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Supplementary Figure 8 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

Supplementary Figure 5 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

Supplementary Figure 7 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

PDF file - 25K, Fig. S5. GTP loading of endogenous Ras isoforms in the DLD1 KRAS wt/- cells expressing oncogenic KRAS phosphomutants.

PDF file - 51K, Fig. S1. Tumor volume of mice grafts from NIH3T3 cells expressing HA-KRAS-G12V phosphomutants.

Supplementary Figure 2 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

PDF file - 646K, Fig. S3. Histological pattern of mice grafts derived from NIH3T3 expressing phosphomutants.

PDF file - 31K, Fig. S4. Treatment with PKC inhibitor Edelfosine inhibits tumor growth of HAKRASG12V in a Ser181 KRAS phosphorylation-dependent fashion.

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors. Cancer Res; 74(4); 1190–9. ©2013 AACR.

Supplementary Methods, Figure Legends 3-8 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

Supplementary Figure 4 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

PDF file - 25K, Fig. S2. Quantitation of apoptosis by cleaved caspase-3 measurement.

Supplementary Figure 6 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

Cell cycle checkpoint abrogation, especially the inhibition of Chk1 in combination with DNA-damaging treatments, has been proposed as a promising way of sensitizing cancer cells. However, less is known about the possibility to selectively affect tumor cells when they are treated with agents that block DNA synthesis in combination with replication checkpoint inhibitors. Here, we present clear insights in the different responses of tumor and non-transformed cells to the inhibition of DNA replication with hydroxyurea in combination with checkpoint abrogation via inhibition of Ataxia telangiectasia–mutated– (ATM) and Rad3-related/ATM (ATR/ATM) and Chk1 kinases. Interestingly, we find that non-transformed cell lines activate ATR/ATM- and Chk1-independent pathways in response to replication inhibition to prevent mitotic entry with unreplicated DNA. In contrast, tumor cell lines such as HCT116 and HeLa cells rely entirely on Chk1 activity for a proper response to replication inhibitors. Our results show that p38 is activated in response to hydroxyurea treatment and collaborates with Chk1 to prevent mitotic entry in non-transformed cell lines by maintaining cyclin B1/Cdk1 complexes inactive. Furthermore, DNA replication arrest down-regulates cyclin B1 promoter activity in non-transformed cells, but not in tumor cells in a Chk1- and p38-independent way. Thus, our data show that non-transformed cells present a more robust DNA replication checkpoint response compared with tumor cells that involves activation of the p38 pathway. We show that some of these responses to replication block can be lost in tumor cells, causing a defective checkpoint and providing a rationale for tumor-selective effects of combined therapies. [Cancer Res 2007;67(24):11648–56]

PDF file - 27K, Figure S6. Detection of the phosphorylated form of oncogenic KRAS in human pancreatic ductal adenocarcinoma by 2-DE.

Supplementary Figure 3 from Different S/M Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition

PDF file - 20K, Table S1. Statistics for the values of volumes (mm3) represented in Fig1 pannel B. Table S2. Statistics for the values of weights (g) represented in Fig1 panel C. Table S3. KRAS mutations I tumor cell lines used to determine PKC inhibition sensitivity and KRAS phosphorylation.

Aim: Calmodulin interacts in many different ways with its ligands. We aim to shed light on its plasticity analyzing the changes followed by the linker region and the relative position of the lobes using conventional molecular dynamics, accelerated MD and scaled MD (sMD). Materials & methods: Three different structures of calmodulin are compared, obtaining a total of 2.5 μs of molecular dynamics, which have been analyzed using the principal component analysis and clustering methodologies. Results: sMD simulations reach conformations that conventional molecular dynamics is not able to, without compromising the stability of the protein. On the other hand, accelerated MD requires optimization of the setup parameters to be useful. Conclusion: sMD is useful to study flexible proteins, highlighting those factors that justify its promiscuity.

Oncogenic mutations of KRAS are found in the most aggressive human tumors, including colorectal cancer. It has been suggested that oncogenic KRAS phosphorylation at Ser181 modulates its activity and favors cell transformation. Using nonphosphorylatable (S181A), phosphomimetic (S181D), and phospho-/dephosphorylatable (S181) oncogenic KRAS mutants, we analyzed the role of this phosphorylation to the maintenance of tumorigenic properties of colorectal cancer cells. Our data show that the presence of phospho-/dephosphorylatable oncogenic KRAS is required for preserving the epithelial organization of colorectal cancer cells in 3D cultures, and for supporting subcutaneous tumor growth in mice. Interestingly, gene expression differed according to the phosphorylation status of KRAS. In DLD-1 cells, CTNNA1 was only expressed in phospho-/dephosphorylatable oncogenic KRAS-expressing cells, correlating with cell polarization. Moreover, lack of oncogenic KRAS phosphorylation leads to changes in expression of genes related to cell invasion, such as SERPINE1, PRSS1,2,3, and NEO1, and expression of phosphomimetic oncogenic KRAS resulted in diminished expression of genes involved in enterocyte differentiation, such as HNF4G. Finally, the analysis, in a public data set of human colorectal cancer, of the gene expression signatures associated with phosphomimetic and nonphosphorylatable oncogenic KRAS suggests that this post-translational modification regulates tumor progression in patients.

Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21‐mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC‐mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability., Hierarchical organization of p21‐mediated G1 arrest and S‐phase DNA damage response signaling ensures genomic stability upon increasing inhibition of nucleotide synthesis enzyme IMPDH.

DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)(2) (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

UV radiation leads to the generation of reactive oxygen species (ROS). These molecules exert a variety of harmful effects by altering key cellular functions and may result in cell death. Several studies have demonstrated that human skin can be protected against UV radiation by using plant-derived antioxidants. Here we evaluated the in vitro capacity of several antioxidant polyphenolic fractions from grape, which differ in their degree of polymerization and percentage of galloylation, to protect HaCaT human keratinocytes against UV-induced oxidative damage. These fractions inhibited both basal and UVB- or UVA-induced intracellular ROS generation in this cell line. Consequently, the same fractions inhibited p38 and JNK1/2 activation induced by UVB or UVA radiation. The highest protective effect was for fractions rich in procyanidin oligomers and gallate esters. These encouraging in vitro results support further research and should be taken into consideration into the clinical pharmacology of plant-derived polyphenolic extracts as novel agents for skin photoprotection.

The small G-protein Ras was the first oncogene to be identified and has a very important contribution to human cancer development (20-23% prevalence). K-RasB, one of the members of the Ras family, is the one that is most mutated and plays a prominent role in pancreatic, colon and lung cancer development. Ras proteins are membrane bound GTPases that cycle between inactive, GDP-bound and active, GTP-bound, states. Most of the research into K-RasB activity regulation has focused on the analysis of how GTP-exchange factors (GEFs) and GTPase activating proteins (GAPs) are regulated by external and internal signals. In contrast, oncogenic K-RasB has a very low GTPase activity and furthermore is not deactivated by GAPs. Consequently, the consensus was that activity of oncogenic K-RasB was not modulated. In this extra view we recapitulate some recent data showing that calmodulin binding to K-RasB inhibits phosphorylation of K-RasB at Ser181, near to the membrane anchoring domain, modulating signaling of both non-oncogenic and oncogenic K-RasB. This may be relevant to normal cell physiology, but also opens new therapeutic perspectives for the inhibition of oncogenic K-RasB signaling in tumors.

