Syrosingopine sensitizes cancer cells to killing by metformin

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer

Degradation of human exonuclease 1b upon DNA synthesis inhibition

In response to DNA damage, signaling pathways are triggered that either block the cell division cycle at defined transitions (G1-S and G2-M) or slow down progression through the S phase. Nucleases play important roles in DNA synthesis, recombination, repair, and apoptosis. In this study, we have examined the regulation of human exonuclease 1 (hEXO1b). The endogenous hEXO1b protein was only detected upon enrichment by immunoprecipitation. We found that hEXO1b was constantly expressed throughout the cell cycle. However, treatment of cells with agents that cause arrest of DNA replication led to rapid degradation of hEXO1b. This effect was fully reversed upon removal of the block. Analysis of synchronized cells showed that degradation of hEXO1b during the S phase was strictly dependent on DNA synthesis inhibition. DNA damage caused by UV-C radiation, ionizing radiation, cisplatin, or the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine did not affect hEXO1b stability. We show that hEXO1b was phosphorylated in response to inhibition of DNA synthesis and that phosphorylation coincided with rapid protein degradation through ubiquitin-proteasome pathways. Our data support the evidence that control of exonuclease 1 activity may be critical for the maintenance of stalled replication forks

Mismatch repair status and the response of human cells to cisplatin

The emergence of resistance to cisplatin is a serious drawback of cancer therapy. To help elucidate the molecular basis of this resistance, we examined matched ovarian cancer cell lines that differ in their DNA mismatch repair (MMR) status and the response to cisplatin. Checkpoint activation by cisplatin was identical in both lines. However, sensitive cells delayed S-phase transition, arrested at G(2)/M and died by apoptosis. The G(2)/M block was characterized by selective disappearance of homologous recombination (HR) proteins, which likely resulted in incomplete repair of the cisplatin adducts. In contrast, resistant cells transiently arrested at G(2)/M, maintained constant levels of HR proteins and ultimately resumed cell cycle progression. The net contribution of MMR to the cisplatin response was examined using matched semi-isogenic (HCT116+/-chr3) or strictly isogenic (293T-Lalpha(-/+)) cell lines. Delayed transition through S-phase in response to cisplatin was also observed in the MMR-proficient HCT116+chr3 cells. Unlike in the ovarian cell lines, however, both HCT116+chr3 and HCT116 permanently arrested at G(2)/M with an intact complement of HR proteins and died by apoptosis. A similar G(2)/M arrest was observed in the strictly isogenic 293T-Lalpha(-/+) cells. This confirmed that although MMR undoubtedly contributes towards the cytotoxicity of cisplatin, it is only one of several pathways that modulate the cellular response to this drug. However, our data highlighted the importance of HR to cisplatin cytotoxicity and suggested that HR status might represent a novel prognostic marker and possibly also a therapeutic target, the inhibition of which would substantially sensitize cells to cisplatin chemotherapy

12-O-Tetradecanoylphorbol-13-acetate activates the Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of SOS involving serine phosphorylation of Shc in NIH3T3 cells

We investigated the activation of the Ras/ERK signaling pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) in NIH3T3 fibroblasts. Interestingly, the activation was suppressed not only by dominant negative Raf-1 but also by dominant negative Ras and SOS. Further analysis revealed that TPA treatment induced, dependently on protein kinase C, the mobility shift of p66(shc) in SDS-polyacrylamide gel electrophoresis, which could be prevented by treatment of the Shc immunoprecipitate with serine/threonine-specific protein phosphatase 1 (PP1) or 2A (PP2A). Phosphoamino acid analysis of Shc showed that unlike growth factor-induced Shc phosphorylation, where Shc is mainly phosphorylated at tyrosine residues, TPA-induced phosphorylation was only at serine residues. Like growth factor-induced Shc phosphorylation, which leads to the association of Shc with Grb2, TPA also induced this association, but, correspondingly to the above results, the TPA-induced association was disrupted by in vitro treatment of the Shc immunoprecipitate with PP1. Taken together, these results suggest that the TPA signal was fed at or upstream of Shc to activate the Ras/ERK signaling pathway involving serine phosphorylation of Shc

Activation and nuclear translocation of mitogen-activated protein kinases by polyomavirus middle-T or serum depend on phosphatidylinositol 3-kinase

Several cellular signal transduction pathways activated by middle-T in polyomavirus-transformed cells are required for viral oncogenicity. Here we focus on the role of phosphatidylinositol 3-kinase (PI 3-kinase) and Ras and address the question how these signaling molecules cooperate during cell cycle activation. Ras activation is mediated through association with SHC.GRB2.SOS and leads to increased activity of several members of the mitogen-activated protein (MAP) kinase family, while activation of PI 3-kinase results in the generation of D3-phosphorylated phosphatidylinositides whose downstream targets remain elusive. PI 3-kinase activation might also ensue as a direct consequence of Ras activation. Oncogenicity of middle-T requires stimulation of both Ras- and PI 3-kinase-dependent pathways. Mutants of middle-T incapable to bind either SHC.GRB2.SOS or PI 3-kinase are not oncogenic. Sustained activation and nuclear localization of one of the MAP kinases, ERK1, was observed in wild type but not in mutant middle-T-expressing cells. Wortmannin, an inhibitor of PI 3-kinase, prevented MAP kinase activation and nuclear localization in middle-T-transformed cells. PI 3-kinase activity was also required for activation of the MAP kinase pathway in normal serum-stimulated cells, generalizing the concept that signaling through MAP kinases requires not only Ras-but also PI 3-kinase-mediated signals

DNA end resection by CtIP and exonuclease 1 prevents genomic instability

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity

14-3-3 proteins regulate exonuclease 1-dependent processing of stalled replication forks

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells