The Cellular Oncogene EWS/Activating Transcription Factor 1

The cellular oncogene EWS/activating transcription factor 1 is unable to activate adenovirus-borne promoters: Implications for cytotoxic prodrug therapy of malignant melanoma of soft parts

Raymond W. M. Lung and Kevin A. W. Lee Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region, China.

The cellular oncoprotein Ewing’s sarcoma oncogene (EWS)/activating transcription factor 1 (ATF1) is a highly specific marker for malignant melanoma of soft parts (MMSP) and is a potent activator of several cAMP-inducible promoters, including the somatostatin promoter. Here we explored the potential for using the somatostatin promoter to direct toxic gene expression in MMSP cells. When introduced into MMSP cells, a somatostatin-herpes simplex virus thymidine kinase fusion gene confers strong and cell-specific sensitivity to the cytotoxic prodrug ganciclovir. Ganciclovir sensitivity requires the ATF-binding site present in the somatostatin promoter, indicating that toxic gene expression is caused by EWS/ATF1. We also tested the efficacy of recombinant adenoviruses adenoviruses for gene delivery and expression in two MMSP cell lines (DTC1 and Su-ccs-1). Surprisingly, several promoters (including somatostatin) that are strongly activated by EWS/ATF1 in transient assays are not activated in DTC1 and Su-ccs-1 cells when present in an adenovirus vector. In summary, our findings demonstrate the potential for using the somatostatin promoter for cytotoxic prodrug therapy for MMSP. However, first-generation adenovirus vectors cannot be used as promoter delivery vehicles for toxic gene expression in MMSP cells. Cancer Gene Therapy (2000) 7, 396–406

Key words: EWS/ATF1 oncogene; malignant melanoma of soft parts; adenovirus; gene therapy.

hromosomal translocations of the N-terminal region combined with chemotherapy and radiotherapy,10,13 but Cof the Ewing’s sarcoma oncogene (EWS) to a recurrence is high and mortality is ϳ45% due to meta- variety of cellular transcription factors produces domi- static disease. The need for more effective therapy of nant oncogenes (EWS fusion proteins (EFPs)) that MMSP and other EFP-related tumors is apparent and cause distinct sarcomas.1,2 In malignant melanoma of emerging gene therapy techniques may offer new ap- soft parts (MMSP), EWS is fused to activating transcrip- proaches. 3 tion factor 1 (ATF1); in Ewing’s sarcoma, EWS is fused In the case of Ewing’s sarcoma5 and desmoplastic 4–8 to the ETS domain family members; and in desmo- small round cell tumors14 the oncogenic properties of plastic small round cell tumors, EWS is fused to the EFPs have been demonstrated directly and deregulated 9 Wilm’s tumor oncogene, WT1. Because EFPs are target genes have been identified.15–20 Furthermore, in strong transcriptional activators, it is thought that EFP- Ewing’s sarcoma, agents that antagonize EWS-Fli1 fu- induced malignancies arise via aberrant gene activation, sion proteins can also inhibit cellular proliferation,22–24 with the tumor type being specified by the EWS fusion indicating the potential for the development of thera- partner. peutic agents that target EWS/Fli. In contrast to EWS/ MMSP (also known as clear cell sarcoma) is typically Fli, the role that EWS/ATF1 plays in tumorigenesis is associated with tendons and aponeuroses and is thought 10,11 poorly characterized. Because ATF1 is a cAMP-induc- to be of neuroectodermal origin. MMSP is a very ible activator,25,26 it is predicted that EWS/ATF1 acts via aggressive, early onset tumor that initially grows slowly, constitutive activation of cAMP-inducible genes. How- resulting in 20% of patients having metastases at the 12 ever, the critical target genes have not yet been identi- time of diagnosis. Current treatment involves surgery fied. In addition, a role for EWS/ATF1 in tumor maintenance has not yet been established. Consequently, it is Received April 30, 1999; accepted July 8, 1999. not known whether therapeutic approaches aimed at adenovirusdress correspondence and reprint requests to Dr. Kevin A. W. antagonizing EWS/ATF1 will be applicable to MMSP. Lee, Hong Kong University of Science and Technology, Clear Water Bay, An alternative therapeutic approach (cytotoxic pro- Kowloon, Hong Kong, S. A. R. China. E-mail address: bokaw@ drug therapy) for MMSP involves the exploitation of usthk.ust.hk tumor-specific promoters to target tumor cells for the

396 Cancer Gene Therapy, Vol 7, No 3, 2000: pp 396–406 LUNG AND LEE: GENE THERAPY FOR MMSP 397 action of cytotoxic agents.27–29 Previous studies have inserting a BamHI/AatII fragment from p⌬(Ϫ71A)SomCAT characterized EWS/ATF1 as a tumor-specific activator and an oligonucleotide to reconstruct the somatostatin pro- 30–32 moter to position Ϫ71 into the multiple cloning site of of promoters containing ATF-binding sites. Tran- ⌬ ⌬ Ϫ scriptional activation is dependent upon the EWS acti- p E1SP1A. p E1( 42A) (used to construct AD42) was ob- 30,31 tained by insertion of an AatII/BamHI blunt fragment from vation domain (EAD) and the DNA-binding domain ⌬ Ϫ 30–32 p ( 71)SomCAT into the EcoRI/BamHI blunt sites of the of ATF1. Most significantly, promoters that can be multiple cloning region of p⌬E1sp1A. p⌬E1(25A) (used to activated by EWS/ATF1 (e.g., the somatostatin pro- construct AD25V) was obtained by inserting a BamHI/AflIII moter) are constitutively active when transiently intro- blunt fragment from pVIP25CAT into the EcoRI/BamHI duced into tumor-derived cell lines (DTC1 and Su-ccs-1, blunt sites of p⌬E1sp1A. p⌬E1(VIPB) (used to construct hereafter referred to as MMSP cells) containing endog- ADVIP) was obtained by inserting a SacI/BamHI blunt frag- enous EWS/ATF1.30 In addition to a cell-specific target- ment from the pVIP4CAT sequence into the EcoRI/BamHI ing agent (such as a tumor-specific promoter), cytotoxic blunt sites of p⌬E1sp1B. prodrug treatment requires a safe and efficient vector to Construction and propagation of recombinant introduce the therapeutic gene and an effective prodrug. adenoviruss Among many available systems, recombinant adenoviruses (adenoviruss)33,34 and the herpes simplex virus Recombinant adenoviruss (adenovirus type 5) were con- thymidine kinase (hsvtk) gene have been well studied. structed by in vivo recombination of plasmids in 293 cells using adenovirus vector construction kit C (Microbix Biosystems, The combined use of hsvtk as a toxic gene and adeno- ⌬ ⌬ virus as a gene delivery vehicle has proven effective for Toronto, Canada). p E1SP1A and p E1SP1B contain the prodrug sensitization of other tumor cell lines.35–37 left-hand end of adenovirus type 5 (from base pair 22 (0 map units) (m.u.) to base pair 5790 (16.1 m.u.)), with a deletion of In the current study, we tested the use of a somatosta- E1 sequences from base pair 342 to base pair 3523 (1.0–9.8 tin promoter-hsvtk fusion gene and adenovirus vectors m.u.), but containing the entire packaging signal. A multiclon- for cytotoxic prodrug treatment of MMSP cell lines. Our ing site is present in the E1 region, allowing for insertion of the results demonstrate the potential of the somatostatin foreign gene. p⌬E1SP1A and p⌬E1SP1B are identical except promoter and hsvtk as cytotoxic agents for MMSP cells. for the orientation of the multicloning site between ClaI and Surprisingly however, several promoters (including so- BglII. The pBHG10 plasmid contains the rest of the adenovirus matostatin) that are strongly activated in MMSP cells type 5 genome and a small overlapping region with both p⌬E1sp1A and p⌬E1sp1B to allow recombination after co- using plasmid-based transient assays are not activated 42 when present in an adenovirus vector. Thus, first-gener- transfection into 293 cells. pBHG10 is noninfectious due to ation adenovirus vectors cannot be used as promoter lack of a packaging signal needed for encapsidation of viral DNA;43 in addition, to allow a bigger insert, the nonessential delivery vehicles for EWS/ATF1-dependent toxic gene E3 region (78.3–85.8m.u.) is deleted. Low-passage 293 cells expression in MMSP cells. (Ͻ40 passages) in a 60-mm dish were set up at 50% confluence. A total of 10 ␮gofp⌬E1(Ϫ71A), p⌬E1(Ϫ42A), MATERIALS AND METHODS p⌬E1(25A), or p⌬E1(VIPB) and 10 ␮g of pBHG10 were then cotransfected into the cells by calcium phosphate precipitation. Plasmids Medium was renewed the following day and on reaching confluence, the cells were trypsinized and diluted. On release p⌬(Ϫ71)SomCAT contains the somatostatin promoter at po- from the cells, recombinant viruses were confirmed by poly- sition Ϫ71, fused to the chloramphenicol acetyl transferase 38 merase chain reaction analysis of isolated viral DNA and (CAT) coding sequences. p⌬(Ϫ71)SomCAT contains a sin- titrated on 293 cells. Purification of viral DNA was performed gle ATF1-binding site, which is required for activation by both 38 30 as follows. Infected 293 cells were harvested and lysed by the cAMP and EWS/ATF1. p⌬(Ϫ42)SomCAT is the same as addition of 400 ␮ p⌬(Ϫ71)SomCAT, except that sequences from position Ϫ71 to L of lysis buffer (20 mM of Tris-Cl (pH 7.5), 30 0.5% Nonidet P-40, 500 mM NaCl, and 1 mM dithiothreitol) Ϫ42 of the somatostatin promoter are deleted. Therefore, on ice for 5 minutes. After centrifugation for 5 minutes in a p⌬(Ϫ42)SomCAT lacks the ATF1-binding site of the soma- 30 ⌬ Ϫ ⌬ Ϫ microfuge, the supernatant was adjusted by the addition of 15 tostatin promoter. p ( 42)SomTK and p ( 71)SomTK are ␮ ␮ identical with p⌬(Ϫ42)SomCAT and p⌬(Ϫ71)SomCAT, re- L of 20% sodium dodecyl sulfate and 150 g of proteinase K spectively, except that the CAT sequence is replaced by the and incubated at 55°C for 30 minutes. The sample was then phenol-extracted, ethanol-precipitated, and finally treated with cDNA sequence of hsvtk. pSVEA contains the complete EWS/ ␮ ATF1 coding sequence with 123 of 5 untranslated sequences from 20 g/mL ribonuclease A for 30 minutes to remove the RNA. EWS and can express EWS/ATF1 under the control of the simian Cell culture and adenovirus infections virus 40 (SV40) early promoter. pRSVCAT contains the Rous sarcoma virus (RSV) long terminal repeat linked to CAT,39 and The JEG3,44 Su-ccs-1,11 and DTC130 cells lines were main- pCAT-TM-control (Promega, Madison, Wis) contains the SV40 tained as monolayers in Dulbecco’s modified Eagle’s medium enhancer and early promoter linked to CAT. pVIP25CAT40 (DMEM) containing 10% fetal calf serum. PC12 cells were contains the vasoactive intestinal peptide (VIP) cAMP-respon- maintained as monolayers in DMEM containing 10% fetal calf sive element containing two ATF-binding sites, fused to the serum and 5% horse serum. RAT1 is a rat embryo fibroblast RSV promoter from positions Ϫ50 to ϩ39 and to CAT. cell line and MEL28 is a human melanoma cell line. Human pVIP4CAT contains 240 bp of the VIP promoter, including the embryo kidney 293 cells (Microbix Biosystems) contain and cAMP-responsive element linked to the reporter CAT gene.40 express the early region 1 (E1) of adenovirus type 5. Clones pCAT-BstN1 (Ref. 41, referred to as pE3CAT in Fig 2) 711, 714, and 716 (collectively called D71TK cells) are inde- contains the adenovirus E3 promoter to position Ϫ105 fused to pendent clones of DTC1 cell lines containing ⌬(Ϫ71)Som/ CAT. p⌬E1(Ϫ71A) (used to construct AD71) was obtained by hsvtk fusion gene in the genome. Clones 422, 423, and 424

