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1、 RESEARCH ARTICLESCIENCE VOL. 274 18 OCTOBER 1996373An Adenovirus Mutant That Replicates Selectively in p53- Deficient Human Tumor CellsJames R. Bischoff，* David H. Kirn, Angelica Williams,Carla Heise, Sharon Horn, Mike Muna，Lelia Ng，* Julie A. Nye，Adam Sampson-Johannes, Ali Fattaey，Frank McCormickt
2、The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumorsuppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ect
3、opic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression o
4、f 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.Deletion or mutation of the p53 tumor suppressor gene occurs frequently in most types of human cancer (I ) Tumors lacking functional p53 are, in many cases, refractory to c
5、hemotherapy or radiation (2). It is therefore critically important to develop therapeutic strategies to treat p5 3-deficient tumors.DNA tumor viruses such as adenoviruses infect quiescent cells and induce them into the S phase of the cell cycle so that viral DNA replication can proceed (3). The El A
6、 protein of human adenoviruses, which binds pRB, p300, and other related proteins, is largely responsible for this forced entry into the S phase (4). The EIB region of the viral genome encodes a 55-kD protein (EIB 55K) that binds and inactivates p53 (5). This binding is essential to virus replicatio
7、n, possibly because El A induces p53-dependent apoptosis- We hypothesized that an adenovirus mutant that does not produce EIB 55K should be unable to replicate in normal cells but would be able to replicate in cells lacking functional p53- The dll520 virus is such a mutant- This human group C adenov
8、irus contains an 827-base pair deletion in the EIB region and a point mutation at codon 2022 that generates a stop codon preventing expression of a truncated protein from the deleted gene (6). The EIB 19Kgene, whose protein product suppresses apoptosis, is not affected by this deletion.We first test
9、ed the ability of this virus toThe authors are with ONYX Pharmaceuticals, 3031 Research Drive, Richmond, CA_94806, USA.Present address: Sugen, Incorporated, 515 Galveston Drive, Redwood City CA 94063, USA.个To whom correspondence should be addressed.grow in cells lacking functional p53. The dll520 vi
10、rus grew as efficiently as wild-type adenovirus in C33A cervical carcinoma cells, which express p53 with an inactivating mutation at codon 273 (7) (Fig- 1A). Under identical conditions, dll520 grew poorly in U20S osteocarcinoma cells, which retain wild-type p53 (8), producing about 100 times less in
11、fectious virus than did wild-type adenovirus- Polymerase chain reaction procedures (9) were used to verify that the virus produced by dll520-infected C33A cells retained the EIB deletion and that the infection was not due to wild-type adenovirus contamination (9).We also performed cytopathic effect
12、(CPE) assays on a panel of human tumor cells and normal cells infected with either dll520 or wild-type adenovirus. The dll520 virus had no detectable cytopathic effect on normal human diploid fibroblasts or on tumor cells retaining wild-type p53, whereas wild-type adenovirus caused compete lysis und
13、er identical conditions (Fig. IB). In contrast, both dll520 and wild-type adenovirus killed C33A cells with high efficiency. This analysis was extended to a variety of tumor cell lines of known p53 status (10), including four cervical carcinoma cell lines expressing human papillomavirus (HPV) E6, wh
14、ich inactivates p53 through ubiq- uitin-mediated protein turnover (7); four colon carcinoma cell lines containing different mutant forms of p53; U373 glioblastoma cells (codon 273 mutation) (11); and HS700T pancreatic adenocarcinoma cells (codon 249 mutation) (12)- In each case, dll520 killed cells
15、with an efficiency comparable to that of wild-type adenovirus (10).These results show a clear correlation between p53 status and susceptibility to dll520 and are consistent with our original hypothesis- However, the dll520 deletion affects genes other than that encoding EIB 55K; it also deletes a co
16、ding sequence from part of the E3 region (11). To test whether the restricted host range of dll 520 is specifically due to loss of EIB 55K, we constructed a U20S cell line that expressed EIB 55K under the control of the human cytomegalovirus immediate-early promoter (13)- We predicted that this would render the U20S cells susceptible to dll520 infection. The EIB 55K protein produced in these transfected cells (designated UFL-A) int