Fine tuning of Ras activity is widely known as a mechanism to induce different cellular responses. Recently, we have shown that calmodulin (CaM) binds to K-Ras and that K-Ras phosphorylation inhibits its interaction with CaM. In this study we report that CaM inhibits K-Ras phosphorylation at Ser181 by protein kinase C (PKC) in vivo, and this is a mechanism to modulate K-Ras activity and signaling. Although CaM inhibition increased the activation of endogenous K-Ras, PKC inhibition decreased its activation status. We demonstrate that K-Ras phosphorylation decreased susceptibility to p120GAP activity. Accordingly, we also observed that non-phosphorylable K-Ras mutant exhibits a less sustained activation profile and do not efficiently activate AKT at low growth factor doses compared with wild-type K-Ras. It is interesting that the physiological responses induced by K-Ras are affected by this phosphorylation; when K-Ras cannot be phosphorylated it exhibits a remarkably decreased ability to stimulate proliferation in non-saturated serum conditions. Finally, we demonstrate that phosphorylation also regulates oncogenic K-Ras functions, as focus formation capacity, mobility and apoptosis resistance upon adriamycin treatment of cells expressing oncogenic K-Ras that cannot be phosphorylated are highly compromised. Moreover, at low serum concentration proliferation and survival is practically inhibited when cells cannot phosphorylate oncogenic K-Ras. In this condition, K-Ras phosphorylation is essential to ensure a proper activation of mitogen-activated protein kinase and PI3K/AKT pathways. In summary, our findings suggest that the interplay between CaM interaction and PKC phosphorylation is essential to regulate non-oncogenic and oncogenic K-Ras activity and functionality.

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

Objective To quantify p27 messenger RNA (mRNA) levels in human arrested and normally developing embryos and nonfertilized oocytes to determine whether the p27 protein abundance, reported in cleavage-stage embryos exhibiting developmental arrest, is regulated at the mRNA expression level. Design Real-time reverse transcription quantitative polymerase chain reaction was used to quantify the expression of p27 in three samples: arrested embryos (group A, n=29), normally developing embryos (group D, n=34), and nonfertilized oocytes (group O, n=20). Setting Research laboratory working closely with a clinical IVF practice. Patient(s) Oocytes and embryos were obtained from patients undergoing assisted fertilization. Intervention(s) None. Main Outcome Measure(s) Quantification of mRNA transcripts. Result(s) The amount of p27 mRNA was statistically significantly higher in group A (mean ± SEM, 86,143 ± 42,496 relative units [RU]) compared with groups D (10,680 ± 3,850 RU) and O (3,555 ± 1,458 RU). Furthermore, in a group of 13 two- to four-cell arrested embryos, high levels of p27 mRNA (51,481 ± 31,120 RU) were found in comparison with the nonfertilized oocyte group (3,555 ± 1,458 RU). Conclusion(s) Cleavage-stage human embryos exhibiting developmental arrest show increased p27 mRNA expression. This probably is due to increased transcriptional activity.

Altres ajuts: AG acknowledges EURATOM (Dark. Risk GA 323216). AB is supported by a Universitat Autònoma de Barcelona fellowship (456-01-1/2012) and AE is supported by a FPI MINECO grant Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16 INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16 INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.

When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM–independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM–dependent and ATR/ATM–independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38α,β are not involved in the ATR/ATM–independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM–independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry. (Cancer Res 2006; 66(17): 8672-9)

Intracellular localization plays an important role in the functional regulation of the cyclin-dependent kinase inhibitor p21. While nuclear functions have been linked to the tumor suppressor activity of p21, cytoplasmatic functions are oncogenic. We have recently shown that Ser153 phosphorylation of p21 by PKC contributes to its cytoplasmatic accumulation, and that this phosphorylation is inhibited by Ca(2+)-dependent calmodulin binding to the C-terminal region of p21. Consequently, PKC and calmodulin/Ca(2+) play diverging roles in the regulation of p21 intracellular localization. Other kinases such as AKT and MIRK/dyrk1B also phosphorylate p21 near the nuclear localization signal, thus inhibiting its nuclear accumulation. We discuss here the effects of such phosphorylations on p21 functionality, as well as its relevance to cell cycle progression and differentiation.

