Regulation of VL30 gene expression by activators of protein kinase C.

Vol. 261, No. 11, Issue of April 15, pp. 5029-5033,1986 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, h e .

Regulation of VL30 Gene Expressionby Activators of Protein Kinase C* (Received for publication, August 13, 1985)

Karin D. Rodland, Shall F. Jue, and Bruce E. Magun From the Departmentof Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201

Themousegenomecontainsa retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenouslysuppliedepidermalgrowthfactor (EGF). Bindingof EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity whichindirectlyleadto activation of protein kinase C. We treated AKR-2Bcells, Swiss 3T3 cells, andthe 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters andwith the synthetic diacylglycerol sn-1,Z-dioctanoylglycerol. Tumor-promoting phorbol esters (e&. 12-O-tetradecanoyl phorbo1 acetate (TPA))increased thelevel of VL30 expression. Stimulationwith either TPA or EGF produced a similar time courseof VL30 expression. TPA induced VL30expressionintheEGF-receptorlessNR6 cell line, indicating that neither EGF ligand-receptor binding norphosphorylationofthe EGF receptor was requiredforinductionofVL30expression.Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30expression was alsostimulatedbytreatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.

The mouse VL30 gene family is represented by100-200 copies of a common 5.6-kilobase pair sequence which is transcribed to produce a 30 S poly(A) mRNA (1). The VL30 sequence has many retroviral characteristics. It is flanked by two long terminal repeat (LTRI) regions which show a high degree of homology to other viral LTRs, including the presence of a 4-base pair direct repeat at each end indicative of transposon-like activity, and a 36-base pair region within the LTR which shows a high degree of homology to theenhancer region of the Moloney sarcoma virus (2). The sequence between the two LTRs does not appear to encode any genes; however, VL30 can be packaged into pseudovirions when cotransfected with a helper virus (3, 4). The VL30 sequence appears to be highly mobile within the mouse genome on an evolutionary time scale, as thegenes are scattered over many * This work was supported by Grants CA39181 and CA39360 from the National Cancer Institute of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’The abbreviations used are: LTR, long terminal repeat; EGF, epidermal growth factor; TPA, 12-O-tetradecanoyl phorbol acetate.

chromosomes and show a high degree of interstrain polymorphism (2). Retroviral enhancer sequences have been implicated in the regulation of gene expression in several systems, including the c-myc and c-erbB proto-oncogenes (5-8), the intracisternal A particle of mouse embryogenesis (9), and the mouse mammary tumor virus (10). Retroviral enhancers may be regulated by other factors: LTR-induced expression of intracisternal A particle is cell cycle linked (11) and the mouse mammary tumor virus enhancer is activated by glucocorticoid treatment (12). Getz and hisco-workers (13) have shown that treatment with epidermal growth factor (EGF) produces a significant increase in VL30 mRNA levels in serum-deprived cells; in fact VL30 was first identified by differential screening of cDNA libraries from EGF-stimulated AKR-2B cells. Although VL30 transcripts corresponding to at least one open reading frame within the VL30 LTR are easily identified, it is notyet known whether transcription of VL30 results inthe appearance of a cellular polypeptide. Nevertheless, because VL30 elements are responsive to EGF and in viewof the known enhancer activity of retroviral repeats (14), VL30 transcription may provide a model system for investigating the regulation of specific gene transcription by polypeptide growth factors. Treatment of responsive cells with EGF results in a large variety of cellular responses, ranging from increased DNA synthesis and mitogenesis to differences in cellular morphology and growth characteristics, including the ability to form colonies in soft agar (e.g. Ref. 15). Phorbol ester tumor promoters such as12-0-tetradecanoyl phorbol acetate (TPA) produce many of the same cellular effects, including the ability to act as strong promoters of transformation (e.g. Ref. 16). While EGF and TPA have similar phenotypic effects on responsive cells, they act via two distinct mechanisms. EGF binds to anintegral membrane protein which acts as specific a EGF receptor. This protein is a tyrosine kinase and shows a high degree of amino acid homology with other cellular tyrosine kinases (17). Binding of EGF to its receptor also results in activation of the phosphatidylinositol messenger system; cellular phospholipase activity is increased, resulting in the production of diacylglycerols and phosphatidylinositol polyphosphates and indirectly stimulating Ca2+influx (18).TPA also binds to a specific receptor molecule, which copurifies with protein kinase C (19). Protein kinase C is a calciumactivated phospholipid-dependent kinase which typically phosphorylates serine and threonine residues (20-22). Cellular diacylglycerols released by phospholipase action on phosphatidylinositol appear to activate protein kinase C by increasing the enzyme’s affinity for both Ca2+and phospholipid (21,22), andTPA treatment also results in the direct activation of protein kinase C (23). Conversely, sn-1,2-dioctanoylglycerol, a highly potent synthetic analog of cellular

