RESUMO
Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.
Assuntos
Transformação Celular Neoplásica/genética , Dano ao DNA/genética , Serina Endopeptidases/fisiologia , Aminopeptidases , Animais , Sobrevivência Celular/genética , Transformação Celular Neoplásica/patologia , Dipeptidil Peptidases e Tripeptidil Peptidases , Fibroblastos/enzimologia , Fibroblastos/patologia , Genes myc , Genes ras , Camundongos , Camundongos Knockout , Serina Endopeptidases/genéticaRESUMO
In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.
Assuntos
Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/fisiologia , Fuso Acromático/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Feminino , Proteínas Mad2 , Meiose , Camundongos , Camundongos Endogâmicos , Mutação , Nocodazol/farmacologia , Proteínas Nucleares , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-mos/metabolismo , Fuso Acromático/química , Fuso Acromático/genética , Estrôncio/farmacologia , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/fisiologiaRESUMO
Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.
Assuntos
Metilação de DNA , DNA/genética , Histonas/metabolismo , Injeções de Esperma Intracitoplásmicas , Espermatozoides/fisiologia , Zigoto/fisiologia , Animais , Núcleo Celular , Feminino , Fertilização , Fertilização in vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Protaminas/metabolismo , Cabeça do Espermatozoide/fisiologiaRESUMO
Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b)-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.
Assuntos
Ciclo Celular/fisiologia , Reprogramação Celular/fisiologia , Aminoácidos/metabolismo , Animais , Arginina/metabolismo , Blastocisto/metabolismo , Análise por Conglomerados , Dano ao DNA , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Imagem com Lapso de Tempo , TransgenesRESUMO
BACKGROUND AND PURPOSE: Stem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors. MATERIALS AND METHODS: Primary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots. RESULTS: SLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe. CONCLUSIONS: Our results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53-deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumor cells.
Assuntos
Neoplasias Encefálicas/radioterapia , Glioma/radioterapia , Mitose/efeitos da radiação , Células-Tronco Neoplásicas/efeitos da radiação , Apoptose , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Sobrevivência Celular , Dano ao DNA/efeitos da radiação , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Raios gama , Glioma/patologia , Histonas/metabolismo , Humanos , Tolerância a Radiação , Radiação IonizanteRESUMO
BACKGROUND: Cancer stem cells are thought to play a pivotal role in tumor maintenance, metastasis, tumor therapy resistance and relapse. Hence, the development of methods for non-invasive in vivo detection of cancer stem cells is of great importance. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe successful in vivo detection of CD133/prominin, a cancer stem cell surface marker for a variety of tumor entities. The CD133-specific monoclonal antibody AC133.1 was used for quantitative fluorescence-based optical imaging of mouse xenograft models based on isogenic pairs of CD133 positive and negative cell lines. A first set consisted of wild-type U251 glioblastoma cells, which do not express CD133, and lentivirally transduced CD133-overexpressing U251 cells. A second set made use of HCT116 colon carcinoma cells, which uniformly express CD133 at levels comparable to primary glioblastoma stem cells, and a CD133-negative HCT116 derivative. Not surprisingly, visualization and quantification of CD133 in overexpressing U251 xenografts was successful; more importantly, however, significant differences were also found in matched HCT116 xenograft pairs, despite the lower CD133 expression levels. The binding of i.v.-injected AC133.1 antibodies to CD133 positive, but not negative, tumor cells isolated from xenografts was confirmed by flow cytometry. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show that non-invasive antibody-based in vivo imaging of tumor-associated CD133 is feasible and that CD133 antibody-based tumor targeting is efficient. This should facilitate developing clinically applicable cancer stem cell imaging methods and CD133 antibody-based therapeutics.
