Transition In Mammalian Cells Research Articles

In vitro and in vivo antimicrobial activity of antimicrobial peptide Jelleine-I against foodborne pathogen Listeria monocytogenes

As a human foodborne pathogen, Listeria monocytogenes can cause severe human listeriosis and develop resistance to antibiotics. Antimicrobial peptides (AMPs) are produced from all kingdoms of life and regarded as promising alternatives to conventional antibiotics. Jelleine-I is an AMP identified from honeybees royal jelly. In this study, we explored the activity and action mechanism of Jelleine-I against L. monocytogenes. We found its minimum inhibitory concentration to be 12.5 μg/mL. Membrane permeability analysis revealed that Jelleine-I increased L. monocytogenes cell membrane permeability, causing calcium leakage. Scanning, transmission electron microscopy and fluorescence microscopy revealed that Jelleine-I destroyed membrane integrity, disrupted intracellular structures and interacted with the bacterial DNA. DNA binding analysis demonstrated that Jelleine-I bound to bacterial genomic DNA. Results of reverse transcription-quantitative PCR revealed that Jelleine-I affected bacterial DNA replication gene expression levels. Moreover, Jelleine-I induced cellular reactive oxygen species (ROS) production from fluorescence intensity analysis, and inhibited bacterial biofilm formation. Results of immunomodulation in Galleria mellonella revealed that Jelleine-I increased host hemocyte counts, upregulated host AMP gene (Gloverin and Cecropin D) expression, and inhibited proinfammatory cytokine (tumor necrosis factor α and interleukin 6) production induced by bacterial infection. It efficiently killed bacteria and increased the survival rate of infected insects to 70 %. Furthermore, Jelleine-I increased the G1 to S phase transition in mammalian cells from cells cycle analysis, and cytotoxicity assay results indicated that it promoted cell proliferation without hemolysis or cytotoxicity. Collectively, Jelleine-I possesses antimicrobial, immunomodulatory and cell proliferative activities, and is a promising candidate for preventing L. monocytogenes emergence and dissemination.

Cul3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E.

Cyclin E and its binding partner Cdk2 control the G1/S transition in mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E-Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and Cul3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which Cul3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how Cul3 mediates cyclin E destruction, we identified a degron on cyclin E that Cul3 targets for ubiquitylation. Recognition of the degron and binding of Cul3 does not require a BTB domain-containing adaptor protein. Additionally, this degron is lacking in N-terminally truncated cyclin E. Our results describe a mechanism whereby N-terminally truncated cyclin E can avoid the Cul3-mediated degradation pathway. This mechanism helps to explain the increased activity that is associated with the truncated cyclin E variants that occurs in some cancers.

Disproportionate feedback interactions govern cell-type specific proliferation in mammalian cells.

In mammalian cells, the decision to maintain quiescence over proliferation commitment during G1 -S transition depends on more than one intertwined feedback interaction and is highly cell-type dependent. However, the precise role played by these individual feedback regulations in organizing such diverse proliferation dynamics is still poorly understood. Herein, we propose a predictive mathematical model of G1 -S transition in mammalian cells that reconciles distinct single-cell experimental observations in a cell-type specific manner. The model analysis reveals that the feedback motifs responsible for the G1 -S transition act in a disparate fashion to organize the cell-type specific proliferation response. Importantly, the proposed model can be effectively tuned to gain insights into the proliferation commitment of diverse mammalian cell types and can find wide applicability.

Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition.

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3-dimensional context of nuclear organization is reflected by two well documented observations. DNA replication-dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone-related requirements for G1/S-phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci.

Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition

From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G1/S-phase transition as a key checkpoint where cells respond to their metabolic status and commit to replicating the genome. However, the mechanism underlying the coordination of metabolism and the G2/M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G2/M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage–induced G2-phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G2/M-phase transition.

Emerging Roles for Ciz1 in Cell Cycle Regulation and as a Driver of Tumorigenesis.