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(collectively called D42TK cells) are independent clones of duced into both MMSP cell lines (DTC1 and Su-ccs- DTC1 cell lines containing ⌬(Ϫ42)Som/hsvtk fusion gene. 1).30 We wanted to test the ability of a somatostatin Polymerase chain reaction analysis of genomic DNA con- promoter-hsvtk fusion gene to express hsvtk in the above firmed the presence of the SomTK fusion gene in each clone cells and thereby confer sensitivity to the cytotoxic (data not shown). Conditions for adenovirus infections were as prodrug GCV. To establish a test system for GCV follows: Cells in a 60-mm culture dish at ϳ50% confluence were infected at a multiplicity of infection (MOI) as indicated sensitivity, we produced DTC1 transfectants containing in the figure legend. adenovirussorption of the virus was a stably integrated somatostatin promoter linked to carried out in 0.5 mL of DMEM buffered with 20 mM CAT as a reporter. Initially, we used CAT instead of N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (pH 7.2) hsvtk as a simpler test for somatostatin promoter activity for 45 minutes, followed by dilution with fresh growth media. under the desired conditions. Two pools of DTC1- derived stable transfectants (D42 and D71) were ob- Transfections, CAT assays, and RNA analysis tained after G418 selection. D42 represents a pool of ⌬ Ϫ All transfections were carried out by calcium phosphate copre- DTC1 cells containing the ( 42)SomCAT fusion gene cipitation, and CAT assays were performed as described (lacking the ATF-binding site in the somatostatin pro- previously.45 Precipitates contained 5 ␮g of reporter plasmid, moter); D71 represents a pool of cells containing the the indicated amount of activator plasmid(s), and 20 ␮gof ⌬(Ϫ71)SomCAT fusion gene (containing the ATF site). total DNA made up with pSP64 as carrier. One-third of the Somatostatin promoter activity was assessed by CAT precipitate was added to ϳ50% confluent JEG3 cells in a assay (Fig 1A). Similar to results obtained in transient 60-mm culture dish. For quantitation of results, the percentage 30 14 assays, there was no detection of somatostatin pro- of conversion of unacetylated to acetylated C-chloramphen- moter activity in D42 cells (or parental DTC1 cells), icol under linear assay conditions was determined by excision whereas strong activity was obtained in D71 cells. PC12 of spots from the thin-layer chromatography plate and quantitation of radioactivity using a liquid scintillation counter. cells containing a stably integrated cAMP-inducible VIP Preparation of total cellular RNA and detection of specific promoter linked to CAT serve as a comparison for transcripts by primer extension was as described previously.46 activated levels of transcription. In the presence of 20 ␮M forskolin (which increases intracellular cAMP lev- Production of DTC1 transfectants and ganciclovir els), the VIP promoter was activated in PC12 cells to a (GCV) sensitivity assays level that was similar to that seen for the somatostatin promoter in D71 cells. The above results show that (on A 50% confluent monolayer of DTC1 cells (ϳ2.5 ϫ 106 cells) was transfected by calcium phosphate coprecipitation39 with 5 average within a pool of transfectants) chromosomal ␮g of the desired plasmid with or without 1 ␮g of polyneo copies of the somatostatin promoter are active in DTC1 plasmid, conferring neomycin resistance. DTC1 cells were cells. Promoter activity is dependent upon the ATF- used because Su-ccs-1 cells do not yield viable colonies due to binding site, consistent with the promoter being acti- a very low transfection efficiency and/or cytotoxicity at low cell vated by endogenous EWS/ATF1. density (K.A.W.L., unpublished observation). Transfected We produced stable transfectants from DTC1 cells cells were washed with phosphate-buffered saline, and old containing somatostatin promoter-hsvtk cDNA fusions medium was replaced by fresh growth medium at 1 day and tested their sensitivity to GCV. GCV is nontoxic and posttransfection. Cells were then split to 25% confluence into ␮ is converted into a cytotoxic DNA synthesis chain ter- medium with 400 g/mL G418 (Life Technologies, Gaithers- minator by hsvtk. D71TK and D42TK represent pools of burg, Md; stored as 40 mg/mL in sterile-filtered 100 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (pH transfectants similar to D71 and D42 cells, except that 7.2)). Selection medium was changed daily for the first 10 days the CAT reporter is replaced by hsvtk cDNA. In the and every 2 days afterward. Cells were set up in duplicate at absence of GCV, D71TK cells exhibit the same growth 30% confluence in 24-well plates the day before the addition of kinetics as parental DTC1 cells (Fig 1B). adenovirusding GCV (Cymevene, 500 mg GCV/546 mg from Ben Venue GCV (5 ␮g/mL) to the cell culture medium has no effect Laboratories, Bedford, Ohio, under license from Syntex Lab- on the survival of parental DTC1 cells but severely oratories, Palo Alto, Calif) to the growth medium. GCV was inhibits the growth of D71TK cells, starting at day 4 and dissolved in autoclaved H2O at a concentration of 0.5 mg/mL ending in cell death by day 12. Individual G418-resistant and stored at 4°C. Appropriate drug concentrations were made clones were isolated and tested for sensitivity to GCV by diluting the stock solution with fresh medium immediately (Fig 1C). Three independent clones (711, 714, and 716) before each experiment. The same volume of medium was added for the control experiment, medium was changed every isolated from D71TK cells and three independent clones (422, 423, and 424) isolated from D42TK cells were 2 days, and viable cell counts were taken using trypan blue ␮ exclusion and a hemocytometer. examined. After treatment with 0.1 g/mL GCV for 10 days, the survival rates of clone 716 (48%), clone 714 (38%), and clone 711 (35%) were lower than the survival RESULTS rates of all three clones (422, 423, and 424, average survival of 97%) from D42TK cells. At 0.3 ␮g/mL GCV, Cytotoxic prodrug treatment of DTC1 cells in vitro the survival rates at 10 days for 716 (8% survival), 714 A truncated version of the somatostatin promoter (here- (5%), and 711 (5%) were quite low, whereas the survival after simply referred to as the somatostatin promoter) of 422, 423, and 424 was only slightly affected (average of containing promoter sequences to Ϫ71 including a single 95%). Thus, D71TK cells are sensitive to GCV at ATF-binding site, is highly active when transiently intro- concentrations previously reported to be toxic for other

