RESUMO
We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Sequência de Bases , Interferon gama , Nefrite Lúpica/imunologia , Deleção de Sequência , Animais , Anticorpos Antinucleares/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interferon gama/genética , Interferon gama/imunologia , Nefrite Lúpica/genética , Macrófagos/imunologia , Macrófagos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos KnockoutRESUMO
The G(1)/S-phase transition is a well-toned switch in the mammalian cell cycle. Cdk2, Cdk4, and the rate-limiting tumor suppressor retinoblastoma protein (Rb) have been studied in separate animal models, but interactions between the kinases and Rb in vivo have yet to be investigated. To further dissect the regulation of the G(1) to S-phase progression, we generated Cdk2(-/-)Cdk4(-/-)Rb(-/-) (TKO) mutant mice. TKO mice died at midgestation with major defects in the circulatory systems and displayed combined phenotypes of Rb(-/-) and Cdk2(-/-)Cdk4(-/-) mutants. However, TKO mouse embryonic fibroblasts were not only resistant to senescence and became immortal but displayed enhanced S-phase entry and proliferation rates similar to wild type. These effects were more remarkable in hypoxic compared with normoxic conditions. Interestingly, depletion of the pocket proteins by HPV-E7 or p107/p130 shRNA in the absence of Cdk2/Cdk4 elicited a mechanism for the G(1)/S regulation with increased levels of p27(Kip1) binding to Cdk1/cyclin E complexes. Our work indicates that the G(1)/S transition can be controlled in different ways depending on the situation, resembling a regulatory network.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/deficiência , Quinase 4 Dependente de Ciclina/deficiência , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fibroblastos/citologia , Interfase/genética , Proteína do Retinoblastoma/deficiência , Animais , Proliferação de Células , Células Cultivadas , Senescência Celular/genética , Fase G1 , Hipóxia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/fisiologia , Fase SRESUMO
Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.
Assuntos
Quinase 2 Dependente de Ciclina/deficiência , Quinase 4 Dependente de Ciclina/deficiência , Embrião de Mamíferos/anormalidades , Proteína do Retinoblastoma/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Quinase 2 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Fatores de Transcrição E2F/antagonistas & inibidores , Fibroblastos/citologia , Inativação Gênica , Hematopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , Fenótipo , Fosforilação , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/químicaRESUMO
Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2(-)(/)(-) and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2(-)(/)(-) quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.
Assuntos
Apoptose , Quinase 2 Dependente de Ciclina/deficiência , Hematopoese/fisiologia , Linfócitos T/citologia , Animais , Células da Medula Óssea/citologia , Camundongos , Baço/citologia , Células-Tronco/citologiaRESUMO
We generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a latent constitutively active TGFbeta type I receptor (TbetaRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken beta-actin) promoter, is repressed by a floxed transcriptional "Stop" (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the "Stop" is excised to allow TbetaRI(CA) transgene expression. We showed that restricted expression of TbetaRI(CA) in T lymphocytes efficiently activates TGFbeta signaling and rescues the T-cell autoimmune disorders of TGFbetaRII conditional knockouts. Unexpectedly, our study reveals that TGFbeta signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFbeta effects on T-cell biology, LSL-TbetaRI(CA) mouse constitutes an attractive tool to address the effect of TGFbeta signaling upregulation in any cell type expressing the cre-recombinase.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Ativação Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Knockout , Receptor do Fator de Crescimento Transformador beta Tipo I , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Ischemia/reperfusion (I/R) injury to the heart is accompanied by the upregulation and posttranslational modification of a number of proteins normally involved in regulating cell cycle progression. Two such proteins, cyclin-dependent kinase-2 (Cdk2) and its downstream target, the retinoblastoma gene product (Rb), also play a critical role in the control of apoptosis. Myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size (IFS), when compared to control mice, associated with a reduction in apoptotic myocytes. To confirm that Rb was the critical target in Cdk2-mediated I/R injury, we determined the consequences of I/R injury in cardiac-specific Rb-deficient mice (CRb(L/L)). IFS was increased 140% in CRb(L/L) mice compared to CRb+/+ controls. TUNEL positive nuclei and caspase-3 activity were augmented by 92% and 36%, respectively, following injury in the CRb(L/L) mice demonstrating that loss of Rb in the heart significantly exacerbates I/R injury. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo and support a cardioprotective role for Rb.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Animais , Apoptose , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Isquemia Miocárdica/patologia , Ratos , Proteína do Retinoblastoma/metabolismoRESUMO
In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27(kip1) or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27(-/-)) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27(+/+) mice. Moreover, in p27(-/-) ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27(-/-) ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27(-/-) ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Folículo Ovariano/metabolismo , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Inibidor de Quinase Dependente de Ciclina p27/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , CoelhosRESUMO
BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.