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C and VL30 Expression

diacylglycerols, can substitute for TPA in inducing leukemic cell differentiation and in promoting mitogenesis (24,25). Since EGF binding results in an increase in intracellular [Ca’+] and diacylglycerol levels,some effects observed following EGF treatmentmay actually result from protein kinase C activation. Conversely, activation of protein kinase C also results in phosphorylation of the EGF receptor and a subsequent decrease in its affinity for EGF (25-27), so that some effects observed following protein kinase C activation may be mediated by the EGFreceptor. However, the ability of phorbol esters to act inEGF-receptorless cells (28-30) implies that at least some of the actions resulting from protein kinase C activation occur via a pathway that does not involve the EGF receptor. In this paper we demonstrate that activation of protein kinase C is responsible for induction of VL30 expression even in the absence of EGF receptors. As in the case of c-myc, this induction occurs in theabsence of protein synthesis.

Fig. 3 indicates that EGF did not induce VL30 expression in the EGF-receptorless NR6 cells; however, TPA stimulation of these cells did cause an increase in VL30 expression. The TPA nonresponsive cell line TNRS was so designated because TPA stimulation failed to induce DNA synthesis andcellular proliferation in these cells, although other TPA-mediated responses were observed (28-30). TNRS cells did show an increase in VL30 expression following exposure to either EGF or TPA. Fig. 3 also shows the response of the parental Swiss 3T3 cell line, which displayed much lower levelsof endogenous VL30 expression than the NR6 or TNRS variants. The parental 3T3 cells showed a weak response to TPA stimulation with an increase in VL30 levels. Although not well demonstrated in this figure, the original autoradiograph did show a small increase in VL30 levels followingtreatment with EGF. Effect of Synthetic Diacylglycerols on VL30 ExpresswnDiacyglycerols produced by the action of cellular phospholipases on phosphatidylinositol are believed to be the physiological activators of protein kinase C (20-23). A variety of MATERIALS AND METHODS~ synthetic diacylglycerols, of which sn-1,2-dioctanoylglycerol RESULTS was the most potent tested, can substitute for phorbol esters in the induction of differentiation by leukemic cells, in stimInduction of VL30 Expression by Phorbol Ester Analogsinhibition of EGF binding (24, Since phorbol ester tumor promoters elicit some of the same ulating mitogenesis, and in the responses produced by EGFstimulation (15, 16, 34), we 25). When sn-l,2-dioctanoylglycerol was applied to theEGFinvestigated whether phorbol esters could also induce expres- receptorless NR6 cells, there was a marked increase in the sion of VL30 mRNA in responsive cell lines. Fig. 1shows the level of VL30 expression within 1 h, and heightened expresresults of an experiment in which AKR-2B cells were exposed sion persisted for 6-10 h (Fig. 4). Treatment with sn-1,2to various phorbol derivatives or EGF for 6 h; Cellular RNA dioctanoylglycerol also produced an increase in VL30 expreswas harvested and analyzed for the presence of VL30 tran- sion in AKR-2B cells (data notshown). The ability of sn-1,2dioctanoylglycerol to increase VL30 expression in the EGFscripts. Those phorbol esters which are potent tumor promoters (i.e. mezerein, TPA, phorbol 12,13-dibenzoate, and receptorless NR6 cells suggests that VL30 induction in these phorbol 12,13-didecanoate) ( 3 5 ) all produced an increase in cells resulted from activation of protein kinase C. Effect of Protein Synthesis on V U 0 Expression-AKR-2B the level of VL30 expression. Phorbol esters which are inactive or weak promoters (i.e. phorbol, phorbol 12,13-diacetate, cells were exposed to either EGF or TPA in the presence or and 4-0-methyl TPA) (35) did not induce VL30 expression. absence of cycloheximide, a potent inhibitor of protein synTheseresults indicate that induction ofVL30 expression thesis (36), to determine whether protein synthesis was recorrelates positively with the tumor-promoting ability of the quired for induction of VL30 expression. The concentration of cycloheximide used in this experiment inhibited the incorcompound. Time Courseof VL30 Induction by EGF and TPA-In order poration of [3H]leucineby 98% (data notshown). Fig. 5 shows to further characterize the respective effects of EGFand that inhibition of protein synthesis by cycloheximide did not phorbol ester stimulation on expression of VL30, the time prevent the appearance of VL30 mRNA in response to either course ofVL30 expression following stimulation by either EGF or TPA. Cycloheximide treatment was associated with EGF or TPA was monitored over a 12-h period. Fig. 2 shows the appearance of a larger fragment hybridizing to the VL30 that stimulation by EGF was maximal at 3 and 6 h, then probe. This larger fragment may represent a precursor of the gradually decreased, although VL30 levels 12 h after stimumature VL30 mRNA,whichwouldbe subject to enzymelation were still elevated over the endogenously expressed mediated processing dependent on protein synthesis. Cyclolevels of VL30 in unstimulated cells. TPA stimulation pro- heximide also appeared to inhibit the degradation ofVL30 duced a similar temporal response. The temporal character- transcripts,as cycloheximide treatment in the absence of istics of VL30induction following treatment with either EGF either EGF or TPA resulted in a modest increase in VL30 or TPA suggest that the two agents effect VL30 expression RNA levels. via similar pathways. DISCUSSION Roleof the EGF Receptor in VL30 Induction-In order to determine whether the presence of EGF receptors was obligProtein kinase C has recently been identified as thecellular atory for induction of VL30 gene expression following TPA receptor for the phorbol ester tumor promoters, and many of stimulation, we monitored VL30 levels in 3T3 cells and in the cellular effects of phorbol esters can be mimicked by EGF-receptorless (NR6) and TPA nonresponsive (TNR9) activation of protein kinase C (24, 37-41). The large number variant cell lines following exposure to either EGF or TPA. of similarities observed between EGF stimulationand phorbol ester treatment (15,16,34,35), coupled with the known effects Portions of this paper (including “Materials and Methods” and of EGF receptor activation on phospholipase activity and Figs. 1-5) are presented inminiprint atthe end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. intracellular calcium levels (18), suggests that many of the Full size photocopies are available from the Journal of Biological cellular responses to EGF treatment may actually be mediated Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- through activation of protein kinase C. It is also possible that ument No. 85M-2700, cite the authors, and include a check or money some TPA-induced effects maybe mediated by the EGF order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from receptor, since activation of protein kinase C results in phosphorylation of the cytoplasmic domain of the EGF receptor Waverly Press.

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(18, 2 5 ) and subsequent alteration of its binding affinity for 11. Augenlicht, L. H., and Halsey, H. (1984) Proc.NatL A d . sei. EGF (26, 27,42, 43). U. S.A. 82, 1946-1949 12. Ponta, H., Kennedy, N., Skroch, P., Hynes, N. E., and Grover, It has been demonstrated that the mouse retroviral-like B. (1985) Proc. Nutl. Acad. Sci. U. S. A. 82, 1020-1024 gene VL30 is sPecificalb induced treatment Of 13. Foste, D. N., Schmidt, L. J., Hodgson, C. P., Moses, H. L., and AKR-2B cells with EGF (2). The observation that transGetz, M. J. (1982) Proc. Nutl. Acud. Sci. U. S. A. 79, 73177321 formed cells have higher levels of endogenous VL30 expres~ E- (1982) Science 2167812-820 sion than their nontransformed counterparis (3, 44, 45) is 14. V a r m Halso significant. The increase in endogenous expression may 15. Carpenter, G., and Cohen, s. (1978) Epidermal Growth Factors in Biochemical Action of Hormones (Litwack, G., ed) Vol. 5, pp. reflect an increased level of protein kinase C activation in 203-244, Academic Press, Orlando, FL transformed perhaps as a Of endogenous expres16. Magun, B. E., and Bowden, G. T. (1979)J. Suprumol. S t r u t . 12, sion of activities normally modulated by external binding of 63-72 ligands to growth factor receptors. The escape from depend- 17. Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255,8363-8365 ence on externally supplied growth factors classically observed 18. Berridge, M. J., and Imine, R. F. (1984) Nature 312,315-321 in transformed cells may result from a transformation-asso19. N i e W J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 8 0 , 36-40 ciated increase in protein kinase C activation. 20. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and The results described in this paper have established the Nishizuka, Y. (1979) J. Biol. Chem. 254,3692-3695 following Points: 1) VL30 expression Was induced bY tumor- 21. Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., and Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 121&1224 promoting phorbol esters; 2) the temporal response of VL30 expression to EGF and TPA stimulation was similar; 3) VL30 22. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka, Y. (1980) J . Biol. Chern. 2 5 5 , 2273-2276 expression could be induced in the absence of EGF receptors M.7 Takai, yv Kaib'Jchi, K.9 Sane, K., Kikkawa, u., and in theabsence of protein synthesis; and 4)the diacylglyc- 23. Castagna, and Nishizuka, Y. (1982) J . Biol. Chem. 257, 7847-7851 erol analog sn-1,2-dioctanoy1g1Ycero1 induced vL30 expres- 24. Ebeling, J, G., Vandenbark, G, R., Kuhn, L. J., Ganong, B.R., sion in the presence or absence of EGF receptors. These Bell, R. M., and Niedel, J. E. (1985) Proc. Natl. Acud.Sci. U. resultsindicate that VL30 expression can occur independent S.A. 82,815-819 of either EGF receptor phosphorylation or ligand binding. 25. Davis, R. J., Ganong, B. R., Bell, R.M., and Czeck, M. P. (1985) However, theseresults do not prove thatEGF induces VL30 J. Biol. Chem. 260, 1562-1566 Fearn, J. C., and King, A. C. (1985) Cell 40,991-1000 expression by a pathwayinvolving diacylglycerols and protein 26. 27. Magun, B.E., Matrisian, L. M., and Bowden, G. T. (1980) J . kinase C. Biol. Chem. 255, 6373-6381 If Protein kinase c is responsible for induction of VL30 28. Butler-Gralla, E., and Herschman, H. R. (1981) J. Cell. Physiol. expression in theabsence of protein synthesis, then an exist107,59-67 ing substrateof protein kinase C may becapable of interacting 29. Butler-Gralla, E., Taplitz, S., and Herschman, H. R. (1983) Biochem. Biophys. Res. Commun. 111,194-199 directly, or indirectly via a cascade mechanism, with genomic DNA sequences to induce ~ ~ expression, 3 0 perhaps by inter- 30. Butler-Gralla, E., and Herschman, H. R. (1983) J. Cell. Physiol. 144,317-320 acting with enhancer sequences within the LTR region Of 31. Auffray, C., and Rougeon, F. (1980) Eur. J. Biochem. 1 0 7 , 303VL30. Identification of the substrates used by protein kinase 307 c in the induction of VL30 expression may contribute to an 32. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 202-203, Cold Spring Harunderstanding of the ways in which protein kinases can bor Laboratory, Cold Spring Harbor, NY regulate the activity of specific genes. 33. Savage, C.R., Jr., and Cohen.. S. (1972) . , J. Biol. Chem. 247. Acknowledgments-We gratefully acknowledge the technical assistance of William Odegard, Joanne Finch, and Thanh-HoaiDinh.

7609-7621 34. Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779 35. Diamond, L., O'Brien, G., and Rovera. G. (1978) . . Life . Sci. 23. 1979-1988 36. Meister, R. K., Hulman, S. E., and Johnson, L. F. (1979) J. Cell. Physiol. 100,531-538 37. Leach, K.L., James, M.L., and Blumberg, P. M. (1983) Proc. Natl. Acud. Sci. U. S.A . 80, 36-40 38. Ashendel, D. L., Staller, J. M., and Boutwell, R. K. (1983) Cancer Res. 43,4333-4337 39. Vandenbark, G. R., Kuhn, L. J., and Niedel, J. E. (1984) J . Clin. Inuest. 73,448-457 40. Nishizuka, Y. (1984) Nature 308, 693-698 41. Sharkey, N. A., Leach, K. L., and Blumberg, P. M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 607-610 42. Lockyer, J. M., Bowden, G. T., Matrisian, L. M., and Magun, B. E. (1981) Cancer Res. 41, 2308-2314 43. King, A. C., and Cuatrecasas, P. (1981)J . Biol. Chem. 257,30533060 44. Besmer, P., Olshevsky, A., Baltimore, D., Dolberg, D., and Fan, H. (1979) J. Virol. 2 9 , 1168-1176 45. Courtney, M.G., Schmidt, L. J., and Getz, M. J. (1982) Cancer Res. 42,569-576 ,

