Lanying Sun

Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation.

Reactive oxygen species (ROS) are supposed to play an important role in hypoxia‐ and ischemia/reperfusion‐mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl2) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl2 and its molecular mechanisms have yet to be elucidated. We report that CoCl2 triggered neuronal PC12 cells apoptosis in a dose‐ and time‐dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl‐XL. To our knowledge, this is the first documentation of the apoptotic induction of CoCl2 on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl2 treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl2‐induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA‐binding activity of AP‐1 in response to CoCl2 and this increase was blocked by antioxidants, showing that CoCl2‐induced apoptosis is accompanied by ROS‐activated AP‐1. CoCl2‐treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS‐linked neuronal disorders. J. Neurosci. Res. 64:646–653, 2001.

Interleukin-2 gene therapy of chronic neuropathic pain

Previous research has revealed an antinociceptive (analgesic) effect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the effect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive effect, but that this only lasted for 10-25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex in contrast also showed dose-dependent antinociceptive effects, but these reached a peak at day 2-3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive effect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not affected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 profile detected by western blot coincided with its mRNA profile except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal fluid could still be detected at day 6, but lower than day 3. The antinociceptive effect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive effect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain.

Interleukin-2 gene has superior antinociceptive effects when delivered intrathecally.

The antinociceptive effect of interleukin-2 gene on rat carrageenan-induced pain was explored using different delivery methods. Intrathecal (i.t.) or plantar s.c. delivery of plasmid harbouring the interleukin-2 gene produced a marked antinociceptive effect, which was maintained up to 6 days; the administration of recombinant human interleukin-2 only had a transitory effect. The antinociceptive effect lasted longer and was more potent when the interleukin-2 gene was administered i.t. than when delivered s.c. The effect of the interleukin-2 gene was related to its protein expression, was dose dependent, and could be potentiated by liposome. The results suggest that the interleukin-2 gene has a good prospect for clinical use.

Combination of Targeting Gene-ViroTherapy with 5-FU Enhances Antitumor Efficacy in Malignant Colorectal Carcinoma

To improve the therapeutic effect of ONYX015, an E1B55kD-deleted replication-competent adenovirus, ZD55 was constructed and armed with the therapeutic gene hTRAIL to form ZD55-hTRAIL, which was used for cancer therapy and which we call Targeting Gene-ViroTherapy. In vitro experiments with SW620, HCT116, and HT29 colorectal carcinoma cell lines demonstrated that they were all sensitive to ZD55-hTRAIL, and especially sensitive to ZD55-hTRAIL plus 5-fluorouracil (5-FU) treatment. In the SW620 xenograft tumor model, various treatment groups showed marked differences at week 11, with the tumor volume for the phosphate-buffered saline (PBS) treatment group >1700 mm3, for 5-FU > 1300 mm3, for ONYX015 1051.3 mm3, for ZD55-hTRAIL 600.05 mm3, and for ZD55-hTRAIL plus 5-FU 230.2 mm3. At the end of week 14, tumor-bearing mice in the other groups almost all died, whereas all the mice in the combined treatment group were alive, with one mouse tumor free. By transmission electron microscopy (TEM) assay, most tumor cells treated with ONYX015 or with ZD55-hTRAIL singly or in combination with 5-FU were lysed due to viral propagation. RT-PCR analysis and immunohistochemistry examination revealed that hTRAIL was expressed in ZD55-hTRAIL-treated SW620 tumor tissue. Furthermore, no detectable hepatoxicity was found by serum enzyme level analysis. These results suggest that ZD55-hTRAIL alone or in combination with 5-FU may have potential clinical implications.

Enhanced suicide gene therapy by chimeric tumor‐specific promoter based on HSF1 transcriptional regulation

Two tandem cassettes, one containing the telomerase reverse transcriptase gene (hTERT) promoter upstream of a constitutively activated form of heat shock transcription factor 1 (cHSF1) and followed by the other containing the heat shock protein 70B (hsp70B) promoter (HSE) upstream of the cytosine deaminase (CD) gene, could greatly enhance the efficiency of CD gene therapy while retaining tumor specificity in vitro and in vivo. This hTERT‐cHSF1/HSE promoter could restrict gene expression in tumor cells and was about 1.5–3‐fold more potent than the cytomegalovirus (CMV) promoter. hTERT‐cHSF1/HSE‐CD transfection led to tumor cells more sensitive to 5‐fluorocytosine compared with hTERT‐CD and its toxicity was comparable to that of CMV‐CD. Besides enhancement of promoter activity, cHSF1 overexpression itself could enhance the bystander effect of CD gene therapy that could be reversed by anti‐Fas antibody. This system also led to activation of stress‐related genes such as hsp70 in tumor cells, which in the presence of cell killing by the cytotoxic gene is a highly immunostimulatory event. Furthermore, a more potent anti‐tumor effect of hTERT‐cHSF1/HSE‐CD was observed in nude mice inoculated with Bcap37 cells. No obvious activity of the hTERT‐cHSF1/HSE promoter was observed in normal tissues after intravenous administration. These results indicate that the hTERT‐cHSF1/HSE promoter is highly tumor‐specific and strong with potential application in targeted gene therapy, and therefore may be useful for construction of vectors for systemic therapy.

Geldanamycin, a heat shock protein 90‐binding agent, disrupts Stat5 activation in IL‐2‐stimulated cells

The 90‐kDa heat shock protein (Hsp90) is the most abundant molecular chaperone in eukaryotic cells. Hsp90 plays a critical role in regulating signal transduction pathways that control cell proliferation since its chaperone function is restricted to a subset of proteins including some signal molecules. Improper function of these proteins can be induced by an anti‐tumor agent geldanamycin (GA) which is the specific inhibitor of Hsp90. In this study, it was demonstrated that GA interferes with IL‐2‐stimulated proliferation of murine CTLL‐2 cells. As to the signaling mechanisms underlying this inhibitory effect, we discovered GA disrupts the IL‐2‐stimulated activation and phosphorylation of the transcription factor Stat5, indicating the proper function of Hsp90 is indispensable for Stat5 activation. This conclusion is validated by the observation that Hsp90 interacts with Stat5 in the immunoprecipitation assay and GA interrupts their interaction. Furthermore, by constructing deletion mutants, we identified the c‐terminal half of Stat5 coiled‐coil region is responsible for binding with Hsp90. J. Cell. Physiol. 198: 188–196, 2004© 2003 Wiley‐Liss, Inc.

Further identification of NSF* as an epilepsy related gene

Previous data proved that NSF* was an epilepsy related gene (ERG1). In this study, using phosphorothioate oligodeoxynucleotide (PS-ODN), an antisense of NSF to downregulate the function of NSF in vitro cultured hippocampus neurons and PC12, this treatment simultaneously induced enhancement of the neurite outgrowth of hippocampal neurons and PC12, a phenomenon similar to the structural changes following epilepsy. Immunocytochemistry analysis showed that the enhancement of neurite outgrowth was in a sequence-specific manner and Northern blot confirmed that the decrease of NSF mRNA levels in PC12 was in a dose-dependent manner. Moreover the expression of NSF was downregulated during differentiation of PC12 induced by NGF and high KCl. Therefore, providing more evidence to support the fact that NSF was an ERG1.

Ref-1 protein enhances the IL-2-stimulated telomerase activity.

Telomerase is an important ribonucleoprotein enzyme involved in cellular proliferation and senescence. Activation of telomerase has been detected in a vast majority of human cancer cells. In this article, we demonstrated that Interleukin‐2 (IL‐2) which is the pivotal cytokine in the immune system could stimulate the activity of telomerase in the cultured BA/F3β cells. It was also found that the level of IL‐2‐induced telomerase activity was decreased by the treatment with chemical oxidant in vitro. Since IL‐2 stimulation produces a oxidative shift of the intracellular environment, the activation and maintenance of telomerase in this oxidative circumstance requires particular protection. Here we proved the redox factor‐1 (Ref‐1) protein was involved in this process. The addition of GST–Ref‐1 protein increased the level of IL‐2‐induced telomerase activity in the TRAP assay, while elimination of the endogenous Ref‐1 protein by immunodepletion decreased it. Consistent with these in vitro results, IL‐2‐induced telomerase activity could be enhanced by transient overexpression of Ref‐1 protein in BA/F3β cells. Taken together, these findings proved that Ref‐1 protein benefits the activation of telomerase activity in the oxidative microenvironment of the BA/F3β cells stimulated by IL‐2. J. Cell. Biochem. 88: 1120–1128, 2003.

126Gln is the residue of human IL-2 binding to IL-2R γ subunit

The 126Gln of human interleukin-2 (IL-2) is a conserved amino acid residue. After substitution of 126Gln with Asp, the binding abilities of this mutant to different composites of IL-2 receptor (R) subunits have been determined. Results show that 126AspIL-2 has higher affinity to IL-2R α β γ complex and normal affinity to IL-2R α β complex, but loses its binding ability to IL-2R β γ complex, demonstrating that the 126Gln is the residue of human IL-2 which binds to IL-2R γ subunit.

Investigating the function of Akt by tet-off inducible expression system

A tet-off inducible cell line named BBT derived from BA/F3β cell line was constructed and the effect of this inducible expression system was significant when detected by tet-off responded luciferase reporter gene assay. Then tet-off responded Akt expression plasmid was transfected into BBT cells, and the stable cell lines were screened. The result of Northern blot showed that the expression ofakt was significantly inducible. The clone with the best inducible effect was selected and named BBA for investigating the function of Akt. We found that Akt could significantly inhibit zinc-induced decrease of cell viability when assayed by MTT method. And the flow cytometric analysis showed that Akt could markedly repress zinc-induced apoptosis.