(2001) 20, 141 ± 146 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc ORIGINAL PAPERS Expression of a down-regulated target, SSeCKS, reverses v-Jun-induced transformation of 10T1/2 murine ®broblasts

Steven B Cohen1, Anke Waha2, Iwin H Gelman3 and Peter K Vogt*,2

1Gen-Probe Incorporated, 10210 Genetic Center Drive, San Diego, California CA 92121, USA; 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California CA 92037, USA; 3Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA

Line 10T1/2 mouse ®broblast overexpressing the v-Jun its cellular counterpart by a 27-amino acid deletion and oncoprotein were morphologically altered, grew into by two amino acid substitutions (Nishimura and Vogt, multilayered foci in culture and formed colonies when 1988). These synergize to make Jun suspended in agar. The growth rate of the v-Jun- independent of cellular regulation and to turn it into transformed 10T1/2 cells was not changed signi®cantly a strong growth promoter (Abate et al., 1990; Boyle et from that of the untransformed parental cells, but the al., 1991; Chida and Vogt, 1992; Hibi et al., 1993; saturation density of the transformed cultures exceeded Morgan et al., 1993; 1994). that of normal controls by a factor of 2. mRNA v-Jun induces oncogenic transformation by aberrant extracted from v-Jun-transformed 10T1/2 cells was regulation of speci®c target . The di€erential analysed for di€erential expression with DNA expression of these targets determines the neoplastic micro-array technology. One of the targets downregu- phenotype of the . An understanding of transforma- lated by v-Jun was identi®ed as SSeCKS (Src-suppressed tion requires the identi®cation and characterization of C kinase substrate). Re-expression of SSeCKS in v-Jun- these targets. Previous work has revealed several genes transformed ®broblasts reversed the transformed pheno- that are up-regulated in v-Jun-transformed avian cells type of the cells. Their ability to form foci was reduced including bkj (Hartl and Bister, 1995), jtab (Hadman et to background levels, the number and size of agar al., 1996), glutaredoxin (Goller et al., 1998), hbegf (Fu colonies was lowered by a factor of 10 and the saturation et al., 1999) and ril (Fu et al., 2000). These genes were density was signi®cantly diminished. However, expres- identi®ed by various forms of substractive hybridiza- sion of SSeCKS had little e€ect on the morphology of v- tion which typically cover only a small fraction of the Jun-transformed 10T1/2 cells. These data suggest that transcriptome. More comprehensive expression pro®l- the SSeCKS protein has growth-attenuating properties. ing is provided by DNA micro-arrays, which are not Down-regulation of SSeCKS may be essential for Jun- yet available for avian genomes. Therefore, we induced transformation. Oncogene (2001) 20, 141±146. established a murine transformation system for Jun. Primary rodent cells are refractory to Jun transforma- Keywords: oncogenic transformation; di€erential gene tion, but some continuous mammalian cell lines are expression: DNA micro-array; tumor suppressor susceptible to the growth-promoting e€ects of Jun (Schutte et al., 1989). We chose murine 10T1/2 cells to generate materials for DNA micro-array analysis. Introduction 10T1/2 cells were stably transformed with a retroviral vector expressing v-Jun. They became refractile, grew The factor Jun belongs to the basic into multi-layered foci at high population densities and region/leucine zipper (bZip) protein family. Jun formed colonies when suspended in nutrient agar. dimerizes with itself or with other bZip proteins to mRNA from these v-Jun-transformed cells and from form the AP-1 complex which their normal 10T1/2 progenitors was used to identify recognizes the DNA target sequence TGACTCA (TRE up- and down-regulated genes in commercial DNA sequence) and controls . Jun can induce micro-arrays (Incyte Pharmaceuticals). This commu- oncogenic transformation; this activity requires the nication will characterize one of the down-regulated three basic functions of the protein: dimerization, genes, SSeCKS (Src suppressed C kinase substrate). DNA binding and transcriptional regulation (Morgan This myristylated protein is