Precise duplication of the genome is a prerequisite for the health and longevity of multicellular organisms. The temporal regulation of origin specification, replication licensing, and firing at replication origins is mediated by the cyclin-dependent kinases. Here the role of Cip1 interacting Zinc finger protein 1 (Ciz1) in regulation of cell cycle progression is discussed. Ciz1 contributes to regulation of the G1/S transition in mammalian cells. Ciz1 contacts the pre-replication complex (pre-RC) through cell division cycle 6 (Cdc6) interactions and aids localization of cyclin A- cyclin-dependent kinase 2 (CDK2) activity to chromatin and the nuclear matrix during initiation of DNA replication. We discuss evidence that Ciz1 serves as a kinase sensor that regulates both initiation of DNA replication and prevention of re-replication. Finally, the emerging role for Ciz1 in cancer biology is discussed. Ciz1 is overexpressed in common tumors and tumor growth is dependent on Ciz1 expression, suggesting that Ciz1 is a driver of tumor growth. We present evidence that Ciz1 may contribute to deregulation of the cell cycle due to its ability to alter the CDK activity thresholds that are permissive for initiation of DNA replication. We propose that Ciz1 may contribute to oncogenesis by induction of DNA replication stress and that Ciz1 may be a multifaceted target in cancer therapy.

Abstract SY34-03: Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer

In spite of the dramatically improved outlook for patients with ER+ breast cancer, a subset of patients are intrinsically non-responsive to anti-hormonal therapy alone, and even the responders invariably acquire resistance to this treatment modality. In the setting of established hormone resistance, most of these patients will receive chemotherapy, with limited benefit and considerable impact on quality of life due to toxicity. Given the sheer numbers of patients affected (approximately two-thirds of all breast cancers are ER+), the unmet need for well-tolerated therapies that overcome resistance to anti-hormonal agents remains very high. Strikingly, the vast majority (>90%) of ER+ breast cancers express a functional retinoblastoma protein (Rb), a tumor suppressor that represents a critical regulator of the G1 to S phase transition in mammalian cells. In its hypo-phosphorylated state, Rb suppresses the expression of proteins that are essential for commitment to S-phase entry and progression through the remainder of the eukaryotic cell cycle. The G1 cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) which function in complexes with the D-type cyclins (collectively termed cyclin D) initiate the phosphorylation of Rb and override the repressive effects of Rb on cell-cycle progression. In ER-driven breast cancers, oncogenic signaling through the ER stimulates the cyclin D-cdk4/6 -dependent phosphorylation of Rb, and this proliferative stimulus is frequently augmented by amplification of cyclin D or loss of expression of the cyclin D-cdk4/6 inhibitor, p16 (encoded by the CDKN2A gene), two alterations that lead to elevated and dysregulated cyclin D-cdk4/6 activity. The requirement for cyclin D-cdk4/6 activity to circumvent the cell-cycle braking action of Rb suggested that ER+ breast cancer cells might be vulnerable to drug-induced inhibition of cdk4/6 activity. PD-0332991 is an orally administered, selective inhibitor of cyclin D-cdk4/6 kinase activities. In preclinical models, this drug showed significant anti-proliferative activity in Rb+ breast cancer cell lines, the majority of which also expressed the ER. The most well established substrate for the cyclin D-cdk4/6 is Rb, which is modified at multiple phosphoacceptor sites (including Ser-780, Ser-795, Ser-807, and Ser-811) by these cdks. The phosphorylation status of the Ser-807 and Ser-811 residues in Rb proved to be the most robust pharmacodynamic readouts for PD-0332991 activity in vivo, and serve as appropriate biomarkers for target modulation by this drug under in vitro and in vivo study conditions. The IC50 for reduction of Rb phosphorylation at these sites in two breast cancer cell lines (MCF7 and T47D) was approximately 20 nM. Similarly, we observed that PD-0332991 inhibits S-phase entry (measured by incorporation of thymidine into newly synthesized DNA) and cellular proliferation in panel of Rb+ cancer cell lines, with IC50 values ranging from 40-170 nM. The antiproliferative effects of PD-0332991 were not limited to transformed cells, reinforcing the idea that this drug was capable of suppressing the proliferation of any human cell type that requires cyclin D-cdk4/cdk6 activity to bypass the G1 restriction point imposed by Rb. Conversely, cancer cells that have inactivated Rb through genetic or epigenetic mechanisms are highly resistant (IC50 > 3 uM) to the inhibitory effects of PD-0332991 on cell-cycle progression and cellular proliferation. Xenograft experiments confirmed the favorable in vivo safety profile and antitumor activity of PD-0332991. The maximum tolerated dose (MTD; defined as the dose that induced These preclinical studies provided a launching pad for a clinical trial in post-menopausal women with locally advanced or metastatic ER+ (HER2-) breast cancer. In this Phase 2 study, PD-0332991 was given in combination with letrozole (an inhibitor of estrogen synthesis), with the control arm receiving letrozole only. An interim analysis showed that the PD-0332991 plus letrozole combination yielded a dramatic improvement in treatment outcomes, more than tripling progression-free survival relative to the letrozole-only treatment arm. The drug was generally well-tolerated, due in part to its high degree of selectivity for cdk4/6 relative to other members of the cyclin-dependent kinase family, as well as other members of the extended kinase superfamily. These positive results led to a Breakthrough Therapy designation in 2013 for PD-0332991 in the treatment of patients with ER+ breast cancer. This presentation will provide information on the mechanisms of antitumor action of PD-0332991, together with updates on the current status of clinical development of this new addition to the armentarium of breast cancer treatments. Citation Format: Robert T. Abraham, Todd VanArsdale, David V. Shields, Nathan V. Lee, Maria Koehler, Kim Arndt. Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY34-03. doi:10.1158/1538-7445.AM2014-SY34-03

