O304 is a mitochondrial uncoupler which extends C. elegans lifespan and induces vasorelaxation of rat mesenteric arteries.

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Cell migration is critical for animal development and physiological as well as pathological responses. One important step during cell migration is the formation of lamellipodia at the leading edge of migrating cells. Here we report that the second messenger cAMP inhibits the migration of mouse embryonic fibroblast cells and mouse breast tumor cells. cAMP acts downstream of the small GTPase Rac and interferes with the formation of lamellipodia. Moreover, cAMP decreases the phosphorylation of the myosin light chain at the leading edge of cells and increases the phosphorylation of the vasodilator-stimulated phosphoprotein. Together with our previous report of a positive role of another second messenger, cGMP, in lamellipodium formation, our data indicate that cAMP and cGMP play opposite roles in modulating lamellipodium formation.

Objective To quantitatively compare the γ-H2AX foci formation between DNA-PKcs+ /+ and DNA-PKcs-/-mouse embryonic fibroblast (MEF) cells, and to investigate the dynamic changes in DNA double-strand breaks (DSBs) in human nasopharyngeal carcinoma SUNE-1 cells exposed to X-ray radiation. Methods The expression of DNA-PKcs was determined by Western blot. The γ-H2AX foci formation induced by 5 Gy X-ray radiation was detected by cell immunofluorescence. The ImageJ software was used to quantitatively analyze the γ-H2AX foci formation. Results The expression of DNA-PKcs was silenced in DNA-PKcs-/-MEF cells and normal in DNA-PKcs+ /+ MEF cells. According to the dynamic analyses of the numbers of γ-H2AX foci/cell and γ-H2AX foci/mm2, a similar tendency was observed in DSB formation in DNA-PKcs+ /+ MEF cells, DNA-PKcs-/-MEF cells, and SUNE-1 cells exposed to X-ray radiation. A large number of γ-H2AX foci formed at 0.5-1.0 h after radiation. DSBs were repaired at 6 h after radiation in DNA-PKcs+ /+ MEF cells and 24 h after radiation in DNA-PKcs-/-MEF cells and SUNE-1 cells. The peak values of γ-H2AX foci/cell and γ-H2AX foci/mm2 were observed at 1.0 and 0.5 h after radiation, respectively. Compared with DNA-PKcs+ /+ MEF cells, DNA-PKcs-/-MEF cells had different numbers of γ-H2AX foci/cell at 0.5, 1.0, 3.0, 6.0, and 12.0 h after radiation, as well as different numbers of γ-H2AX foci/mm2 at 3.0, 6.0, and 12.0 h after radiation. Conclusions Quantitative measurement of the number of γ-H2AX foci/cell or γ-H2AX foci/mm2 by cell immunofluorescence provides new insights into the quantitative and dynamic study of DSB damage and repair. Key words: Non-homologous terminal connection; DNA dependent protein kinase catalytic subunit; γH2AX foci formation; SUNE-1 cell line

Tumor suppressor gene PTEN is highly mutated in a wide variety of human tumors. To identify unknown targets or signal transduction pathways that are regulated by PTEN, microarray analysis was performed to compare the gene expression profiles of Pten null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts. Expression of a heparin binding growth factor, pleiotrophin (Ptn), was found to be up-regulated in Pten-/- MEFs as well as Pten null mammary tumors. Further experiments revealed that Ptn expression is regulated by the PTEN-PI3K-AKT pathway. Knocking down the expression of Ptn by small interfering RNA resulted in the reduction of Akt and GSK-3beta phosphorylation and suppression of the growth and the tumorigenicity of Pten null MEFs. Our results suggest that PTN participates in tumorigenesis caused by PTEN loss and PTN may be a potential target for anticancer therapy, especially for those tumors with PTEN deficiencies.

Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational C-terminal processing of prenylated proteins, suppresses tumor cell growth and induces cell death. Icmt inhibition by either a small molecule inhibitor termed as cysmethynil or inhibitory RNA induces marked autophagy leading to cell death. HepG2 cells were used to investigate the function of autophagy in tumor cell death. Suppression of autophagy, either pharmacologically or through knockdown of the autophagy essential proteins, Atg5 or Atg1, inhibits not only cysmethynil-induced autophagy, but also apoptosis in HepG2 cells. The dependence of cysmethynil-induced apoptosis on autophagy was further shown using autophagy-deficient mouse embryonic fibroblast (MEF) cells. Atg5(-/-) MEF cells were found to be resistant to cysmethynil-induced apoptosis, whereas wild-type MEFs showed high sensitivity to apoptosis induction. These data indicate that inhibition of Icmt can elicit cell death through two linked mechanisms, autophagy and apoptosis, and that autophagy can be an active player upstream of apoptosis in cell types capable of apoptotic cell death, such as HepG2 and MEFs. Further, treatment of mice-bearing HepG2-derived tumors with cysmethynil resulted in marked inhibition of tumor growth; analysis of tumor tissue from these mice revealed markers consistent with autophagy induction and cell growth arrest.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