rapidly translocated from et al., 1992). An oncogenic version of Jun is transduced the cortical cytoskeletal matrix to the perinucleus in by the avian retrovirus ASV17 and is referred to as v- response to treatment with phorbol esters (Lin et al., Jun (Bos et al., 1990; Maki et al., 1987). It di€ers from 1996). SSeCKS is down-regulated in rodent ®broblasts transformed by Src, Ras, Fos or but shows unchanged levels of expression in cells transformed by Raf, Mos or Neu (Lin et al., 1995). SSeCKS expression *Correspondence: PK Vogt Received 7 August 2000; revised 25 October 2000; accepted 1 has also been found to be down-regulated in human November 2000 breast cell lines as compared to normal breast V-Jun down regulated SSeCKS in murine fibroblasts SB Cohen et al 142 epithelial cells (Zhang et al., 1995). Expression of Jun-expressing cells were round and refractile, ap- SSeCKS in Src-transformed NIH3T3 cells has the peared less adherent and grew in an irregular pattern. e€ect of attenuating the transformed phenotype (Lin and Gelman, 1997). In the current study, expression of v-Jun expressing 10T1/2 cells lose contact inhibition of SSeCKS in v-Jun-transformed 10T1/2 cells induced a growth and gain the ability to form colonies of agar dramatic reduction of their oncogenic growth proper- ties. These observations suggest that SSeCKS can 10T1/2 cells transformed by Jun multiplied initially at function as a tumor suppressor. the same rate as control cells but they reached a far higher saturation density (Figure 2), forming sheets of multiple cell layers which have lost contact inhibition of growth. Focus formation on plastic support was Results assayed by plating various dilutions of v-Jun-trans- formed 10T1/2 cells together with a constant number Expression of v-Jun in 10T1/2 cells causes morphological of vector-transfected controls (Brugarolas et al., 1998). transformation Highly ecient focus formation was observed with v- v-Jun is a highly e€ective oncogene for chicken embryo Jun-expressing 10T1/2 cells; vector controls produced ®broblasts (CEF), inducing morphological transforma- only a low background number of foci (Figure 3). tion and robust anchorage-independent growth (Bos et In order to examine anchorage-independent growth, al., 1990; Maki et al., 1987). In primary mammalian v-Jun-expressing cells were suspended in 0.35% cell cultures expressing v-Jun, such transformation is nutrient agar. The number of colonies was determined not observed, but continuous rodent cell lines are after 4 weeks (Table 1 and Figure 4). Vector-infected susceptible to transformation (Schutte et al., 1989). In cells did not grow in suspension, while v-Jun- a comparison of several murine cell lines, 10T1/2 cells transformed 10T1/2 cells produced colonies with an emerged as most sensitive to v-Jun-induced transforma- eciency of greater than 30%. tion and were therefore chosen for expression-pro®ling using commercially available DNA micro-array tech- SSeCKS expression is repressed in v-Jun-transformed nology. For production, the chicken v-Jun cDNA 10T1/2 cells without Gag sequences (VJ1), was subcloned into the pBabe-puro vector and transfected into the BOSC23 The identi®cation of genes with altered expression ecotropic packaging cell line. The replication-defective patterns is critical to an understanding of oncogenic infectious were then used to infect the transformation. Expression-pro®ling was therefore amphotropic PA317 packaging cell line, and virus performed with the v-Jun-transformed 10T1/2 cells. producers were selected with puromycin. Harvests from Four individual agar colonies were picked, expanded these cells were used to infect 10T1/2 cells, inducing a and used to generate poly(A)+ RNA. DNA micro- characteristic morphological transformation (Figure 1). arrays identi®ed 221 genes that are di€erentially Compared to controls infected with empty vector, v- expressed by a factor of greater than two in v-Jun- transformed cells; 147 were up-regulated, and 74 were down-regulated. Initial attention focused on the down- regulated genes, because the signi®cance of the down- regulation for transformation can be readily tested by reexpressing the gene in question. A partial listing of

