In vivo regulation of the early embryonic cell cycle in Xenopus

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.     

The lurcher mutation and ionotropic glutamate receptors: contributions to programmed neuronal death in vivo

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

Cloning and characterization of the Xenopus cyclin-dependent kinase inhibitor p27XIC1

We have isolated a gene encoding Xic-1, a 27-kDa cyclin-dependent kinase (Cdk) inhibitor from Xenopus ovary that shares significant homology with both mammalian CIP1 and Kip1/Kip2. The N- and C-terminal halves of Xic-1 are sufficient for interacting with Cdks and proliferating cell nuclear antigen, respectively. Recombinant Xic-1 inhibits Xenopus cyclin E/Cdk2, cyclin A/Cdk2 and cyclin B/Cdc2 activities, although with quite different IC50 values. Truncation of the N terminus of Xic-1 increases the IC50 value for cyclin A/Cdk2 50-fold with no effect on the inhibition of cyclin E/Cdk2 or cyclin B/Cdc2.Xic-1 inhibits both single-stranded and nuclear DNA synthesis in egg extracts, an effect reversed by proliferating cell nuclear antigen or cyclin E/Cdk2, respectively. These results suggest a function for Xic-1 in the control of DNA synthesis by cyclin E/Cdk2.     

Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.     

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.     

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.     

The risk of persistent paresthesia is not increased with repeated axillary block

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.     

Cyclin D2 arrests Xenopus early embryonic cell cycles

Xenopus cyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. When Xenopus cyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However, Xenopus cyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced by Xenopus cyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. Radiolabeled Xenopus cyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest that Xenopus cyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.     

Transcription factor E2F and cyclin E-Cdk2 complex cooperate to induce chromosomal DNA replication in Xenopus oocytes

Although no chromosomal DNA replication actually occurs during Xenopus oocyte maturation, the capability develops during the late meiosis I (MI) phase in response to progesterone. This ability, however, is suppressed by Mos proteins and maturation/mitosis promoting factor during the second meiosis phase (meiosis II; MII) until fertilization. Inhibition of RNA synthesis by actinomycin D during early MI prevented induction of the replication ability, but did not interfere with initiation of the meiotic cell cycle progression characterized by oscillation of the maturation/mitosis promoting factor activity and germinal vesicle breakdown. Microinjection of recombinant proteins such as dominant-negative E2F or universal Cdk inhibitors, p21 and p27, but not wild type human E2F-1 or Cdk4-specific inhibitor, p19, into maturing oocytes during MI abolished induction of the DNA replication ability. Co-injection of human E2F-1 and cyclin E proteins into immature oocytes allowed them to initiate DNA replication even in the absence of progesterone treatment. Injection of cyclin E alone, which was sufficient to activate endogenous Cdk2 kinase, failed to induce DNA replication. Moreover, the activation of Cdk2 was not affected under the conditions where DNA replication was blocked by actinomycin D. Thus, like somatic cells, both activities of E2F and cyclin E-Cdk2 complex are required for induction of the DNA replication ability in maturing Xenopus oocytes, and enhancement of both activities enables oocytes to override DNA-replication inhibitory mechanisms that specifically lie in maturing oocytes.     

Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells

The G1 cyclins, cyclin D1 and E, are rate limiting for progression through G1 phase of the cell cycle in breast epithelial cells and are oncogenic when expressed in the mammary epithelium of transgenic mice. These genes are frequently overexpressed in clinical breast cancer where overexpression appears to be associated with specific disease phenotypes, altered responsiveness to therapeutic intervention and patient survival. In order to investigate the functional correlates of cyclin D1 and cyclin E overexpression we employed a panel of normal, immortalized and neoplastic breast epithelial cell lines to examine the relationships between cyclin gene expression, cyclin-CDK complex formation and CDK activity. In agreement with earlier studies cyclin D1 and E expression varied over an approximately tenfold range among the 18 cell lines studied. There was no apparent relationship, however, between cyclin D1 expression and the in vitro activity of its major kinase partner, Cdk4, although MDA-MB-134 cells displayed the highest level of both cyclin D1 expression and Cdk4 activity. Similarly, there was no significant relationship between cyclin E expression and cyclin E-Cdk2 activity. Fractionation of whole cell lysates by gel filtration chromatography revealed that approximately 90% of the cyclin E protein was present in inactive complexes containing the CDK inhibitors p21 and p27. Much of the small fraction of active cyclin E protein was of very high apparent molecular mass, >400 kDa, suggesting that formation of these complexes is a more important determinant of cyclin E-Cdk2 activity than cyclin E abundance. These data suggest that properties of cyclins D1 and E in addition to their ability to activate Cdk4 and Cdk2 may contribute to the effects of overexpression on the breast cancer phenotype.     

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.     

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.     

Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins

The passage of mammalian cells through the restriction point into the S phase of the cell cycle is regulated by the activities of Cdk4 and Cdk6 complexed with the D-type cyclins and by cyclin E/Cdk2. The activities of these holoenzymes are constrained by CDK inhibitory proteins. The importance of the restriction point is illustrated by its deregulation in many tumour cells and upon infection with DNA tumour viruses. Here we describe the properties of cyclins encoded by two herpesviruses, herpesvirus saimiri (HVS) which can transform blood lymphocytes and induce malignancies of lymphoid origin in New World primates, and human herpesvirus 8 (HHV8) implicated as a causative agent of Kaposi's sarcoma and body cavity lymphomas. Both viral cyclins form active kinase complexes with Cdk6 that are resistant to inhibition by the CDK inhibitors p16(Ink4a), p21Cip1 and p27Kip1. Furthermore, ectopic expression of a viral cyclin prevents G1 arrest imposed by each inhibitor and stimulates cell-cycle progression in quiescent fibroblasts. These results suggest a new mechanism for deregulation of the cell cycle and indicate that the viral cyclins may contribute to the oncogenic nature of these viruses.     

Identification of a new coiled body component

The cyclin-dependent kinase (Cdk) inhibitor p27 interrupts progression of the cell cycle by inhibiting various cyclin/Cdk activities. Since the protein level of p27 does not correlate with its mRNA level or protein synthesis rate in most cases, it is suggested that degradation of the protein may be regulated via an unidentified mechanism(s) involving a post-translational modification(s). We present evidence here that p27 phosphorylation is cell cycle-dependent and peaks in the late G1 phase and that the level of p27 protein is inversely correlated with its phosphorylation. Although both cyclin D1- and cyclin-E-dependent kinases are active in the late G1 phase in human fibroblasts, cyclin E/Cdk2 specifically phosphorylates p27 on threonine-187 in vitro. Interestingly, ectopic expression of T187A revealed that it was far more stable in vivo than wild type p27. Thus, phosphorylation of p27 by cyclin E/ Cdk2 may affect the stability of its protein and play a role in how the protein functions.     

Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- promoting complex at mitosis

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction in Xenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.