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The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.

The tumor suppressor p21 regulates cell cycle progression and peaks at mid/late G1. However, the mechanisms regulating its expression during cell cycle are poorly understood. We found that embryonic fibroblasts from p27 null mice at early passages progress slowly through the cell cycle. These cells present an elevated basal expression of p21 suggesting that p27 participates to its repression. Mechanistically, we found that p27 represses the expression of Pitx2 (an activator of p21 expression) by associating with the ASE-regulatory region of this gene together with an E2F4 repressive complex. Furthermore, we found that Pitx2 binds to the p21 promoter and induces its transcription. Finally, silencing Pitx2 or p21 in proliferating cells accelerates DNA replication and cell cycle progression. Collectively, these results demonstrate an unprecedented connection between p27, Pitx2 and p21 relevant for the regulation of cell cycle progression and cancer and for understanding human pathologies associated with p27 germline mutations.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies., This work was supported by grants from Fundación Tatiana Pérez de Guzman el Bueno, from Ministerio de Economia y competitividad (MINECO) SAF2015- 64244-R, La Caixa2016-052.407 and from the Instituto de Salud Carlos III CB16/12/00244. AB is supported by grants from the Fondation ARC pour la Reserche sur le Cancer, Ligue National contre le Cancer and Institut National du Cancer. Work in the laboratory of IF is supported by Fundación Botín-Banco Santander.

Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.

Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40 S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF., Ministerio de Economía y Competitividad (MINECO) [SAF2012-38078 to O.B. and BFU2015-65311-R to R.A.]; Instituto de Salud Carlos III [RD12/0036/0054 to O.B. and CB16/12/00244 (CIBERONC) to A.B.]; ‘La Caixa’ Foundation 2016-052407 (to R.A.). Funding for open access charge: MInisterio the Economia y Competitividad, Spain La Caixa Foundation. Spain.

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G2/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5., This work was supported by Grants SAF2009-07769 from the Ministerio de Ciencia e Innovación of Spain and Reticc RD06/0020/0010 from the Istituto de Salud Carlos III.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27.

Background/Aims: The cell cycle inhibitors p21 Cip1 and p27 Kip1 regulate liver regeneration by modulating the activity of cyclin-dependent kinases (CDKs). However, the specific role of these inhibitors in the regulation of CDK2 activity during liver regeneration remains unknown. The aim of this study was to examine the association of p21 Cip1 and p27 Kip1 with cyclin E-CDK2 and cyclin A-CDK2 complexes during rat liver regeneration and to correlate the association of both inhibitors with CDK2 activity. Methods: The association of p21 Cip1 or p27 Kip1 with cyclin E-CDK2 or cyclin A-CDK2 and the activities of these complexes were analyzed by immunoprecipitation of rat liver homogenates obtained at different times after a partial hepatectomy (PH), followed by Western blotting or kinase assays. Results: High amounts of p27 Kip1 bound to cyclin E-CDK2 were observed during the first 13 h after PH, when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound-p27 Kip1 decreased strongly. The amount of p21 Cip1 bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both inhibitors were detected and at 28 h, a significant increase in p21 Cip1 and p27 Kip1 associated with cyclin A-CDK2 was observed. Conclusions: p27 Kip1 acts as a brake on cyclin E-CDK2 activity during the first 13 h after a PH. Both p21 Cip1 and p27 Kip1 down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.

α 1 -Adrenergic agonists are known to be involved in the regulation of hepatocyte proliferation after a partial hepatectomy. The blockade of α 1 -adrenergic receptors with the specific antagonist prazosin inhibits DNA synthesis which peaks at 24 h after surgery. In this report we have studied the effects of prazosin administration on several events occuring during liver regeneration. The results show that the nuclear volume and nucleolar volume density of hepatocytes were increased and that the relative amount of heterochromatin decreased at 24 h. The increase in hepatocyte nucleolar volume density and the decrease in the relative amount of heterochromatin were partially abolished by prazosin administration while the increase in the nuclear volume was not affected. The relative amount of the histone H1 variant H1° was reduced in 24-h regenerating liver and prazosin treatment prevented this reduction.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping glycolitic enzyme that recently has been implicated in cell signaling. Under apoptotic stresses, cells activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The GAPDH-Siah interaction depends on the integrity of lysine 227 in human GAPDH, being the mutant K227A unable to associate with Siah. As lysine residues are susceptible to be modified by acetylation, we aimed to analyze whether acetylation could mediate transport of GAPDH from cytoplasm to the nucleus. We observed that the acetyltransferase P300/CBP-associated factor (PCAF) interacts with and acetylates GAPDH. We also found that over-expression of PCAF induces the nuclear translocation of GAPDH and that for this translocation its intact acetylase activity is needed. Finally, the knocking down of PCAF reduces nuclear translocation of GAPDH induced by apoptotic stimuli. By spot mapping analysis we first identified Lys 117 and 251 as the putative GAPDH residues that could be acetylated by PCAF. We further demonstrated that both Lys were necessary but not sufficient for nuclear translocation of GAPDH after apoptotic stimulation. Finally, we identified Lys 227 as a third GAPDH residue whose acetylation is needed for its transport from cytoplasm to the nucleus. Thus, results reported here indicate that nuclear translocation of GAPDH is mediated by acetylation of three specific Lys residues (117, 227 and 251 in human cells). Our results also revealed that PCAF participates in the GAPDH acetylation that leads to its translocation to the nucleus. © 2010 Elsevier Ltd., This research was supported by grants SAF2006-05212 and SAF2009-07769 from the Ministerio de Educación y Ciencia of Spain and RETICS RD06/0020/0010 from the Instituto de Salud Carlos III.

Cyclin A must be degraded at prometaphase in order to allow mitosis progression. Nevertheless, the signals that trigger cyclin A degradation at mitosis have been largely elusive. In the present paper, we review the status of cyclin A degradation in the light of recent evidence indicating that acetylation plays a role in cyclin A stability. The emerging model proposes that the acetyltransferase PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] [perhaps also its homologue GCN5 (general control non-derepressible 5)] acetylates cyclin A at Lys54, Lys68, Lys95 and Lys112 during mitosis, leading to its ubiquitylation by the anaphase-promoting factor/cyclosome and its subsequent degradation via proteasome. Interestingly, these four lysine residues in cyclin A also participate in the regulation of cyclin A–Cdk (cyclin-dependent kinase) activity by modulating its interaction with Cdks.

In the present study, a relationship between convulsant activity and two cellular events, changes in calmodulin (CaM) concentration and proto-oncogene c-fos expression has been considered. c-fos has been found activated after the administration of the organochlorine insecticide lindane, the Ca2+ channel agonist Bay K, and N-methyl-D-aspartate (NMDA). The administration of the voltage-dependent Ca2+ channel antagonist nifedipine was able to block the expression elicited by lindane. The effect of lindane on c-fos expression could not be blocked by prior administration of MK-801, a non-competitive antagonist of the NMDA receptor. These results suggest a possible role for the voltage-dependent Ca2+ channels in the mechanism of action of lindane. By means of in situ hybridization, the different patterns of c-fos expression after the administration of the mentioned compounds have been described. A possible modification of the levels of CaM has also been investigated. Among all the subcellular fractions considered, only levels of nuclear CaM appeared to be affected after the different treatments. The changes observed seemed to follow a similar pattern to that described for c-fos induction. Calcium entry through these voltage-dependent calcium channels would be the link between membrane depolarizing events and expression of c-fos and/or increase in nuclear CaM.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.

Background & Aims: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the beta-catenin umion domain. Our aim was to determine its functional relevance to establish its pathogenicity. Methods: N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal. cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on beta-catenin binding, beta-catenin/Tcf-4 -mediated transcription and beta-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation. Results: N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded beta-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present Expression of APC N1026S in SW480 and DLD-1 cells did not diminish beta-catenin/Tcf-4 -mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear beta-catenin in SW480 cells as effectively as WT. Conclusions: These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.

We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts in vitro and in vivo with the protein SET. This interaction is performed through the acidic domain of SET located at the carboxy terminal region. On analysing the functional relevance of SET-GAPDH interaction, we observed that GAPDH reverses in a dose-dependent manner, the inhibition of cyclin B-cdk1 activity produced by SET. Similarly to SET, GAPDH associates with cyclin B, suggesting that the regulation of cyclin B-cdk1 activity might be mediated not only by the interaction of GAPDH with SET but also with cyclin B. To analyse the putative role of GAPDH on cell cycle progression, HCT116 cells were transfected with a GAPDH expression vector. Results indicate that overexpression of GAPDH does not affect the timing of DNA replication but induces an increase in the number of mitosis, an advancement of the peak of cyclin B-cdk1 activity and an acceleration of cell cycle progression. All these results suggest that GAPDH might be involved in cell cycle regulation by modulating cyclin B-cdk1 activity.

The SET protein and the cell cycle inhibitor p21(Cip1) interact in vivo and in vitro. We identified here the domain (157)LIF(159) of p21(Cip1) as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21(Cip1), one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21(Cip1) co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G(2)/M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21(Cip1) cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G(2)/M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G(2)/M transition by modulating cyclin B-CDK1 activity.

We have investigated the messenger RNAs expressed from the three calmodulin genes during rat liver regeneration. The results revealed that all the calmodulin transcripts increased from 8 hr after a partial hepatectomy, although differences in the timing and the level of expression from the three genes were observed. Calmodulin I transcripts peaked at 16 hr, whereas calmodulin II and calmodulin III progressively increased from 8 to 24 hr. At 24 hr after surgery, calmodulin I, calmodulin II and the 2.3 kb calmodulin III transcripts reached values of a 6-fold increase, whereas the 0.8 kb product of calmodulin III increased 25-fold. At 30 hr the levels of all the calmodulin transcripts were similar to those observed at 24 hr. The transcription rates of the three calmodulin genes augmented after hepatectomy (calmodulin I and calmodulin II twofold and calmodulin III fourfold), indicating that the elevation of the calmodulin transcripts could be, at least partially, the result of this increase in the transcription rates. The total calmodulin concentration also increased twofold at 24 hr after hepatectomy. We also report that the administration of the β-adrenergic blocker, D, L-propranolol inhibited the accumulation of calmodulin protein without significantly affecting the increase of the messenger RNAs. These results indicate that the expression of calmodulin observed during liver regeneration could be regulated by cyclic AMP at the translational or post-translational level. (Hepatology 1994;20:1002–1008).

By using a 125I-calmodulin overlay assay, three major high-affinity calmodulin-binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60- and 50-kDa proteins have been previously identified as subunits of the calmodulin-dependent protein kinase II. We report here the immunoblot identification of the 135-kDa cal modulin-binding protein as myosin light chain kinase. We also show that the calmodulin-dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.

As has been previously reported one surge in cytosolic calmodulin is produced between 4 and 12 h after a partial hepatectomy. Moreoever, a surge in cytosolic cyclic AMP and another in cyclic AMP-dependent protein kinase activity can be detected during the late period of the prereplicative phase of liver regeneration after a partial hepatectomy. It is known that these three surges are involved in triggering DNA synthesis. By kinetic studies and by injecting transcription (actinomycin D) and translation (cycloheximide) inhibitors into hepatectomized rats we have demonstrated that the cyclic AMP-dependent protein kinase surge is produced by new synthesis of the enzyme. In thyroparathyroidectomized rats subjected to a partial hepatectomy the calmodulin surge was similar to that observed in normal hepatectomized rats whereas the surge in cyclic AMP-dependent protein kinase activity was strongly decreased suggesting that the cyclic AMP-dependent protein kinase surge is not generated by the previous surge in calmodulin.

When rats were infused with a solution containing triiodothyronine, amino acids, glucagon and heparin (solution A) the hepatocytes increased DNA synthesis and decreased plasma membrane sialic acid. In order to study whether the reduced levels of sialic acid in the plasma membrane were associated with hepatocyte proliferation, different mixtures of three components of solution A were infused into rats and the DNA synthetic activity as well as the sialic acid content measured. Results reported here show a correlation between DNA synthetic activity and sialic acid reduction suggesting that the decrease in the plasma membrane sialic acid can be a pre-replicative step associated to cell proliferation.

A transient peak of cytosolic calmodulin (CaM) was produced during the prereplicative phase of rat liver cell proliferation following partial hepatectomy. After accumulating in the cytosol, CaM apparently translocated into the nuclei, associating with the nuclear matrix. The administration of alpha 1-adrenergic blockers to hepatectomized rats prevented the association of CaM with the nuclear matrix without affecting the increase in the total nuclear CaM. The inhibitory effect of the alpha 1-antagonists was reversed by the simultaneous injection of the alpha-agonist noradrenaline. Since the activation of alpha 1-adrenergic receptors results in the release of Ca2+ from endoplasmic reticulum stores, the results suggest that the association of CaM with the nuclear matrix during proliferative activation is mediated by Ca2+ released from endoplasmic reticulum and show that the association with the matrix is independent of its intranuclear accumulation.

Enzyme biochemical and histochemical assays during chemical carcinogenesis in rat liver have revealed that several adult enzyme activities are lost and some fetal enzyme activities are re-expressed in the hyperplastic foci as well as in the developed hepatomas. How these enzyme alterations are acquired and to what extent these changes are specifically related to the growth alterations leading to neoplastic development are decisive questions. Using a carcinogenesis protocol that combines a single dose of diethylnitrosamine and phenobarbital given continuously as the promoting agent and by assaying serial liver sections for glucose-6-phosphatase, adenosines-5′-triphosphatase and 5′-nucleotidase, we have identified the three-enzyme pattern of 1,746 islands and measured their section areas. We found a clear trend that clones with more deviated enzyme pattern grow faster than less deviated ones.

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G. 2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G. 2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity. © The Author(s) 2009. Published by Oxford University Press., Grants SAF2006-05212 and SAF2007-60491 from the Ministerio de Ciencia e Innovación of Spain and Rticc RD06/0020/0010 from the Instituto de Salud Carlos III. Funding for open access charge: Grant from the Spanish Government