Figure 2 v-Jun-transformation of 10T1/2 cells leads to increased Figure 1 v-Jun-infection of 10T1/2 cells causes morphological saturation density that is attenuated by SSeCKS expression. transformation. 10T1/2 cells were infected with either p-Babe- 10T1/2 cells were infected with p-Babe-hygro-SSeCKS and VJ1 puro or p-Babe-puro-VJ1 virus and then selected with puromycin 10T1/2 cells were super-infected with SSeCKS as described in as described in Materials and methods. Cells were allowed to Materials and methods. Cells were plated at 26104 cells in grow to con¯uency and photographed at 1006magni®cation with 35 mm plates, and counted every day for 9 days. The experiment phase contrast optics was run three times, and a representative example is shown

Oncogene V-Jun down regulated SSeCKS in murine fibroblasts SB Cohen et al 143

Figure 4 v-Jun-expression in 10T1/2 cells leads to anchorage- independent growth that is attenuated by SSeCKS expression. The cells used were the same as described in Figures 1 and 2. 1.256104 cells were suspended in 2 ml of 0.35% nutrient agar in 60 mm plates that contained 3 ml of pre-cast 0.7% agar. Cells were fed every 5 days, and after 4 weeks were photographed. Table 1 shows the quanti®cation of this experiment

for SSeCKS in the DNA micro-arrays was 3.9-fold. A Northern blot with poly(A) RNA from v-Jun-trans- formed and from vector-transfected 10T1/2 cells showed an even greater repression, more than 10-fold (Figure 5). Northern blots of other down-regulated targets revealed that the data from the DNA micro- arrays generally understate the degree of di€erential expression. A Western blot was also prepared using cell lysates from v-Jun infected and control cells, and the quantity of SSeCKS protein observed was signi®cantly Figure 3 v-Jun-expression in 10T1/2 cells leads to focus formation which can be attenuated by reexpression of SSeCKS. reduced in v-Jun transformed 10T1/2 cells (Figure 6). The cells used were the same as described in Figures 1 and 2. These cells were diluted with p-Babe-infected cells 1 : 50 and 1 : 200. A total of 2.56105 cells were plated per 35 mm well. Cells Expression of SSeCKS in v-Jun-transformed 10T1/2 cells were fed every 3 days and stained and photographed after 10 days attenuates the transformed phenotype as described in Materials and methods The role of SSeCKS in Jun-induced transformation was examined by re-expressing the down-regulated protein in v-Jun-transformed cells. The murine Table 1 Soft agar cloning eciency of 10T1/2 cell infected with SSeCKS cDNA was cloned into the p-Babe-hygro di€erent constructs vector (Lin and Gelman, 1997) and v-Jun-transformed Virus Cloning efficiencya 10T1/2 cells were superinfected and selected as were Babe 50.1 cells infected with empty p-Babe-hygro 10T1/2. SSeCKS 50.1 Western blots were performed to con®rm that SSeCKS VJ1 30+3 was being expressed (Figure 6). v-Jun-infected cells VJ1+SSeCKS 4.6+0.1 were also superinfected with p-Babe-hygro virus, followed by hygromycin selection to ensure that results aPercentage of cells forming colonies after 4 weeks were not an artifact of the hygromycin selection process. The morphology of v-Jun-transformed 10T1/ 2 cells was not altered by reexpression of SSeCKS. down-regulated v-Jun targets is given in Table 2. One However, the saturation density of SSeCKS-expressing of these targets is the gene coding for SSeCKS. It was v-Jun-transformed 10T1/2 cells was signi®cantly re- selected for characterization because it is known to be duced. Control 10T1/2 cells expressing SSeCKS also down-regulated in cells transformed by Src, Ras, showed the same saturation density as p-Babe-infected Fos, and Myc (Lin et al., 1995) and in cell lines derived 10T1/2 (Figure 2) v-Jun 10T1/2 cells expressing from breast cancer (Zhang et al., 1995). It has been SSeCKS also formed signi®cantly fewer foci than v- classi®ed as tumor suppressor gene; re-expression leads Jun transformants with down-regulated SSeCKS. to a reversion of the transformed phenotype in Src- Focus-forming eciencies of SSeCKS expressers were expressing cells without causing growth arrest (Lin and similar to those of control p-Babe infected 10T1/2 cells, Gelman, 1997). The degree of repression determined or of 10T1/2 cells expressing SSeCKS alone. These