beta1 integrins bind to the extracellular matrix and stimulate signaling pathways leading to crucial cellular functions, including proliferation, apoptosis, cell spreading and migration. Consequently, control of beta1 integrin function depends upon its subcellular localization, and recent studies have begun to unravel the complex regulatory mechanisms involved in integrin trafficking. We report that the C-terminal Eps15-homology (EH) domain-containing protein EHD1 plays an important role in regulating beta1 integrin transport. Initially, we demonstrated that RNAi-knockdown of Ehd1 results in impaired recycling of beta1 integrins and their accumulation in a transferrin-containing endocytic recycling compartment. Mouse embryonic fibroblast (MEF) cells derived from EHD1-knockout mice (Ehd1(-/-) MEF) exhibited lower overall levels of beta1 integrins on the plasma membrane, but higher cell-surface-expressed activated beta1 integrins, and larger, more prominent focal adhesions resulting from slower kinetics of focal adhesion disassembly. In addition, both migration and cell spreading on fibronectin were impaired in Ehd1(-/-) MEF cells, and these defects could be similarly induced by EHD1-RNAi treatment of normal Ehd1(+/+) MEF cells. They could also be rescued by transfection of wild-type EHD1 into Ehd1(-/-) MEF cells. Our data support a role for EHD1 in beta1 integrin recycling, and demonstrate a requirement for EHD1 in integrin-mediated downstream functions.

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.

Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9 -/- MES and Mdc1 -/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9 -/- MES. As the exposure to SMG was prolonged, Rad9 -/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9 -/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1 -/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1 -/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.

In the present study, we show that treatment of wild-type (p53+/+) mouse embryonic fibroblast (MEF) cells with a DNA-alkylating agent, N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), resulted in increased levels of adenomatous polyposis coli (APC) mRNA compared to p53 gene-knocked out (p53-/-) MEF cells, indicating that p53 is required for APC expression after alkylation damage. By using HCT-116 colon cancer cells (containing wild-type p53 gene) or p53-/- MEF cells transfected with a pCMV-p53 overexpression plasmid [p53-/-(CMV-p53)], we show that p53 is a labile factor for APC gene expression, and that pretreating HCT-116 cells with a protein synthesis inhibitor, cycloheximide (CHX), inhibited MNNG-induced APC mRNA levels by inhibiting p53 protein synthesis. The effect of CHX on p53 protein synthesis was reversible, as the withdrawal of CHX permitted p53 protein synthesis to resume with a concomitant increase in APC mRNA levels after MNNG treatment. To examine whether p53 regulates APC gene expression at the transcriptional level, we treated HCT-116 or p53-/-(CMV-p53) MEF cells with 5,6-dichloro-1-beta-D-ribofuranosylbenzamidazole (DRB; a transcriptional inhibitor), before the MNNG treatment. Although treatment of cells with DRB resulted in increased p53 protein levels, that the APC mRNA levels were decreased suggests that p53 may enhance APC gene expression upstream of the transcriptional machinery where DRB interacts. That the withdrawal of DRB, and subsequent MNNG treatment, increased the level of APC mRNA indicated that the binding of DRB to the transcriptional machinery was reversible.

Ca(+) sparks are vascular smooth muscle cell (VSMC) Ca(2+)-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca(2+)-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H2S)-induced dilation. Because we also observed that H2S can increase Ca(2+) spark activity, we hypothesized that loss of H2S after IH exposure reduces Ca(2+) spark activity and that blocking Ca(2+) spark generation reduces H2S-induced dilation. Ca(2+) spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K(+)] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H2S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca(2+) spark activity were due to increased sparks per site, whereas H2S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca(2+) spark activity with ryanodine (10 μM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H2S in arteries from both sham and IH rats. Our results suggest that H2S regulates RyR activation and that H2S-induced dilation requires Ca(2+) spark activation. IH exposure decreases endogenous H2S-dependent Ca(2+) spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) has been successfully used in the treatment of cancers. However, the mechanism of 5-ALA transportation into cancer cells is still not fully elucidated. Previous studies have confirmed that the efficiency of 5-ALA-PDT could be affected by the membrane skeleton protein 4.1R. In this study, we investigated the role of 4.1R in the transport of 5-ALA into cells. Wild-type (4.1R+/+ ) and 4.1R gene knockout (4.1R-/- ) mouse embryonic fibroblast (MEF) cells were incubated with 1mm 5-ALA and different concentrations of specific inhibitors of GABA transporters GAT (1-3). Our results showed thatthe inhibition of GAT1 and GAT2 in particular markedly attenuated the intracellular PpIX production, reactive oxygen species (ROS) level and 5-ALA-induced photodamage. However, the inhibition of GAT3 did not show such effects. Further research showedthat 4.1R-/- MEF cells had a lower expression of GAT1 and GAT2 than 4.1R+/+ MEF cells. Additionally, 4.1R directly bound to GAT1 and GAT2. Taken together, GAT1 and GAT2 transporters are involved in the uptake of 5-ALA in MEF cells. 4.1R plays an important role in transporting 5-ALA into cells via at least partly interaction with GAT1 and GAT2 transporters in 5-ALA-PDT.