Shyng-Shiou F. Yuan

Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products.

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mre11. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mre11/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

Nuclease Activities in a Complex of Human Recombination and DNA Repair Factors Rad50, Mre11, and p95

Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganese-dependent single-stranded DNA endonuclease and 3′ to 5′ exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability.

Ionizing radiation-induced Rad51 nuclear focus formation is cell cycle-regulated and defective in both ATM(-/-) and c-Abl(-/-) cells.

In eukaryotes, DNA double-strand breaks (DSBs) can be repaired by either non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways. Rad50 protein is a component of the Rad50/NBS1/Mre11 nuclease complex that functions in both the NHEJ and recombinational repair of DNA DSBs. On the other hand, Rad51 protein, a homolog of bacterial RecA and a member of the Rad52 epistasis group, plays a crucial role exclusively in the recombinational repair pathway. We analyzed the effects of cell cycle progression and genetic background on the ionizing radiation (IR)-induced Rad51 and Rad50 repair focus formation. Herein, we demonstrated that IR-induced Rad51, but not Rad50, nuclear focus formation was cell cycle-dependent. Furthermore, IR-induced Rad51 focus formation was defective in AT and c-Abl(-/-) cells, but not wild type or NBS cells. A decreased and delayed formation of Rad51 foci-containing nuclei was observed in AT cells upon IR, whereas in c-Abl(-/-) cells a decreased but not delayed formation of Rad51 foci-containing nuclei was observed. In conclusion, effective and prompt IR-induced Rad51 focus formation is cell cycle-regulated and requires both ATM and c-Abl.

Correlations between umbilical and maternal serum adiponectin levels and neonatal birthweights

Objective.  To measure adiponectin levels in maternal serum and umbilical cord serum at delivery, and examine whether or not there are correlations between adiponectin levels and neonatal birthweights, maternal body weights and body mass indexes.

lc3 Over-expression Improves Survival and Attenuates Lung Injury Through Increasing Autophagosomal Clearance in Septic Mice

Objective: To clarify the role of autophagy in sepsis-induced lung injury. Background: The role of autophagy as a protective or maladaptive response in lung cells during sepsis has not yet been determined. The lack of specificity of the autophagic process has driven the development of new approaches that assessautophagosomes from formation to fusion with lysosomes. Methods: Sepsis was induced by cecal ligation and puncture (CLP). The autophagic process was manipulated using the pharmacological inhibitors of the autophagy pathway. Green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice were further used to determine the role of autophagy. Results: The formation of autophagosomal protein LC3-II progressively accumulated in the lungs over 24 hours after CLP, with the Lc3 gene expression returning to baseline levels at 24 hours. Autophagosome-lysosome fusion, however, gradually decreased from 8 to 24 hours after CLP, suggesting impaired clearance of autophagosomes rather than upregulation of autophagy in the septic lung. In contrast, transgenic mice overexpressing the Lc3 gene exhibited increased clearance of autophagosomes and improved survival after CLP. This protective effect was also seen in decreased cell death, inflammatory responses, neutrophil accumulation, albumin leakage, and edema formation. However, blockade of autophagosome-lysosome fusion with bafilomycin A1 abolished the protective effects in transgenic mice. This indicates that Lc3 transgene attenuates lung injury/inflammation in sepsis, possibly through increasing the clearance of autophagosomes. Conclusions: Autophagy in the septic lung represents a protective response. However, autophagy, by virtue of excessive autophagosome accumulation, may play a maladaptive role in the late stage of sepsis, leading to acute lung injury.

High Visfatin Expression in Breast Cancer Tissue Is Associated with Poor Survival

Background: Adipocytokines, adipocyte-secreted hormones, play a critical role in breast cancer development. The expression of visfatin, a newly discovered adipocytokine, in breast cancer tissues was determined and correlated with patient clinicopathologic variables. Methods: Visfatin expression in breast cancer tissues was analyzed by immunohistochemistry. Visfatin expression was correlated with clinicopathologic variables as well as recurrence rates, using the χ2 test. The prognostic value of visfatin for disease-free and overall survival was evaluated by Kaplan–Meier estimates, and the significance of differences between curves was evaluated by the log-rank test. Results: High visfatin expression in breast cancer tissues was significantly correlated with tumor size, estrogen receptor (ER) negativity, and progesterone receptor (PR) negativity. Hormone therapy, but not radiotherapy or chemotherapy, decreased the recurrence rate in patients with high visfatin expression. Whereas high visfatin expression alone was associated with poor disease-free and overall survival, worse disease-free and overall survival was observed when high visfatin expression was combined with ER- and PR-negative status. Cox regression analysis also revealed that visfatin is an independent predictor of disease-free and overall survival. Conclusion: High visfatin expression in breast cancer tissue is associated with more malignant cancer behavior as well as poor patient survival. Impact: Visfatin is an independent prognosis predictor for breast cancer. Cancer Epidemiol Biomarkers Prev; 20(9); 1892–901. ©2011 AACR.

