1. Volume 13, Issue 5, Pages 928-937 (May 2006)
Selective Targeting of Checkpoint Kinase 1 in Tumor Cells with a Novel Potent Oncolytic Adenovirus 
Qinglei Gao, Jianfeng Zhou, Xiaoyuan Huang, Gang Chen, Fei Ye, Yunping Lu, Kanyan Li, Liang Zhuang, Mei Huang, Gang Xu, Shxuan Wang, Ding Ma 
Molecular Therapy 
Volume 13, Issue 5, Pages (May 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Characterization of M2 mutant adenovirus. (A) A 27-bp sequence from wt-Adv5 (bp 920 to 946) was deleted to generate Adv5/dE1A. A single region corresponding to E3 6.7K and gp19K of wt-Adv5 bp 28530–29360 was excised from the Adv5/dE1A genome and substituted with a fragment of reverse chk1 cDNA (bp 853–250) using a ClaI restriction site introduced at each end to generate M2. (B) Immunoprecipitation of the E1A/host protein complex from Adv-TK-, M2-, or wt-Adv5-infected A549 cells was blotted with anti-human Rb antibody. (C) Genomic structure of M2 was confirmed by ClaI digestion. Since the native ClaI restriction site (bp 917 to 922) was disrupted, the genome of M2 contained only two ClaI restriction sites located at the 5′ end and 3′ end of the reverse chk1 cDNA. The appearance of a 0.6-kb band verified the presence of the inserted chk1 cDNA and deletion in the E1A region. (D) A549 cells were infected with Adv-TK, M2, or wt-Adv5 at an m.o.i. of 5. PBS was included as a mock control. Twenty-four hours after infection, the total mRNA was isolated and tested for amplification of fusion mRNA containing sequences of antisense chk1 cDNA and the ADP cDNA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Replication of M2 in cancer and normal cells. (A) Normal cells (Q-MVEC, P-MVEC, and h Nheps) and tumor cells (HeLa, A2780, and MCF-7) were infected with various viral mutants at an m.o.i. ranging from 0.01 to 500 and cultured for the indicated days prior to the CPE assay. Viable cells were stained with crystal violet for the CPE assay. (B) Viral production of M2, wt-Adv5, and Adv-TK was determined using viral replication assays in cancer (A549 and MDA-MB-231) and normal cells (Q-MVEC and P-MVEC) at 48 h per input virus at an m.o.i. of 5. Values are logarithms of viral titers (pfu/ml). (C) A549 cells or Q-MVEC cells were infected with M2, wt-Adv5, or Adv-TK at an m.o.i. of 5 and cultured for 24 h. Total RNA was extracted and tested for levels of viral fiber mRNA by real-time quantitative PCR. Results are the means of three independent experiments and expressed as the ratio to GAPDH. Control, amplification performed with total RNA from cells without any treatment.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Effects of M2 on levels of chk1 protein in tumor and normal cells. (A) HeLa cells were infected with M2 or Adv5/dE1A at an m.o.i. of 1 and cultured for the designated time. Protein was then extracted and 20 μg of total protein was subjected to Western blot analysis. Adv5/dE1A is the parent mutant of M2 with a 27-bp deletion from bp 920 to 946 of wt-Adv5. Chk2 is checkpoint kinase 2. (B) Q-MVEC were infected with M2 or Adv5/dE1A at an m.o.i. of 100 and cultured for the depicted times. Protein was then extracted and 40 μg of total protein subjected to Western blot analysis. (C) Tumor (A549 and HepG2) or normal cells (Q-MVEC) were infected with M2, wt-Adv5, or Adv-TK at an m.o.i. of 5 and cultured for 24 h. Total RNA was extracted and tested for levels of viral E3 14.7K mRNA by real-time quantitative PCR. Results are the means of three independent experiments and expressed as the ratio of E3 14.7K to GAPDH. (D) A549 cells were infected with M2 at an m.o.i. of 5 and cultured in the presence or absence of Ara-C. Levels of antisense chk1 cDNA/ADP fusion mRNA at the various times postinfection were determined by real-time quantitative PCR. M2 + Ara-C, M2-infected A549 cells were cultured in the presence of 1-β-d-arabinofuranosylcytosine at a concentration of 20 μg/ml.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
M2 sensitized tumor cells to DNA-damaging agents. (A) Cancer cells (MCF-7, A2780, and HeLa) and MVEC were infected with virus at an m.o.i. of 1 and cultured for 24 h. The cells were then treated with either 10 μM cisplatin (for A2780) or 6 Gy irradiation (for MCF-7, HeLa, or MEVC) and cultured for another 46 h for HeLa, 24 h for MCF-7, 28 h for A2780, and 24 h for MEVC before being subjected to apoptosis analysis. Results are the means of three independent experiments. IR/DDP, treated with 6 Gy irradiation (for MCF-7, HeLa, or MVEC) or 10 μM cisplatin DDP (for A2780). **P < 0.01, M2 plus IR/DDP compared with Adv5/dE1A plus IR/DDP. (B) A549 cells were infected with M2 or Adv5/dE1A at an m.o.i. of 5. Levels of total ADP mRNA at the various times postinfection were determined by real-time quantitative PCR.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Antitumoral effects of intratumoral injection of M2. (A) Intratumoral viral replication was detected throughout the tumor by in situ hybridization staining for viral fiber 40 days after direct intratumoral injection with M2 or Adv-TK (1 × 108 pfu once daily for five doses). Cells that contained replicative virions were stained dark blue (arrows). These representative sections depict similar data observed in subcutaneous tumor samples from two mice for each group (Adv-TK or M2). (B) Levels of chk1 protein 40 days after intratumoral injection of virus were determined by Western blot analysis. PBS was included as mock control. (C) Kaplan–Meier survival curves following intratumoral injection of M2, Adv5/dE1A, Adv-TK, or PBS (negative control) in BALB/c athymic subcutaneous human-tumor-bearing mice. P = 0.180, M2 compared to Adv5/dE1A.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Antitumoral potency of intravenous administration of M2 in the presence of cisplatin. (A) Viral particles were detected by immunohistochemistry (IHC) staining. Cells containing viral particles were stained brown (black arrows) after intravenous injection of M2 at a dose of 2 × 108 pfu for 5 consecutive days. Viral replication was detected by in situ hybridization staining for viral fiber. Cells that contained replicative virions were stained dark blue (red arrows). (B) Levels of chk1 protein were determined by Western blot after five doses of intravenous injection of virus. (C) Kaplan–Meier survival curves following intravenous administration of M2 plus DDP (cisplatin) to orthotopic HepG2 human hepatic carcinoma mice. P = 0.039, M2 plus DDP compared with Adv5/dE1A plus DDP.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions