Differential Regulation of the Stability of Cyclic AMP ... · Differential Regulation of the Stability of Cyclic AMP-dependent Protein Kinase Messenger RNA in Normal versus Neoplastic

(CANCER RESEARCH 51, 6699-6703, December 15, 1991]

Advances in Brief

Differential Regulation of the Stability of Cyclic AMP-dependent Protein KinaseMessenger RNA in Normal versus Neoplastic Mouse Lung Epithelial Cells1

Carol A. Lange-Carter and Alvin M. Malkinson

Molecular and Environmental Toxicology Program, Colorado Cancer Center, School of Pharmacy, University of Colorado, Boulder, Colorado 80309-0297

Abstract

Neoplastic mouse lung epithelial cells contain greatly diminishedactivity, protein, and mRNA for the type I isozyme of cyclic AMP-dependent protein kinase (PKA I), while expression of the type II isozyme(PKA II) is similar to that of normal lung cells. A time course of PKAmRNA content in transcriptionally inhibited cells indicated that mostPKA mRNAs are more stable in the neoplastic E9 cell line than inrelated nontumorigenic CIO cells. To address the basis of this differentialstability, we treated both cell lines with cycloheximide, an inhibitor ofprotein synthesis, in the presence or absence of the transcriptionalinhibitor, 5,6-dichloro-l-e-ribofuranosyl-benzimidazole (DRB). The rateof PKA II regulatory subunit a mRNA decay in the presence of DRBwas unaffected by cycloheximide treatment in E9 cells but decreasedupon the addition of cycloheximide to DRB-treated CIO cells. Thecombination of these two agents markedly destabilized PKA II mRNAs(PKA catalytic subunit a and PKA II regulatory subunit a) relative toDRB treatment alone in neoplastic E9 cells, causing them to decay at arate equal to that in CIO cells. PKA II mRNA may be specificallystabilized by a protein with a relatively short half-life in neoplastic E9cells. These results suggest the involvement of tumor-specific factor(s) inthe regulation of PKA mRNA stability, a potential mechanism forconferring the observed differential responsiveness of normal and neoplastic lung cells to cyclic AMP.

Introduction

PKAs2 are important regulators of cellular growth and dif

ferentiation (1). The type I (PKA I) and type II (PKA II)isozymes exist as inactive tetrameric holoenzymes consisting oftwo C subunits and either a RI or RII regulatory subunit dimer,respectively. The binding of cAMP to R subunits dissociatesthe holoenzyme, thereby activating the C subunits (1, 2). Distinct genes exist for each PKA subunit, encoding «and ßisoforms of both RI and RII and the a, ßand y isoforms of theC subunit (3). Expression of these isoforms varies among species, is tissue specific and developmentally regulated, and fluctuates upon cellular perturbation (4-7). PKA II is predominantly associated with changes in cellular differentiation, whilevariations in PKA I often correlate with proliferative status (8,9). However, no consistent role in these processes can yet beclearly assigned to either isozyme.

Alterations in cAMP-mediated signal transduction are associated with neoplastic transformation of mouse lung epithelium,an animal model of human bronchioloalveolar carcinoma (10).Chemically induced mouse lung tumors are characterized by

Received 10/4/91 ; accepted 10/29/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by USPHS Grant ES02370.2The abbreviations used are: PKA, cAMP-dependent protein kinase; cAMP,

cyclic AMP; PKA I, the type I isozyme of PKA; PKA II, the type II isozyme ofPKA; C, the catalytic subunit of PKA; RI. the regulatory subunit of PKA I; RII,the regulatory subunit of PKA II; DRB, 5,6-dichloro-l-e-ribofuranosylbenzimi-dazole; Chx, cycloheximide; ARE, AU-rich element; cDNA, complementaryDNA; poly(A), polyadenylate.

carcinogen-specific activating mutations of the K-ras protooncogene (11). These tumors also contain functional anomalies inthe stimulatory GTP-binding protein, Gs«, associated withreceptor-coupled adenylate cyclase (12). Smith et al. derivedimmortalized nontumorigenic cell lines from normal mouselung epithelium (13) and selected spontaneous transformantsof these cells (14). CIO is a nontumorigenic cell line withbiochemical characteristics at early passage that resembledthose of alveolar type 2 cells (13), one of the cell types of lungtumor origin (15). E9 is a tumorigenic variant of similar histológica!origin. Neoplastic characteristics can thus be directlycompared to those of the nontumorigenic cells, providing anexcellent "normal" control. Tumorigenic E9 cells mimic mouse

lung tumors in exhibiting similar biochemical changes in bothK-rai (16) and Gs«(17), further emphasizing their significanceas an important in vitro model system.

Several independently derived tumorigenic cell lines fromboth spontaneous and carcinogen-induced mouse lung tumorsexpress decreased PKA I (18); this isozyme is nearly absentfrom neoplastic E9 cells. One important physiological consequence of this decreased PKA I expression is altered PKA I-dependent phosphorylation of several cytosolic proteins (19).The mechanism of this aberration most likely resides at thetranscriptional level, since neoplastic E9 cells actually containmore stable PKA mRNAs than normal CIO cells (20). Differential mRNA stability is now widely recognized as an importantmeans by which cells control gene expression (reviewed in Ref.21). Little is known about how PKA mRNA levels are post-transcriptionally regulated. Herein, the mechanism of differential PKA mRNA stability in CIO and E9 cells is examinedvia studies using transcriptional and translational inhibitors.We find that otherwise stable PKA II mRNAs are markedlydestabilized by these treatments in neoplastic E9 cells. Theseresults suggest the involvement of a tumor-specific factor(s) inthe regulation of PKA mRNA stability.

Materials and Methods

Materials. Chx and DRB were obtained from Sigma and Calbiochem,respectively.

Cell Culture. CIO, a nontumorigenic, immortal cell line, and E9, atumorigenic, spontaneous in vitro transformant, were derived fromnormal adult BALB/cJ mouse lung epithelium (13, 14). CIO cells growslowly to confluence and are then contact inhibited, whereas E9 cellsgrow more rapidly and do not display density-dependent inhibition ofgrowth at confluence (14). Both cell lines were maintained using CM RL1066 (Irvine) medium supplemented with 10% fetal bovine serum,grown in a humidified atmosphere of 5% CO2/95% air, and harvestedbefore confluence.

Preparation of Total RNA and Northern Analysis. The proceduresused for preparing cDNA probes, isolating total cellular RNA, andNorthern blotting were described by Shannon et al. (22). Expressionvectors containing mouse RI«,RII«,and C«cDNA inserts were kindlyprovided by Dr. G. Stanley McKnight, University of Washington,Seattle. Human /3-actin cDNA insert was generously supplied by Dr.

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STABILIZATION OF PKA II mRNA IN NEOPLASTIC LUNG CELLS

John Shannon, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO.

Results

To determine whether decreased mRNA stability could account for the lack of PKA I in E9 cells, the relative rates ofPKA subunit decay in CIO and E9 cells were assessed using thetranscriptional inhibitor, DRB. The half-lives of PKA subunitmRNAs were estimated by examining the relative abundanceof each message following Northern blot analysis of total cellular RNA after 0-12 h of DRB treatment (Table 1; Fig. 1).There was no evidence of cytotoxicity in either cell line after12 h of treatment with 100 fiM DRB.

The half-life of 2.4-kilobase Ca mRNA was approximately 8h in transcriptionally inhibited CIO cells, while this mRNA didnot significantly decay over a 12-h period of DRB treatment inE9 cells (Table 1). The three species of Ria mRNA were slightlymore stable in E9 cells compared to CIO cells. The 3.2- and3.0-kilobase doublet Ria mRNAs exhibited a 7-8-h half-life inCIO cells, while at least one-half of these mRNAs remainedafter 11 h in E9 cells. The 1.8-kilobase RI«mRNA species wasstable in both cell lines, as was 0-actin mRNA. RII«exhibitedthe shortest half-life of the PKA mRNAs examined; less than50% remained after 4 h in CIO cells, while 40% was still presentafter 12 h in E9 cells. Thus, following transcriptional inhibitionthe mRNAs which encode both PKA isozymes decay less rapidly in neoplastic E9 cells than in normal CIO cells, suggestingthat these mRNAs are more stable in E9 cells relative to CIOcells.