REFERENCES 1. Keshet, E., and Itin, A. (1982) J. Virol. 43, 50-58 2. Hodgson, C. P., Elder, P. K., Tetsuya, O., Foster, D. N., and Getz, M. J. (1983) Mol. Cell. Biol. 3,2221-2230 3. Sherwin, S. A., Rapp, U. R., Benveniste, R. E., Sen, A., and Todaro, G. J. (1978) J . Virol. 2 6 , 257-275 4. Scolnick, E. M., Vass, W . C., Howk, R. S., and Duesberg, P. H. (1979) J. Virol. 29, 964-972 5. Neel, B. G., Hayward, W . S., Robinson, H. L., Fang, J., and Astrin, S. M. (1981) Cell 2 3 , 323-334 6. Hayward, W. S., Neel, B. J., and Astrin, S. M. (1981) Nature 290,475-479 7. Payne, G. S., Courtneidge, S. A., Crittenden, L. B., Fadly, A. M., Bishop, J. M., and Varmus, H. E. (1981) Cell 23,311-322 8. Fung, Y.-K. T., Lewis, W . G., Crittenden, L. B., and Kung, H.-J. (1983) Cell 3 3 , 357-368 9. Royston, M. E., and Augenlicht, L. H. (1983) Science 222,13391341 10. Nusse, R., and Varmus, H. E. (1982) Cell 31,99-109

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Supplementary Material to: Regulation of VL30 Gene Expression By Acivators of P r o t e i n K i n a s e C K a r i n D. Rodland, Shall F.J u e a n d B r u c e E. M a g u n D e p a r t m e n t of Cell Biology and Anatomy The Oregon Health Sciences University Portland,Oregon97201 Material and Methods .Cell C u l t u r q The mouse embryo-derived AKR-ZB cell line (supplied by Harold Moses), the Swiss mouse 3T3 cell line, the TPA nonresponsive cell line TNR9, and the EGF-receptorless 3T3 variant designated NR6 (generously supplied by H. Herschman) were cultured in Dulbecco's modified Eagles' medium (DMEM) containing 10% defined calf serum (HyClone) at 37' C in a humidified 5% C02/95% air atmosphere. Prior to stimulation, nearly confluent cultures were washed twice in a serumfree deprivation medium consisting of a 1:3 mix of DMEM and IO mM glucose, 3 mM KCI, 130mM NaCI, 1.0 mM Na PO 00033 mM phenol red, 30 mM HEPES, pH 7.6 (2). then incubatedTn t t s medium for 24 hours. Cells were stimulated by the addition of either epidermal growth factor (EGF) at IO ng/ml final concentration, phorbol esters at a final concentration of 100 ng/ml, or s1t-1,2-dioctanoylglycerol (DOG) at a final concentration of 0.1 mM. When cycloheximide treatment was used, the appropriate culture plates were exposed to freshly-prepared cycloheximide at a concentration of 25pg/ml of medium for IO min before the addition of EGF or TPA.

Figure 1: Effect of ohorbol and ohorbol analoas on VL30 exoression. Confluent IO cm plates of AKR-ZB cells were serum-deprived 5 plates for 24 hr as described in Methods. Experimental groups of were then exposed to the indicated compounds for 6 hr before total cellular RNA was harvested as described. A Northern blot was constructed using lOpg of each experimental RNA per lane. The blot was hybridized to 32P-labeled DNA from the plasmid BVL-I, which contains the entire VL30 sequence including both LTRs. The resulting autoradiograph shows the response obtained from EGF (lane 2). TPA (lane 3). MEZ (lane 4), PDB (lane 5). PDD (lane 6), PDA (lane 7), 4-0methyl TPA (lane 8) and phorbol (lane 9). Lane I contains RNA obtained after the 24 hr serum-deprivation, without experimental additives (the 0 time control).

Analvsis of RNA: At appropriate intervals after stimulation, total cellular RNA was extracted essentially as described by Auffray and Rougeon (31). Total cellular RNA was fractionated by electrophoresis in 1.2% agarose gels containing formaldehyde and formamide as described by Maniatis et al. (32) and immobilized on nitrocellulose membranes (Schleicher & Schuell BA85, 45 my pore size) by capillary transfer. The plasmid BVL-I (containing the entire VL30 coding sequ ce and obtained from M. Getz) was labelled by incorporating dCTP "P (New England Nuclear, Boston, MA), using a commercial nick translation kit (Amersham Corp, Arlington, Ill). The labeled probe was heat-denatured and added to fresh hybridization buffer. The pre-hybridization solution was replaced with the radioisotopically-labeled hybridization buffer, and the membranes were incubated at 42' C for 16-24 hr. T h e membranes were then washed twice in 0.2 M NaCI, 30 mM Na citrate, 0.1% NaDodS04 at room temperature for 30 min. The membranes were then autoradiographed by exposure to Kodak X-Omat X-ray film in the presence of DuPont Cronex intesifying screens. Chemicals: 12-0-tetradecanoylphorbol-13-acetate (TPA), phorbol-12.13 dibenzoate (PDB); phorbol 12.13-diacetate (PDA), mezerein (MEZ), 4-0methyl-12-0-tetradecanoylphorbol-13-acetate(4-0-methyl-TPA) and phorbol were purchased from Dr. Peter Borchert, Chemical Carcinogenesis, Eden Prairie, Minn. All diterpene derivatives were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.1 mg/ml and added to specified culture dishes at a final concentration of 100 ng/ml of medium. Control culture dishes were exposed to DMSO alone at a final concentration of 0.1%. L-sn- 1.2 dioctanoylglycerol (DOG) was purchased from Avanti Polar Lipids, Inc., Birmingham, Ala. as a solution in chloroform. The chloroform was evaporated under a stream of nitrogen and the DOG was then dissolved in DMSO to a final concentration of 100 mM. The DOG was stored under nitrogen at -80' C until use. EGF was prepared from mouse submaxillary glands as described by Savage & Cohen (33).