Assuntos
Antígenos CD/biossíntese , Antígenos CD/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Animais , Citometria de Fluxo/métodos , Glioma/metabolismo , Humanos , Hibridomas/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas , RecidivaRESUMO
The giant cytosolic protease tripeptidyl peptidase II (TPPII) was recently proposed to play a role in the DNA damage response. Shown were nuclear translocation of TPPII after gamma-irradiation, lack of radiation-induced p53 stabilization in TPPII-siRNA-treated cells, and complete tumor regression in mice after gamma-irradiation when combined with TPPII-siRNA silencing or a protease inhibitor reported to inhibit TPPII. This suggested that TPPII could be a novel target for tumor radiosensitization and prompted us to study radiation responses using TPPII-knockout mice. Neither the sensitivity to total body irradiation nor the radiosensitivity of resting lymphoid cells, which both strongly depend on p53, was altered in the absence of TPPII. Functional integrity of p53 in TPPII-knockout cells is further shown by a proper G(1) arrest and by the accumulation of p53 and its transcriptional targets, p21, Bax, and Fas, on gamma-irradiation. Furthermore, we could not confirm radiation-induced nuclear translocation of TPPII. Nevertheless, after gamma-irradiation, we found slightly increased mitotic catastrophe of TPPII-deficient primary fibroblasts and increased apoptosis of TPPII-deficient activated CD8(+) T cells. The latter was accompanied by delayed resolution of the DNA double-strand break marker gammaH2AX. This could, however, be due to increased apoptotic DNA damage rather than reduced DNA damage repair. Our data do not confirm a role for TPPII in the DNA damage response based on nuclear TPPII translocation and p53 stabilization but nevertheless do show increased radiation-induced cell death of selected nontransformed cell types in the absence of the TPPII protease.
Assuntos
Serina Endopeptidases/deficiência , Proteína Supressora de Tumor p53/metabolismo , Aminopeptidases , Animais , Núcleo Celular/metabolismo , Dano ao DNA , Dipeptidil Peptidases e Tripeptidil Peptidases , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Fase G1/fisiologia , Fase G1/efeitos da radiação , Raios gama , Histonas/metabolismo , Linfócitos/enzimologia , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Tolerância a Radiação , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Irradiação Corporal TotalRESUMO
During meiotic maturation, the majority of oocytes from LT/Sv mice arrest at metaphase I. However, anaphase may be induced through parthenogenetic activation. If this happens within the ovary, it often results in the development of ovarian teratomas. Here, we show that the induction of first meiotic anaphase in LT/Sv oocytes results in incorrect chromosome segregation. In search of the molecular basis of this complex phenotype, we analyzed the localization/destruction of cohesins, as well as the function of the components of the spindle assembly checkpoint (SAC). Both localization and removal of meiotic cohesin REC8 from chromosomes are unperturbed. In contrast, there is prolonged localization of SAC proteins BUB1 and MAD2L1 (MAD2) at the metaphase I kinetochores in mutant oocytes compared with the wild-type. Interfering with BUB1 function through expression of a dominant-negative mutant protein resulted in the increase of the number of LT/Sv oocytes completing the first meiosis, which indicates SAC involvement in metaphase I arrest. These data show for the first time that there is a direct link between the SAC function and the heritable meiotic incompetence of a mammalian oocyte.
Assuntos
Metáfase/fisiologia , Oócitos/fisiologia , Fuso Acromático/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Anáfase/fisiologia , Animais , Antineoplásicos/farmacologia , Calcimicina/farmacologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos/fisiologia , Cromossomos/efeitos dos fármacos , Cromossomos/ultraestrutura , Feminino , Imunofluorescência , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , RNA/biossíntese , RNA/genética , Fuso Acromático/ultraestrutura , CoesinasRESUMO
We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Núcleo Celular/fisiologia , Ciclina B/biossíntese , Ciclina B/genética , Meiose , Oócitos/fisiologia , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Células Cultivadas , Ciclina B1 , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fator Promotor de Maturação/metabolismo , Meiose/genética , Mesotelina , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , RNA Mensageiro/biossínteseRESUMO
We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.