Oncogene V-Jun down regulated SSeCKS in murine fibroblasts SB Cohen et al 144 Table 2 Genes that exhibit the greatest down-regulation in 10T1/2 Jun-transformed 10T1/2 cells were also super-infected ®broblasts in response to VJ1 expresssion with p-Babe-hygro virus, followed by hygromycin Gene Fold repressiona selection. These cells showed the growth and transfor- Serine protease inhibitor 4 14 mation phenotype of the v-Jun-10T1/2 cells, demon- Pentaxin-related gene 5.6 strating that hygromycin selection did not contribute to Biglycan 5.1 the observed e€ects of SSeCKS (data not shown). IMAGE EST 423177 5.0 Pleiotrophin 4.7 MARCKS 4.1 Secreted phosphoprotein 1 4.0 Discussion SSeCKS 3.9 Oncogenic transcription factors induce transformation aCompared to vector infected 10T1/2 cells by altering the normal controls of gene expression in the cell. Aberrant regulation of speci®c target genes initiates and maintains cellular properties that constitute the oncogenic phenotype. Identi®cation and characteriza- tion of these critical oncogenic targets has become the Holy Grail in basic . DNA micro-array technology o€ers powerful tools for the identi®cation of genes that are di€erentially expressed in cancer cells. At present, the application of these tools is limited to cells of mouse and man. For other vertebrate species, the available genomic information is as yet insucient to produce comprehensive arrays. Yet oncogenic transcrip- tion factors that are potent transformers of avian cells generally fail to a€ect primary mouse cells, although they can transform continuous rodent cell lines that presumably have undergone some preneoplastic changes (Alt and Grassmann, 1993; Colledge et al., 1989; Land Figure 5 Transcription of ssecks is down-regulated in v-Jun- et al., 1986; Schreiber-Agus et al., 1993; Sterneck et al., transformed 10T1/2 cells. A Northern blot was prepared using 1992; Weinstein et al., 1986). There is therefore an 2 mg of poly(A)+ RNA from 10T1/2 cells infected with (1) p- Babe-puro or (2) p-Babe-puro-VJ1. Sample 3 is from an urgent need for murine transformation systems driven individual agar colony of v-Jun-transformed 10T1/2 cells by transcription factor oncoproteins. The 10T1/2 cells infected by the v-Jun expressing vector have all cells. They are morphologically altered, grow in multi-layered foci, show increased saturation density and have acquired the ability of anchorage-independent growth. In the course of these studies, several types of murine ®broblasts carrying null mutations in key tumor suppressor genes were also tested for their susceptibility to transformation by v-Jun. v-Jun induced anchorage-independent growth in cells lacking the gene but failed to transform murine cells defective in the Rb or genes. v-Jun was also poorly Figure 6 SSeCKS expression is down-regulated in v-Jun trans- expressed in these latter cell types, in contrast to 10T1/ formed 10T1/2 cells. A Western blot was prepared for 50 mgof 2 cells which showed high levels of v-Jun (SB Cohen, whole cell lysates from 10T1/2 cells infected with (1) p-Babe-puro, 2000, unpublished observations). (2) p-Babe-hgro-SSeCKS, (3) p-Babe-puro-VJ1, or (4) p-Babe- The number of di€erentially expressed genes detected puro-VJ1 and p-Babe-hygro-SSeCKs by DNA arrays in cancer cells is typically very large (Alizadeh and Staudt, 2000; Pollack et al., 1999; Wang et al., 2000; Xu et al., 2000), and the v-Jun-transformed results suggest that SSeCKS can partially reestablish 10T1/2 cells are no exception. Probably only a small the contact inhibition of growth that is lost during v- set of these targets is in control of neoplastic proper- Jun-induced transformation. v-Jun 10T1/2 cells expres- ties. Identifying these critical actors and distinguishing sing SSeCKS cells also showed an about 10-fold them from innocent bystanders with functional tests is reduced plating eciency in agar. The colonies formed a laborious and time-consuming task. One justi®able by these cells were also signi®cantly smaller than criterion for the initial selection of targets to be colonies of v-Jun-transformed 10T1/2 cells (Figure 4 characterized is the degree of di€erential expression. and Table 1). Expression of SSeCKS by itself had no Genes that are highly up or down-regulated are e€ect on anchorage-independent growth; SSeCKS cells perhaps more likely to a€ect the cellular phenotype. behaved identical to p-Babe-infected 10T1/2 cells. v- SSeCKS is one such highly misregulated gene; North-

Oncogene V-Jun down regulated SSeCKS in murine fibroblasts SB Cohen et al 145 ern blots show its expression lowered by a factor of (MARCKS). Like SSeCKS, it has an attenuating e€ect more than 10 in v-Jun-transformed 10T1/2 cells. on the transformed cellular phenotype (SB Cohen, Previous studies had also identi®ed the product of 2000, in preparation). Interference with PKC signals the SSeCKS gene as a potential tumor suppressor (Lin may therefore be an important aspect of Jun-induced and Gelman, 1997). The SSeCKS protein of rodents transformation. shows homology to a human protein recognized by an auto-antibody in myasthenia gravis and termed `gravin' (Nauert et al., 1997). These proteins share two domains, one mediating interaction with protein kinase Materials and methods C (PKC) and the other with the regulatory subunit of protein kinase A. SSeCKS is therefore likely to play a Recombinant vectors role in several signaling cascades; it could also function pBabe-puro-VJ1 was generated by excising the ClaI fragment as a bridge connecting two pathways. SSeCKS from RCAS-VJ1 (Bos et al., 1990) and subcloning into the expression is also down-regulated in rodent cells BstB1 site of a modi®ed pBabe-puro vector (Morgenstern transfected by Src, Ras, Fos or Myc, but shows no and Land, 1990) that contains the multi-cloning site of MCS5 change in expression in cells expressing Raf, Mos or (BamHI to HpaI) cloned into BamHI/SnaBI. The pBabe- hygro-SSeCKS vector has been described previously (Lin and Neu (Lin et al., 1995). These facts suggest a Gelman, 1997). convergence of di€erent oncogenic pathways and reinforce the role of SSeCKS as a tumor suppressor. Whereas certain can transform without Cell culture, transfection, saturation density, focus assays and a€ecting SSeCKS expression Jun, Src, Ras, Fos, and soft agar cloning Myc may perhaps require down regulation of SSeCKS BOSC23 and PA317 cells are ecotropic and amphotopic to e€ect transformation. In the case of Src and Jun, the retroviral packaging lines, respectively (Miller and Rosman, re-introduction of SSeCKS can, at least in part, block 1989; Pear et al., 1993). 10T1/2 cells are a continuous line of transformation. normal murine ®broblasts (Rezniko€ et al., 1973). All three Re-expression of SSeCKS partially reverses the cell lines were obtained from the laboratory of Edward Stavnezer. BOSC23, PA317 and 10T1/2 cells were grown in transformed phenotype of v-Jun-transformed 10T1/2 DMEM/high glucose medium with glutamine (Gibco) cells. This result suggests that down-regulation of containing 10% fetal calf serum, 10 mg/ml gentamicin, 2 mg/ SSeCKS is a requirement for Jun-induced oncogenic ml amphotercin B (Sigma), 10 mM HEPES. Transient transformation. SSeCKS functions as a tumor suppres- transfections of BOSC23 cells were performed using the sor counteracting the oncogenic e€ect of Jun as well as DOTAP reagent according to the manufacturer's Src (Lin and Gelman, 1997). SSeCKS is the second instructions (Boehringer Mannheim Corp.). Infectious virus identi®ed Jun target that by itself can a€ect the growth was harvested 24 h post transfection, passed through a 0.2 m behavior of the cell. The other one, heparin binding syringe ®lter, and applied to either PA317 (VJ1) or 10T1/2 epithelial (HB-EGF), is up-regulated in cells (SSeCKS). PA317-VJ1 cells were selected with puromy- Jun-transformed CEF and induces a partial transfor- cin (2.5 mg/ml) for 5 days, and from the surviving cells infectious virus was again harvested and used to infect 10T1/ mation when overexpressed in CEF. Over-expression of 2 cells. 10T1/2 cells were selected in either puromycin (VJ1) at HB-EGF in 10T1/2 cells does not cause transformation 3.0 mg/ml or hygromycin B (SSeCKS) at 75 mg/ml. SSeCKS (SB Cohen, 1999, unpublished observations). expression had a toxic e€ect on PA317, therefore virus was Down-regulation of SSeCKS is probably not su- used directly from the BOSC23 packaging line. 10T1/2 cells cient to induce cellular transformation; di€erential expressing v-Jun were superinfected with SSeCKS virus or expression of other Jun targets may be required for pBabe-hygro virus in an analogous manner, as were this process. The attenuating e€ect of reexpressing individual p-Babe-puro and pBabe-hygro controls. SSeCKS on the transformed phenotype is reminiscent Saturation density was determined by plating 26104 cells in of other tumor suppressor genes. Colon lines 12 35 mm plates. Each day the cells in one plate were carry mutations in several growth-regulatory genes trypsinized and counted. Focus assays were performed by diluting the appropriate cells at 1/50 or 1/200 with p-Babe including both loss and gain of functions, yet reversion infected cells, and seeding 2.56105 total cells in 35 mm plates. of transformation can be achieved by reintroducing a Medium was changed every third day, and cells were allowed single tumor suppressor, the adenomatous polyposis to grow for 10 days when they were stained in 2% crystal coli (APC) gene, into these cells (Groden et al., 1995). violet in 20% methanol for 1 min and destained in 1% acetic Loss of APC function is an early event in acid, 1% methanol for 2 min. Soft agar cloning followed formation and is insucient to cause colon cancer. published techniques, using 1.256104 cells in a 60 mm plate Progression from polyp to malignant tumor requires (Colmenares et al., 1991). Cells were fed every 5 days, and additional mutations including loss of function in p53 colonies were counted after 4 weeks. Saturation density, focus and gain of function in Ras (Cho and Vogelstein, assays and soft agar cloning were repeated at least once using 1992). Similarly, Jun-transformed cells will contain a VJ1/p-Babe-hygro co-infected 10T1/2 cells to ensure that e€ects seen were not the result of hygromycin selection. other up and down-regulated genes that are necessary, but individually are not sucient for transformation. SSeCKS is a substrate of PKC. The DNA micro- Northern blots arrays also identi®ed another down-regulated PKC The preparation of total RNA, poly(A)+ RNA and Northern substrate, myristylated alanine-rich C kinase substrate blots has been previously described (Fu et al., 1999).

