Volume 140, Issue 1, Pages 332-343 (January 2011) HIV-Tat–Mediated Delivery of an LPTS Functional Fragment Inhibits Telomerase Activity and Tumorigenicity of Hepatoma Cells  Guangming Chen, Liang Da, Hongfei Wang, Ying Xu, Guoyuan Chen, Chengfu Sun, Leiming Wang, Jing Zhao, Fang Zhang, Jian Feng, Yifei Wang, Pierre Tiollais, Tsaiping Li, Mujun Zhao  Gastroenterology  Volume 140, Issue 1, Pages 332-343 (January 2011) DOI: 10.1053/j.gastro.2010.08.046 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Expression and telomerase inhibitory function of LPTS proteins. (A) Expression of LPTS proteins in human HCC tissues (C) and adjacent nontumor tissues (N) was analyzed by Western blotting. β-tubulin was loaded as an internal control. (B) Identification of the regions of LPTS proteins effectively inhibiting telomerase activity. (C) Twelve percent polyacrylamide gel showed telomerase activities inhibited by GST-fused LPTS and LPTS deletions. Various concentrations of GST fusion proteins were used as indicated. GST without LPTS and RNase A acted as negative and positive controls, respectively. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 The TAT-LPTS-LC protein is delivered into cells and inhibits telomerase activity in vitro. (A) Schematic representation of TAT-LPTS-LC protein. (B) Ten percent sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of recombinant TAT-LPTS-LC fusion protein. Lane 1, protein molecular weight marker; lane 2, cell lysate of E coli BL21 before or after (lane 3) IPTG induction; lane 4, total proteins in the supernatant; lane 5, affinity chromatography purified TAT-LPTS-LC protein. (C) Various concentrations of TAT-LPTS-LC as indicated were used to measure the inhibition of telomerase activity. (D) Western blotting analysis of transduction of TAT-LPTS-LC proteins into BEL-7404 and Saos-2 cells. The cells were incubated with proteins for the indicated times. (E) Immunofluorescence of BEL-7404 and Saos-2 cells transduced with TAT-LPTS-LC proteins. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 TAT-LPTS-LC inhibits telomerase-positive cell proliferation. (A) The inhibition of cell growth was evaluated by colorimetric tetrazolium salt (MTT) assay. (B) Percentages of dying cells were counted and presented. (C) Cell death was measured by the flow cytometry. The cell populations in sub-G1 were referred to as necrotic cells. All the data are presented as mean ± SD from at least 3 independent experiments. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 TAT-LPTS-LC induces cell crisis and shortens telomere length. (A) Photographs of the cells treated with TAT-LPTS-LC or PBS. (B) Photographs of the treated cell nuclei stained by 4',6-diamidino-2-phenylindole. The white arrows indicate the crisis cells and the red arrows show apoptotic cells. (C) Southern blotting analysis of telomere length of BEL-7404 and L02 cells after treatment. The a-32P-labeled TTAGGGA repeats was used as a probe. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 TAT-LPTS-LC decreases tumorigenicity of BEL-7404 cells in nude mice. (A) Calculation of tumor appearance at injected sites weekly. Seven mice were used per group. (B) Representative photographs of one mouse in each group. (C) Tumors were harvested and weighed 7 weeks after injection. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 TAT-LPTS-LC suppresses the xenograft growth of BEL-7404 cells in nude mice. (A) The TAT-GFP protein was delivered into tumors and organs analyzed by GFP imaging. (B) Western blotting analysis of the TAT-LPTS-LC proteins reached to the tumors and organs. (C) The tumor volumes were calculated weekly. Data are presented as means ± standard deviation (n = 5). (D) At 7 weeks after xenografting, the tumors were removed and photographed. The average tumor weights were counted. Statistical significance was set at a P value of less than .05. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Evaluation of toxicity of TAT-LPTS-LC. (A) The main organs collected from normal and xenografting mice after treatment. (B) The serum biochemistry parameters and complete blood counts were analyzed. BUN, blood urea nitrogen; CRE, creatinine; HGB, hemoglobin; PLT, platelet count; RBC, red blood cell; WBC, white blood cell. The values represent averages for 5 mice. (C) The median lethal dose (LD50) of TAT-LPTS-LC on Kunming mice. Gastroenterology 2011 140, 332-343DOI: (10.1053/j.gastro.2010.08.046) Copyright © 2011 AGA Institute Terms and Conditions