The oncoprotein SET participates in a diversity of cellular functions including cell proliferation. Its role on cell cycle progression is likely mediated by inhibiting cyclin B-cdk1 and the protein phosphatase 2A (PP2A). On identifying new SET cellular partners, we found that SET interacts in vitro and in vivo with the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2); a protein involved in various aspects of mRNA biogenesis. The SET-binding region of hnRNPA2 is the RNP1 sequence that belongs to the RNA-binding domain (RBD) of this protein. We also found that hnRNPA2 has much higher affinity for single-standed DNA than for SET. On analysing the effect of hnRNPA2 on PP2A inhibition by SET, we observed that hnRNPA2 cooperates with SET on PP2A inhibition. This is because we found that hnRNPA2 is also a PP2A inhibitor. HnRNPA2 interacts with PP2A by the RNP1 sequence; however, to inhibit PP2A activity, the complete RBD is needed. We also observed that overexpression of hnRNPA2 inhibits PP2A activity and stimulates cell proliferation. Interestingly, the overexpression of the complete RBD is sufficient to stimulate proliferation. As hnRNPA2 is overexpressed in a variety of human tumors, our results suggest that hnRNPA2 might participate in oncogenesis by stimulating cell proliferation.

The DNA replication checkpoint is an inhibitory pathway ensuring that mitosis occurs only after completion of DNA synthesis. Its function may be relevant to the stability of the genome. The essential elements of this checkpoint are ATM/ATR kinases that indirectly lead to the phosphorylation and inhibition of the mitosis-promoting factor (Cdc2/cyclin B1). The function of this checkpoint was analysed in diverse nontransformed and tumour-derived cell lines. All cell lines tested arrested mitosis entry when DNA synthesis was inhibited by hydroxyurea (HU) treatment. But, unlike what has been described in yeast and Xenopus, in normal rat kidney (NRK) cells and NIH 3T3 fibroblasts, the arrest induced by HU treatment was not abrogated by caffeine, an ATM and ATR inhibitor. This indicated the presence of an ATM/ATR-independent response to DNA synthesis inhibition in these nontransformed mammalian cell lines. Interestingly, the behaviour of different tumour cell lines after caffeine treatment varied. While SW480, NP29, NP18 and HeLa cells did not enter mitosis in the presence of caffeine after HU treatment, in CaCo2, DLD1, HCT116 and HT29 caffeine abrogated the checkpoint response. In nontransformed cell lines, lack of cyclin B1 accumulation was observed when DNA synthesis was inhibited. This response was not abrogated by caffeine. In the tumour cell lines, a good correlation between the ability to arrest cell cycle when DNA synthesis was inhibited in the presence of caffeine and the lack of cyclin B1 accumulation was observed. Thus, there is an ATM/ATR-independent checkpoint response that leads to a decrease in cyclin B1 accumulation. However, this response is not functional in some tumour cell lines. Using inhibitors of p38alpha and beta, Mek1, 2 and p53-/- knocked-out fibroblasts, we showed that these proteins were also not involved in this particular checkpoint response. Lack of cyclin B1 accumulation after DNA synthesis inhibition in NRK cells was not due to increased degradation of the protein, but correlated with a decrease in mRNA accumulation.

Overexpression of p21(cip1) induces cell cycle arrest. Although this ability has been correlated with its nuclear localization, the evidence is not conclusive. The mutants that were used to inhibit its nuclear translocation could no longer bind to several proteins known to interact with the last 25 amino acids of p21(cip1). Here we used point mutation analysis and fusion of the proteins to DsRed to identify which amino acids are essential for the nuclear localization of p21(cip1). We conclude that amino acids RKR(140-142) are essential for nuclear translocation of p21(cip1). While wild-type DsRed-p21 induces cell cycle arrest in 95% of transfected cells, overexpression of cytoplasmatic p21AAA(140-142) arrested only 20% of transfected cells. We conclude that cytoplasmatic p21, with no deletion in the C-terminal region, had a much lower capacity to arrest the cell cycle.

