[CANCER RESEARCH 63, 760 –765, February 15, 2003] Advances in Brief Adenoviral-mediated Retinoblastoma 94 Produces Rapid Telomere Erosion, Chromosomal Crisis, and Caspase-dependent Apoptosis in Bladder Cancer and Immortalized Human Urothelial Cells but not in Normal Urothelial Cells 1 Xinqiao Zhang, Asha S. Multani, Jain-Hua Zhou, Jerry W. Shay, David McConkey, Li Dong, Chang-Soo Kim, Charles J. Rosser, Sen Pathak, and William F. Benedict 2 Departments of Genitourinary Medical Oncology [X. Z., J-H. Z., L. D., C-S. K., W. F. B.], Cancer Biology [A. S. M., D. M., S. P.], and Urology [C. J. R.], The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas Texas 75390 [J. W. S.] Abstract Retinoblastoma (RB)94, which lacks the NH 2 -terminal 112 amino acid residues of the full-length RB protein (RB110), is a more potent tumor and growth suppressor than RB110. In this study, Ad-RB94, but not Ad-RB110, produced marked growth inhibition, cytotoxicity, caspase-dependent apo- ptosis, and G 2 -M block in the human RB-negative, telomerase-positive blad- der cancer cell line UM-UC14. This effect was completely inhibited by pre- treatment with caspase inhibitors (P < 0.0001). Similar results were seen in RB-positive and other RB-negative bladder cancer cell lines. Ad-RB94 pro- duced rapid telomere length shortening and loss of telomere signal, which was associated with polyploidy and chromosomal aberrations (P < 0.001). Ad-RB94, however, showed no cytotoxicity to telomerase-negative human normal urothelium cells but was highly cytotoxic to telomerase-positive human E6 and E7 immortalized urothelial cells (P < 0.0001). In addition, telomerase-negative cells, which maintain their telomere length through an alternative lengthening of telomeres DNA recombination pathway, showed no cytotoxicity to RB94. These results suggest that the induction of rapid telomere erosion and chromosomal crisis by RB94 in telomerase-positive cancer and in telomerase-expressing immortalized human cells is a major factor in its selective and potent tumor suppression and cytotoxic activity. The lack of cytotoxicity to normal cells should also provide a high therapeutic index when used in gene therapy protocols for the treatment of bladder and other cancers. Introduction RB94 3 is produced by translation of the wild-type RB gene from the second in-frame AUG codon and lacks the NH 2 -terminal 112 amino acids present in RB110 (1). RB94 has markedly more tumor suppressor potency than wild-type RB110 and is active against both RB+ and RB- tumors, including bladder cancer (1, 2). Because RB94 retains the same key functional domains (such as A, B, and C pockets, nuclear signaling) as RB110, it becomes critical to under- stand the mechanism(s) of increased potency for RB94. Differences reported thus far include the observation that RB94 has a longer half-life than RB110, remains in its active hypophosphorylated form for an extended period of time, and causes unusual nuclear morpho- logical changes (1, 2). Although these differences may be important to the unique properties of RB94, they do not address the mechanism(s) of increased tumor suppression and cell kill produced by RB94. An adenoviral construct containing RB94 under tetracycline control enabled us to examine changes that are RB94 specific because the protein is not produced in the presence of doxycycline, although adenoviral proteins are still made. Therefore, changes seen after Ad-RB94 treatment and that are blocked by doxycycline can be considered to be RB94 specific. Focus was given initially to the effect of RB94 on RB-negative bladder cancer cells and immortalized urothelial cells (telomerase positive) as well as normal urothelial cells (telomerase negative) because gene therapy trials using RB94 are being planned for bladder cancer. We report that Ad-RB94 is cytotoxic to every bladder cancer cell expressing RB94, and this is associated with rapid telomere erosion and chromosomal crisis. Ad-RB110 treatment does not produce these changes. In addition, Ad-RB94 suppresses the growth of E6 and E7 immortalized human urothelial cells and produces similar telomere and chromosomal changes. In contrast, no cytotoxicity, telomere attrition, or chromosomal abnormalities were seen in normal urothe- lial cells after Ad-RB94 treatment. On the basis of these results, we present one hypothesis to explain the basis for the specificity of RB94 cytotoxicity to human cancer cells and to genetically altered, immor- talized cells but not to normal cells. Materials and Methods Cell Lines. The bladder cancer cell lines 5637, UM-UC9, UM-UC11, and UM-UC14, the immortalized urothelial cells -E6-1, -E6-2, and  E7, and the normal human urothelial cells were obtained from Dr. Barton Grossman (Department of Urology, University of Texas M. D. Anderson Cancer Center). The -E6-1, -E6-2, and -E7 cells were originally obtained from Catherine Reznikoff (3, 4). The immortalized telomerase-negative SUSM1 fibroblasts have been described previously (5). Except for normal urothelial cells, all of the other cells were grown in T-75 tissue culture flasks in modified minimum essential medium supplemented with 10% FCS and incubated at 37°C in 5% CO 2 and 95% air. The normal urothelial cells were grown in keratinocyte growth medium without serum as described previously (6). Ad-RB94 Construction. The RB94 gene was inserted into an expression cassette under a tTA-inducible cytomegalovirus promoter. This was, in turn, inserted into plasmid p1sp1A obtaining shuttle plasmid pEW22. We also inserted a tTA protein expression cassette into plasmid pBHG11 through plasmid pABS.4, obtaining master plasmid pEW23. By cotransfection of pEW22 and pEW23 into 293 cells, we obtained the recombinant adenovirus expressing RB94 under the control of the tetracycline system. After initial harvesting, the viral vector underwent plaque to plaque purification. Subse- Received 11/11/02; accepted 1/7/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by a grant from the Retina Research Foundation and the Tobacco Settlement Funds as Appropriated by the Texas State Legislature. It was also supported by the Bladder SPORE CA091846 and Lung SPORE CA70907. C. J. R. was funded by the American Foundation of Urological Disease. 2 To whom requests for reprints should be addressed, at Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-4013; Fax: (713) 794-4019; E-mail: wbenedic@mdanderson.org. 3 The abbreviations used are: RB, retinoblastoma; RB110, full-length wild-type RB protein of M r 110,000; Ad-RB110, adenoviral-mediated RB110; RB94, RB protein of M r 94,000; Ad-RB94, adenoviral-mediated RB94; BOC, BOC-Asp(Ome)-fluoro-methyl ketone; zVADfmk, benzyloxycarbonyl-val-ala-Asp(OMe)-fluoro-methyl ketone; FISH, fluorescence in situ hybridization; Q-FISH, quantitative-FISH; MTT, 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; TRF2, telomeric-repeat binding factor 2; ALT, alternative lengthening of telomere. 760 Research. on February 17, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from