI rats and resistant Brown Norway (BN) rats. A panel of unique congenic rat strains has now been generated and characterized to confirm the existence of these quantitative trait loci, designated Emca3 through Emca9, and to quantify their individual effects on susceptibility to 17ß-estradiol-induced mammary cancer. Each congenic strain carries BN alleles spanning an individual Emca locus, introgressed onto the ACI genetic background. Data presented herein indicate that BN alleles at Emca3, Emca4, Emca5, Emca6, and Emca9 reduce susceptibility to 17ß-estradiol-induced mammary cancer, whereas BN alleles at Emca7 increase susceptibility, thereby confirming the previous interval mapping data. All of these Emca loci are orthologous to regions of the human genome that have been demonstrated in genome-wide association studies to harbor genetic variants that influence breast cancer risk. Moreover, four of the Emca loci are orthologous to loci in humans that have been associated with mammographic breast density, a biomarker of breast cancer risk. This study further establishes the relevance of the ACI and derived congenic rat models of 17ß-estradiol-induced mammary cancer for defining the genetic bases of breast cancer susceptibility and elucidating the mechanisms through which 17ß-estradiol contributes to breast cancer development.
Assuntos
Predisposição Genética para Doença , Neoplasias Mamárias Animais/genética , Locos de Características Quantitativas , Animais , Animais Congênicos , Estradiol , Estrogênios , Feminino , Humanos , Neoplasias Mamárias Animais/induzido quimicamente , Fenótipo , Ratos Endogâmicos BN , RiscoRESUMO
The ACI rat model of 17ß-estradiol (E2)-induced mammary cancer has gained wide use in the study of breast cancer etiology, prevention, and genetics. Emca8, a QTL that determines susceptibility to E2-induced mammary cancer, was previously mapped to rat chromosome 5 (RNO5) in an intercross between resistant Brown Norway (BN) and susceptible ACI rats. In this study, a panel of congenic rat strains, each of which carries BN alleles across a defined segment of RNO5 on the ACI genetic background, was generated and used to map more precisely the Emca8 determinants of mammary cancer susceptibility. Three distinct genetic determinants were localized within Emca8, and two of these were mapped to intervals of less than 15 megabases. Emca8.1 harbors Cdkn2a, Cdkn2b, and other genes and is orthologous to the 9p21 breast cancer locus identified in genome-wide and candidate gene association studies. Emca8.2 harbors Cdkn2c and other genes and is orthologous to the 1p32 locus in humans that is frequently deleted in breast cancers. Both Emca8.1 and Emca8.2 harbor copy number variants that are orthologous to copy number variant regions in humans. Gene expression profiles were defined for mammary tissues from E2-treated ACI and ACI.BN-Emca8 rats to define the impact of Emca8 on gene expression and identify differentially expressed genes residing within Emca8.1 and Emca8.2. This study further illustrates the relevance of the ACI rat model of E2-induced mammary cancer for identifying novel genetic determinants of breast cancer susceptibility and defining the mechanisms through which estrogens contribute to breast cancer development. Cancer Prev Res; 6(1); 59-69. ©2012 AACR.
Assuntos
Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Neoplasias Mamárias Animais/genética , Alelos , Animais , Animais Congênicos , Mapeamento Cromossômico/métodos , Hibridização Genômica Comparativa , Cruzamentos Genéticos , Estradiol/metabolismo , Feminino , Dosagem de Genes , Variação Genética , Genótipo , Fenótipo , Locos de Características Quantitativas , Ratos , Fatores de TempoRESUMO
Inhibition of the insulin-like growth factor 1 receptor (Igf1r) is an approach being taken in clinical trials to overcome the dismal outcome for metastatic alveolar rhabdomyosarcoma (ARMS), an aggressive muscle cancer of children and young adults. In our study, we address the potential mechanism(s) of Igf1r inhibitor resistance that might be anticipated for patients. Using a genetically engineered mouse model of ARMS, validated for active Igf1r signaling, we show that the prototypic Igf1r inhibitor NVP-AEW541 can inhibit cell growth and induce apoptosis in vitro in association with decreased Akt and Mapk phosphorylation. However, drug resistance in vivo is more common and is accompanied by Igf1r overexpression, Mapk reactivation, and Her2 overexpression. Her2 is found to form heterodimers with Igf1r in resistant primary tumor cell cultures, and stimulation with Igf2 leads to Her2 phosphorylation. The Her2 inhibitor lapatinib cooperates with NVP-AEW541 to reduce Igf1r phosphorylation and to inhibit cell growth even though lapatinib alone has little effect on growth. These results point to the potential therapeutic importance of simultaneous targeting of Igf1r and Her2 to abrogate resistance.
Assuntos
Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Rabdomiossarcoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/patologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Lapatinib , Camundongos , Fosforilação/efeitos dos fármacos , Codorniz , Quinazolinas/farmacologia , Interferência de RNA , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Adulto JovemRESUMO
Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.
RESUMO
Genetically engineered mouse models (GEMM) of cancer are of increasing value to preclinical therapeutics. Optical imaging is a cost-effective method of assessing deep-seated tumor growth in GEMMs whose tumors can be encoded to express luminescent or fluorescent reporters, although reporter signal attenuation would be improved if animals were fur-free. In this study, we sought to determine whether hereditable furlessness resulting from a hypomorphic mutation in the Hairless gene would or would not also affect immune competence. By assessing humoral and cellular immunity of the SKH1 mouse line bearing the hypomorphic Hairless mutation, we determined that blood counts, immunoglobulin levels, and CD4+ and CD8+ T cells were comparable between SKH1 and the C57Bl/6 strain. On examination of T-cell subsets, statistically significant differences in naïve T cells (1.7 versus 3.4 x 10(5) cells/spleen in SKH1 versus C57Bl/6, P = 0.008) and memory T cells (1.4 versus 0.13 x 10(6) cells/spleen in SKH1 versus C57Bl/6, P = 0.008) were detected. However, the numerical differences did not result in altered T-cell functional response to antigen rechallenge (keyhole limpet hemocyanin) in a lymph node cell in vitro proliferative assay. Furthermore, interbreeding the SKH1 mouse line to a rhabdomyosarcoma GEMM showed preserved antitumor responses of CD56+ natural killer cells and CD163+ macrophages, without any differences in tumor pathology. The fur-free GEMM was also especially amenable to multiplex optical imaging. Thus, SKH1 represents an immune competent, fur-free mouse strain that may be of use for interbreeding to other genetically engineered mouse models of cancer for improved preclinical studies.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Engenharia Genética , Imunocompetência/imunologia , Alopecia/imunologia , Alopecia/patologia , Animais , Contagem de Células Sanguíneas , Modelos Animais de Doenças , Feminino , Imageamento Tridimensional , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunização , Subpopulações de Linfócitos/imunologia , Masculino , Camundongos , Camundongos Pelados , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologiaRESUMO
Estrogens are important regulators of growth and development and contribute to the etiology of several types of cancer. Different inbred rat strains exhibit marked, cell-type-specific differences in responsiveness to estrogens as well as differences in susceptibility to estrogen-induced tumorigenesis. Regulation of pituitary lactotroph homeostasis is one estrogen-regulated response that differs dramatically between different inbred rat strains. In this article we demonstrate that the growth response of the anterior pituitary gland of female ACI rats to 17beta-estradiol (E2) markedly exceeds that of identically treated female Brown Norway (BN) rats. We further demonstrate that pituitary mass, a surrogate indicator of absolute lactotroph number, behaves as a quantitative trait in E2-treated F(2) progeny generated in a genetic cross originating with BN females and ACI males. Composite interval mapping analyses of the (BNxACI)F(2) population revealed quantitative trait loci (QTLs) that exert significant effects on E2-induced pituitary growth on rat chromosome 4 (RNO4) (Ept5) and RNO7 (Ept7). Continuous treatment with E2 rapidly induces mammary cancer in female ACI rats but not BN rats, and QTLs that impact susceptibility to E2-induced mammary cancer in the (BNxACI)F(2) population described here have been mapped to RNO3 (Emca5), RNO4 (Emca6), RNO5 (Emca8), RNO6 (Emca7), and RNO7 (Emca4). Ept5 and Emca6 map to distinct regions of RNO4. However, Ept7 and Emca4 map to the same region of RNO7. No correlation between pituitary mass and mammary cancer number at necropsy was observed within the (BNxACI)F(2) population. This observation, together with the QTL mapping data, indicate that with the exception of the Ept7/Emca4 locus on RNO7, the genetic determinants of E2-induced pituitary growth differ from the genetic determinants of susceptibility to E2-induced mammary cancer.
Assuntos
Mapeamento Cromossômico , Cruzamentos Genéticos , Estrogênios/fisiologia , Lactotrofos/patologia , Doenças da Hipófise/genética , Adeno-Hipófise/patologia , Locos de Características Quantitativas/genética , Animais , Estradiol/fisiologia , Feminino , Predisposição Genética para Doença , Hiperplasia , Lactotrofos/metabolismo , Masculino , Doenças da Hipófise/metabolismo , Doenças da Hipófise/patologia , Adeno-Hipófise/metabolismo , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BNRESUMO
Estrogens play an important role in breast cancer etiology and the ACI rat provides a novel animal model for defining the mechanisms through which estrogens contribute to mammary cancer development. In crossing experiments between the susceptible ACI strain and two resistant strains, COP (Copenhagen) and BN (Brown Norway), several quantitative trait loci (QTL) that affect development of 17beta-estradiol (E2)-induced mammary tumors have been defined. Using comparative genomic hybridization (CGH), we have analyzed cytogenetic aberrations in E2-induced mammary cancers and have found clear patterns of nonrandom chromosomal involvement. Approximately two thirds of the tumors exhibited copy number changes. Losses of rat chromosome 5 (RNO5) and RNO20 were particularly common, and it was found that these two aberrations often occurred together. A third recurrent aberration involving proximal gain and distal loss in RNO6 probably defined a distinct subgroup of tumors, since it never occurred in combination with RNO5 loss. Interestingly, QTL with powerful effects on mammary cancer development have been mapped to RNO5 and RNO6. These findings suggest that there were at least two genetic pathways to tumor formation in this rat model of E2-induced mammary cancer. By performing CGH on mammary tumors from ACI rats, F1 rats from crosses between the ACI and COP or BN strains and ACI.BN-Emca8 congenic rats, which carry the BN allele of the Emca8 QTL on RNO5 on the ACI genetic background, we were able to determine that the constitution of the germ line influences the pattern of chromosomal aberrations.