It is accepted that approximately 50% of embryos obtained after IVF arrest during the first week. Traditionally, chromosome abnormality and suboptimal culture conditions have been proposed as factors commonly associated with embryo arrest. However, even when considering 'ideal' conditions and embryos of only excellent morphology in vitro, there is still a significant incidence of embryonic arrest. There is considerable evidence that the nuclear protein p27, a member of the Cip/Kip family of CDK inhibitors, plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis. The present investigation, using immunocytochemical techniques coupled with confocal microscopy, was undertaken to determine whether p27 could play a role in the arrest of 4-8-cell human embryos. A total of 28 preimplantation embryos at the 4-8-cell stage were investigated. Of these, 16 were diploid embryos showing cleavage arrest with no further progression, and 12 were normally developing embryos. There was a 2-fold increased expression of the cell-cycle inhibitor p27 in arrested embryos compared with control normally developing embryos. This study represents the first demonstration of an increased expression of p27 in cleavage-stage human arrested embryos.

Ras activation induces a variety of cellular responses that depend on the specific activated effector, the intensity and amplitude of its activation, and the cellular type. Transient activation followed by a sustained but low signal of the Ras/Raf/MEK/ERK pathway is a common feature of cell proliferation in many systems. On the contrary, sustained, high activation is linked with either senescence or apoptosis in fibroblasts and to differentiation in neurones and PC12 cells. The temporal regulation of the pathway is relevant and not only depends on the specific receptor activated but also on the presence of diverse modulators of the pathway. We review here evidence showing that calcium (Ca(2+)) and calmodulin (CaM) are able to regulate the Ras/Raf/MEK/ERK pathway. CaM-binding proteins (CaMBPs) as Ras-GRF and CaM-dependent protein kinase IV (CaMKIV) positively modulate ERK1/2 activation induced by either NGF or membrane depolarisation in neurones. In fibroblasts, CaM binding to EGF receptor and K-Ras(B) may be involved in the downregulation of the pathway after its activation, allowing a proliferative signalling.

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13–mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Liver cells from p21(Cip1-/-) mice subjected to partial hepatectomy (PH) progress into DNA synthesis faster than those from wild-type mice. These cells also show a premature induction of cyclin E/cyclin-dependent kinase (CDK) 2 activity. We studied the mechanisms whereby cells lacking p21(Cip1) showed a premature induction of this activity. Whereas the levels of CDK2, cyclin E, and p27(Kip1) were similar in both wild-type and p21(Cip1-/-) mice, those of the activator CDC25A were much higher in p21(Cip1-/-) quiescent and regenerating livers than in wild-type animals. Moreover, p21(Cip1-/-) cells also showed a premature translocation of CDC25A from cytoplasm into the nucleus. The ectopic expression of p21(Cip1) into mice embryo fibroblasts from p21(Cip1-/-) mice decreased the levels of CDC25A and delayed its nuclear translocation. The levels of CDC25A messenger RNA in p21(Cip1-/-) cells were higher than in wild-type cells, suggesting that this increase might be responsible, at least in part, for the high levels of CDC25A protein in these cells. Thus, the results reported here indicate that p21(Cip1) regulates the levels and the intracellular localization of CDC25A. We also found a good correlation between CDC25A nuclear translocation and cyclin E/CDK2 activation. In conclusion, premature translocation of CDC25A to the nucleus might be involved in the advanced induction of cyclin E/CDK2 activity and DNA replication in cells from animals lacking p21(Cip1).

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors. © 2014 American Association for Cancer Research., This study was supported by MICINN-Spain (SAF2010-20712) and RTICC (MINECO-Spain; groups RD 12/0036/0049 and RD 12/0036/0008). C. Barceló and N. Paco are recipients of predoctoral fellowships from MEC-Spain and the Catalan Government, respectively., http://dx.doi.org/10.1158/0008-5472.CAN-13-1750

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

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.