Assuntos
Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclo Celular/fisiologia , Células Germinativas/citologia , Meiose/fisiologia , Animais , Quinases relacionadas a CDC2 e CDC28/fisiologia , Mapeamento Cromossômico , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Gônadas/citologia , Técnicas Histológicas , Histonas/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Sonda Molecular , Testes de Precipitina , Baço/químicaRESUMO
The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.
Assuntos
Proteínas/fisiologia , Espermatogênese , Animais , Exorribonucleases , Células Germinativas/patologia , Haploidia , Imuno-Histoquímica , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Fenótipo , Proteínas/genética , Proteínas Repressoras , Ribonucleases , Túbulos Seminíferos/patologia , Células de Sertoli/patologia , Fatores de TranscriçãoRESUMO
It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina E/fisiologia , Fase S/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Camundongos , Camundongos KnockoutRESUMO
PURPOSE: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. EXPERIMENTAL DESIGN: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. RESULTS: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. CONCLUSIONS: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Fluoruracila/administração & dosagem , Regulação Neoplásica da Expressão Gênica , Humanos , Irinotecano , Masculino , Camundongos , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , RatosRESUMO
The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G(1) deregulation or a combination of these effects. In order to determine the role of proteins involved in G(1) control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G(1)/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2(-/-) p53(-/-) mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53(-/-) mice. In contrast, the genotypes Cdk4(-/-) p53(-/-) were mostly lethal, with few exceptions, and Cdk2(-/-) Cdk4(-/-) p53(-/-) mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53(-/-), Cdk2(-/-) p53(-/-), Cdk4(-/-) p53(-/-), and Cdk2(-/-) Cdk4(-/-) p53(-/-) MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/fisiologia , Transformação Celular Neoplásica , Quinase 2 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclinas/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Transplante de Neoplasias , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genéticaRESUMO
Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle-associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.
Assuntos
Apoptose , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Mitocôndrias/fisiologia , Mutação , Proteínas Serina-Treonina Quinases/genética , Animais , Transporte Biológico/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células , Ceramidas/metabolismo , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Genótipo , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Organogênese/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de SinaisRESUMO
Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2(-/-) mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication and survival. Cdk2(-/-) hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2(-/-) liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2(-/-) hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2(-/-) mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.
Assuntos
Ciclo Celular , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/fisiologia , Hepatócitos/enzimologia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Centrossomo/metabolismo , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Regeneração Hepática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Purinas/farmacologia , Roscovitina , Fatores de Tempo , TransfecçãoRESUMO
It was believed that Cdk2-cyclin E complexes are essential to drive cells through the G1-S phase transition. However, it was discovered recently that the mitotic kinase Cdk1 (Cdc2a) compensates for the loss of Cdk2. In the present study, we tested whether Cdk2 can compensate for the loss of Cdk1. We generated a knockin mouse in which the Cdk2 cDNA was knocked into the Cdk1 locus (Cdk1Cdk2KI). Substitution of both copies of Cdk1 by Cdk2 led to early embryonic lethality, even though Cdk2 was expressed from the Cdk1 locus. In addition, we generated Cdk2-/- Cdk1+/Cdk2KI mice in which one copy of Cdk2 and one copy of Cdk1 were expressed from the Cdk1 locus and the Cdk2 gene was deleted from the endogenous Cdk2 locus. We found that both male and female Cdk2-/- Cdk1+/Cdk2KI mice were sterile, similar to Cdk2-/- mice, even though they expressed the Cdk2 protein from the Cdk1 locus in testes. The translocational and cell cycle properties of knockin Cdk2 in Cdk2-/- Cdk1+/Cdk2KI cells were comparable to those of endogenous Cdk2, but we detected premature transcriptional activation of Cdk1 during liver regeneration in the absence of Cdk2. This study provides evidence of the molecular differences between Cdk2 and Cdk1 and highlights that the timing of transcriptional activation and the genetic locus play important roles in determining the function of Cdk proteins in vivo.
Assuntos
Proteína Quinase CDC2/genética , Quinase 2 Dependente de Ciclina/genética , Genes Letais , Meiose/genética , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Quinase 2 Dependente de Ciclina/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Imuno-Histoquímica , Masculino , CamundongosRESUMO
We investigated the function of cyclin-dependent kinase 2 (Cdk2) in neural progenitor cells during postnatal development. Chondroitin sulfate proteoglycan (NG2)-expressing progenitor cells of the subventricular zone (SVZ) show no significant difference in density and proliferation between Cdk2(-/-) and wild-type mice at perinatal ages and are reduced only in adult Cdk2(-/-) mice. Adult Cdk2(-/-) SVZ cells in culture display decreased self-renewal capacity and enhanced differentiation. Compensatory mechanisms in perinatal Cdk2(-/-) SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2(-/-) cells coincides with lower NG2+ proliferation and self-renewal capacity similar to adult levels. Cdk4 silencing in perinatal Cdk2(-/-) SVZ cells abolishes Cdk4 up-regulation and reduces cell proliferation and self- renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal through age-dependent regulation of Cdk4.
Assuntos
Ventrículos Cerebrais/citologia , Quinase 2 Dependente de Ciclina/fisiologia , Neurônios/citologia , Células-Tronco/citologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Ventrículos Cerebrais/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Quinase 4 Dependente de Ciclina/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco/metabolismoRESUMO
Granule neurons of the dentate gyrus (DG) of the hippocampus undergo continuous renewal throughout life. Among cell cycle regulators, cyclin-dependent kinase 2 (Cdk2) is considered as a major regulator of S-phase entry. We used Cdk2-deficient mice to decipher the requirement of Cdk2 for the generation of new neurons in the adult hippocampus. The quantification of cell cycle markers first revealed that the lack of Cdk2 activity does not influence spontaneous or seizure-induced proliferation of neural progenitor cells (NPC) in the adult DG. Using bromodeoxyuridine incorporation assays, we showed that the number of mature newborn granule neurons generated de novo was similar in both wild-type (WT) and Cdk2-deficient adult mice. Moreover, the apparent lack of cell output reduction in Cdk2(-/-) mice DG did not result from a reduction in apoptosis of newborn granule cells as analyzed by TUNEL assays. Our results therefore suggest that Cdk2 is dispensable for NPC proliferation, differentiation and survival of adult-born DG granule neurons in vivo. These data emphasize that functional redundancies between Cdks also occur in the adult brain at the level of neural progenitor cell cycle regulation during hippocampal neurogenesis.
Assuntos
Quinase 2 Dependente de Ciclina/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Animais , Apoptose , Diferenciação Celular , Quinase 2 Dependente de Ciclina/genética , Giro Denteado/citologia , Giro Denteado/enzimologia , Giro Denteado/fisiologia , Hipocampo/citologia , Hipocampo/enzimologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Progression through the mammalian cell cycle is associated with the activity of four cyclin dependent kinases (Cdc2/Cdk1, Cdk2, Cdk4, and Cdk6). Knockout mouse models have provided insight into the interplay of these Cdks. Most of these models do not exhibit major cell cycle defects revealing redundancies, and suggesting that a single Cdk might be sufficient to drive the cell cycle, similar as in yeast. Recent work on Cdk2/Cdk4 double knockouts has indicated that these two Cdks are required to phosphorylate Rb during late embryogenesis. The lack of Rb phosphorylation is progressive and associated with reduced E2F-inducible gene expression. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis. However, proliferation in early embryogenesis appears to be independent of Cdk2 and Cdk4. We discuss these observations and propose molecular mechanisms that establish the requirement for Cdk2 and Cdk4 at the G1/S transition. We are considering that the balance between proliferation and differentiation is disturbed, which affects especially heart development and leads to embryonic lethality in Cdk2-/- Cdk4-/- mutants. We also discuss the specific functions of Cdk4 and Cdk6, which ironically do not compensate for each other.
RESUMO
In the vertebrate genome, methylation of deoxycytosine residues of CpGs dinucleotide has been associated with transcriptional silencing of genes, parental imprinting, X-inactivation and chromatin remodelling. In human somatic tissues, the 5' end of the BRCA1 CpG island is methylated, whereas this region is unmethylated in mature germ cells and early embryos. In gametes, as in somatic tissues, the CpG sites in the coding region are methylated. We took advantage of this bimodal distribution as a model to analyse the epigenetic reprogramming of coding regions during early human embryogenesis using the bisulphite-based genomic sequencing method. During preimplantation divisions, exon 11 of BRCA1 was slowly demethylated and retained approximately 30% of its methylated residues at the blastocyst stage. Moreover, the change in the distribution of methylated residues was not restricted to the BRCA1 gene, since for another gene, p53, a relatively high level of methylation (50%) of exon 4 was observed in blastocysts. Taken together, these data suggest that a significant part of the methylated residues of coding sequences might be conserved during preimplantation development.
Assuntos
Blastocisto/metabolismo , Metilação de DNA , Genes BRCA1 , Proteína Supressora de Tumor p53/genética , Animais , Ilhas de CpG/genética , Desenvolvimento Embrionário e Fetal/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cinética , Masculino , Camundongos , Oócitos/metabolismo , Espermatozoides/metabolismoRESUMO
BACKGROUND: Several recent reports have connected protein methylation with differentiation. Furthermore, the BTG/TOB proteins have also been implicated in such control. BTG1 and 2 have been shown to interact with PRMT1 (predominant cellular arginine N-methyltransferase of type I). RESULTS: First, we have studied the interaction between PRMT1 and the proteins of the BTG/TOB family. We show that boxC, a sequence present only in BTG1 and BTG2, is essential for this association. Using boxC peptide, we have investigated the importance of PRMT1/BTG protein association during type I protein methylation reactions. Finally, we show that the addition of boxC fused to penetratin interferes with the neuronal differentiation of PC12 cells and ES cell-derived neurones. CONCLUSIONS: Taken together, these results indicate that PRMT1/BTG proteins could play a key role in the arginine methylation-mediated signalling pathway as well as in neuronal differentiation.