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THE COUNCIL FOR TOBACCO RESEARCH - U.So.~., |NC Supporti~Lz B*or~cdicai InvcstiEat~on Research Grant Applicat, ion~l ~t CURRENT DATE: 11/19/93 ,, The Role of Prostaglandin EP3 Receptors in Breast Ca,'ncec -, as~s ~ . KEY WOF, DS (UM.IT TO FIVE} ! prostaglandin, receptor, PROPOSED PROPOSED DURATION START (YRS) DA~E 1, 2 OR 3 07/01/94 3 murine, mammary/carcinoma FUNDS REQUESTED (INCLUDING INDIRECT COSTS} YEAR 1 YEAR 2 YEAR ;3 TOTAl REQUEST (page 5): 64,244 67,029 67,167 PERM. EQU;PMENT (page 5): 3,322 3,060 - 0 - PRINCIPAL INVESTIGATOR (do not indicate any co-P.I, on this page) NAME (LAST, FIRST M.I.) AND DEGREES: Breyer, Richard M., Ph.D. ACADEMIC OR PROFESSIONAL TITLE: Assistant [D~ofessor DEPARTMENT; Nephrology and Pharmacology ,~S~IZUTION: Vanderbilt University STREEt" ADDF-~S$: 1161 21st. Avenue South CITY, STATE (OR COUNTRY) ZIP CODE: Nashville, TN 37232 MAIUNn ADDRESS IF DIFFERENT FROM ABOVE: Division of Nephrology S-3223 MCN Nashville, TN 37232-2372 TELEPHONE NUMBER: (615) 343-8496 INSTITUTIONAL OFFICIAL {accepUn=q for the institution) NA,~: John H. Hash, Ph.D. POS,nON ~,~t£:: Associate Dean for Biomedical Sciences Vanderbilt University Biomedical Sciences CCC-3322 MCN Nashville, TN 37232-2]03 FINANCIAL ADMINISTRATOR (person to comae, for budder information} NAME: Stephen M. To~d POSITION TIT~: Assistant Director of Financial Management MAIUNG ADDRESS: Vandecbilt University CCC-4322 MCN Nashville, TN 37232-2270 TELEPHONENUMBER: (615) 322-2301 IMAKECHECKSPAYABLETO: Vanderbilt University Division of Nephrology MAIL CHECKS TO {NAME AND POSITION TIT~): Stephen M. TrOd, Assistant Director of Finanical Management I~IUNGADDRESS: Vanderbilt University CCC-4322 MCN Nashville, TN"~ 37232-2270 ?RL~CIPAL ]~%'£STICATOR. I h=v¢ r¢.~d P~U¢? ~d =~ree Io {~ te~ ~4 condit;o~. A~,I *c¢¢p¢ r¢~;b;{i~" for ~c sclend6c conducK of ~ project ~d will provide progress rc~ when rcqu:~cd. I ~11 =c~nwlcdgc ~ppon by ~c COIJN~ FOR TOBACCO ~SEAR~I ~ publ;catlo~ regaling from ~=~ 40008886

I. a. Pl. Richard M. Breyer, Ph.D. 5. ~:u:~s Vand-=rbilt U~-xiversity ~::2.~ical Center c.TiflcofProj=cz : Yhe Role of Prostaglandin EP3 Receptors in Breast Cancer t,~_~tastasis d. CMafly s~.u'arn~rize BACKGROUND, %VO/LI, LING HY~OTI-~SIS and BROAD GOALS of |h,- project. Do not u_~e addilion~1 _Da~es. BACKGROUND. Prostag]andin £2 (PeH2) is a potent modulator of a wide variety of physio~ogiea~ responses including inflammation i, Sastdc acid secretion 2 vascular tone 3, ~ipo~ysis 4 and water and ion transport s. PGH2a~so modulates ce~i grov~h and division, and increased P~£2 synthes|s has been associated with proliferation of tumor ceils in human rung cancer, breast cancer and colon cancer as we]] as in rodent model systems 6,7,8. In patients with run%cancer, there is an increased PeR2 synthesis in cancerous versus normal tissue . This increase was more pronounced in ~ung cancers of patients who smoked cigarettes as compared with nonsmokers. ~n other studies an inverse correlation between elevations in P~H2 synthesis and mean surv|va[ time has been observed for patients with breast cancer, and to a ~esser extent in rung and colonic cancers i0. Interestingly, in some se~ings, ~nhibit]on of prostag[and}n synthesis can alter the course o~ malignant disease. In patients with familial polyposis, a genetic predisposition to malignant colon cancer, tl'ie administration of nonsteroidal anti-inflammatory drugs (NSAIDs) which are potent inhibitors of PGE2 synthesis have been shown to cause polyp regression, and withdrawal of the NSAIDs reverse this effect n,n. These findings support the notion that PGE2 mediated effects may play an important role in cell transformation, tumor growth and/or metastasis m Unlike classical circulating hormones, such as insulin and corticosteroids, prostaglandins are autacoids, acting locally on the tissues in which they are synthesized or on adjacent tissues. Accumulating evidence suggests that PGE2 exerts its cellular action through specific receptors coupled to their intracellular effectors via guanine nucleotide regulatory proteins (G-proteins) ~,~s,~. Based upon their pharmacological ligand binding properties and physiologic effects in smooth muscle at least three classes of PGE2 receptor have been proposed ~,3,~7. These three E-Prostanoid receptors are designated EP1, EP2 and EP3. It is of interest that PGE2 receptors have been shown to couple to three separate signalling pathways, via G-protein mediated PI (EP1) turnover and cAMP generation via Gs (EP2) and Gi. (EP3) ~s,m,20,2z. Recently a family of alternatively spliced variants of the EP3 receptor have been cloned in our laboratory. Potential functional differences among these alternatively spliced isoforms is one aspect of the current proposal. WORKING HYPOTHESIS In the model of mouse mammary tumor metastasis cell lines 66 and 4526 have been demonstrated to express a high affinity PGE2 receptor. Stimulation of this receptor leads to an increase in cAMP levels (Le. it is EP2- like). Blockade of this receptor with PGE2 selective antagonists leads to a decrease in cAMP generation and an increase in the lung metastasis of this tumor in vivo. s. In principle, stimulation of the Gi coupled EP3 receptor should lower cAMP levels in the same manner as EP2 receptor blockade. We will test the hypothesis that stimulation of EP3 receptors transfected into cell lines 66 and 4526 will lead to a decrease in intracellular cAMP levels and an increase metastatic potential of these tumor cells. Moreover, different isoforms of the EP3 receptor may have distinct effects on tumor growth and metastasis, and we will determine the effects of expression of each of the EP3 isoforms. BROAD GOALS The mechanism of action of PGE2 in metastatic disease remains unclear. It has recently become evident that there exist a large.number of specific cell surface receptors for PGE2 and activation of these receptors may have different and often conflicting effects on cell function. Our goal is to define the role of PGE2 in tumor metastasis; and in particular to understand how the activation of individual PGE2 receptor subtypes impacts the metastatic process. ~^-~LDOC ~,~ CONFID]LNTIAL 40008887

SPECIFIC AltdS of this research prq~=ct (~ o~F~-_d to th-~ broad gon|~ pr~=-nted in |to~ra l) To test the hypothesis that activation of the EP3 receptor will increase the metastatic potential via a cAMP dependent mechanism, we will : I. Express EP3 receptor subtype clones 72A, 74A 77A, and 80A in murine mammary tumor lines 66 and 4526 and characterize their signal . transductlon properties. To accomplish this aim we will establish permanently transfected cell lines with plasmids expressing each receptor individually. These cell Fines will be assayed for receptor message expression, ligand binding, and second messenger generation. II. Determine the changes in metastasis of these transfected cell lines in the presence or absence of pretreatment with PGE2 analogs. Cell lines established in Specific Aim one will be injected into mice and their ability to estab!ish metastatic lesions in vivo evaluated. These studies will allow direct correlation of EP3 receptor expression on cell metastasis. 3. SUPPORTING DATA, EXPER.IlVflENTAL DESIGN and PROCEDURES. Do not attach more than six addit~nal pagei(3.a.-3. O. All figures, charts, tables and references must fit within pages 3 - 3f. ~umor ue. ~ines 66 and 4526 Tumor lines 66 and 4526 are both derived a spontaneously arising mammary tumor of a Balb/cfC3H mouse 2~. These lines may be maintained indefinitely in vitro in tissue culture. When injected intravenously into Balb/c mice these cell lines are highly metastatic and form multiple colonies in the lung within three weeks ~. Importantly, the metastatic potential of these cell lines is modulated PGE2. Several studies have shown that these cell lines synthesize PGE2 and have demonstrated that there exists a high affinity PGE2 receptor on these cell lines 6,23 Stimulation of this receptor leads to increases in cAMP; Le. it is an EP2 receptor subtype. Saturation isotherm binding analysis suggests that these is only a single class of binding sites on these cells ~; with a Kd of 6.9 nM. Functionally, blockade of this high affinity receptor with PGE receptor antagonists inhibits PGE2 mediated increases in cAMP in vitro, and leads to an increase in tumor metastasis in vivo. Preliminary reports suggest that these cell lines are devoid of endogenous EP3 receptors ~. As discussed in the Experimental Design section, we will confirm this finding using EP3 receptor probes, and then transfect the cloned EP3 receptor(s) into these cell lines. Stimulation of the EP3 receptor leads to decreases in cAMP levels and we predict that this will have the same consequences as blockade of the EP2 receptor: decreases in intracellular cAMP levels in vitro and increasing tumor metastasis in vivo. Characterization of the EP3 rece0tor Initial efforts to clone and characterize PGE2 receptors focused on the kidney. Not only is PGE2 the major renal cyclooxygenase metabolite of arachidonic acid, but the kidney is one of the richest sources of PGE2 receptors and as described by Narumiya had the highest message levels in the mouse as well ~g. Moreover, the rabbit kidney represent~ well characterized system with respect to EP3 receptor function. We amplified rabbit cDNA sequences by reverse transcription PCR (RT-PCR) using poly (A)+ RNA isolated from rabbit renal cortex and oligonucleotide primers derived from the cloned mouse EP3c~ receptor !9. Overlapping PCR proc~..e~r~[:~p~sL.enting putative transmembrane regions III NA-&P£L~D 0C ~13/93 40008888

3a P.I. : Ri~.hsrd ~.~. Breyer, Ph.D. ~hrough V|~ were generated, cloned and sequenced. Using these PCR fragments as probes, northern hybridization analysis revealed multipIe transcripts of 1.8, 2.6 and 7 kb expressed in both renal cortex and medulla. Moreover the levels of e×pression of these transcripts were modulated by in vivo pretreatment of animals with indomethacin, suggesting feedback modulation of receptor message levels by depletion of the endogenous ligand. Moreover the relative abundance of the various isoforms may be regulated by the physiologic status of the cell. A family of alternatively s.oliced EP3 receDtors. We probed a rabbit renal cortex cDNA library with PCR fragments of the rabbit EP3 receptor and isolated five independent clones. The deduced amino acid sequence of one clone, 72A, is shown in Figure 1. This clone contains a cDNA of 2061 nt and has an open reading frame of 361 amino acids. Analysis of the deduced amino acid sequence indicates that the polypeptide encoded by this cDNA has seven transmembrane regions as represented in Figure 1 and is 82% homologous to the mouse EP3 receptor. Sequencing of the remaining c~o ~o clones indicates that they fall into ~,~ four classes (Fig. 2)° Each class ~n'~CE,,U~RSUm=ACE ~O appears to be nearly identical up to nucleotide 1065 (using the clone 72A numbering system) where the four classes of receptors clearly diverge in sequence. At the amino acid level, the divergent region corresponds to amino acid 355, ten amino acids C-terminal to transmembrane VII on the cytoplasmic surface. Sequence analyses indicate that the receptors are very nearly identical throughout the seven CYTOPlaSMIC SURFACE transmembrane domain region, and diverge at the C-terminal tail 24 Genomic Southern blot analysis with probe EP 342-816, which lies within the common Figure 1. The deduced amino acid sequence of the region of the four cDNA clones, rabbit EP receptor 72A, showing the typical seven hybridizes to a single DNA transmembrane structure of a G-protein coupled receptor. The transmembrane spanning regions of G-protein coupled receptors fragment in- each restriction largely determine the ligand binding specificity. The intracellular digest tested. This is consistent third loop and the C-terminal tail have been implicated in with the existence of a single coupling specilicity and regulation. common region gene which is alternatively spliced. Sequence analyses of rabbit genomic clones demonstrate the existence of an intron/exon boundary at nucleotide 1065, the position where the proposed alternative splice junction of the EP3 cDNAs occurs. The genomic sequence immediately preceding each of the unique 3' exons closely match the consensus splice acceptor sequences, consistent with alternative 3' splice site selection 25 While we do not know the functional significance of the unique region of the EP3 receptor, this segment of the amino acid sequence has been implicated in regulation of other G-protein coupled receptors by a number of protein kinases 25,27,2S. It may be that the functional effect of these different C-terminal sequences is to provide differential

P.I. : R~zhard M. e~eyar, Ph.D. EP3 receptor modulation by the regulatory kinases. Functionally this would imply that the ligand binding portion of each receptor is similar and only the signalling differs. Differential regulation of these receptor subtypes may be of potential functional significance in determining the response of a given cell type of tissue to PGE2. One receptor clone, 72A, has the C-terminal sequence RKILLRKFCQEEFWEK. This C- terminal 'hail" is notable in that it is exceptionally short and is different from each of the other receptor clones in that it contains no serine or threonine residues. Both serine and threonine are targets for G- protein coupled receptor regulation via phosphorylation by a number of protein kinases including receptor- specific kinases z6,zT. It might be predicted that changes in the relative fraction of the 72A receptor subtype will change the susceptibility of PGE2 receptors to regulation by protein kinases. Liaand binding of the EP~ re(~ep~or in cell culture. When transfected into COS1 cells, clone 72A confers [3H] PGE2 binding to cell membranes, with a KD of 300 pM, and an order of agonist affinity typical of an EP3 receptor: M&B28767 > sulprostone > PGE2 = PGE1 > PGF2(~ > PGD2 > 8-epi PGF2o~. Clone 77A has also been shown to confer [3H] PGE2 binding to cell membranes when transiently transfected into COS1. Signal transduction of the EP$ receptor in cell culture. The murine L-cell derivative LVIP.OZc contains a l~-gal reporter gene construct which allows convenient measurement of intracellular cAMP using a colorimetric assay 2{) Two of our EP3 receptor clones 72A and 77A have been transfected into LVIP.Ozc and permanent transfected lines established. These 80A Fig 2 . upper panel A schematic alignment of the four EP receptor cDNA clones isolated from rabbit renal cortex. The approximate position of the seven transmembrane domains is shown above, lower panel The deduced amino acid sequence of the divergent portion of the four EP3 receptor subtypes. The underlined regions indicate the seventh transmembrane domain, vertical lines indicate residues conserved between EP receptor isolorms. 0.4 0.3 0.2 0.I [Sulprostone] Fig 3. Inhibition of forskolin stimulated cAMP levels by sulprostone in LVIP.OZc cells transfected with either EP3 receptor 77A • or pRCCMV vector alone C~ cAMP levels are determined by measuring the level o! expression of the reporter gene J~gal, where higher levels of cAMP lead to increase I]gal activity as determined by product accumulation at A405. receptor isoforms couple to Gi in LVIP.Ozc and demonstrate the ability to decrease forskolin stimulated intracellular cAMP levels (Figure 3). Clones expressing 77A are much more potent in inhibiting cAMP than are clones expressing 72A. Neither of these clones demonstrate coupling to Gs in mouse L-cells. 40008890

3c P.I. : R~.~h.~rd M. Breyer, Ph.D. Thus far, studies on the effects of prostanoids and cancer have focused on the generation of cyclooxygenase metabolites of arachidonic acid, and their inhibition by NSAIDs. With the cloning of indMdual PGE2 receptors it is now possible to determin~ the effects of PGE2 at specific receptor subtypes, and to characterize the relat[ve abundance of the different receptor isoforms under various conditions Understanding the effects of stimulation or btockade of particular EP receptor subtypes on cell growth in normal and pathophysiologic states may ultimately lead our understanding of the role PGE2 plays in tumor promotion, growth and metastasis. EXPERIMENTAL DESIGN AND PROCEDURES Specific Aim I Evaluate the effect of expression of EP3 receptor subtypes 72A, 74A 77A, and 80A in murine mammary tumor lines 66 and 4526 on second messenger generation. Analysis of the complement of endogenous EP receptor:;. Based upon Scatchard analysis cell lines 66 and 4526 appear to have a single EP receptor and functional studies indicate that this is an EP2 subtype. We will contirm the absence of EP3 receptor message in cell lines 66 and 4526 by Northern Blot using a rabbit EP3 common region probe. This probe has previously been shown in our hands to cross- hybridize to the mouse EP3 receptor message present in normal tissues. Cells will be evaluated for the effects of EP3 selective agonists. Initially we will utilize M&B 28767 (EP3 selective ) and sulprostone (EPI/3 selective). Both of these ligands have a high affinity for the EP3 receptor (see accompanying manuscript). Cells will be pretreated with these compounds at saturating concentrations (~0.1 mM) and assayed for their ability to stimulate cAMP formation, or inhibit forskolin stimulated cAMP generation. The presence of the endogenous EP2 receptorS,23 will be confirmed by RT-PCR using oligonucleotides derived from the cloned mouse EP2 receptor as primers 18, Initially we will characterize cell lines 66 and 4526, though the results of the above experiments may lead us to focus preferentially on one line: a cell line which contains the EP2 but not the EP3 receptor subtype. Transfection of murine cell lines with EP3 receptor isoforms. Cell lines 66 and 4526 will be transfected with each of the full length receptor constructs, 72A, 77A, 80A and 74A individually. Expression plasmid constructs (pRCCMV; Invitrogen) expressing clones 77A and 72A exist in the laboratory. We have recently isolated full length clones of 80A and 74A by PCR and these will be subcloned into pRCCMV using standard methodology. Expression plasmid constructs will be transfected into mammary tumor cell lines by the lipofectin method (Gibco BRL) and cells assayed for receptor expression. As a control pRCCMV without a cDNA insert will be transfected into each of the parental cell lines. Cell lines will be assayed for i) the presence of the transfected receptor message by RNase protection; ii) their ability to bind PGE2 and EP3 selective ligands, and iii) the ability to inhibit forskolin stimulated cAMP generation. RNAse protection RNAse protection assays will be carried out as described in the accompanying manuscript 24, using RNA isolated from candidate transfected cell lines. We will use both common region probes e.g. EP 506-963, and isoform selective probes to confirm the existence of expression of the correct EP3 isoform. RNAse protection assays will be carded out on both polyclonal and subcloned cell populations (see below). .P_..~]~irld[[~ Initially we will analyze polyclor, al transient transfectants 48 to 72 hours after transfection to determine [3H]PGE2 binding which is displaceable by M&B 28767. Permanent transfectants will be selected for by resistance to G-418. G-418 resistant clones which demonstrate significant binding of M&B 28767, will be subcloned. 40008891

P.I. : R~chard M. Breyer, Ph.D. in general cells will be subjected to two or three rounds of subcloning to ensure ctonal purity. Saturation isotherms for [3H]PGE2 will be determined (using EP3 selective ligands to displace for "nonspecific" binding) to ascertain the affinity and level of expression of the PGE2 receptor. K~s will be determined for the panel of PGE2 iigands (PGE2, PGD2, PGF2c~, M&B 28767, sulprostone) in order to determine the relative abundance of the EP2 and EP3 receptor subtypes. Signal Transduction Clones demonstrating either high or low levels of expression will be assayed for their ability to raise cAMP levels in a PGE2 dependent manner. We will also attempt to suppress cAMP stimulation. For example cells have endogenous adenylate cyclase activity which may be stimulated by the addition of forskolin. Forskolin stimulated cells will be incubated with or without M&B 28767 and assayed for agonist dependent attenuation of cAMP generation. We will assay cells to determine if this effect is Ptx reversible by pretreating cells with 1 p.g/ml Ptx for 4 hours at 37°C prior to assay. Reversal by Ptx would suggest a Gi coupled mechanism of signal transduction. It has been reported 3o that in some in vitro transfection systems EP3 can couple through an IP3 mediated pathway. IP3 generation will be monitored as described in "General Methods". These studies will characterize binding and signaling properties of homogeneous populations of PGE2 receptors. Specific Aim II Determine the changes in metastasis of these transfected cell lines in the presence or absence of pretreatment with PGE2 analogs. Untransfected lines, or cell lines transfected with EP3 receptors will be injected into Balb/c mice in PBS i. v. in the tail vein. Three weeks later, mice will be sacrificed and lung tissue will be examined for the presence of surface colonies and the total lung mass determined 6. Cells will be assayed initially without any drug pretreatment, and the effects of expression of the EP3 receptor isoforms on cell metastasis determined. For some experiments cells will be grown in medium containing the cyclooxygenase inhibitor indomethacin, to eliminate endogenous PGE2 production. This has been reported to cause inhibition in metastasis by untransfected cells, for both 66 and 4526 presumably by increasing the EP2 receptor density by relieving ligand mediated tonic down-regulation. This treatment will affect both the endogenous EP2 and transfected EP3 receptor and the net effect of this treatment will be determined empirically. In some cases, EP3 selective agonists will be added to the culture medium pdor to injection into the test animals. Initially we will use the EP3 selective agonist M&B 287676 and the EPI/3 selective agonist sulprostone. Detailed methods are presented below in the General Methods section. Cells with or without pretreatment are injected into the tail vein of Balb/o mice. Eighteen to 21 days later mice are sacrificed and the lungs removed and analyzed for both total lung mass and the presence of surface colonies. Each EP3 receptor isoform will be evaluated for its effect on the metastatic potential of the tumor cell lines in terms of both number of surface colonies on the lung and increases in total lung mass. GENERAL METHODS Cell culture Cell lines 66 and 4526 are grown in Waymouth's medium (GibcoBRL) containing 10% FBS (GibcoBRL); 2 mM glutamine; and pen/strep. Full length coding regions encoding clones 72A, 74A, 77A, and 80A subcloned into the mammalian expression vector pRCCMV (Invitrogen) are transfected into these cells using the lipofectin method (Gibco BRL), using 10 l~g plasmid DNA and 40 I~g lipofectin as initial conditions. Cells are cultured for 72 hr, changing the medium every 24 hours and assayed for the expression of receptor specific RNA and [3H]PGE2 binding which is displaceable by EP3 selective ligands. 40008892

3e P.I. : RL~hard M. Breyer, Ph.D. Liaand Binding Studies. Ceils are scraped into buffer containing 15 mM HEPES, 5 mM EGTA, 5 mM EDTA, 40 p.M indomethacin and 2 mM phenylmethyl sulfonyl fluodde and lysed by passage through a 21 ga needle. The lysate is centrifuged at 200,000 x g for I hour and total membranes collected on a 60% sucrose cushion. For saturation isotherm experiments, 40 p.g of membrane protein is incubated in Buffer A (25 mM KPO4 pH 6.2, 10 mM MgCI2, and 1 mM EDTA) for 2 hours at 30° C with various concentrations of [3H]PGE2. Nonspecific binding is determined in the presence of 50 I~M unlabeled PGE2. Reactions are stopped by addition of three ml ice cold Buffer A followed by rapid filtration on Whatman GF/F glass fiber filters. Filters are washed three times with Buffer A, dried and counted in Dupont 989 fluor. For competition assays, 80 p.g of membrane protein is incubated with 1 nM [3H]PGE2 and vadous concentrations of unlabeled competitor and reactions carried out as above. Measurement of cAMP generation, cAMP generation will be measured by RIA of acetylated cAMP. All assays will be performed in the presence of 0.1 mM IBMX, a potent inhibitor of phosphodiesterase, and 50 ~M indomethacin to block endogenous cyclooxygenase. Measurement of inositol phosphate _~eneratigr~. Inositol phosphate generation by transfected cells will be measured using a competitive IP3 binding assay kit (Amersham). Cells will be resuspended at a density of 1 to 2 x105 cells/1001~l of stimulation buffer (containing 20 mM LiCI and 50 p.M indomethacin), incubated for 15 minutes at 37°0 and then varying concentrations of agonists added to each tube in 100~1. The reaction will be stopped with 200~1 of chilled 10% trichloroacetic acid, cells freeze-thawed, spun at 2000 xg 10 min. and extracted with 3 volumes of water saturated diethylether. The water phase will then be titrated to pH 7.5 with 1 M NaHCO3. 100p.I of 3H inositol 1,4,5 triphosphate is added to 1001~1 sample, 100~1 binding protein and 100~1 buffer. The mixture incubates on ice 15', is centrifuged and the pellet counted, Decreased 3H inositol 1,4,5 triphosphate counts in the pellet reflect increased sample IP3 production. The results are compared to a standard curve. Ribonuclease Drotecti0n. For generation of RNAse probes, fragment EP 506-963 (common region) or fragments containing both common and C-terminal specific sequences were cloned in the transcription vector pCRII. Ribonuclease protection assays are performed essentially as described 24. Antisense RNA is transcribed from the flanking T7 or SP6 promoters, depending upon the fragment orientation, in the presence of [~,_32p] UTP. The antisense RNA (5 x105 cpm) is hybridized to 10 ~g total cellular RNA from transfected or untransfected cell lines at 45° C for 14 to 18 hours. Ribonuclease A digestion (20 p.g/ml) is carried out at 30° C for 45', and protected fragments are separated on a 4% polyacrylamide/7 M urea gel, followed by autoradiography. RNA Analysis by Northern Hybridization. Total and poly (A)+ RNA is size fractionated on 1% agarose gels containing 2.2 M formaldehyde, 20 mM MOPS pH 7.0, 8 mM NaOAc, 1 mM EDTA. After electrophoresis, the RNA containing gel is treated with 50 mM NaOH, neutralized with 50 mM Tris CI pH 7.5, 1.5 M NaCI and transferred to nitrocellulose (Schleicher & Schuell). DNA probes are prepared by random hexanucleotide priming, using r~ 32p labeled dCTP (Dupont NEN). Hybridization conditions (stringency) will be determined empirically. .In vivo metastasis assay. Normal or transfected cell lines are grown in medium. as described above, in some cases, 48 hours prior to harvesting cells will be treated with 40 p.gfml indomethacin (Sigma) to inhibit endogenous PGE2 production. For stimulation of the EP3 receptor, cells will be grown in the presence of EP3 selective 40008893

3f P.I. : R,;=h~rd M. Breyer. Ph.D. agonists (e.g. 0.1 ~M M&B28767) for six to eight hours. Cells are trypsinized, washed 3 times in lx PBS and counted. Balb/c mice (Jackson Laboratories) 6 to 12 weeks of age are injected in the lateral tail vein with 2 x 105 cells in a volume of 0.1 ml PBS. Eighteen to 21 days later, mice are killed by CO2 asphyxiation and the lungs removed. Lungs are then weighed and the total number of surface colonies counted. Data are analyzed by the Mann-Whitney Utest. Literature Cited 1. Armstrong, R.A., Matthews, J.S., Jones, R.L. & Wilson, N.H. Adv. Prostaglandhz, Thromboxane and Leukotriene Res. 21, 375-378 (1990). 2. Barr, D.B., Duncan, J.A., Kierman, J.A., Soper, B.D. & Tepperman, B.L.J. Physiol. 405, 39-55 (1988). 3. Coleman, R.A., Kennedy, I., Humphrey, P.P.A., Bunce, K. & Lumley, P. in Comprehensive Medicinal Chemistry (eds. Emmet, J.C.) Vol. 3, 643-714 (Pergammon Press, Oxford, 1990). 4. Richelson, B. & Beck-Neilsen, H. ]. of Lipid Res. 26, 127-134 (1985). 5. Dunn, M.J. & Hood, V.L. Am.J.Physiol. 233, F169-F18~ (1977). 6. Fulton, A.M., Zhang, S.-z. & Chong, Y.C. Cancer Res. 51, 2047-2050 (1991). 7. Thun, M.J., Namboociiri, B.S. & Heath Jr., C.W.N. EngI. J. Med. 325, 1593-1596 (1991). 8. Noguchi, M., et al. Cancer Res. 51, 2683-2689 (1991). 0. McLemore, T.L., et al. Cancer Res. 48, 3140-3147 (1988). 10. Bennett, A., Stamford, I.F. & Cooper, D. Adv. Prostaglandin, Thromboxane and Leukotriene Res. 21, 875-878 (1990). 11. Klein, W.A., Miller, H.H., Anderson, M.A. & DeCosse, ~.J. Cancer 60, 2863-2868 (1987). 12. Rigau, J., et al. Ann. Intern. Med. 115, 952-954 (1991). 13. Honn, K.V., Bockman, R.S. & Marnet~, L.J. ProstagIandins 21, 833-865 (1981). 14. Haga, T., Ross, E.M., Anderson, H.J. & Gilman, A.G. Proc. Natl. Acad. Sci. 74, 2016- 2020 (1977). 15. Lefkowitz, R.J., Mulliken, D., Wood, C.L., Gore, T.B. & Mukherjee, C. J. Biol. Chem. 252, 5295-5303 (1977). 16. Watanabe, T., Umegaki, K. & Smith, W.L.J. Biol. Chem. 261, 13430-13439 (1986). 17. Gardiner, P.J. Adv. in Prostaglandin , Thromboxane, and Leukotriene Res. 20, 110-118 (1990). 18. Honda, A., et at. J. Biol. Chem. 268, 759-762 (1993). 10. Sugimoto, ¥., Namba, T., Negishi, M., Ichikawa, A. & Narumiya, S. ]. Biol. Chem. 267, 6463-6466 (1992). 20. Watabe, A., et al. ]. Biol. Chem. 268, 20175 (1993). 21. Funk, C., et al.]. Biol. Chem. (in press) (1993). 22. Miller, F.R., Miller, B.E. & H., H.G. Invasion Metastasis 3, 22-31 (1983). 23. Fulton, A.M., Laterra, J.J. & Hanchin, C.M.J. Cell. Physiol. 139, 93-99 (1989). 24. Breyer, R.M., et al. ]. Biol. Chem. fin press) (1994). 25. Mount, S.M. Nucl. Acids Res. (1982). 26. Hausdorf, W.P., Caron, M.G. & Let'ko~itz, R.J. FASEB J. 4, 2881-2889 (1990). 27. Bouvier, M., et al. Nature 333, 370-373 (1988). 28. Hausdorf, ~.r.p., et al. Proc. Natl. Acad. Sci. 88, 2979-2983 (1991). 29. Konig, M., Mahan, L.C., Marsh, J.W., Fink, J.S. & Brownstein, M.~. J. Molec. ceil. NeuroscL 2, 331-337 (1991). 30. Namba, T., et aI. Nature 355, 166-170 (1993). 40008894

4. L.~BOP_~.TOP.Y SPACE ~ FACILITIES a~ala~Ie to the apph~t. ~ ~y fazili~i~ ~at ~e required bul ~ n~l ~ntl~ a~lable. The main laboratory is 513 square feet, with an adjoining office of 120 square feet. The laboratory is equipped with research material for molecular biology and pharmacology. Aside from the items listed in the proposed budget, all the necessary equipment will be readily available. This includes a shaking water baths and an Inotech 96-well Harvester for ligand binding assays in the laboratory; Beckman Ultracentrifuges (Optima and L- 8M), Beckman J2-21 and Sorval RC5B high speed centrifuges, and a Sorval Low speed RC3B centrifuge are available in the Nephrology Division core facility. Other Nephrology core facilities located on the same floor as the main laboratory include: a tissue culture .facility located near the main laboratory equipped with two Baker Sterileguard laminar flow hoods, two Forma double-stack incubators, as well as a clinical centrifuge. There is also a darkroom equipped with a Polaroid MP-4 camera system, UV transilluminator and automated X-ray development system. In addition there is a shared cold room of 132 square feet. 5. BUDGET JUSTIFICATION - Use this space Io explain specific needs for items described on budget pages. Personnel: Salary support (15%) is requested for Richard Breyer who will serve as Principal Investigator and direct all of the proposed studies, as well as carry out construction of mammalian expression vectors. Salary support (100 %) is requested for Cuilan Nian, a Research Assistant !! in the laboratory. Ms. Nian will carry out all mammalian cell culture, as well as the in vivo metastasis assay. Supplies: Radiochemi~,l~; 3H PGE2 for ligand binding displacement experiments; 3~p tz UTP for RNAse protection studies; 32p a dCTP for northern hybridizations; 35S ct dATP for sequencing Pi3E2 receptor constructs. Mice will be used for assesing the metastatic potential of thb transfected cell lines; 100 animals per year. Chemical6 for bacterial media, reagents for the preparation of stock solutions and buffers for molecular biology. Cell Culture SupDlies Include G418 antibiotic for selecting permanent transfectants.;. culture medium and Fetal Calf Serum for growth of mammalian cell lines. Equipment: Funds are requested for a small inverted microscope (Nikon TMS) for use in cell culture. There is no microscope available in our culture room, and we currently borrow a microscope in Dr. R. Harris' lab; the intensive tissue culture required by this project will make this arrangement unworkable. Year Two. A liquid Nitrogen storage freezer is r~.q~J~t.ad.for.storaoe of oermanertt cell lines (]enerated exoressing the EP receptors. • A~'.~t~'~A.: ~'lace u~ appenmx materials aner the ofigYnal andeach COlSy of the application form ~ indicated m the Instructions for New Applications. a. Biographical Sketches of the professional personnel to be ~sociated with the project• Each sketcl'i should be NO MORE THAN TWO (2) PAGES. The NIH format is acceptable. The P.]. should include and indicate by an asterisk the FI\rE (5) most significant publications whether or not they relate directly to this application. b. Supporting material (such as letters of collaboration). c. Copies of not more than FIVE (5) of the applicant's publications or manuscripts that arc penlncnt to the project. 7. ABSTP~.CTS of PUBLICATIONS : Only one set is required. See Instructio:~s for New Applications. Submit ONE PHOTOCOPY of the abstract page of each "pertinent publication" included in ~l~e appendix (6.c.) above; For each manuscript, submit a sirlgle composite page that includes authors, title, journal, abstract and publication status if or example, "submitted for publication"). .NA-APFLD~ CONFIDENWIAL 40008895