STAT3 ser727 phosphorylation and its association with negative estrogen receptor status in breast infiltrating ductal carcinoma.

Although it is known that STAT3 transcriptional activity is modulated by phosphorylation at serine residue 727, the role of STAT3 serine phosphorylation in breast cancer remains mostly unexplored. In this study, we examined the expression patterns of serine residue 727‐phosphorylated STAT3 (p‐ser727‐STAT3) in breast infiltrating ductal carcinoma tissues and nearby noncancer tissues by using immunoblotting techniques, and correlated the expression profiles with clinicopathological characteristics. A significantly elevated p‐ser727‐STAT3 expression was observed in 61.8% (42/68) of breast cancer tissues as compared to corresponding noncancer tissues (p < 0.001). Further, immunohistochemical analysis also showed an increased nuclear p‐ser727‐STAT3 staining in cancer lesions. The increased p‐ser727‐STAT3 expression in breast infiltrating ductal carcinoma tissues correlated significantly with negative estrogen receptor (ER) status, increased stage of cancer and increased tumor size (p = 0.001, 0.024 and 0.014, individually). Intriguingly, we noticed that the expression levels of p‐ser727‐STAT3 in ER‐negative breast cancer cell lines were higher than those in ER‐positive breast cancer cell lines. In ER‐positive MCF7 cells, treatment with ERα‐specific siRNA increased, whereas treatment with anticancer drug tamoxifen decreased the expression of p‐ser727‐STAT3, phenomena not observed in ER‐negative MDA‐MB‐231 cells. In conclusion, our results suggest that p‐ser727‐STAT3 may be involved in the pathogenesis of breast cancer in an ER‐dependent manner.

Amniotic fluid and maternal serum leptin levels in pregnant women who subsequently develop preeclampsia.

OBJECTIVES To study the correlation between amniotic fluid leptin levels and maternal serum leptin levels during the early second trimester, and to determine whether the ratios of amniotic fluid leptin levels to maternal serum leptin levels are elevated in pregnant women who subsequently develop preeclampsia. STUDY DESIGN Samples from 120 pregnant women were included in this prospective study, of which 20 were from pregnant women who subsequently developed preeclampsia and 100 were from normal pregnant women. Both the amniotic fluid and the maternal serum leptin levels were ascertained by radioimmunoassay (RIA). RESULTS A strong correlation between amniotic fluid leptin levels and maternal serum leptin levels was observed in both preeclamptic and normal pregnant women. In addition, the ratios of amniotic fluid leptin levels to maternal serum leptin levels were positively correlated to amniotic fluid leptin levels, but negatively correlated to maternal serum leptin levels. Furthermore, the ratios of amniotic fluid leptin levels to maternal serum leptin levels in preeclamptic women were significantly higher than those in normal pregnant women. CONCLUSIONS Amniotic fluid leptin levels correlated with maternal serum leptin levels during the early second trimester. The ratios of amniotic fluid leptin levels to maternal serum leptin levels were elevated in preeclamptic women. However, the maternal serum leptin levels themselves showed no such elevation. Therefore, this elevated ratio may be a marker at the early stage of pregnancy in preeclamptic women.

Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells.

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.

The synthetic β-nitrostyrene derivative CYT-Rx20 induces breast cancer cell death and autophagy via ROS-mediated MEK/ERK pathway.

The β-nitrostyrene family has been shown to suppress cancer cell proliferation and induce programmed cell death. However, mechanisms underlying β-nitrostyrenes remain less evaluated. Here, we synthesized a β-nitrostyrene derivative, CYT-Rx20, and characterized its anticancer effect and involving mechanisms in breast cancer. We found that CYT-Rx20 arrested breast cancer cells at G2/M phase and decreased cell viability by activating the caspase cascade, accompanying with increases of poly (ADP-ribose) polymerase (PARP) cleavage and γ-H2AX expression. On the other hand, up-regulation of Beclin-1, ATG5, and LC-3 was observed in CYT-Rx20-induced autophagy, which was evidently shown by transmission electron microscopy. In addition to these, CYT-Rx20-induced breast cancer cell death, intracellular reactive oxygen species (ROS) formation and expression of phospho-ERK1/2, Beclin-1, and LC-3 were significantly reversed in the presence of N-acetyl-l-cysteine (NAC), a thiol antioxidant. Furthermore, the cytotoxicity of CYT-Rx20 was enhanced by co-treatment with the autophagy inhibitor chloroquine or bafilomycin A1, suggesting that an incomplete autophagy process could deteriorate CYT-Rx20-induced cytotoxicity. In nude mice xenograft study, CYT-Rx20 significantly reduced orthotopic tumor growth. Immunohistochemical analysis revealed elevated expression of phospho-ERK1/2 and LC-3 in tumor tissues of the mice treated with CYT-Rx20. Together, we propose that CYT-Rx20 may have potential to be further developed into a β-nitrostyrene-based anticancer compound for the treatment of breast cancer.