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Figure 1. Cytotoxicity of a somatostatin-hsvtk fusion gene in DTC1 cells. A: Somatostatin promoter activity in stably transfected DTC1 cells. Cell extracts were prepared from parental DTC1 cells (DTC1) or from pools of G418-selected cells containing the somatostatin promoter linked to CAT as a reporter. D42 is a pool of cells containing p⌬(Ϫ42)SomCAT (which lacks the ATF-binding site in the somatostatin promoter); D71 is a pool of cells containing p⌬(Ϫ71)SomCAT (including the ATF-binding site). PC12 represents a stable transfectant containing a chromosomal cAMP-inducible VIP-CAT reporter. A representative autoradiogram for CAT assays (conversion of chloramphenicol (c) to acetylated chloramphenicol (Ac)) is shown. B: GCV sensitivity of DTC1 cells containing a somatostatin-hsvtk fusion gene. Growth curves are shown for parental DTC1 cells or for a pool of stable DTC1 transfectants (D71TK) containing a stably integrated somatostatin-hsvtk fusion gene. GCV was included in the cell culture medium at a concentration of 5 ␮g/mL. C: GCV sensitivity of individual transfectants. Cells were treated with the indicated concentrations of GCV, and cell survival was determined by viable cell counting after 10 days of GCV treatment. Three independent clones (711, 714, and 716) isolated from D71TK cells and three independent clones (422, 423, and 424) isolated from D42TK cells were examined. Survival rates for the three control (EWS/ATF1-negative) cell lines (MEL28, JEG3, and RAT1) are also shown.

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Figure 2. A: Transcriptional activation of selected promoters by EWS/ATF1 in JEG3 cells. Cells were transfected with 5 ␮gof reporter plasmid containing the test promoter (shown above) in the absence or presence of pSVEWS/ATF1 (5 ␮g) (shown below) made up to a total of 20 ␮g DNA with pSP64. CAT assays were performed at 40 hours posttransfection. Reporter plasmids are described in Materials and Methods. B: Structure of recombinant adenoviruss. The open box at the top represents the entire 36-kb adenovirus genome. ITR is the in- verted terminal repeat sequence, the E3 gene is deleted (⌬E3), and foreign promoter- CAT inserts into the E1 region to produce different recombinant viruses (AD42, AD71, AD25V, and ADVIP) are shown below. The transcription orientation for the CAT reporter gene is indicated by an arrow, and the test promoter sequences are indicated in black boxes with coordinates relative to the transcription start site (ϩ1) indicated.

hsvtk-expressing tumor cells.35,47 As an additional test of EWS/ATF1 in JEG3 cells is shown (Fig 2A). VIP25CAT speciﬁcity, we examined the effect of GCV (after intro- (containing the cAMP-response-element from the VIP duction of the SomTK fusion gene) in three other promoter linked to the RSV TATA box), VIP4CAT mammalian cell lines (MEL28, JEG3, and RAT1) that (containing the intact VIP promoter to position Ϫ94), do not express EWS/ATF1 (Fig 1C). Similar to clones and ⌬(Ϫ71)SomCAT are all strongly trans-activated 422, 423, and 424, MEL28 (90% survival), JEG3 (87%), (ϳ100-fold) by EWS/ATF1. In addition, the adenovirus and RAT1 (85%) were all only slightly affected by GCV E3 promoter, which contains an ATF site required for treatment. In summary, the above results show that a trans-activation by cAMP,48–50 is strongly activated by somatostatin promoter-hsvtk fusion can confer GCV- EWS/ATF1 in this assay (Fig 2A). We constructed dependent cytotoxicity to DTC1 cells. This effect is recombinant adenoviruss containing the above promot- cell-speciﬁc and (as indicated by the requirement for the ers (and a control virus derived from ⌬(Ϫ42)SomCAT) ATF site in the somatostatin promoter) most likely linked to CAT (Fig 2B). Again, we initially used CAT results from somatostatin promoter activation by endog- instead of hsvtk as a simpler test for promoter activity, enous EWS/ATF1. and we refer to the above promoters (AD71, AD42, AD25V, and ADVIP) as virus-borne ATF-dependent Design of recombinant adenoviruss for toxic gene promoters (VAPs). delivery VAP activity during adenovirus infection In a transient cotransfection assay EWS/ATF1 can activate several promoters that contain ATF-binding sites.30 DTC1 and Su-ccs-1 were infected with AD71 and AD42 Trans-activation of four such promoters by exogenous at different MOIs, and CAT assays performed at 48

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Figure 3. A: Viral promoter activity during infection of MMSP cells. The same samples of recombinant adenoviruss (shown in Fig 2) were used to infect DTC1, Su-ccs-1, and 293 cells at different MOIs. CAT assays were performed at 48 hpi unless otherwise indicated, and representative autoradio- grams of CAT assays are shown. B: Trans- activation using DTC1 cells in a transient assay. Test promoters were assayed after the transfection of DTC1 and JEG3 cells. Transfection conditions were as described in the legend to Figure 2, and the same precipitate for each test promoter was evenly divided and added to either DTC1 and JEG3 cells. CAT assays were performed at 40 hours posttransfection.

hours postinfection (hpi) (Fig 3A). At MOIs up to 100 AD71 and analyzed VAP activity by CAT assay (Fig (or 1000, data not shown), both AD71 and AD42 3A). 293 cells are useful for the above purposes because showed no detectable promoter activity in DTC1 or they express an endogenous adenovirus E1A gene that is Su-ccs-1 cells. Using the transient assay described pre- an activator of many ATF-dependent promoters.46,48 viously,30 we tested the ability of the DTC1 cells used in For AD71, promoter activity is readily detectable at low the above experiment to support activation of the soma- MOIs and reaches a maximum at an MOI of between 20 tostatin promoter (Fig 3B). As shown previously,30 the and 100 (14.7% conversion in the CAT assay at an MOI somatostatin promoter has high activity in DTC1 cells of 100). For AD42, promoter activity was also detectable (comparable or greater than the activity of the SV40 and and saturated at an MOI of between 100 and 400 (1.4% RSV promoters) compared with the near background conversion at an MOI of 400). The maximal promoter levels observed in JEG3 cells (that lack endogenous activity for AD71 is therefore ϳ10-fold higher than EWS/ATF1). Thus, the lack of VAP activity observed in AD42. The results show that AD42 and AD71 are DTC1 cells is not due to loss of trans-activation capacity infectious in 293 cells, and that the somatostatin pro- of the batch of DTC1 cells used. To conﬁrm the integrity moter can be activated in these cells either by E1A or by of the recombinant viruses, to test their infectivity, and other viral activators produced during a viral infection. to test whether activators other than EWS/ATF1 can Similar to AD42 and AD71, AD25V and ADVIP exhib- activate VAPs, we infected 293 cells with AD42 and ited no VAP activity in either DTC1 or Su-ccs-1 cells,

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Figure 4. adenovirus replication and gene expression in MMSP cells. A: Cells were infected with WT or dl312 adenoviruss as described in Materials and Methods. At different times after infection, culture medium and remaining cells were freeze/thawed three times, the debris was removed by centrifugation, and viral titers were determined by plaque assay using 293 cells. B: E3 gene expression during adenovirus infection of MMSP cells. DTC1 and Su- ccs-1 cells were infected with WT adenovirus (unless otherwise indicated) at different MOIs or for different times (as indicated above). Total RNA was extracted, and correctly initiated E3 transcripts (indicated to the side) were detected by primer extension using 32P- labeled primers as described in Materials and Methods. Experiments were repeated twice. C: Trans-activation of the somatostatin promoter by WT adenovirus. DTC1 or 293 cells were infected with recombinant AD71 virus (MOI ϭ 100) in the absence or presence of WT adenovirus (MOI ϭ 50). CAT assays were performed at 24 hpi, and a representative CAT assay is shown. Ex- periments were repeated twice. whereas VAP activity was readily detected after infec- were infected for virus replication experiments, and the tion of 293 cells (Fig 3A). Thus, the inability of endog- virus produced was quantitated by plaque assay at enous EWS/ATF1 to activate VAPs in MMSP cells does different times after infection (Fig 4A). As a control for not reflect a general repression of VAPs in infected cells. viral infections and plaque assays, 293 cells were in- Viral replication and gene expression in cluded. WT virus propagates to high titer in 293 cells, MMSP cells and due to the presence of endogenous E1A in these cells, the replication of an E1A mutant virus (dl312) is The low activity of VAPs in MMSP cells could be caused similar to WT virus. Replication in 293 cells confirms the either by inefficient delivery of adenovirus to the nucleus presence of infectious viruses for the experiments in of MMSP cells or the inability of EWS/ATF1 to activate MMSP cells. For both DTC1 and Su-ccs-1 cells, WT transcription in the context of viral chromatin. To dis- virus replicates to high titer (comparable with 293 cells), tinguish between these possibilities, we examined viral although the kinetics of replication are delayed relative replication and gene expression in MMSP cells (Fig 4). to 293 cells. Maximal levels of virus production take 4 Replication of wild-type (WT) adenovirus serves as a days for both DTC1 and Su-ccs-1 cells but only 2 days for positive control for normal (E1A-dependent) replica- 293 cells. As expected, dl312 showed limited ability to tion, and an E1A-negative virus (dl312) is very similar to replicate in both DTC1 and Su-ccs-1 cells, and cell the recombinant viruses (AD42, AD71, ADVIP, and viability was not obviously affected even after 14 days of AD25V) used in our gene transfer experiments. Cells infection with dl312. The inability of dl312 to replicate in

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MMSP cells indicates that the E1A-negative recombi- ment of MMSP tumor cells in vitro. Using an integrated nant viruses used in our experiments do not replicate in somatostatin promoter linked to hsvtk, GCV sensitivity MMSP cells. can be conferred to DTC1 cells. This cytotoxic effect is Consistent with the efficient replication of adenovirus specific for DTC1 cells and requires the ATF-binding in MMSP cells, the viral E1A protein is expressed in the site in the somatostatin promoter, indicating that cyto- nucleus (data not shown); in addition, we have shown toxicity is caused by EWS/ATF1. The finding that the previously that two adenovirus early promoters (E2 and somatostatin promoter can be activated in MMSP cells E3) are active during a WT infection of MMSP cells, (presumably by EWS/ATF1) when present in a chromo- depending upon the viral E1A protein (Ref. 51, Fig 4B). somal context is significant, because D71TK cells pro- To further characterize the behavior of adenovirus dur- vide a cell system for the further evaluation of the ing infection of MMSP cells, we examined E3 promoter cytotoxic prodrug approach for MMSP therapy. For activity in more detail. Consistent with the delayed example, in the future, D71TK cells can be used for kinetics of viral replication in MMSP cells, the E3 tumor production in nude mice and subsequent testing promoter is also activated later than in 293 cells. In 293 ϳ of GCV sensitivity of tumors in intact animals. The cells, the E3 promoter is typically activated at 6 hpi, above findings indicate that the somatostatin-hsvtk fu- whereas in MMSP cells the promoter has only low sion tested (or hsvtk fusions to other promoters that are activity at 20 hpi and is strongly activated only at later strongly activated by EWS/ATF1) has great potential for times (between 20 and 40 hpi). Titration experiments cytotoxic prodrug treatment of MMSP tumor cells in using different MOIs show that E3 gene expression is vivo. saturated at a relatively low MOI (saturation is reached In contrast to previous studies that exploited adeno- at an MOI of Ͻ20, Fig 4B), indicating that MMSP cells 35,36,52–55 virus as a vector for cellular promoters, promot- take up the virus efficiently. The late onset of E3 gene ers that can be activated by EWS/ATF1 in transient expression is therefore probably not due to inefficient assays are not activated in the context of adenovirus uptake of virus, but more likely reflects delayed kinetics for chromatin. VAPs are efficiently delivered to the nucleus early viral gene expression. Because the E3 promoter contains an ATF1-binding site and is strongly activated by of MMSP cells but cannot be activated by EWS/ATF1. EWS/ATF1 in the transient assay (see Fig 2A), it is also of Therefore, our findings indicate that first-generation significance that, like other VAPs, the E3 promoter is not adenovirus vectors (such as the one used in our study) activated by endogenous EWS/ATF1 in the context of a may have limited potential as a gene delivery vehicle for viral chromatin. cytotoxic prodrug treatment of MMSP cells. Taken together, the above data indicate that VAPs are The reason for the inability of EWS/ATF1 to activate effectively delivered to the nucleus of MMSP cells but that VAPs is unclear. A variety of VAPs are refractory to endogenous EWS/ATF1 is unable to activate these pro- EWS/ATF1 and (as far as our studies go) this block is moters. To test whether VAPs are generally repressed or not restricted to position or orientation within the viral whether they are specifically refractory to activation by genome. In addition, the inability of EWS/ATF1 to EWS/ATF1, we asked whether E1A (which can activate activate resident viral promoters (E3 and probably E2) VAPs in 293 cells (Fig 3A)) can activate VAPs in MMSP indicates that the lack of response of cellular promoters cells (Fig 4C). DTC1 cells were coinfected with WT is not due to the absence of putative cis elements that adenovirus (containing E1A) and AD71, and CAT assays are required for transcription in adenovirus chromatin. were performed at 24 hpi. Infection of 293 cells with AD71 Efficient replication of adenovirus together with E3 was performed as a positive control for viral infection and promoter activation in MMSP cells demonstrates that CAT assays. As shown earlier, AD71 infection alone these cells can support E1A-dependent transcription of produces no detectable promoter activity in DTC1 cells, VAPs. Therefore, our results are consistent with an whereas coinfection with WT virus results in significant inability of EWS/ATF1 to activate VAPs. In infected promoter activation. Thus, E1A protein (or possibly some cells, viral chromatin is thought to be a distinct nucleo- other viral proteins) can activate VAPs in DTC1 cells. protein complex (the adenovirus-core) containing the Activation during viral infection of DTC1 cells is not as viral core proteins VII and V.56 In vitro transcription great as that observed in 293 cells. This might reflect a from adenovirus-core templates is repressed and re- lower ability of E1A to activate in DTC1 cells or the quires a protein, template-activating factor-1, which has delayed kinetics for VAP activation as shown for the E3 been suggested to modify the adenovirus-core to allow promoter (Fig 4B). In summary, the above results show functional interactions with both replication and tran- that the inability of EWS/ATF1 to activate VAPs is not due scription factors.57 Therefore, it is possible that, in the to a general repression mechanism (because E1A can absence of E1A, template-activating factor-1 (or other activate VAPs), but rather is specific for EWS/ATF1 similar factors) is unable to modify the adenovirus-core, and/or MMSP cells. resulting in a block to activation by EWS/ATF1. How- ever, such an effect would be relatively specific for DISCUSSION EWS/ATF1 and/or MMSP cells, because many other promoters can be activated by their corresponding cel- We have evaluated the use of tumor-specific promoters lular activators (independently of E1A) when present and an adenovirus vector for cytotoxic prodrug treat- in the adenovirus-core.35,36,52–55 Future in vitro studies

Cancer Gene Therapy, Vol 7, No 3, 2000 404 LUNG AND LEE: GENE THERAPY FOR MMSP will be required to test the effect of adenovirus-core 3. Zucman J, Delattre O, Desmaze C, et al. EWS and ATF1 templates on trans-activation by EWS/ATF1. gene fusion induced by t(12;22) translocation in malignant Irrespective of the protein factors involved, it is likely melanoma of soft parts. Nat Genet. 1993;4:341–345. that cis-acting viral DNA sequences play a role (indi- 4. Delattre O, Zucman J, Plougastel B, et al. Gene fusion rectly, due to their implied role in viral chromatin with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992;359:162– assembly) in preventing VAPs being activated by EWS/ 165. ATF1. Therefore, it is possible that vector modification 5. May WA, Gishizky ML, Lessnick SL, et al. Ewings sar- could overcome the effect. The use of larger regions of coma 11–22 translocation produces a chimeric transcrip- EWS/ATF1-dependent promoter sequences instead of tion factor that requires the DNA-binding domain en- the small truncated promoters used in our study is one coded by fli1 for transformation. Proc Natl Acad Sci USA. option. Second-generation adenovirus vectors58,59 con- 1993;90:5752–5756. taining less of the viral genome enable this possibility to 6. May WA, Lessnick SL, Braun BS, et al. The Ewing’s be tested by allowing larger foreign DNA inserts. An- sarcoma EWS/FLI-1 fusion gene encodes a more potent other option would be to test so called “gutless vectors” transcriptional activator and is a more powerful transform- lacking all adenovirus genes.60,61 If the adenovirus-core ing gene than FLI-1. Mol Cell Biol. 1993;13:7393–7398. 7. Zucman J, Melot T, Desmaze C, et al. 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The EAD has 1994;54:2837–2840. 10. Chung EB, Enzinger FM. Malignant melanoma of soft a highly repetitive primary structure4,32 and makes dis- 62,63 parts. Am J Surg Pathol. 1983;7:405–413. tinctive contacts with the transcriptional machinery, 11. Epstein AL, Martin AO, Kempson R. Use of a newly indicating that the EAD is a likely target for small established human cell line (SU-CCS-1) to demonstrate molecule inhibitors. However, because it is currently not the relationship between clear cell sarcoma to malignant known whether EWS/ATF1 is required for MMSP tu- melanoma. Cancer Res. 1984;44:1265–1274. mor maintenance, the potential that EAD inhibitors 12. Hicks MJ, Saldivar VA, Chintagumpala MM, Cooley LD. would hold for MMSP therapy is unclear. In view of this, Malignant melanoma of soft parts involving the head and the properties that we have described for tumor-specific neck region: review of literature and case report. 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