Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors

Biochimica et Biophysica Acta 1392 Ž1998. 85–100

Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors Randolph A. Hennigar ) , Mildred Pochet, Dirk A Hunt, Aron E. Lukacher, Virginia J. Venema, Elizabeth Seal, Mario B. Marrero Department of Pathology and Laboratory Medicine, Emory UniÕersity School of Medicine, Atlanta, GA 30322, USA Received 1 December 1997; accepted 15 January 1998

Abstract The lipogenic enzyme fatty acid synthase ŽFAS. is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium ŽNMuMG. and from mammary tumors induced by either rodent polyoma ŽPy. virus or murine mammary tumor virus ŽMMTV.. Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera Žand vice versa. were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose Ž10y4 M. dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations Ž10y8 , 10y6 M. of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate Ža primary end-product of FAS. prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate Ža monounsaturated fatty acid synthesized from palmitate. synergized cerulenin’s cytotoxic effects. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Fatty acid synthase; Lipid metabolism; Polyoma virus; Murine mammary tumor virus; Breast cancer

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Corresponding author. Department of Pathology and Laboratory Medicine, Rm 7105B WMRB, Emory University School of Medicine, Atlanta, GA 30322. Fax: q1-404-727-8540; E-mail: [email protected]

1. Introduction Endogenous lipogenesis involves the elongation of acetyl-CoA to fatty acid. Cytoplasmic acetyl-CoA

0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 8 . 0 0 0 2 3 - X

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generated by the citric acid cycle is diverted away in times of need to acetyl-CoA carboxylase ŽACC.. ACC converts acetyl-CoA to malonyl-CoA which serves as the primary carbon substrate for fatty acid synthase ŽFAS.. FAS is a large multienzyme complex which facilitates a complicated series of condensations, transacylations, reductions, and dehydrations w1–5x. Mammalian FAS contains seven catalytic sites, all of which occur along a single protein encoded by a single gene. FAS resides in the cytoplasm as a homodimer consisting of two 270 kDa proteins arranged head-to-tail. The enzyme’s activity is NADPH-dependent and catalyzes the sequential addition of carbon pairs until a medium- or long-chain saturated free fatty acid is formed. Palmitate, a longchain fatty acid containing 16 carbons, is the most common product of the enzyme’s activity. Recent immunohistochemical studies report elevated amounts of FAS protein in human primary carcinomas of breast, prostate, colorectum and ovary relative to normal w6–14x. The finding of excessive enzyme in these common tumors is associated with adverse clinical outcome and serves as an independent prognostic indicator. Thus, there is an implied relationship between FAS elevation and increased tumor aggression. Human cell lines established from metastatic breast adenocarcinomas also show increased rates of lipogenesis commensurate with elevated levels of FAS w15–17x. The enzyme is functional in these cancer cells and is vital since its inhibition is cytotoxic w16,18x. This dependence of some human cancers upon accelerated lipogenesis differs from normal human tissues where de novo fatty acid synthesis and FAS occur constitutively at very low levels w19x. Hence, elevated FAS has important clinical implications: it portends worsened prognosis in patients with certain cancers, and shows potential as a chemotherapeutic target. In contrast to the growing implications of elevated FAS in human neoplasia, little is known about the enzyme in experimental tumors. We have characterized FAS in a panel of cell lines derived from normal murine mammary epithelium and mouse mammary tumors induced by either murine mammary tumor virus ŽMMTV. or rodent polyoma Ž Py. virus. The primary advantage of using the virus-transformed cells is that unlike human breast cancer, the intracellular events involved in neoplastic transformation

by these viruses are better understood w20x. Certainly, these virus-induced mammary tumor systems have furthered our understanding of the relationships between breast cancer and viral, hormonal, immunological and genetic factors. The RNA virus MMTV and DNA virus Py offer two distinct mechanisms of transformation and serve as comparative models of tumorigenesis Ž for reviews, see Refs. w20–23x.. We exploited these virus transformation systems to find potential experimental models of elevated FAS in breast cancer. To meet this end, cell lines derived from mammary tumors induced by MMTV or Py, and one established from normal mouse mammary epithelium were compared with regard to Ž 1. constitutive FAS content and activity; Ž 2. hormonal response of FAS content and activity; Ž 3. qualitative differences in the phosphorylation state of FAS; Ž 4. effects of the FAS inhibitor cerulenin upon cell growth; and Ž5. effects of exogenous lipids upon growth of cerulenin-treated cells.

2. Materials and methods 2.1. Cell lines We chose a total of five experimental breast cancer lines, two of which were transformed by rodent Py virus and three transformed by MMTV. The two Py-transformed lines were recently established and characterized by one of the authors ŽA.L... Line A1-1 was derived from a murine mammary tumor induced by wild-type Py strain A2. The wild-type strains induce infected host cells to produce a series of proteins called the small, middle and large tumor Ž T. antigens. Middle T serves as the primary initiator of transformation by activation of host regulatory proteins, including Tyr kinase pp60 c-src w20x. Phosphorylation of specific tyrosine residues in both src and middle T result in activation of signal transduction pathways mediated by phosphatidylinositol 3X-OH kinase Ž PI3K. and Shc w20,23–25x. Small, middle and large T also interact with other cellular components to further undermine signal transduction pathways and enhance cell proliferation w20x. Line T1 was established from a mouse mammary tumor transformed by Py virus strain PTA315YF Žoriginally termed PTA1178T. described previously w24x. Cells

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infected with this virus produce a middle T capable of activating pp60 c-src but substitution of Tyr315 by phenylalanine prevents binding and activation of PI3K w25x. Lines A-1 and T1 were cloned by limiting dilution from mammary tumors arising in C3HrBiDa mice inoculated at birth with wild-type A2 or the PTA315YF mutant virus, respectively w26,27x. Western immunoblot analysis demonstrated that both cell lines expressed similar levels of Py T proteins. Immune complexes of middle T immunoprecipitated from A1-1 and T1 mediated Tyr autophosphorylation of denatured rabbit muscle enolase, thereby confirming activation of middle T-associated pp60 c-src by wild-type and mutated middle T proteins. Immune complexes from A1-1 possessed PI3K activity while those from T1 failed to activate the enzyme. Also, the p85 subdomain of PI3K coimmunoprecipitates with middle T from A1-1 but not T1 Žnot shown.. The three MMTV-transformed mouse mammary tumor cell lines Mm5MT, RIIIMT and MMT060562 were obtained from The American Type Culture Collection ŽATCC, Rockville, MD.. MMTV is a retrovirus that inserts itself as a provirus into the host cell’s genome, and with appropriate hormonal simulation, induces the cell to become transformed w21,22x. Information derived from our studies using these MMTV- and Py-transformed cell lines is compared with data obtained from cell lines NMuMG established from normal murine mammary epithelium or BNL.CL2 from normal mouse hepatocytes. Both normal cell lineages were also purchased from ATCC. Human breast adenocarcinoma cell lines T47D and SKBr3 Ž ATCC. were employed as positive controls for studies outlined below. All cell lines propagate as monolayered cultures. A1-1 and T1 were maintained in Dulbecco’s modified eagle medium ŽDMEM. supplemented with 1 grl glucose; BNL.CL2 in DMEM with 4.5 grl glucose; Mm5MT, RIIIMT and SKBr3 in Roswell Park Memorial Institute Ž RPMI. media formulation a1640; NMuMG and T47D in RPMI 1640 with 0.2 IU bovine insulin; and MMT060562 in Cell Repository Culture Media ŽCRCM. 30. DMEM and RPMI 1640 were purchased from Gibco ŽGaithersburg, MD. whereas CRCM 30 was purchased from Sigma ŽSt. Louis, MO.. All media were supplemented with 10% fetal bovine serum ŽFBS. Ž Atlanta Biologicals, Atlanta, GA., and 100 unitsrml penicillin G and 100

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m grml streptomycin sulfate Ž Gibco.. All lines except MMT060562 were propagated in a 95% O 2r5% CO 2 humidified atmosphere at 378C. MMT060562 was maintained in room air in a 378C incubator. 2.2. SDS-PAGE and Western immunoblot analysis of FAS To assess relative FAS content among the various cell lines, confluent cells were washed, scraped, pelleted and homogenized in modified RIPA buffer Ž 2.5 mM EDTA, 50 mM NaF, 10 mM Na 2 P2 O 7 , 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10% glycerol, 20 mM Tris, pH 7.4. with a protease inhibitor cocktail ŽPIC. Ž1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3VO4 , 2.5 mM EDTA, 10 m grml leupeptin, 10 m grml pepstatin A and 10 m grml aprotinin. . After microcentrifugation and determination of protein concentrations with Pierce’s Ž Rockford, IL. BCA Protein Assay kit, 75 m g of crudely prepared cytosolic protein was fractionated by 6% SDS-PAGE. Gels were stained with Coomassie blue and dried. For Western immunoblot analysis, 25 m g of crudely prepared cytosolic protein was fractionated by 6% SDS-PAGE. Proteins were electroblotted overnight onto nitrocellulose membranes. Membranes were blocked with 5% dried nonfat milk then probed with 150 ngrml of polyclonal rabbit anti-human FAS antisera Žanti-OA-519 FAS e antisera w7,8,10,12–14x, generously provided by Dr. Sheldon Broedel, ChekTec, Baltimore, MD. for 1 h. Membranes were washed with 5% nonfat milk in tris-buffered saline ŽpH 7.4. with 0.05% Tween 20 Ž TTBS. . Membranes were incubated with goat anti-rabbit IgG–horseradish peroxidase Ž HRP. Ž1:3333. in TTBS and developed using the ECLe detection kit from Amersham ŽArlington Heights, IL. or incubated with goat antirabbit IgG–alkaline phosphatase Ž1:3000. in TTBS and developed using the Immun-Blote assay kit from Bio-Rad ŽHercules, CA. . Band intensities were quantitated by computerized densitometric analysis. 2.3. [ 14 C]Malonyl-CoA incorporation assay for specific actiÕity of FAS Specific activity of FAS in cell lines was determined by w2 y14Cxmalonyl-CoA incorporation into

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total cellular lipids, according to the method of Kuhajda et al. w16x. This assay is considered more sensitive than the spectrophotometric quantitation of NADPH oxidation during fatty acid synthesis w28x. Specific activity is reported as nanomoles malonyl-CoA incorporated per minute per microgram cell lysate. All incorporations of radiolabeled malonyl-CoA were standardized with w2 y14Cxmalonyl-CoA from Amersham lot a51 Žexcept in experiments studying dexamethasone effects below.. In preliminary experiments, we investigated the effects of different commercially prepared media formulations Žincluding serum-free media. upon FAS activity in selected cell lines, and found that media had only minimal influence on activity Ž not shown. .

and incubated overnight at 48C. Protein–ArG Ž1:1. agarose Ž Gibco. was added to lysate and incubated for 3 h at 48C. Protein–agarose complexes were then isolated by centrifugation and washed several times in 50 mM Tris ŽpH 8.r500 mM NaClr0.1% Triton-X 100rPIC with a final rinse in 50 mM Tris ŽpH 8.0.r150 mM NaClrPIC. Entire samples were boiled in sample buffer and electrophoresed by 6% SDSPAGE. The dried gel containing 32 P-labeled phosphoprotein was developed autoradiographically at y808C. Control studies for immunoprecipitations employed nonimmune rabbit serum Ž1:500. in place of anti-OA-519 FAS antisera. 2.6. Immunoprecipitation and Western immunoblot analysis of phosphorylated forms of FAS

2.4. Hormonal effects upon FAS content and actiÕity Changes in FAS behavior were studied with the synthetic glucocorticoid dexamethasone Ž Sigma. and the synthetic progestin R5020 Žkindly provided by Roussel-Uclaf, Romaineville, France. . Cell lines were grown to confluency in serum-supplemented media containing 10y4 , 10y6 and 10y8 M dexamethasone or 10, 25 and 50 nM R5020. At confluency, cells were lysed and FAS activity and content assessed as above, and compared with untreated cells. Incorporations of radiolabeled malonyl-CoA in this experiment were standardized with w2 y14Cxmalonyl-CoA from Amersham lot a58. 2.5. In Õitro 32P labeling and immunoprecipitation of phosphoprotein Selected cell lines were grown to near-confluency in their optimal medium containing FBS and antibiotics, washed in serum-free phosphate-free DMEM, then incubated in 1400 m Ci of w32 Pxorthophosphate ŽDuPontrNEM, Boston, MA. in 5 ml serum-free phosphate-free DMEM for 3 h w29x. Cells were washed with PBS ŽpH 7.2. with PIC, then lysed in modified RIPA buffer with PIC. Total cell lysates were shaken on ice for 30 min and centrifuged. Supernatants were precleared with 50% Immunoprecipitine Ž Gibco. at 48C and centrifuged. Anti-OA519 FAS antisera Ž5 m grml. were added to supernatant

Crudely prepared cytosolic protein from selected cell lines was generated with modified RIPA containing PIC, precleared with Immunoprecipitin, and immunoprecipitated overnight with 10 m grml of either polyclonal anti-phosphoserine antisera Ž Zymed, South San Francisco, CA., monoclonal anti-phosphothreonine antibodies Ž Zymed. , or monoclonal antiphosphotyrosine antibodies PY20 ŽTransduction, Lexington, KY.. After complexing with protein–ArG agarose, centrifugation and washing, immunoprecipitated phosphoproteins were then fractionated by SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 5% dried nonfat milk and immunoblotted with anti-OA-519 FAS antisera, as above. Inversely, cell lysates were immunoprecipitated with 5 m grml anti-OA-519 FAS antisera, fractionated by SDS-PAGE, and electroblotted onto nitrocellulose membranes. Membranes were blocked with TTBS in 10% dried nonfat milk for 2 h and then immunoblotted with either 2 m grml anti-phosphoserine antisera, 5 m grml anti-phosphothreonine antibodies, or 1 m grml PY20, in TTBSrmilk overnight at 48C. After washing, immunoblots using anti-phosphoserine antisera were incubated with donkey anti-rabbit IgG– HRP Ž Amersham. at 1:2500 in TTBSrmilk. Immunoblots using anti-phosphothreonine antibodies or PY20 were incubated with sheep anti-mouse IgG– HRP ŽAmersham. at 1:2500 in TTBSrmilk. All immunoblots were washed and developed employing

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Amersham’s ECL Detection kit according to manufacturer’s protocol. Cell lysates immunoprecipitated with polyclonal anti-OA-519 FAS or anti-phosphoserine antisera were controlled by replacing the primary antisera with nonimmune rabbit serum Ž Sigma. at 1:500. Cell lysates immunoprecipitated with monoclonal antiphosphoamino acid antibodies were controlled by replacing the primary antibodies with 10 m grml mouse IgG ŽSigma.. In PY20-related experiments, FAS from the human breast adenocarcinoma cell line SKBr3 was used as a positive control for phosphotyrosine-containing FAS, as previously described by us w30x. 2.7. Inhibition of FAS actiÕity in BNL.CL2 cells Inhibition of FAS in cell lines was facilitated by the drug cerulenin Ž 2 S .,Ž 3 R .-2,3-epoxy-4-oxo-7,10dodecadienoylamide4 Ž Sigma. . To demonstrate cerulenin-induced inhibition of FAS in cultured cells, BNL.CL2 hepatocytes propagated to confluency were treated with 7.5 m grml cerulenin in serum-supplemented media plus 0.5% DMSO. Control cells were treated with serum-supplemented media plus 0.5% DMSO and no drug. After 6 h, cells were scraped, pelleted, lysed and assayed for FAS activity. Cerulenin-treated cells were compared with control cells in their ability to incorporate w14Cxmalonyl-CoA into total cellular lipids, as described above. 2.8. Cell growth inhibition assays with cerulenin All mammary cell lines, as well as BNL.CL2 hepatocytes, were tested for cerulenin-induced growth inhibition. For each line, four 96-well plates were seeded with 20,000 cellsrwell on day 0 and propagated in their appropriate media containing 10% FBS. On the following day Ž day 1. , media was removed from one of the four plates and replaced with serumsupplemented media containing 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, 15.0 and 20.0 m grml cerulenin dissolved in 100% DMSO Žfinal DMSO concentration - 0.5%.. Control cells were switched to serum-supplemented media with 0.5% DMSO and no drug. Six hours later, the cells were washed with Hanks’s balanced salt solution, fixed for 30 min in 1% glutaraldehyde, washed, and stained with 1% crystal violet for 30 min

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to assess cell density. After air drying, stain was solubilized in 10% acetic acid and absorbance determined at both 490 nm and 595 nm ŽBioRad 3550 Automated Microplate Reader. . Values from blanked wells containing only serum-supplemented media and 0.5% DMSO, without cells or drug, were subtracted from all readings. The second, third and fourth plates of each cell line were allowed to propagate to days 2, 3 and 4, respectively. On the morning of each of these days, cells in a given plate were treated with the various concentrations of cerulenin and cell density determined by crystal violet staining. Percent cell density was defined as the ratio of crystal violetstained treated cells to control cells =100 and the inhibitory concentration at the 50th percentile of cell growth ŽIC 50 . determined. A decrease in density of treated cells Ž as compared to control cells. is interpreted as growth inhibition. 2.9. Effects of exogenous fatty acids upon cerulenininduced growth inhibition Mm5MT, RIIIMT, T1 and A1-1 cells at 20,000 cellsrwell were seeded into 96-well plates and propagated in serum-supplemented media. After 2 days, cells were replenished with mediarFBS containing free palmitate Ž Sigma. at concentrations of 25, 50, 100 and 150 m M and incubated for 6 h. Cells were then treated overnight with 5, 10 or 15 m grml cerulenin in mediarFBS. Cells propagated in mediarFBS with 0.5% DMSO served as controls. Percent cell density was determined using crystal violet. T1 cells at 20,000 cellsrwell were seeded into 96-well plates and propagated in serum-supplemented media. After 2 days, cells were replenished with mediarFBS containing either Ž1. water-soluble oleate ŽSigma. at 50, 100, 200 or 300 m M, Ž2. 5, 10 or 15 m grml cerulenin, or Ž 3. both oleate and cerulenin at various concentrations. Control cells were propagated in mediarFBS with 0.5% DMSO. Percent cell density was determined using Crystal violet. To study the effects of oleate preconditioning of T1, cells were incubated overnight in 50 m M oleate and on the following day, treated again with oleate, cerulenin or oleatercerulenin, according to the format outlined above.

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2.10. Statistics Statistical analysis was performed using Microsoft Excele and Jandel Scientific Sigma Plote software. P values were computed from comparative t tests using Microsoft Excel. Error bars were the standard deviation as calculated by Microsoft Excel. Activity ranges were determined at a 95% confidence level using Student’s t test. Graphs and charts were constructed on Sigma Plot and Microsoft Excel.

3. Results 3.1. RelatiÕe FAS content among mouse lines Coomassie blue-stained gels and Western blotting were employed for relative analysis of constitutive levels of FAS protein in normal cell lineage NMuMG and mouse mammary tumor cell lines transformed by MMTV Ž Mm5MT, RIIIMT, MMT060562. or Py Ž T1, A1-1.. With Coomassie blue, protein bands delineated at 270 kDa were intensely stained in lanes loaded with T1 and A1-1 lysates Žnot shown. . In contrast, bands at the same molecular mass were only weakly stained in lanes loaded with lysates from NMuMG and the MMTV-transformed lines. Computerized densitometry of Western immunoblots using anti-OA-519 FAS antisera ŽFig. 1A. confirmed the results with Coomassie blue-stained gels in showing much smaller amounts of enzyme in NMuMG and MMTV-transformed lines than the Py-transformed lines. Enzyme levels were 2.5- to 4-fold higher in T1 and A1-1 than in NMuMG or MMTV-transformed lines. 3.2. Specific actiÕity of FAS in mouse lines Specific activity of FAS was determined by w2 y14Cxmalonyl-CoA incorporation into total cellular lipids w16x. Fig. 1B relates constitutive FAS content to its specific activity in each of the mouse lines. Activity was highest in T1 and correlated with the elevated FAS content. A1-1 also contained large amounts of enzyme but unexpectedly exhibited only about 1r3 the FAS activity of Py-transformed counterpart T1. Furthermore, activity in A1-1 was ex-

Fig. 1. Comparison of FAS content and specific activity among mouse cell lines derived from normal mammary epithelium ŽNMuMG., MMTV-transformed mammary tumors ŽMm5MT, RIIIMT, MMT060562., and Py-transformed mammary tumors ŽA1-1, T1.. ŽA. Cytosolic protein was prepared from each mouse cell line by lysing cell pellets in modified RIPA buffer containing a protease inhibitor cocktail ŽPIC., followed by centrifugation and fractionation by 6% SDS-PAGE. Gel-immobilized protein was electroblotted onto nitrocellulose membrane, immunoblotted with 150 ngrml anti-OA-519 FAS antisera, and colorized with BioRad’s Immun-Blot assay kit. ŽB. The specific activity of FAS in each mouse line was determined by w14Cxmalonyl-CoA incorporation into total cellular lipids according to the procedure of Kuhajda et al. w16x. Specific activity is measured along the x-axis as nanomolars of malonyl-CoA incorporation per minute per microgram cell lysate. Band density on the y-axis was determined by computerized densitometric analysis of the Western blot depicted in ŽA.. MMT s MMT060562. P values: NMuMG vs. mean activity of MMTV-transformed lines Ži.e., 0.072., P 0.0001; NMuMG vs. A1-1, P s 0.0027; NMuMG vs. T1, P 0.00001; MMTV-transformed lines vs. A1-1, n.s.; MMTV-transformed lines vs. T1, P - 0.00001; A1-1 vs. T1, P s 0.00015 Ž ns 4..

ceeded by NMuMG by about 30%, even though A1-1 cells contained nearly 3 times as much enzyme. Indeed, FAS activity in A1-1 was not significantly different from the mean activity of MMTV-transformed cells Ž i.e., 0.072. , which were low in their content of FAS. Of note, enzymatic activity in NMuMG was about 40% higher than the mean activ-

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ity of MMTV-transformed cells, despite similar amounts of FAS. 3.3. Effects of synthetic progestin and glucocorticoid on FAS content and actiÕity Human breast cancer line T47D and mouse lines were propagated to confluency in media containing R5020. Previous studies showed that FAS is progesterone-responsive in hormone-dependent human breast cancer cell lines, as evidenced by increased FAS mRNA, protein content and enzymatic activity induced by the synthetic progestin, R5020 w31–36x. We confirmed these findings after stimulating T47D

Fig. 2. FAS content and activity in MMT060562 cells treated with R5020. ŽA. MMT060562 cells were propagated to confluency in medium supplemented with 10, 25 and 50 nM R5020, then lysed in modified RIPA buffer with PIC. Crudely prepared cytosolic protein Ž25 m g. from untreated Ž0 nM R5020. and treated Ž10–50 nM R5020. was fractionated by 6% SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with antiOA-519 FAS antisera, and colorized with BioRad’s Immun-Blot assay kit. ŽB. Enzymatic activity Ždetermined by radiolabeled malonyl-CoA incorporation into total cellular lipids according to the procedure of Kuhajda et al. w16x. was measured in untreated Ž0 nM R5020. and treated Ž10–50 nM. MMT060562 cells. The extent of malonyl-CoA incorporation is measured along the y-axis Ž ns 3..

Fig. 3. Dexamethasone effects upon FAS content in mouse breast lines. Cells were propagated to confluency in media supplemented with 10y4 M dexamethasone. Lysates were fractionated by 6% SDS-PAGE, transferred to nitrocellulose, immunoblotted with anti-OA-519 FAS antisera, and colorized with BioRad’s Immun-Blot assay kit. Untreated Žy. cells were compared with treated Žq. cells. MMT s MMT060562 Ž ns 3..

cells with 10 nM R5020 and saw a 6-fold increase in FAS content by immunoblot analysis and a doubling of enzymatic activity Ž not shown. . In contrast to the T47D control line, 10, 25 and 50 nM R5020 failed to induce FAS content or activity in most of the mouse lines, except MMT060562 Ž Fig. 3A and B. . Here, only a very slight but reproducible rise in FAS protein was observed with 10 nM but not 25 or 50 nM R5020 ŽFig. 2A. . Likewise, FAS activity was only slightly increased and not in a dose-dependent manner, as enzyme activity increased by only about 10% with 10 and 50 nM but not at all with 25 nM R5020 ŽFig. 2B. . Thus, FAS responsiveness to progestin was nil in Mm5MT and RIIIMT and nominal in MMT060562. By itself, the synthetic glucocorticoid dexamethasone reportedly has no affect upon FAS content or gene expression in human breast cancer cell lines w31,32x. However, it is a potent activator of the MMTV long terminal repeat ŽLTR. promoter in MMTV-transformed cell lines w21,22x. We tested the possibility that dexamethasone may also regulate FAS expression in mouse mammary tumors. Accordingly, mouse lines were propagated to confluency in media containing 10y4 , 10y6 and 10y8 M hormone, then lysed and analyzed for FAS content and activity. While no significant increase of FAS content occurred in lines treated with 10y6 M or 10y8 M

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Fig. 4. Dexamethasone effects upon FAS activity in NMuMG and MMTV-transformed mammary tumor lines Žbeyond 95% confidence interval.. Cells were propagated to confluency in optimal media containing 10y8 M Žwhite bars., 10y6 M Žstippled bars., or 10y4 M Žblack bars. dexamethasone. FAS activity was determined by radiolabeled malonyl-CoA incorporation into total cellular lipids, according to the procedure of Kuhajda et al. w16x. Results are plotted along the y-axis as percentage of activity in untreated versus treated cells. MMTs MMT060562 Ž n s 3..

dexamethasone, 10y4 M hormone doubled the amount of enzyme in NMuMG and MMTV-transformed cells ŽFig. 3.. In contrast, FAS in the Py-transformed cells was not upregulated by dexamethasone, even at 10y4 M ŽFig. 3.. Though FAS protein was not induced by 10y6 M or 10y8 M dexamethasone, FAS activity was significantly increased Ž beyond 95% confidence interval. at these concentrations in NMuMG and MMTV-transformed cell lines ŽFig. 4. . NMuMG showed a classic dose-dependent response to increasing hormonal concentrations whereas MMTV-transformed lines exhibited a nonlinear dose-dependent response Ž Fig. 4. . In contrast to NMuMG and the MMTV-transformed lines, FAS activity in Py-transformed lines was unaffected by dexamethasone Žnot shown. . 3.4. EÕidence for phosphorylated forms of FAS in NMuMG and mammary tumor cell lines The marked discrepancy between enzymatic activity in Py-transformed cell lines despite similar content, as well as induction of enzyme activity but not protein content with lower concentrations of dexa-

methasone in MMTV-transformed cells, implied that FAS may undergo some kind of covalent modification, namely phosphorylation. To test this hypothesis, cell lines were labeled in vitro with w32 Pxorthophosphate followed by immunoprecipitation of cell lysates using anti-OA-519 FAS antisera. Fig. 5 shows a single

Fig. 5. In vitro labeling of FAS by w32 Pxorthophosphate. The incubation of 1=10 5 cells with 1400 m Ci w32 P xorthophosphate was according to the procedure of Paxton et al. w29x, lysed with modified RIPA buffer with PIC, cleared with Immunoprecipitin, immunoprecipitated with 5 m grml anti-OA-519 FAS antisera, and fractionated by 6% SDS-PAGE. Control studies consisted of replacement of anti-OA-519 FAS antisera with nonimmune rabbit sera ŽNRS.. The dried gels were developed autoradiographically overnight at y808C Ž ns 2..

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band in each lane loaded with immunoprecipitated FAS from NMuMG, Mm5MT, T1 and A1-1. Intense bands were identified in the region corresponding to 270 kDa, the known molecular mass of FAS w1–4x. Control cells immunoprecipitated with nonimmune rabbit sera failed to produce such a signal ŽFig. 5..

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Hence, in vitro radiolabeling of the enzyme with w32 Pxorthophosphate was consistent with but not definitive for phosphorylated FAS in NMuMG and mouse mammary tumor cell lines. To determine which amino acid residues might be phosphorylated in FAS, the enzyme was immunoprecipitated with anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine ŽPY20. antibodies, electrophoresed by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antiOA-519 FAS antisera. A single band at 270 kDa representing phosphoserine- and phosphothreoninecontaining FAS was seen with NMuMG, T1 and SKBr3 but not Mm5MT and A1-1 ŽFig. 6A and B. . Similar results were obtained when FAS was immunoprecipitated with anti-OA-519 FAS antisera and then immunoblotted with anti-phosphoserine antisera or anti-phosphothreonine antibodies. When phosphoprotein was immunoprecipitated with PY20, phosphotyrosine-containing FAS was detected in SKBr3 but not in the mouse lines ŽFig. 6C. . Inversely, immunoprecipitation of FAS with anti-OA-519 FAS antisera, followed by immunoblotting with PY20 showed similar results. In control studies, replacement of polyclonal antisera with nonimmune rabbit sera or monoclonal antibodies with nonspecific mouse IgG in the primary immunoprecipitation step failed to immunoprecipitate FAS from cell lysates. 3.5. Cerulenin effects in mouse cell lines

Fig. 6. Immunoprecipitation of phosphorylated FAS using antiphosphoamino acid antibodies. Crudely prepared cytosolic protein from RIPA-lysed NMuMG, Mm5MT, T1, A1-1 and SKBr3 cells was precleared with Immunoprecipitin, and then immunoprecipitated with 10 m grml of either anti-phosphoserine antisera ŽA., anti-phosphothreonine antibodies ŽB., or anti-phosphotyrosine antibodies ŽC.. After electrophoresis, proteins were electroblotted onto nitrocellulose, immunoblotted with anti-OA519 FAS antisera, and developed with Amersham’s ECL kit. Arrows delineate phosphorylated enzyme Ž ns 4..

Cerulenin is derived from the fungus Cephalosporium caerulens and is a well-characterized inhibitor of FAS activity w37,38x. The drug binds irreversibly to the catalytic binding site of the b-ketoacyl-acyl carrier protein synthase in the multienzyme FAS complex w38x. To demonstrate cerulenin-induced inhibition of FAS in cultured mouse cells, BNL.CL2 hepatocytes propagated to confluency were treated with 7.5 m grml cerulenin. After 6 h, total FAS activity was measured and compared with untreated BNL.CL2 cells. Specific activity in untreated BNL.C2 was 0.133 " 11 versus 0.052 malonyl-CoA incorporated miny1 m gy1 cell lysate in cerulenin-treated cells Ž P s 0.004.. Despite this significant drop in FAS activity, BNL.CL2 retained ) 95% viability Žsee ‘Day 4’ in Fig. 7A. .

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Fig. 7. Growth inhibition curves of mouse cell lines treated with varying concentrations of the FAS inhibitor cerulenin Ž95% confidence interval.. For each line, four microtiter plates were seeded with 20,000 cellsrwell on day 0 and propagated in optimal serum-supplemented media. On days 1, 2, 3 and 4, media in plates 1, 2, 3 and 4, respectively, were replaced with fresh media containing 0.5–20 m grml cerulenin in DMSO and incubated for 6 h. Cells were then washed, fixed, washed again, and stained with Crystal violet. Control cells were also propagated in optimal serum-supplemented media containing DMSO only and no drug. Absorbance of solubilized stain was measured at 490 nm and response curves calculated as outlined in Section 2. Percent cell density was defined as the ratio of Crystal violet-stained treated cells to control cells =100. A decrease in % cell density is interpreted as growth inhibition. Dose-dependent response curves from normal cell lineages BNL.CL2 and NMuMG are shown in ŽA., MMTV-transformed lines Mm5MT, MMT060562, and RIIIMT are shown in ŽB., and Py-transformed lines A1-1 and T1 in ŽC. Ž n s 5..

Previous reports have shown that FAS inhibition by cerulenin is cytotoxic to human cancer cells in vitro and in vivo which implicates FAS as a potential

chemotherapeutic target w16,18,39–41x. We sought to extend this same concept to mouse mammary tumors by incubating our lines with various concentrations of

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Table 1 IC 50 s of mouse lines treated with cerulenin 1, 2, 3 and 4 days after seeding Ž95% confidence interval.

3.6. Effects of exogenous palmitate upon cerulenininduced growth inhibition

Cell lines

In previous reports, replenishment of media with palmitate Žone of the primary end-products of FAS. succeeded in ‘rescuing’ cell lines from the growth inhibitory effects of cerulenin w16,39,41x. This maneuver implied the specificity of the drug’s antagonistic effects upon lipogenesis. Similarly, Mm5MT, RIIIMT, T1 and A1-1 cells were preincubated with various concentrations Ž 0–150 m M. of palmitate prior to treatment with 5, 10 or 15 m grml of cerulenin. Palmitate alone imparted a slight proliferative effect at 25 and 50 m M to cells but inhibited growth at 100 and 150 m M ŽFig. 8.. When A1-1 cells were preconditioned with 25 and 50 m M palmitate and then treated with 5 m grml cerulenin, there was a slight but statistically significant increase in cell density Ž Fig. 8. . It is unclear, however, whether the rise in cell density is attributable to a proliferative or rescuing effect imparted by 25 and 50 m M palmitate. No such effect occurred in A1-1 cells treated with palmitate and higher does Ž 7.5 and 10 m grml. of

Day 1

Day 2

Day 3

Day 4

Normal cell lineages BNL.CL2 8.3"1.5 NMuMG 7.5"0.9

13.0"1.1 10.5"2.3

19.5"1.5 15.0"0.7

) 20 17.8"4.3

MMTV-transformed Mm5MT 7.1"5.3 RIIIMT 4.4"1.1 MMT060562 6.0"1.5

14.3"0.9 4.2"0.8 10.0"3.6

14.3"1.4 6.8"0.2 12.0"0.4

14.5"1.3 11.1"0.7 8.1"0.1

Py-transformed T1 4.8"3.5 A1-1 3.6"0.7

7.6"0.7 6.1"0.3

13.2"0.7 6.8"0.2

11.9"0.3 8.6"0.1

cerulenin, establishing dose–response curves and determining IC 50 s. Normal cell lineages BNL.CL2 and NMuMG were most sensitive to the growth inhibitory effects of cerulenin when treated 24 h after seeding Žday 1. and least sensitive when treated 96 h Žday 4. after seeding ŽFig. 7A. . Accordingly, dose– response curves shifted to the right and IC 50 s increased as these cell lines progressed toward confluency ŽFig. 7A, Table 1.. Cerulenin’s effects were minimal on day 4, as evidenced by mean IC 50 s of 17.8 m grml for NMuMG and ) 20 m grml for BNL.CL2 ŽTable 1.. In general, mammary tumor cell lines resembled the normal cell lineages by showing right-sided shifts of dose–response curves and increasing IC 50 s as cells became more confluent Ž Fig. 7B,C; Table 1. . However, most of the mammary tumor cell lines differed from normal cell lineages by showing greater overall sensitivity to the growth inhibitory effects of cerulenin by days 3 and 4 Ž Table 1.. During the first 48 h after seeding Ž days 1 and 2. , only the IC 50 s for RIIIMT and A1-1 differed significantly from BNL.CL2 and NMuMG Ž Table 1.. By day 3, these statistically meaningful differences extended to MMT060562 and T1 so that by day 4, the IC 50 s of all mammary tumor lines except Mm5MT were significantly different from the normal cell lineages ŽTable 1.. Cell lines on day 4 exhibited mean IC 50 s Ž in parentheses. in the following order; BNL.CL2 Ž ) 20. ) NMuMG Ž 17.8. ) Mm5MT Ž14.5. ) T1 Ž11.9. ) RIIIMT Ž11.1. ) A1-1 Ž8.6. ) MMT060562 Ž8.1..

Fig. 8. Effects of exogenous palmitate on growth inhibition of A1-1 cells treated with 5 m grml cerulenin. Microtiter plates were seeded with 20,000 cellsrwell and propagated for 2 d in serum-supplemented media. Cells were then pretreated with 25, 50, 100 or 150 m M free palmitate for 6 h, followed by overnight incubation in 5 m grml cerulenin in DMSO. Control cells were propagated in mediarFBS with 0.5% DMSO. Percent cell density was defined as the ratio of Crystal violet-stained treated cells to control cells =100. A decrease in % cell density is interpreted as growth inhibition Ž ns 3..

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cerulenin Žnot shown.. Likewise, palmitate failed to rescue Mm5MT, RIIIMT and T1 cells from cerulenin’s anti-tumor effects Žnot shown. .

3.7. Effects of exogenous oleate upon cerulenininduced growth inhibition of T1 Pizer et al. w39x reported that acute addition of oleate to media rescues the human promyelocyte cell line HL-60 from cerulenin-induced cytotoxicity. To document a similar effect for mouse mammary tumor lines, T1 was propagated in serum-supplemented media. After 2 days, cells were replenished with media containing various concentrations of oleate or cerulenin or both. Addition of 100, 200 and 300 m M oleate to T1 was markedly cytotoxic to the cells Ž not shown.. In contrast, 50 m M oleate did not inhibit cell growth in experiments where lipid was added acutely Ž1st pair of bars, Fig. 9A.. Acute addition of oleate failed to rescue T1 from the growth inhibitory effects of 5 and 10 m grml cerulenin Ž2nd and 3rd pair of bars, Fig. 9A.. However, mild but statistically meaningful rescue occurred with 50 m M oleate added with 15 m grml of the drug Ž4th pair of bars, Fig. 9A. Ž P s 0.003.. T1 cells preconditioned overnight with 50 m M oleate were retreated on the following day with oleate, cerulenin or a mixture of both. As above, oleate by itself did not inhibit cell growth Ž1st pair of bars, Fig. 9B.. In contrast to above where lipid was added acutely, cerulenin’s growth inhibitory effects were dramatically synergized in dose-dependent fashion by oleate preconditioning and retreatment Ž2nd, 3rd and 4th pair of bars, Fig. 9B. .

4. Discussion Fig. 9. Effects of exogenous oleate on cerulenin-induced growth inhibition of T1 cells. ŽA. Microtiter plates were seeded with 20,000 cellsrwell and propagated for 2 d in serum-supplemented media. Cells were replenished with the same media containing 50 m M water-soluble oleate and 0, 5, 10 or 15 m grml cerulenin, and incubated overnight. ŽB. Microtiter plates were seeded with 20,000 cellsrwell and propagated for 2 d in serum-supplemented media. Cells were replenished with the same media containing 50 m M water-soluble oleate and incubated overnight. On the next day, cells were treated with 50 m M oleate plus 0, 5, 10 or 15 m grml cerulenin, and incubated overnight. Percent cell density was defined as the ratio of crystal violet-stained treated cells to control cells =100. A decrease in % cell density is interpreted as growth inhibition. In both experiments ŽA and B., control cells were propagated in mediarFBS with 0.5% DMSO Ž ns 3..

Our analysis of FAS in experimental mammary tumors is the first of its kind and shows that fundamentally dissimilar mechanisms of viral transformation may influence the enzyme differently. The MMTV-transformed lines exhibit significantly lower enzymatic activity than mouse lines derived from normal cells. Comparison of FAS specific activity in MMTV-transformed lines Ž average of the meanss 0.072. with NMuMG Ž0.113. and BNL.CL2 Ž0.133. suggests suppression of FAS in the former. In contrast to MMTV, Py-transformed lines showed elevated amounts of FAS. Enzyme content was signifi-

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cantly increased in A1-1 and T1 over that of NMuMG and MMTV-transformed lines. Interestingly, FAS activity paralleled enzyme amount in T1 but not A1-1. Phosphorylation of FAS via Py-facilitated signal transduction was implicated as the reason for this disparity, since these lines differed in phosphatidylinositol 3X-OH kinase activation. Although an earlier study describes a phosphorylated isoform of FAS in pigeon liver w42x, evidence for direct phosphorylation of the mammalian enzyme has never been described, to the best of our knowledge. We demonstrated in vitro labeling of FAS with radioactive phosphate but showed no qualitative difference between A1-1 and T1. While consistent with FAS phosphorylation, most of the in vitro labeling seen in our study was probably secondary to rapid metabolic turnover of the 4X-phosphopantetheine prosthetic group, rather than by direct phosphorylation of the enzyme. Here, the phosphate group is introduced at the pantothenate kinase step and then transferred from CoA as 4X-phosphopantetheine to a serine residue in the acyl carrier protein w43x. Evidence for more direct phosphorylation of FAS, however, is provided by our immunoprecipitation studies using anti-phosphoamino acid antibodies which detect phosphoserine and phosphothreonine in FAS from some of the mouse lines. Phosphoserine- and phosphothreonine-containing FAS is detected in T1 but not A1-1, so that a qualitative difference in phosphorylation exists between these lines which is not explained by 4X-phosphopantetheine turnover. Moreover, expression of phosphoserine and phosphothreonine in mouse FAS correlates with higher enzyme activity, as phosphoamino acid was detected in enzyme from T1 and NMuMG but not A1-1 or Mm5MT. While these results suggest that the phosphorylated form may represent a more active form of FAS, the effects of phosphorylation upon enzyme activity require further investigation. Enzyme from SKBr3 contains readily detectable amounts of phosphoserine, phosphothreonine and, in contrast to the mouse lines, phosphotyrosine. SKBr3 is a human breast adenocarcinoma cell line which exhibits extremely high levels of FAS content and activity w15x. We have found phosphotyrosinecontaining FAS in other human breast cancer lines w30x but not cell lines derived from normal human mammary epithelium Ž unpublished data. . Thus, phos-

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photyrosine-containing FAS may represent a human cancer-specific isoform of the enzyme. Confirmatory studies are ongoing. Though the significance of FAS phosphorylation is not yet fully understood, there are important metabolic implications. In normal cells, the major regulatory burden of endogenous lipogenesis falls primarily on acetyl-CoA carboxylase which controls lipid synthesis in the short-term via specific allosteric effectors and hormone-induced covalent modification via phosphorylation–dephosphorylation w43,44x. Previously, FAS was thought to regulate lipid metabolism primarily in the long-term w1–4,43x. Our discovery of phosphorylated FAS impacts this belief because it implies an active role for the enzyme in the more immediate control over lipogenesis. Given that FAS is vital to certain cancers as evidenced by its marked elevation and cytotoxic consequences if inhibited, phosphorylated FAS is presumed to have even greater importance to tumor cells than normal cells. Studies are underway to further characterize phosphorylated FAS in normal and cancerous mouse mammary cells. We expect A1-1 and T1 to be particularly valuable for investigating phosphorylated FAS and its role in cancer-related lipid metabolism. The synthetic progestin R5020 was not found to be a potent regulator of FAS expression in NMuMG or mouse breast cancer lines, as it was in the progesterone receptor ŽPR.-positive human breast cancer line T47D. Studies on progesterone’s effects upon MMTV expression may provide some insight as to why FAS resists upregulation by R5020, at least in the MMTV-transformed lines. Progesterone neither stimulates DNA polymerase activity w45x nor effectively induces MMTV particle synthesis in the virally transformed mammary tumors w45–48x, despite the presence of a strong progesterone response element in the MMTV-LTR w49x. Marginal synthesis of viral particles and protein by progesterone in these cells is mediated primarily by a cross-reaction of the hormone with cytoplasmic glucocorticoid receptors ŽGRs., rather than by PRs w47,48x. Progesterone binds strongly to the cytoplasmic GRs but dissociates easily so that translocation to nuclear GRs is inefficient and results in minimal induction of viral protein synthesis w45–48,50,51x. Furthermore, the defect in progesterone responsiveness is not attributed to absence of or inherently weak progesterone response elements,

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since MMTV-LTR:reporter gene chimeras transfected into T47D cells Ž where PRs are intact and active. and induced with progesterone exhibit high rates of activity w49x. Thus, data strongly implies that either unavailable or dysfunctional PRs, a defective PR–estrogen receptor Ž ER. axis, or both, are present to varying degrees in MMTV-transformed mammary cells w52x. Whatever the reasonŽ s. , defective PR function is consistent with the unimpressive FAS response to R5020 in our MMTV-transformed mammary tumor cell lines. The rodent FAS promoter harbors glucocorticoid and progesterone response elements, similar to MMTV-LTR w53x. However, defective PRs or ERs in certain MMTV-transformed lines would preempt progesterone-mediated upregulation of the enzyme. Long-term addition of dexamethasone to culture media increased FAS activity and content in NMuMG and relieved suppression of the enzyme in MMTVtransformed lines. Significant elevations in activity occurred at lower concentrations of hormone Ž 10y8 M to 10y6 M. without detectable increases in FAS amount. Commensurate elevation of FAS content was not seen until extremely high concentrations Ž 10y4 M. of hormone were employed. Presumably, glucocorticoid induction of FAS in NMuMG and MMTVtransformed mammary tumor lines is facilitated by intact glucocorticoid receptors and regulatory mechanisms activating the FAS gene via the promoter’s glucocorticoid response element w53,54x. This scenario contrasts with certain human breast cancer cell lines where dexamethasone by itself reportedly has little or no effect upon FAS w31,32x. Interestingly, hormone also failed to induce enzyme in the Pytransformed lines. Reasons for the lack of FAS responsiveness to dexamethasone in certain human breast cancer cell lines and the Py-transformed cells are unclear but may include disruption or absence of glucocorticoid receptors andror alteration of regulatory mechanisms downstream of the receptor. The FAS inhibitor cerulenin has been employed in previous studies elucidating the role of endogenous lipogenesis in various cellular processes w55–62x. The latter reports and our results with mouse hepatocyte line BNL.CL2 show that cerulenin significantly inhibits FAS activity in lines derived from normal cells but with only minimal loss of viability. While clearly significant, FAS inhibition is not complete in

BNL.CL2. Despite partial inhibition by cerulenin, the enzyme continues to function at a level adequate enough to fulfil critical metabolic needs. Apparently, the metabolic needs of mouse mammary tumor lines differ from those of normal cell lineage, since the growth inhibitory effects of cerulenin are significantly greater in the former. These results recapitulate the drug’s effects upon certain human breast cancer lines, but with some differences. Our study showed that the drug inhibits growth of murine mammary tumor lines in a dose-dependent manner, similar to human breast cancer cell lines w16x. In the human lines, however, the extent of growth inhibition was generally commensurate with FAS activity; i.e., lines with high constitutive enzymatic activity were generally more sensitive to the drug w16x. Growth inhibition by cerulenin in mouse lines does not necessarily correlate with constitutive FAS activity and varied markedly among the MMTV-transformed lines, despite their similarities in enzyme activity. Also, T1 harbored significantly more FAS activity than any other tumor line in our study. Yet, T1 cells were not the most sensitive to cerulenin’s growth inhibitory effects. These discrepancies led us to pretreat selected mouse lines with exogenous palmitate prior to drug exposure, in an attempt establish the specificity of cerulenin in mouse breast cancer cell lines. Since palmitate represents one of the primary products of the FAS reaction, reversal of cerulenin-induced growth inhibition by addition of this fatty acid would implicate a role for lipogenesis in tumor cell proliferation. We were unable to definitively demonstrate palmitate rescue of mouse mammary tumor cells from cerulenin’s anti-tumor effects. The reasons why may be related to Ž1. viable contributions of other FAS end-products such as myristate and stearate which were not replenished like palmitate before cerulenin treatment, and Ž2. the drug’s possible interference with cellular pathways other than lipogenesis. While cerulenin’s ability to inhibit FAS is well-characterized w38,55–62x, its epoxide moiety serves as a highly reactive alkylating agent that could interfere with a number of enzymes containing active thiol groups. Such events might explain in part the lack of palmitate rescue in mouse lines. However, our results should not trivialize the concept that a drug whose primary effect is inhibition of FAS retards the growth

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of cancer cells more so than normal cells. Indeed, more significant palmitate rescue from cerulenin’s effects has been reported in experiments designed similar to ours on several human cancer lines, including SKBr3 w16x, promyelocyte leukemia line HL-60 w39x, neuroblastoma line SK–N–SH w41x, and medulloblastoma line Daoy w41x. These studies support the contention that cerulenin’s inhibition of FAS hinders human cancer cell growth. Nevertheless, our study raises the possibility that cerulenin facilitates its antitumor effect via pathways other than endogenous lipogenesis and signifies a need to develop more specific FAS inhibitors. Another lipid precursor, oleate, added at the time of cerulenin treatment partially rescues T1 cells at higher dosages of the drug. This finding was similar to that of human promyelocytic leukemia line HL-60, although the latter showed a greater degree of rescue w39x. Interestingly, preconditioning tumor cells with oleate prior to cerulenin and oleate retreatment results in significant growth inhibition rather than rescue of tumor cells. Here, oleate synergizes cerulenin’s antitumor effects. This finding could not be explained on the basis of toxic accumulation of oleate in culture media since cells treated similarly with lipid but not cerulenin went unaffected. Tumor cells apparently undergo some kind of metabolic adaptation in response to their lipid environment which alters their sensitivity to FAS inhibition, as hypothesized by Pizer et al. w39x. Oleate pretreatment may prove clinically useful should anti-FAS Žor anti-lipogenic. drugs be fully developed as anti-cancer reagents.

Acknowledgements The authors wish to thank Dr. Hilary Lane for her editorial comments. This study was funded in large part by The Elsa U. Pardee Foundation and by an Institutional Grant from The American Cancer Society. The study was presented in part at the 5th Annual International Symposium on Hormones and Cancer, Quebec City, Quebec, Canada, August, 1995, and The Annual Meeting of The American Association for Cancer Research, Washington, DC, April, 1996.

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