Systematic reverse engineering of network topologies: a case study of resettable bistable cellular responses.

A focused theme in systems biology is to uncover design principles of biological networks, that is, how specific network structures yield specific systems properties. For this purpose, we have previously developed a reverse engineering procedure to identify network topologies with high likelihood in generating desired systems properties. Our method searches the continuous parameter space of an assembly of network topologies, without enumerating individual network topologies separately as traditionally done in other reverse engineering procedures. Here we tested this CPSS (continuous parameter space search) method on a previously studied problem: the resettable bistability of an Rb-E2F gene network in regulating the quiescence-to-proliferation transition of mammalian cells. From a simplified Rb-E2F gene network, we identified network topologies responsible for generating resettable bistability. The CPSS-identified topologies are consistent with those reported in the previous study based on individual topology search (ITS), demonstrating the effectiveness of the CPSS approach. Since the CPSS and ITS searches are based on different mathematical formulations and different algorithms, the consistency of the results also helps cross-validate both approaches. A unique advantage of the CPSS approach lies in its applicability to biological networks with large numbers of nodes. To aid the application of the CPSS approach to the study of other biological systems, we have developed a computer package that is available in Information S1.

Abstract 203: Differences Between Mammalian and Drosophila Mitochondrial Calcium Handling Help Identify Mechanisms Regulating the Permeability Transition.

Studies of cardiomyocyte death during calcium overload induced by ischemia-reperfusion injury or heart failure have implicated the mitochondrial permeability transition as a key pathway. During the permeability transition, an opening of a channel in the inner membrane leads to mitochondrial depolarization and swelling. Despite extensive studies in mammalian systems, the machinery responsible for this phenomenon remains only partially identified. If present in non-mammalian species, the components of the permeability transition may be further elucidated, given potential advantages within these systems for high-throughput screens. However, the existence of a permeability transition remains controversial in non-mammalian organisms. In Drosophila , prior studies have documented calcium-induced mitochondrial depolarization, but no obvious swelling. Here we show that Drosophila S2R+ cells do possess the machinery for permeability transition, but that the threshold for a calcium trigger is significantly higher than in mammalian systems. Using a calcein-loading method, we show that Drosophila permeability transition can be triggered by calcium overload, using ionomycin, and by cysteine oxidation, using phenylarsine oxide. As in mammalian systems, blockade of mitochondrial cyclophilin or the ATP/ADP transporter appears to inhibit the Drosophila permeability transition. Finally, we examine three alternative hypotheses that may explain these differences in permeability transition. First, we test if perturbing the pathways for calcium influx into S2R+ mitochondria can trigger this phenomenon. Second, we test if the discrepancy in the calcium threshold is due to structural differences in the key regulators, particularly the mitochondrial cyclophilin. Third, we compare Drosophila and human genomes to see if any novel molecules may be responsible for setting the lower threshold for calcium-induced permeability transition in mammalian cells. Since the Drosophila cells possess such significant resistance to permeability transition, the results of our investigations suggest potential new strategies for the development of therapeutics inhibiting mitochondrial permeability transition in cardiac calcium-induced injury.