1 2 3 4 5 6 7 8 9

Figure 2: Time course of EGF and TPA induction of VL3Q. Confluent IO cm plates of AKR-2B cells were serum deprived for 24 hr then exoosed to either EGF or TPA for varying time intervals, as described. Total cellular RNA was used to construct a No hern blot (2Opg RNA per lane). and was subsequently hybridized 1 to j5P-labeled VL30 DNA as described, then autoradiographed. Lane contains RNA from the 0 time control. Lanes 2-5 contain RNA obtained following EGF stimulation for 3 hr (lane 2). 6 hr (lane 3). 9 hr (lane 4) and I2 hr (lane 5). Lanes 6-9 contain RNA obtained after TPA stimulation for 3 hr (lane 6). 6 hr (lane 7) 9 hr (lane 8) and 12 hr (lane 9). ~

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Figure 3 Effect of EGF or TPA stimulation on VL30 exoression in NR6. TNR9. and NIH 3T3 cells. Confluent IO cm plates containing either NR6, TNR9, or NIH 3T3 cells were serum deprived for 24 hr as described. Experimental groups of 5 plates each were then exposed to either EGF (IOng/ml) or 24 h r TPA lor 3 hr. Control groups were harvested immediately after a serum deprivation. Total cellular RNA was extracted and used to conIObg of RNA from each group. The struct a Northern blot con ining blot was hybridized with "P-labelled VL30 DNA, then autoradiographed. The original autoradiograph indicates a faint positive response to EGF in NIH 3T3 cells, although the response is not I: N R 6 + TPA; Lane 2 N R 6 + EGF; Lane clear in the photograph. Lane 3: control NR6; Lane 4: T N R 9 + TPA; Lane 5: T N R 9 + EGF; Lane 6: control TNR9; Lane 7: 3T3 + TPA; Lane 8: 3T3 + EGF; Lane 9: control 3T3.

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Figure 4: Eflect of DOG on VL30 exoression in NR6 cells. Confluent IO cm plates of NR6 cells were serum deprived for 24 hr as described, then exposed to 0.1 mM DOG for varying time intervals. Total cellular RNA was obtained from groups of 5 plates for 2 0 p g of each time point and sed to construct a Northen blot with RNA in each lane. 3'P-labeled VL30 DNA was hybridized to the 1 contains conmembrane, and an autoradiograph was obtained. Lane trol RNA after serum deprivation for 24 hr. RNA obtained after 1 hr (lane 2). 3 hr (lane 3). 6 hr (lane 4) end IO hr (lane 5 ) of DOG stimulation was used for lanes 2-5. Lane 6 contains RNA from cells exposed to the solvent DMSO for IO hr.

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Figure 5: Effect of CHX on VL30 exoression induced bv TPA or EGF. Confluent IO cm plates of AKR-2B cells were serum-deprived as described for 24 hr, then exposed to either TPA or EGF in the Presence or absence of CHX for 6 hr. Total cellular RNA was obtained from experimental groups of 5 plates each, and used to construct a Northern blot. RNA species containing2equences homologous to VL30 were detectedbyhybridizationtoP-labeledBVL-IDNA.Asecond,higher molecular weight VL30 RNA was visible following CHX treatment. I: Control The experimental treatment of the cells was as follows: lane RNA before stimulation; lane 2 TPA; lane 3: TPA + CHX; lane 4: EGF; lane 5: E G F + CHX: lane 6: CHX.