Expression of a Down-Regulated Target, Ssecks, Reverses V-Jun-Induced Transformation of 10T1/2 Murine ®Broblasts

Oncogene (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 mutations 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 genes. The dierential analysed for dierential gene expression with DNA expression of these targets determines the neoplastic micro-array technology. One of the targets downregu- phenotype of the cell. 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 eect 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; dierential gene tion, but some continuous mammalian cell lines are expression: DNA micro-array; tumor suppressor susceptible to the growth-promoting eects 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 transcription 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 transcription factor 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 gene expression. 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 diers from 1996). SSeCKS is down-regulated in rodent ®broblasts transformed by Src, Ras, Fos or Myc 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 cancer 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. eect 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 properties. 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-transformed 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 ecient focus formation was observed with v- v-Jun is a highly eective 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- eciency 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 virus 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 viruses 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 dierentially 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.

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