Chk1 is required for the metaphase–anaphase transition via regulating the expression and localization of Cdc20 and Mad2

AimsThe checkpoint kinase 1 (Chk1) functions not only in genotoxic stresses but also in normal cell cycle progression, particularly in the initiation, progression and fidelity of unperturbed mitosis. In this study, we investigated the role of Chk1 in regulating the metaphase–anaphase transition in mammalian cells. Main methodsThe mitotic progression was monitored by flow cytometry analysis. The levels of cyclin B1, Cdc20 and Mad2 were measured by Western blotting. Metaphase chromosome alignment and the subcellular localization of Cdc20 and Mad2 were analyzed by immunofluorescence and confocal microscopy. Key findingsCyclin B1 degradation and the metaphase–anaphase transition were severely blocked by Chk1 siRNA. Depletion of Chk1 induced chromosome alignment defect in metaphase cells. The kinetochore localization of Cdc20, Mad2 was disrupted in Chk1 depleted cells. Chk1 abrogation also dramatically reduced the protein expression levels of Cdc20 and Mad2. SignificanceThese results strongly suggest that Chk1 is required for the metaphase–anaphase transition via regulating the subcellular localization and the expression of Cdc20 and Mad2.

RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B

Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.

A Myc–microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes

The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. However, the interplay between transcription factors and miRNAs in modulating gene regulatory networks involved in human cell proliferation is largely unknown. Here we show that the miRNA miR-22 promotes proliferation in primary human cells, and through a combination of Argonaute-2 immunoprecipitation and reporter assays, we identified multiple novel targets of miR-22, including several cell-cycle arrest genes that mediate the effects of the tumor-suppressor p53. In addition, we found that miR-22 suppresses interferon gene expression by directly targeting high mobility group box-1 and interferon regulatory factor (IRF)-5, preventing activation of IRF3 and NF-κB, which are activators of interferon genes. The expression of interferon genes is elevated in quiescent cells and their expression is inhibitory for cell proliferation. In addition, we find that miR-22 is activated by the transcription factor Myc when quiescent cells enter proliferation and that miR-22 inhibits the Myc transcriptional repressor MXD4, mediating a feed-forward loop to elevate Myc expression levels. Our results implicate miR-22 in downregulating the anti-proliferative p53 and interferon pathways and reveal a new transcription factor–miRNA network that regulates the transition of primary human cells from quiescence to proliferation.

Identification of preferentially reactivated genes during early G1 phase using nascent mRNA as an index of transcriptional activity

During mammalian mitosis, transcription is silenced due to dissociation of transcription factors from DNA and chromosome condensation. At the end of mitosis, transcription is reactivated through chromosome relaxation and reloading of these factors to the DNA. Early G1 genes, which are preferentially reactivated during M/G1 transition, are important for maintenance of cellular function and are known to be strictly regulated. As only few early G1 genes have been identified to date, screening for early G1 genes by genome-wide analysis using nascent mRNA could contribute to the elucidation of the regulatory mechanisms during early G1. Here, we performed a detailed expression analysis for the M/G1 transition of mammalian cells by microarray analysis of nascent mRNA, and identified 298 early G1 genes. Analysis of these genes provides two important insights. Firstly, certain motifs are enriched in the upstream sequences of early G1 genes; from this we could predict candidate cognate transcription factors, including Sp1, which is recruited to the DNA in the early G1 phase. Secondly, we discovered that neighboring genes of early G1 genes were also frequently up-regulated in the G1 phase. Information about these numerous newly identified early G1 genes will likely contribute to an understanding of the regulatory mechanisms of the early G1 genes.

Lowered Nudix type 5 (NUDT5) expression leads to cell cycle retardation in HeLa cells

The molecule 8-oxo-7,8-dihydroguanine (8-oxoGua), an oxidized form of guanine, can pair with adenine or cytosine during nucleic acid synthesis. Moreover, RNA containing 8-oxoGua causes translational errors, thus leading to the production of abnormal proteins. Human NUDT5, a MutT-related protein, catalyzes the hydrolysis of 8-oxoGDP to 8-oxoGMP, thereby preventing misincorporation of 8-oxoGua into RNA. To investigate the biological roles of NUDT5 in mammalian cells, we established cell lines with decreased level of NUDT5 expression. In NUDT5 inhibited cells, the RNA oxidation was not significantly higher than that of normal cells. However, the cell cycle G1 phase was significantly delayed, and cell numbers in both S and G2/M phases were reduced, indicating that cell proliferation was hampered by NUDT5 suppression. Key proteins for preventing the G1-S transition, including p53, p16, and Rb were increased, while the Rb phosphorylation was decreased. These results suggested that the NUDT5 protein may play significant roles in regulating the G1-S transition in mammalian cells.

Specific GATA Factors Act as Conserved Inducers of an Endodermal-EMT

The epithelial-to-mesenchymal transition (EMT) converts cells from static epithelial to migratory mesenchymal states (Hay, 1995). Here, we demonstrate that EMT in the Drosophila endoderm is dependent on the GATA-factor Serpent (Srp), and that Srp acts as a potent trigger for this transition when activated ectopically. We show that Srp affects endodermal-EMT through a downregulation of junctional dE-Cadherin (dE-Cad) protein, without a block in its transcription. Moreover, the relocalization of dE-Cad is achieved through the direct repression of crumbs (crb) by Srp. Finally, we show that hGATA-6, an ortholog of Srp, induces a similar transition in mammalian cells. Similar to Srp, hGATA-6 acts through the downregulation of junctional E-Cad, without blocking its transcription, and induces the repression of a Crumbs ortholog, crb2. Together, these results identify a set of GATA factors as a conserved alternative trigger to repress epithelial characteristics and confer migratory capabilities on epithelial cells in development and pathogenesis.

NFBD1/MDC1 participates in the regulation of G2/M transition in mammalian cells

NFBD1/MDC1 is a large nuclear protein involved in the early cellular response to DNA damage. Upon DNA damage, NFBD1 has an ability to facilitate the efficient DNA repair. In the present study, we have found that, in addition to DNA damage response, NFBD1 plays a critical role in the regulation of G2/M transition. Expression study using synchronized HeLa cells demonstrated that, like the mitotic kinase Plk1, NFBD1 expression level is maximal in G2/M-phase of the cell cycle. siRNA-mediated knockdown of NFBD1 resulted in G2/M arrest as well as simultaneous apoptosis in association with a significant increase in the amounts of γH2AX and pro-apoptotic p73. Since a remarkable down-regulation of mitotic phospho-histone H3 was detectable in NFBD1-knocked down cells, it is likely that knocking down of NFBD1 inhibits G2/M transition. Taken together, our present findings suggest that NFBD1 has a pivotal role in the regulation of proper mitotic entry.

Abstract C23: Therapy of triple-negative breast tumors by targeting the MYC oncogene pathway

Abstract Human breast cancers that lack expression of the estrogen and progesterone nuclear receptors and with low HER2 expression are known as “gtriple-negative” tumors. These are amongst the most aggressive breast cancers and no targeted therapeutics exists for this subclass of human tumors. MYC is a proto-oncogene transcription factor that plays a key role in cell cycle transition of mammalian cells. Increased expression of MYC is estimated to occur in ∼ 20–50% of human breast cancers. Genomic amplification of the MYC locus, 8q24, is correlated with decreased patient survival, and MYC-over-expression in breast cancer cells has been associated with a resistance to anti-hormonal therapies that seek to inhibit ER signaling. Furthermore, MYC has recently been shown to be one of the genes unique to high-grade mammary tumors. Thus, these prior studies suggest that the level of MYC expression might be useful as a biomarker and that the inhibition of MYC function may be a useful therapeutic approach against this difficult-to-treat type of breast cancer. However, while MYC is strongly implicated in breast tumorigenesis, no direct small molecule inhibitors of its transcriptional activity are available in clinical practice. An alternative approach to directly inhibiting MYC is to find “synthetic-lethal” combinations that selectively kill those cells with elevated MYC levels without affecting normal cells. The term “synthetic-lethality” refers to a genetic context in which a mutation in a gene, which itself does not cause lethality, can cause lethality when combined with a mutation in another gene. Using engineered cells, we have previously demonstrated that inhibiting cyclin-dependent kinase 1 (CDK1) can kill cells that over-express MYC. We have now used a panel of breast cancer cell lines established from human patients to show that (1) those breast cancers that are receptor-negative have significantly higher levels of MYC protein, (2) that the MYC pathways are critical for the survival and proliferation of this tumor subset, (3) CDK1 inhibition in triple-negative breast cancer cells results in the initiation of apoptosis mediated by mitochondria-dependent intrinsic pathway, and that (4) this apoptotic event is specifically achieved by altering the balance between pro-survival and pro-apoptotic members of the Bcl-2 family proteins. Our results present a novel treatment strategy for “triple-negative” breast cancers by targeting the MYC pathways via CDK inhibitors. Citation Information: Cancer Res 2009;69(23 Suppl):C23.

