Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis Xiao-Feng Le, Sadeq Vallian, Zhao-Mei Mu, Mien-Chie Hung and Kun-Sang Chang Division of Laboratory Medicine and the Department of Tumor Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA Our previous studies demonstrated that the promyelocy- tic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing eect in breast cancer cells were examined. We showed that Ad-PML eec- tively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post- infection and a detectable level remained at day 16. Ad- PML signi®cantly suppressed the growth rate, clono- genicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the eect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a signi®cant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphory- lated 48 h post-infection. These ®ndings indicate that PML exerts its growth suppressing eects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppres- sing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy. Keywords: Ad-PML; breast cancer; tumorgenicity; G1 arrest; apoptosis Introduction The promyelocytic leukemia (PML) gene was ®rst identi®ed in the nonrandom chromosomal transloca- tion t(15;17)(q22;q12) associated with acute promye- locytic leukemia (APL) (Goddard et al., 1991; de The et al., 1991; Kakizuka et al., 1991; Pandol® et al., 1991; Chang et al., 1992). The PML's gene product belongs to a novel family of proteins characterized by a C3HC4 RING-®nger, two additional Cys/His-rich motifs, and an a-helical coiled-coil region (Freemont et al., 1991; Borden et al., 1995). PML protein was found to be the major component of a novel form of nuclear body designated PML oncogenic domain (POD) (Dyck et al., 1994). In addition to PML, POD was found to contain at least six other cellular proteins such as Sp-100 (Szostecki et al., 1990), NDP55 (Ben-Chetrit et al., 1988), a 65-kd protein (Weis et al., 1994), NDP52 (Korioth et al., 1995), PIC1 (Boddy et al., 1996), and Int-6 (Desbois et al., 1996). The integrity of the POD is disrupted in leukemic blasts from patients with APL and the APL- derived NB4 cell line. All trans-retinoic acid (ATRA) or arsenic trioxide treatment of APL cells induced dierentiation and apoptosis which is associated with a reorganization of wild-type POD structure (Dyck et al., 1994; Weis et al., 1994; Chang et al., 1995). Some viral proteins such as ICPO (Maul et al., 1993), the adenoviral protein E1A, E4-ORF3, and E1B (Car- valho et al., 1995; Doucas et al., 1996). Epstein-Barr nuclear protein 5 (Szekely et al., 1996), and a novel ubiquitin-speci®c protease (Everett et al., 1997) are shown to target and reorganize PODs. These studies demonstrated that after virus infection a dramatic structural change in the PODs was found, suggesting that PODs are the target of tumor virus oncoproteins. Interestingly, during adenovirus infection, Sp-100 and NDP55 but not PML relocated from the POD to a unique nuclear elongated ®brous structures (viral inclusion bodies) (Doucas et al., 1996), indicating that viral oncoproteins, e.g. E4-ORF, may release PML-associated factors from POD and possibly promote transformation. It was hypothesized that POD may play a role in sequestration of important cellular regulatory proteins. Studies on the biologic function of PML in our laboratory demonstrated that PML is a growth and transformation suppressor (Mu et al., 1994, 1996; Liu et al., 1995). Our previous study further showed that the nuclear localization domain, the dimerization domain, and RING-®nger motif are necessary for suppressing transformation of NIH3T3 by activated neu oncogene (Le et al., 1996). Recently, we established stable transfectant of PML in HeLa cells and further showed that PML exerts a strong negative growth control over these cells (Mu et al., 1996). We also extend our studies to other human cancer cell lines and found that overexpression of PML in these cells such as HeLa, PC-3, DU145, LNCaP, SK-OV3 and SW-13 remarkably inhibited their growth in vitro (He et al., 1997; Mu et al., 1997 and our unpublished data). This growth suppressor function of PML was also reported by other groups which showed that overexpression of Correspondence: K-S Chang Received 18 September 1997; revised 3 November 1997; accepted 4 November 1997 Oncogene (1998) 16, 1839 ± 1849  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc