Chi-Chen Fan

Antibody detection of SARS-CoV spike and nucleocapsid protein

Abstract Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6× His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy.

Curcumin Inhibits Invasiveness and Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma Through Reducing Matrix Metalloproteinase 2, 9 and Modulating p53-E-Cadherin Pathway

Hypotheses. Epithelial-mesenchymal transition (EMT) and invasion play a critical role in cancer progression and metastasis. We have shown that low E-cadherin and high Twist expression are significantly correlated with prognostic survival prediction in oral squamous cell carcinoma (OSCC). This study aimed to determine the anti-invasive effect of curcumin on the expression of matrix metalloproteinases (MMPs) and of EMT regulators in OSCC. Methods. SCC-25 cells were treated with curcumin, and cell proliferation, invasion, and expression of MMPs and EMT regulators were assessed for cell viability by trypan blue exclusion, for invasion by Matrigel invasion chamber, and for EMT regulators and MMP changes in the levels of proteins by immunoblotting. Results. Our data showed that curcumin treatment not only decreased the expression of MMP-2 and MMP-9 to inhibit invasiveness in oral cancer but also modulated the expression of EMT markers, such as Snail, Twist, and E-cadherin, and induced p53 expression that is crucial to EMT repression. Conclusion. Curcumin has the potential to become an adjunctive regimen for the prevention of cancer progression and metastasis in oral cancer.

Disruption of murine mp29/Syf2/Ntc31 gene results in embryonic lethality with aberrant checkpoint response.

Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29GT/GT) using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29GT/GT mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf). A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.

hPuf-A/KIAA0020 Modulates PARP-1 Cleavage upon Genotoxic Stress

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3.

Hemodynamic and Neuropathological Analysis in Rats with Aluminum Trichloride-Induced Alzheimer's Disease

Background and Aims Hemodynamic normality is crucial to maintaining the integrity of cerebral vessels and, therefore, preserving the cognitive functions of Alzheimers disease patients. This study investigates the implications of the hemodynamic changes and the neuropathological diversifications of AlCl3-induced AD. Methods The experimental animals were 8- to 12-wk-old male Wistar rats. The rats were randomly divided into 2 groups: a control group and a (+)control group. Food intake, water intake, and weight changes were recorded daily for 22 wk. Synchronously, the regional cerebral blood flow (rCBF) of the rats with AlCl3-induced AD were measured using magnetic resonance imaging (MRI). The hemorheological parameters were analyzed using a computerized auto-rotational rheometer. The brain tissue of the subjects was analyzed using immunohistological chemical (IHC) staining to determine the beta-amyloid (Aβ) levels. Results The results of hemodynamic analysis revealed that the whole blood viscosity (WBV), fibrinogen, plasma viscosity and RBC aggregation index (RAI) in (+)control were significantly higher than that of control group, while erythrocyte electrophoresis (EI) of whole blood in (+)control were significantly lower than that of control group. The results of acetylcholinesterase-RBC (AChE-RBC)in the (+)control group was significantly higher than that of the control group. The results also show that the reduction of rCBF in rats with AlCl3-induced AD was approximately 50% to 60% that of normal rats. IHC stain results show that significantly more Aβ plaques accumulated in the hippocampus and cortex of the (+)control than in the control group. Conclusion The results accentuate the importance of hemorheology and reinforce the specific association between hemodynamic and neuropathological changes in rats with AlCl3-induced AD. Hemorheological parameters, such as WBV and fibrinogen, and AChE-RBC were ultimately proven to be useful biomarkers of the severity and progression of AD patients. In addition, the parameters can be substituted for invasive inspection in therapeutic intervention.

Cloning, expression, and genomic organization of mouse mp29 gene.

Human p29 has been demonstrated in the yeast two-hybrid method and in vitro GST pull-down assay to associate with GCIP, a cyclin D interacting protein. In this study, we describe the cloning and genomic structure of the mouse homologue, mp29. The overall mouse mp29 amino acid sequence is highly identical (91%) to human p29. Polyclonal antibody against mp29 was raised and the subcellular localization of mp29 was identified to be in the nucleus. Genomic clones containing mp29 gene were isolated and this gene was divided into seven exons spanning 9kb of genomic DNA. The transcription initiation site of mp29 gene was determined to be 94bp upstream of the translation initiation codon and the first 140bp proximal TATA-less promoter region is required to activate minimal transcription of mouse mp29 gene in mammalian cells.

Upregulated hPuf-A promotes breast cancer tumorigenesis

AbstracthPuf-A is a member of RNA-binding PUF family that regulates mRNA translation. Redistribution of hPuf-A from the nucleolus to the nucleoplasm upon genotoxic stress modulates the poly(ADP-ribosyl)ation activity of PARP-1. Here, we report a novel function of hPuf-A involved in promoting breast cancer progression. Immunohistochemical studies showed higher expression levels of hPuf-A in stage I, II, III, and IV breast cancer specimens in contrast with those of hPuf-A in ductal carcinoma in situ. The presence of hPuf-A is highly associated with colony formation capacities in breast cancer T47D and MDA-MB-231 cells. Xenograft growth of hPuf-A-silenced and hPuf-A overexpressing MDA-MB-231 cells in nude mice was substantially in concert with colony formation capacities. This promoting effect of hPuf-A in tumorigenesis might be correlated with the regulation of its associated mRNAs, such as RbAp48 and DDX3. Collectively, hPuf-A may have diagnostic values in breast cancer progression.

Loss of GDF10/BMP3b as a prognostic marker collaborates with TGFBR3 to enhance chemotherapy resistance and epithelial-mesenchymal transition in oral squamous cell carcinoma

Growth differentiation factor‐10 (GDF10), commonly referred as BMP3b, is a member of the transforming growth factor‐β (TGF‐β) superfamily. GDF10/BMP3b has been considered as a tumor suppressor, however, little is known about the molecular mechanism of its roles in tumor suppression in oral cancer. Clinical significance of GDF10 downregulation in oral squamous cell carcinoma (OSCC) was evaluated using three independent cohorts of OSCC patients. The molecular mechanisms of GDF10 in the suppression of cell survival, cell migration/invasion and epithelial‐mesenchymal transition (EMT) were investigated by using oral cancer cell lines. The present study shows that GDF10 is downregulated during oral carcinogenesis, and GDF10 expression is also an independent risk factor for overall survival of OSCC patients. Overexpression of GDF10 attenuates cell proliferation, transformation, migration/invasion, and EMT. GDF10‐inhibited EMT is mediated by ERK signaling but not by typical TGF‐β signaling. In addition, overexpression of GDF10 promotes DNA damage‐induced apoptosis and sensitizes the response to all‐trans retinoic acid (ATRA) and camptothecin (CPT). Intriguingly, the expression of GDF10 is induced by type III TGF‐β receptor (TGFBR3) through TGF‐β‐SMAD2/3 signaling. Our findings suggest that TGFBR3 is an upstream activator of GDF10 expression and they share the same signaling to inhibit EMT and migration/invasion. These results support that GDF10 acts as a hinge to collaborate with TGFBR3 in the transition of EMT‐MET program. Taken together, we illustrated the clinical significance and the molecular mechanisms of tumor‐suppressive GDF10 in OSCC.

Cdc7-Dbf4-mediated phosphorylation of HSP90-S164 stabilizes HSP90-HCLK2-MRN complex to enhance ATR/ATM signaling that overcomes replication stress in cancer

Cdc7-Dbf4 kinase plays a key role in the initiation of DNA replication and contributes to the replication stress in cancer. The activity of human Cdc7-Dbf4 kinase remains active and acts as an effector of checkpoint under replication stress. However, the downstream targets of Cdc7-Dbf4 contributed to checkpoint regulation and replication stress-support function in cancer are not fully identified. In this work, we showed that aberrant Cdc7-Dbf4 induces DNA lesions that activate ATM/ATR-mediated checkpoint and homologous recombination (HR) DNA repair. Using a phosphoproteome approach, we identified HSP90-S164 as a target of Cdc7-Dbf4 in vitro and in vivo. The phosphorylation of HSP90-S164 by Cdc7-Dbf4 is required for the stability of HSP90-HCLK2-MRN complex and the function of ATM/ATR signaling cascade and HR DNA repair. In clinically, the phosphorylation of HSP90-S164 indeed is increased in oral cancer patients. Our results indicate that aberrant Cdc7-Dbf4 enhances replication stress tolerance by rewiring ATR/ATM mediated HR repair through HSP90-S164 phosphorylation and by promoting recovery from replication stress. We provide a new solution to a subtyping of cancer patients with dominant ATR/HSP90 expression by combining inhibitors of ATR-Chk1, HSP90, or Cdc7 in cancer combination therapy.