Assuntos
Aberrações Cromossômicas/efeitos dos fármacos , Estradiol/farmacologia , Neoplasias Mamárias Experimentais/genética , Animais , Árvores de Decisões , Feminino , Mutação em Linhagem Germinativa , Cariotipagem , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/fisiopatologia , Ratos , Ratos Endogâmicos ACIRESUMO
Unilateral renal agenesis (URA) is a common developmental defect in humans, occurring at a frequency of approximately 1 in 500-1,000 births. Several genetic syndromes include bilateral or unilateral renal agenesis as an associated phenotype. However, URA frequently occurs in individuals not afflicted by these syndromes and is often asymptomatic. Although it is clear that genetic factors contribute to the etiology of URA, the genetic bases of URA are poorly defined at this time. ACI rats, both males and females, exhibit URA at an incidence of 5%-15%. In this article we characterize the incidence of URA in female and male F(1), F(2), and backcross (BC) progeny from reciprocal genetic crosses between the ACI strain and the unaffected Brown Norway (BN) strain. Through interval mapping analyses of 353 phenotypically defined female F(2) progeny, we mapped to rat Chromosome 14 (RNO14) a genetic locus, designated Renag1 (Renal agenesis 1), that serves as the major determinant of URA in these crosses. Further genotypic analyses of URA-affected female and male F(2) and BC progeny localized Renag1 to a 14.4-Mb interval on RNO14 bounded by markers D14Rat50 and D14Rat12. The data from these genetic studies suggest that the ACI allele of Renag1 acts in an incompletely dominant and incompletely penetrant manner to confer URA.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Mamíferos/genética , Rim/anormalidades , Ratos Endogâmicos ACI/genética , Animais , Feminino , Masculino , Ratos , Ratos Endogâmicos BN/genéticaRESUMO
In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17beta-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August x Copenhagen Irish (ACI) strain. In a (BN x ACI)F(2) population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN x ACI)F(2) population.
Assuntos
Cromossomos de Mamíferos/genética , Endometrite/induzido quimicamente , Endometrite/genética , Estradiol/toxicidade , Predisposição Genética para Doença , Ratos Endogâmicos BN/genética , Animais , Mapeamento Cromossômico , Feminino , Masculino , Ratos , Ratos Endogâmicos ACIRESUMO
BACKGROUND: : Previously, we have observed that highly unsaturated dietary (n-3) fatty acids inhibit cell proliferation in conjunction with stimulation of insulin-like growth factor-binding protein (IGFBP)-6 secretion in Caco-2 cells, a human colon carcinoma cell line. METHODS: : To test the converse hypothesis that inhibition of endogenous IGFBP-6 secretion stimulates Caco-2 cell proliferation, cells were transfected with the antisense IGFBP-6 expression construct or pcDNA3 vector only, and single colonies resistant to G418 sulfate were isolated. RESULTS: : Our initial studies indicated that three antisense clones grew faster and produced less IGFBP-6 than two pcDNA3 clones, so antisense IGFBP-6 #5 and pcDNA3 #8 were selected for further detailed analysis. Both the control and antisense clones grew in serum-free medium reaching a plateau density at day eight. However, the antisense clone grew at a rate faster than that of the control and reached a final density that was 31 +/- 3% higher than the control. Northern blot, ligand blot and immunoblot analyses revealed that accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide produced by the antisense clone were decreased by 80-90% compared to the control. The doubling times of the antisense and control clones were 21.9 +/- 0.4 and 24.8 +/- 0.3 h (P < 0.05), respectively. Exogenous IGF-I and IGF-II (0.2-200 nmol/L) stimulated proliferation of both the control and antisense clones in a dose-dependent manner, but the relative potency and efficacy of IGF-II was higher in the antisense clone compared to the control. These results indicate that suppression of IGFBP-6 secretion correlates with an increase in the basal rate of Caco-2 cell growth. CONCLUSIONS: : Our findings are consistent with the hypothesis that IGFBP-6 inhibits cell growth by binding to endogenously produced IGF-II, thereby preventing IGF-II from interacting with the IGF-I receptor to stimulate cellular proliferation by an autocrine mechanism.