RESUMO
Neonatal treatment of hamsters with diethylstilbestrol (DES) induces uterine hyperplasia/dysplasia/neoplasia (endometrial adenocarcinoma) in adult animals. We subsequently determined that the neonatal DES exposure event directly and permanently disrupts the developing hamster uterus (initiation stage) so that it responds abnormally when it is stimulated with estrogen in adulthood (promotion stage). To identify candidate molecular elements involved in progression of the disruption/neoplastic process, we performed: (1) immunoblot analyses and (2) microarray profiling (Affymetrix Gene Chip System) on sets of uterine protein and RNA extracts, respectively, and (3) immunohistochemical analysis on uterine sections; all from both initiation stage and promotion stage groups of animals. Here we report that: (1) progression of the neonatal DES-induced hyperplasia/dysplasia/neoplasia phenomenon in the hamster uterus involves a wide spectrum of specific gene expression alterations and (2) the gene products involved and their manner of altered expression differ dramatically during the initiation vs. promotion stages of the phenomenon.
Assuntos
Dietilestilbestrol/toxicidade , Neoplasias Uterinas/induzido quimicamente , Útero/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Caderinas/fisiologia , Feminino , Perfilação da Expressão Gênica , Cobaias , Hiperplasia , Proteínas Substratos do Receptor de Insulina/análise , Mesocricetus , Oncogenes , Gravidez , Antígeno Nuclear de Célula em Proliferação/análise , Receptores Androgênicos/análise , Neoplasias Uterinas/metabolismo , Útero/metabolismo , Útero/patologiaRESUMO
The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Resistência ao Cisalhamento , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fibrinogênio/farmacologia , Hemorreologia/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/deficiência , Adesividade Plaquetária/efeitos dos fármacos , Resistência ao Cisalhamento/efeitos dos fármacos , Trombose/metabolismo , Trombose/patologia , Trombose/fisiopatologia , Fatores de TempoRESUMO
We assessed neonatal diethylstilbestrol (DES)-induced disruption at various endocrine levels in the hamster. In particular, we used organ transplantation into the hamster cheek pouch to determine whether abnormalities observed in the post-pubertal ovary are due to: (a) a direct (early) mechanism or (b) an indirect (late) mechanism that involves altered development and function of the hypothalamus and/or pituitary. Of the various disruption endpoints and attributes assessed: (1) some were consistent with the direct mechanism (altered uterine and cervical dimensions/organization, ovarian polyovular follicles, vaginal hypospadius, endometrial hyperplasia/dysplasia); (2) some were consistent with the indirect mechanism (ovarian/oviductal salpingitis, cystic ovarian follicles); (3) some were consistent with a combination of the direct and indirect mechanisms (altered endocrine status); and (4) the mechanism(s) for one (lack of corpora lutea) was uncertain. This study also generated some surprising observations regarding vaginal estrous assessments as a means to monitor periodicity of ovarian function in the hamster.
Assuntos
Animais Recém-Nascidos , Dietilestilbestrol/toxicidade , Estrogênios não Esteroides/toxicidade , Genitália Feminina/efeitos dos fármacos , Animais , Colo do Útero/anatomia & histologia , Cricetinae , Ciclo Estral , Tubas Uterinas/anatomia & histologia , Tubas Uterinas/efeitos dos fármacos , Tubas Uterinas/fisiologia , Feminino , Genitália Feminina/anatomia & histologia , Genitália Feminina/fisiologia , Hormônios/sangue , Hipotálamo/fisiologia , Mesocricetus , Ovariectomia , Ovário/anatomia & histologia , Ovário/fisiologia , Ovário/transplante , Hipófise/fisiologia , Gravidez , Maturidade Sexual , Útero/anatomia & histologia , Vagina/fisiologiaRESUMO
BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)ß(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.