Oncogene V-Jun down regulated SSeCKS in murine fibroblasts SB Cohen et al 146 Acknowledgments Western blots This work was supported by grant CA42564 from the 10T1/2 cells were lysed in RIPA bu€er (150 nM NacL/ National Cancer Institute; S Cohen is the recipient of a 100 mM Tris HCl pH 8.0, 1%. Triton X-100, 1% deoxycholic fellowship from NIH training grant 5 T32 DK07022, and acid, 5 mM EDTA, 1 mM PMSF, 0.5 mM DTT). The lysates A Waha is supported by the Deutsche Forschungsge- were clari®ed by centrifugation at 15 0006g for 10 min. meinschaft (WA1428/1-1). The p-Babe-puro vector and Aliquots containing 50 mg of protein were resolved by SDS ± PA317, BOSC23 and 10T1/2 cells were a gift from Edward PAGE and transferred to PVDF in 20 mM TRIS-HCl Stavnezer (Department of Biochemistry, Case Western pH 8.0, 150 mm glycine, 120% (vol/vol) methanol. Mem- Reserve University). The p537/7 and Rb7/7 mouse embryo branes were blocked with 5% nonfat dry milk in PBS, 0.05% ®broblasts were a gift from (Department of Tween 20 bu€er and incubated with a rabbit antibody Biology, Massachusetts Institute of Technology). This is speci®c for SSeCKS (Lin et al., 1996). Protein bands were manuscript number 13353-MEM at The Scripps Research visualized by incubating with a horseradish peroxidase Institute. conjugated secondary antibody (Amersham) and were detected by incubating with a chemiluminescent substrate per manufacturer's protocol (Dupont/NEN).

