Purpose: Abnormal spindle-like microcephaly associated (ASPM) plays an important role in neurogenesis and cell proliferation. This study is to elucidate its role in hepatocelllular carcinoma (HCC), particularly early tumor recurrence (ETR) and prognosis. Experimental Design: We used reverse transcription-PCR assays to measure the ASPM mRNA levels in 247 HCC and correlated with clinicopathologic and molecular features. Results: ASPM mRNA levels were high in fetal tissues but very low in most adult tissues. ASPM mRNAwas overexpressedin162 HCC (66%) but not in benignliver tumors. ASPM overexpression correlated with high a-fetoprotein (P = 1 Â10 ). ETR is the most critical unfavorable clinical prognostic factor. Among the various independent histopathologic (tumor size, tumor grade and tumor stage) and molecular factors (p53 mutation, high a-fetoprotein, and ASPM overexpression), tumor stage was the most crucial histologic factor (odds ratio, 14.7; 95% confidence interval, 6.65-33.0; P = 1 Â10 -8), whereas ASPM overexpression (odds ratio, 6.49; P = 1 Â10 -8 ) is the most important molecular factor associated with ETR. ASPM overexpression was associated with vascular invasion and ETR in both p53-mutated (all P values = 1 Â10 -8) and non-p53-mutated HCC (P = 1 Â10 -8 and 0.00088, respectively). Hence, patients with APSM-overexpressing HCC had lower 5-year survival (P = 0.000001) in both p53-mutated (P = 0.00008) and non-p53-mutated HCC (P = 0.0027). In low-stage (stage II) HCC, ASPM overexpression also correlated with higher ETR (P = 0.008). Conclusion: ASPM overexpression is a molecular marker predicting enhanced invasive/metastatic potential of HCC, higher risk of ETR regardless of p53 mutation status and tumor stage, and hence poor prognosis.

Reactive oxygen species may cause oxidative damage in the placenta, yet some mechanisms must exist to reduce or prevent such damage. We investigated whether oxidative injury to placental endothelial cells is inhibited by activation of antioxidant enzymes by paracrine factors secreted by human placental multipotent mesenchymal stromal cells (hPMSC). hPMSC-conditioned medium and umbilical endothelial cells were assayed for cytokines and cytokine receptor expression by immunoassay and real-time PCR. Endothelial cell survival was evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and caspase 3 activity assay. tert-Butyl hydroperoxide was used to induce oxidative injury in endothelial cells, with fluorescent microscopy and flow cytometry used to detect intracellular peroxides and cell apoptosis. Western blot, real-time PCR, STAT3 DNA-binding activity assay, and STAT3 siRNA were used to assess endothelial cell antioxidant enzymes. hPMSC-conditioned medium supported endothelial cell survival and reduced endothelial cell intracellular peroxides and apoptosis. hPMSCs expressed the transcripts of the interleukin (IL) 6 cytokine family, including IL6 and leukemia-inhibitory factor. hPMSC-conditioned medium activated STAT3 expression in endothelial cells, which was inhibited by neutralizing antibody to interleukin 6 signal transducer (IL6ST) but not to IL6 or leukemia-inhibitory factor. STAT3 siRNA or manganese superoxide dismutase (SOD2) siRNA transfected into endothelial cells inhibited the antiapoptotic effect of conditioned medium. SOD2 was significantly upregulated in endothelial cells by conditioned medium via STAT3 activation that, in turn, was inhibited by IL6ST-neutralizing antibody or STAT3 siRNA. Paracrine factors secreted by hPMSCs support endothelial cell survival. STAT3 activation and SOD2 production protect against oxidative stress-induced endothelial cell damage.

Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.

<div>Abstract<p><b>Purpose:</b><i>Abnormal spindle-like microcephaly associated</i> (<i>ASPM</i>) plays an important role in neurogenesis and cell proliferation. This study is to elucidate its role in hepatocelllular carcinoma (HCC), particularly early tumor recurrence (ETR) and prognosis.</p><p><b>Experimental Design:</b> We used reverse transcription-PCR assays to measure the <i>ASPM</i> mRNA levels in 247 HCC and correlated with clinicopathologic and molecular features.</p><p><b>Results:</b><i>ASPM</i> mRNA levels were high in fetal tissues but very low in most adult tissues. <i>ASPM</i> mRNA was overexpressed in 162 HCC (66%) but not in benign liver tumors. <i>ASPM</i> overexpression correlated with high α-fetoprotein (<i>P</i> = 1 × 10^-8), high-grade (grade II-IV) HCC (<i>P</i> = 2 × 10^-6), high-stage (stage IIIA-IV) HCC (<i>P</i> = 1 × 10^-8), and importantly ETR (<i>P</i> = 1 × 10^-8). ETR is the most critical unfavorable clinical prognostic factor. Among the various independent histopathologic (tumor size, tumor grade and tumor stage) and molecular factors (<i>p53</i> mutation, high α-fetoprotein, and <i>ASPM</i> overexpression), tumor stage was the most crucial histologic factor (odds ratio, 14.7; 95% confidence interval, 6.65-33.0; <i>P</i> = 1 × 10^-8), whereas <i>ASPM</i> overexpression (odds ratio, 6.49; <i>P</i> = 1 × 10^-8) is the most important molecular factor associated with ETR. <i>ASPM</i> overexpression was associated with vascular invasion and ETR in both p53-mutated (all <i>P</i> values = 1 × 10^-8) and non-p53-mutated HCC (<i>P</i> = 1 × 10^-8 and 0.00088, respectively). Hence, patients with <i>APSM</i>-overexpressing HCC had lower 5-year survival (<i>P</i> = 0.000001) in both p53-mutated (<i>P</i> = 0.00008) and non-p53-mutated HCC (<i>P</i> = 0.0027). In low-stage (stage II) HCC, <i>ASPM</i> overexpression also correlated with higher ETR (<i>P</i> = 0.008).</p><p><b>Conclusion:</b><i>ASPM</i> overexpression is a molecular marker predicting enhanced invasive/metastatic potential of HCC, higher risk of ETR regardless of p53 mutation status and tumor stage, and hence poor prognosis.</p></div>

Supplementary Data from <i>ASPM</i> Is a Novel Marker for Vascular Invasion, Early Recurrence, and Poor Prognosis of Hepatocellular Carcinoma