Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

The crucial role of DNA-dependent protein kinase and myelin transcription factor 1-like protein in the miR-141 tumor suppressor network.

Glioblastoma is the most aggressive brain tumor. Although miR-141 has been demonstrated to primarily function as a tumor suppressor in numerous malignancies, including glioblastoma, the mechanisms involved remain poorly understood. Here, it is shown that miR-141 is downregulated in glioblastoma cell lines and tissues and may exert its biological function via directly targeting myelin transcription factor 1-like (MYT1L). Using two glioblastoma cell lines that differ from each other by the functionality of DNA-dependent protein kinase (DNAPK), a functional involvement of DNAPK in the miR-141 tumor suppression network was observed. In M059K cells with a normal function of DNAPK, the enforced expression of miR-141 attenuated MYT1L expression and suppressed cell proliferation. Conversely, the inhibition of miR-141 expression promoted cell proliferation; however, in M059J cells with a loss-of-function DNAPK, miR-141 constitutively inhibited cell proliferation upon ectopic overexpression or inhibition. An overexpression of miR-141 suppressed M059J cell migration, while it had no effect on M059K. Furthermore, the ectopic expression of miR-141 induced an S-phase arrest in both cell lines, whereas the inhibition of miR-141 caused a G1 arrest in M059J and accelerated the S phase in M059K. An overexpression and suppression of miR-141 resulted in an aberrant expression of cell-cycle proteins, including p21. Moreover, MYT1L may be a transcription factor of p21 in p53-mutant cells, whereas DNAPK may function as a repressor of MYT1L. The findings revealed the crucial role of DNAPK in miR-141-mediated suppression of gliomagenesis and demonstrated that it may be a target molecule in miR-141-associated therapeutic interventions for glioblastoma.

Dicranopteris linearis extract inhibits the proliferation of human breast cancer cell line (MDA-MB-231) via induction of S-phase arrest and apoptosis.

CONTEXT: Dicranopteris linearis (Burm.f.) Underw. (Gleicheniaceae) has been scientifically proven to exert various pharmacological activities. Nevertheless, its anti-proliferative potential has not been extensively investigated. OBJECTIVE: To investigate the anti-proliferative potential of D. linearis leaves and determine possible mechanistic pathways. MATERIALS AND METHODS: MTT assay was used to determine the cytotoxic effects of D. linearis methanol (MEDL) and petroleum ether (PEEDL) extracts at concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 µg/mL against a panel of cancer cell lines (breast [MCF-7 and MDA-MB-231], cervical [HeLa], colon [HT-29], hepatocellular [HepG2] and lung [A549]), as compared to negative (untreated) and positive [5-fluorouracil (5-FU)-treated] control groups. Mouse fibroblast cells (3T3) were used as normal cells. The mode of cell death was examined using morphological analysis via acridine orange (AO) and propidium iodide (PI) double staining. Cell cycle arrest was determined using flow cytometer, followed by annexin V-PI apoptosis detection kit. RESULTS: MEDL demonstrated the most significant growth inhibition against MDA-MB-231 cells (IC50 22.4 µg/mL). PEEDL showed no cytotoxic effect. Induction of apoptosis by MEDL was evidenced via morphological analysis and acridine orange propidium iodide staining. MEDL could induce S phase cell cycle arrest after 72 h of incubation. Early apoptosis induction in MDA-MB-231 cells was confirmed by annexin V-FITC and PI staining. Significant increase in apoptotic cells were detected after 24 h of treatment with 15.07% cells underwent apoptosis, and the amount escalated to 18.24% with prolonged 48 h incubation. CONCLUSIONS: MEDL has potential as a potent cytotoxic agent against MDA-MB-231 adenocarcinoma.

G1 and S phase arrest in Candida albicans induces filamentous growth via distinct mechanisms.

Candida albicans is an opportunistic fungal pathogen. In immunocompromised individuals, it can cause bloodstream infections with high mortality rates. The ability to switch between yeast and hyphal morphologies is a critical virulence factor of C. albicans. In response to diverse environmental cues, several signaling pathways are activated resulting in filamentous growth. Interestingly, cell cycle arrest can also trigger filamentous growth although the pathways involved are not well-understood. Here, we demonstrate that the cAMP-PKA pathway is involved in the filamentous growth caused by G1 arrest due to the depletion of the G1 cyclin Cln3 and S phase arrest due to hydroxyurea treatment. The downstream mechanisms involved in filamentation are different between the two cell cycle arrest phenomena. Cln3-depleted cells require HGC1 and UME6 for filamentous growth, but hydroxyurea-induced filamentation does not. Also, the hyphal repressor Nrg1 is not involved in the suppression of Cln3-depletion and hydroxyurea-induced filamentous growth. The findings highlight the complexity of the signaling networks that control filamentous growth in which different mechanisms downstream of the cAMP-PKA pathway are activated based on the nature of the inducing signals.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints.

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.

G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFßTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFßTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.

ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells.

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 (Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein4 (Igfbp4) expression and decreased hyaluronan synthase 2 (Has2), prostaglandin-endoperoxide synthase 2 (Ptgs2), and prostaglandin F receptor (Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

Induction of acetylation and bundling of cellular microtubules by 9-(4-vinylphenyl) noscapine elicits S-phase arrest in MDA-MB-231 cells.

Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6µM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

PURPOSE: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. METHODS AND MATERIALS: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. RESULTS: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. CONCLUSION: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

NONO regulates the intra-S-phase checkpoint in response to UV radiation.

The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.

Disruption of follistatin by RNAi increases apoptosis, arrests S-phase of cell cycle and decreases estradiol production in bovine granulosa cells.

Follistatin (FST), a local regulator of gonadal functions is a powerful inhibitor of follicle stimulating hormone (FSH) secretion. In the present study, the expression of FST was partially silenced at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors in bovine granulosa cells (bGCs). The results showed that transfection with FST-1 and FST-2 vectors significantly down-regulated mRNA and protein expressions of follistatin by 51% (P = 0.0093) and 72% (P = 0.0078) respectively. After down-regulation of FST in bGCs, cell cycle was arrested at S-phase (9.2 ± 0.6 vs 12.5 ± 0.2, P = 0.0055), and apoptosis was significantly (21.3 ± 2.7 vs 13.9 ± 2.5, P = 0.0051) increased. These findings were further verified by down-regulation of protein level of B-cell leukemia/lymphoma 2 (Bcl2, P = 0.0423), and up-regulation of caspase-3 (P = 0.0362), p21 (P = 0.0067) and mRNA levels of Bcl2-associated X protein (Bax, P = 0.041). Knockdown of FST in bGCs significantly increased activin A concentration in culture medium, while level of estradiol (E2) was suppressed without affecting progesterone production. In addition, mRNA levels of all activin receptor subtypes [activin receptor types I (ACRI) and II (ACRIIA and ACRIIB)] and inhibin α-subunit were augmented (P < 0.05) without altering both inhibin ß-subunits. These findings suggest that follistatin may participate in caspase3-dependent apoptosis through Bcl2/Bax gene family in bovine GCs, whereas, activin and its receptors are associated with its regulation. Activin-induced up-regulation of inhibin-α subunit in bGCs seems to be involved in the regulation of steroidogenesis.

SIRT1 deacetylates TopBP1 and modulates intra-S-phase checkpoint and DNA replication origin firing.

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength.

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that Rad54B--a SNF2 helicase-like DNA-repair protein--limits the strength of both the G1/S and G2/M checkpoints. We find that Rad54B functions as a scaffold for p53 degradation via its direct interaction with the MDM2-MDMX ubiquitin-ligase complex. During the early phases of the DDR, Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the p53-mediated checkpoint is established, Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

The RASSF6 tumor suppressor protein regulates apoptosis and the cell cycle via MDM2 protein and p53 protein.

Ras association domain family (RASSF) 6 is a member of the C-terminal RASSF proteins such as RASSF1A and RASSF3. RASSF6 is involved in apoptosis in various cells under miscellaneous conditions, but it remains to be clarified how RASSF6 exerts tumor-suppressive roles. We reported previously that RASSF3 facilitates the degradation of MDM2, a major E3 ligase of p53, and stabilizes p53 to function as a tumor suppressor. In this study, we demonstrate that RASSF6 overexpression induces G1/S arrest in p53-positive cells. Its depletion prevents UV- and VP-16-induced apoptosis and G1/S arrest in HCT116 and U2OS cells. RASSF6-induced apoptosis partially depends on p53. RASSF6 binds MDM2 and facilitates its ubiquitination. RASSF6 depletion blocks the increase of p53 in response to UV exposure and up-regulation of p53 target genes. RASSF6 depletion delays DNA repair in UV- and VP-16-treated cells and increases polyploid cells after VP-16 treatment. These findings indicate that RASSF6 stabilizes p53, regulates apoptosis and the cell cycle, and functions as a tumor suppressor. Together with the previous reports regarding RASSF1A and RASSF3, the stabilization of p53 may be the common function of the C-terminal RASSF proteins.

Nek1 interacts with Ku80 to assist chromatin loading of replication factors and S-phase progression.

NIMA-related kinases (Neks) play divergent roles in mammalian cells. While several Neks regulate mitosis, Nek1 was reported to regulate DNA damage response, centrosome duplication and primary cilium formation. Whether Nek1 participates in cell cycle regulation is not known. Here we report that loss of Nek1 results in severe proliferation defect due to a delay in S-phase of the cell cycle. Nek1-deficient cells show replication stress and checkpoint activation under normal growth conditions. Nek1 accumulates on the chromatin during normal DNA replication. In response to replication stress, Nek1 is further activated for chromatin localization. Nek1 interacts with Ku80 and, in Nek1-deficient cells chromatin localization of Ku80 and several other DNA replication factors is significantly reduced. Thus, Nek1 may facilitate S-phase progression by interacting with Ku80 and regulating chromatin loading of replication factors.

Platelet-derived growth factor-A regulates lung fibroblast S-phase entry through p27(kip1) and FoxO3a.

BACKGROUND: Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation. METHODS: Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/G0 to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27(kip1) and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27(kip1) in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry. RESULTS: In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27(kip1) correlated with reduced LF entry into S-phase. CONCLUSIONS: Nuclear localization of FoxO3a, an important regulator of p27(kip1) gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.

SMC1-mediated intra-S-phase arrest facilitates bocavirus DNA replication.

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction.

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts.

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which are mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that is separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition.

The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G 1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.