Inactivation of Rho GTPases withClostridium difficileToxin B Impairs Centrosomal Activation of Aurora-A in G2/M Transition of HeLa Cells

During G2 phase of cell cycle, centrosomes function as a scaffold for activation of mitotic kinases. Aurora-A is first activated at late G2 phase at the centrosome, facilitates centrosome maturation, and induces activation of cyclin B-Cdk1 at the centrosome for mitotic entry. Although several molecules including HEF1 and PAK are implicated in centrosomal activation of Aurora-A, signaling pathways leading to Aurora-A activation at the centrosome, and hence mitotic commitment in vertebrate cells remains largely unknown. Here, we have used Clostridium difficile toxin B and examined the role of Rho GTPases in G2/M transition of HeLa cells. Inactivation of Rho GTPases by the toxin B treatment delayed by 2 h histone H3 phosphorylation, Cdk1/cyclin B activation, and Aurora-A activation. Furthermore, PAK activation at the centrosome that was already present before the toxin addition was significantly attenuated for 2 h by the addition of toxin B, and HEF1 accumulation at the centrosome that occurred in late G2 phase was also delayed. These results suggest that Rho GTPases function in G2/M transition of mammalian cells by mediating multiple signaling pathways converging to centrosomal activation of Aurora-A.

Overexpression of the N-terminal end of the p55γ regulatory subunit of phosphatidylinositol 3-kinase blocks cell cycle progression in gastric carcinoma cells

pRb and its family members p130 and p107 regulate cell cycle progression and direct G1/S transition in mammalian cells through interaction with the transcription factor E2F. Phosphatidylinositol 3-kinase (PI3K) is an essential component of growth factor-regulated pathways and plays a crucial role in the regulation of cellular proliferation and differentiation. It has been demonstrated that PI3K can regulate cell cycle progression via Akt-mediated pathway. However, the possible interactions between PI3K and Rb pathways remain to be defined. It was reported that the unique 24-amino-acid N-terminal end of the p55 regulatory subunits of PI3K is an Rb-binding domain and affects Rb action or Rb-E2F interaction. The 24 N-terminal amino acids of p55gamma encoded by a cDNA construct could compete with the endogenous p55gamma for binding to Rb, which influences Rb-mediated signaling and blocks cell cycle progression. In the current study, we investigated the effects of this 24-peptide on cell proliferation in human gastric carcinoma MKN-28 cells by means of cell cycle analysis, BrdU incorporation, and determining the levels of cell cycle regulatory molecule expression. Our results showed that p55gammaPI3K and the Rb family members p130 and p107 exist in MKN-28 cells, while the p110PI3K was not detected. Moreover, p55gammaPI3K was found binding to p130/p107 in these cells. We demonstrated that the introduction of the plasmid N24p55-GFP (harboring the cDNA for the 24 N-terminal amino acids of p55gamma) into MKN-28 cells caused cell cycle arrest at G1. Furthermore, we showed that the over-expression of the 24-peptide in MKN-28 cells decreased the population of cells incorporating BrdU and reduced the levels of cyclin D1 and cyclin A. These observations suggest that PI3K can regulate cell cycle progression and cell proliferation in human gastric tumor cells via Rb-mediated pathway, and that this effect of PI3K is mediated through a direct association with Rb via the N-terminal end of its p55 kDa regulatory subunits and modulating Rb-E2F interactions. Taken together with previous studies, our data provide a new therapeutic target in human stomach cancer. Strategies targeting PI3K signal transduction or the association of PI3K with Rb, or regulating PI3K-Rb interactions could be employed for gene therapy or chemotherapy of gastric cancer and other tumors.

DNA topoisomerase II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast.

We previously reported that DNA topoisomerase II (topo II) is required for the G(0)-to-S phase transition in mammalian cells [Hossain et al. (2002) ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells. Genes Cells 7, 285-294]. In this study, we examined whether the requirement for topo II is evolutionarily conserved in Drosophila and yeast. ICRF-193, a catalytic inhibitor of topo II, inhibited DNA synthesis in Drosophila Schneider cells released from the G(0) (stationary) phase, whereas the drug did not inhibit DNA synthesis in Schneider cells released from the M phase. Depletion of topo II mRNA by RNA-interference (RNAi) in G(0)-phase Schneider cells resulted in significant inhibition of DNA synthesis after release from G(0)-arrest. In the yeast topo II temperature-sensitive (ts) mutant, the initial cycle of DNA synthesis occurred at a restrictive temperature after release from starvation-induced G(0) phase and doubling of the DNA content in the cells was confirmed by both flow cytometry and fluorescence spectrophotometry. DNA synthesis in yeast cells after release from the G(0) phase was also observed in the presence of ICRF-193. Doubling of the DNA content was observed during spore germination of topo II ts mutant yeast at a restrictive temperature as determined by fluorescence spectrophotometry. These results indicate that topo II is required for the G(0)-to-S phase transition in Drosophila Schneider cells, but not in yeast.