References

Abate C, Patel L, Rauscher FJD and Curran T. (1990). Maki Y, Bos TJ, Davis C, Starbuck M and Vogt PK. (1987). Science, 249, 1157 ± 1161. Proc. Natl. Acad. Sci. USA, 84, 2848 ± 2852. Alizadeh AA and Staudt LM. (2000). Curr. Opin. Immunol., Miller AD and Rosman GJ. (1989). Biotechniques, 7, 980 ± 12, 219 ± 225. 982, 984 ± 986, 989 ± 990. Alt M and Grassmann R. (1993). Oncogene, 8, 1421 ± 1427. Morgan IM, Asano M, Havarstein LS, Ishikawa H, Hiiragi Bos TJ, Monteclaro FS, Mitsunobu F, Ball Jr AR, Chang T, Ito Y and Vogt PK. (1993). Oncogene, 8, 1135 ± 1140. CH, Nishimura T and Vogt PK. (1990). Genes Dev., 4, Morgan IM, Havarstein LS, Wong WY, Luu P and Vogt PK. 1677 ± 1687. (1994). Oncogene, 9, 2793 ± 2797. Boyle WJ, Smeal T, De®ze LH, Angel P, Woodgett JR, MorganIM,RansoneLJ,BosTJ,VermaIMandVogtPK. Karin M and Hunter T. (1991). Cell, 64, 573 ± 584. (1992). Oncogene, 7, 1119 ± 1125. Brugarolas J, Bronson RT and Jacks T. (1998). J. Cell. Biol., Morgenstern JP and Land H. (1990). Nucleic Acids Res., 18, 141, 503 ± 514. 3587 ± 3596. Chida K and Vogt PK. (1992). Proc. Natl. Acad. Sci. USA, Nauert JB, Klauck TM, Langeberg LK and Scott JD. (1997). 89, 4290 ± 4294. Curr. Biol., 7, 52 ± 62. Cho KR and Vogelstein B. (1992). Cancer, 70, 1727 ± 1731. Nishimura T and Vogt PK. (1988). Oncogene, 3, 659 ± 663. Colledge WH, Gebhardt A, Edge MD and Bell JC. (1989). Pear WS, Nolan GP, Scott ML and Baltimore D. (1993). Oncogene, 4, 753 ± 757. Proc. Natl. Acad. Sci. USA, 90, 8392 ± 8396. Colmenares C, Sutrave P, Hughes SH and Stavnezer E. Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Perga- (1991). J. Virol., 65, 4929 ± 4935. menschikov A, Williams CF, Je€rey SS, Botstein D and Fu S, Bottoli I, Goller M and Vogt PK. (1999). Proc. Natl. Brown PO. (1999). Nat. Genet., 23, 41 ± 46. Acad. Sci. USA, 96, 5716 ± 5721. Rezniko€ CA, Brankow DW and Heidelberger C. (1973). Fu S-L, Waha A and Vogt P. (2000). Oncogene, 19, 3587 ± Cancer Res., 33, 3231 ± 3238. 3545. Schreiber-Agus N, Torres R, Horner J, Lau A, Jamrich M Goller ME, Iacovoni JS, Vogt PK and Kruse U. (1998). and DePinho RA. (1993). Mol. Cell. Biol., 13, 2456 ± 2468. Oncogene, 16, 2945 ± 2948. Schutte J, Minna JD and Birrer MJ. (1989). Proc. Natl. Acad. Groden J, Joslyn G, Samowitz W, Jones D, Bhattacharyya Sci. USA, 86, 2257 ± 2261. N, Spirio L, Thliveris A, Robertson M, Egan S, Meuth M Sterneck E, Muller C, Katz S and Leutz A. (1992). EMBO J., and R White. (1995). Cancer Res., 55, 1531 ± 1539. 11, 115 ± 126. Hadman M, Gabos L, Loo M, Sehgal A and Bos TJ. (1996). Wang T, Hopkins D, Schmidt C, Silva S, Houghton R, Oncogene, 12, 135 ± 142. Takita H, Repasky E and Reed SG. (2000). Oncogene, 19, Hartl M and Bister K. (1995). Proc. Natl. Acad. Sci. USA, 92, 1519 ± 1528. 11731 ± 11735. WeinsteinY,IhleJN,LavuSandReddyEP.(1986).Proc. Hibi M, Lin A, Smeal T, Minden A and Karin M. (1993). Natl. Acad. Sci. USA, 83, 5010 ± 5014. Genes Dev., 7, 2135 ± 2148. Xu J, Stolk JA, Zhang X, Silva SJ, Houghton RL, Land H, Chen AC, Morgenstern JP, Parada LF and Matsumura M, Vedvick TS, Leslie KB, Badaro R and Weinberg RA. (1986). Mol. Cell. Biol., 6, 1917 ± 1925. Reed SG. (2000). Cancer Res., 60, 1677 ± 1682. LinXandGelmanIH.(1997).Cancer Res., 57, 2304 ± 2312. Zhang M, Zou Z, Maass N and Sager R. (1995). Cancer Res., Lin X, Nelson PJ, Frankfort B, Tombler E, Johnson R and 55, 2537 ± 2541. Gelman IH. (1995). Mol. Cell. Biol., 15, 2754 ± 2762. LinX,TomblerE,NelsonPJ,RossMandGelmanIH. (1996). J. Biol. Chem., 271, 28430 ± 28438.

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