RESUMEN
Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.
Asunto(s)
División Celular , Aberraciones Cromosómicas , Células Madre Embrionarias Humanas/citología , Selección Genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 17 , Variaciones en el Número de Copia de ADN , Humanos , Hidroxiurea , Estrés Fisiológico , TranscriptomaRESUMEN
Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Q(sp)) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Q(sp) in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Q(sp) in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.
Asunto(s)
Proteínas de Transporte de Anión/genética , Células Ciliadas Auditivas Externas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Encéfalo/metabolismo , Cóclea/anatomía & histología , Cóclea/efectos de los fármacos , Cóclea/fisiología , Colchicina/farmacología , Cartilla de ADN , Transferencia Resonante de Energía de Fluorescencia , Gerbillinae , Cobayas , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/efectos de los fármacos , Especificidad de Órganos , Órgano Espiral/fisiología , Reacción en Cadena de la Polimerasa , Proteínas/genética , Proteínas/metabolismo , Ratas , Transportadores de SulfatoRESUMEN
Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We show that Vpus from SIVgsn, SIVmus, and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpus from nonpandemic HIV-1 O strains are poor tetherin antagonists, whereas those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the global HIV/AIDS pandemic.