Degradation rates of eukaryotic mRNAs are believed to bedetermined by interactions between mRNA secondary structureand specific nucleotide sequences with cellular proteins, although interactions with other RNAs have not been ruled out(21). While most of the known determinants of mRNA stabilityare located within 3'-untranslated regions of mature messages,sequences in 5'-untranslated regions as well as the beginningor end of the protein-coding regions appear to be important incontrolling mRNA degradation (21). To examine whether themechanism of differential PKA mRNA stability in CIO and E9cells involved a protein factor(s), we treated cells with DRB inthe presence and absence of the translational inhibitor, Chx(Fig. 1).

Chx alone increased the concentrations of all PKA messagesabove that of DRB alone; this was most apparent for the threeRia mRNAs, which rose well above untreated control levels, ß-Actin mRNA content also increased slightly in CIO (data not

Table 1 PKA mRNA half-lives in DRB treated CIO and E9 cellsCells were treated with 100 „¿�,„DRB for 0-12 h and total cellular RNA was

isolated every 3 h and analyzed by Northern blotting as described in "Materialsand Methods." Blots were subjected to densitometric scanning and mRNA half-

lives were determined from linear regression of the resulting cunes. Data represent the average of two independent experiments ±range.

Half-life (h)

MessageCa

(2.4 kilobases)Ria (3.2)'

Ria (3.0 kilobases)Ria (1.8 kilobases)Rila (6.0 kilobases)0-ActinCIO

cells8.4

±1.56.8 ±0.07.7 ±0.7(Stable)4.1 ±0.6(Stable)E9

cells(Stable)"

11.3 ±1.811.2± 1.9(Stable)

9.5 ±0.3(Stable)

"(Stable) indicates that mRNA concentration did not change over 12 contin

uous h of DRB treatment.* Mouse Ria mRNA consists of three different sized species of 3.2, 3.0, and

1.8 kolobases.

shown) and E9 cells (Fig. 3). Interestingly, RII«mRNA decayedmore rapidly in CIO cells that had been treated with Chx aloneas compared to control levels, while this mRNA remained stableover 12 h in Chx-treated E9 cells.

In contrast to the results with Chx alone, addition of Chx toDRB-treated E9 cells markedly destabilized both Ca and RIIomRNAs but had no effect on the rate of decay of these messagesin DRB-treated CIO cells (Figs. 1 and 2). Thus, in the presenceof both DRB and Chx, the mRNAs which encode Ca and RHaare equally labile in the two cell lines (Fig. 2). Ria mRNA inDRB-treated E9 cells was increased slightly by Chx treatment(Fig. 2F). This mRNA was stabilized in CIO cells relative to itshalf-life in the presence of DRB alone and failed to decay over12 hours (Fig. 2C). Chx had no effect on ß-actinmRNA levelsin DRB-treated E9 cells (Fig. 3), suggesting that the changes in

Ca and Rila mRNA decay rates were specific.

Discussion

We previously demonstrated differential PKA mRNA stability in CIO and E9 cells (20). Herein we expanded this observation and examined the basis of these differences under conditions used widely to study mRNA stability/regulation (23-25).DRB interferes specifically with RNA polymerase II-mediatedtranscription by enhancing premature transcriptional termination, thus blocking the synthesis of heterogeneous nuclear RNA(26). Inhibition is rapid and reversible (27), and protein synthesis is largely unaffected (26). In CHO cells, DRB caused littlecytotoxicity, and the rate of "A" mRNA turnover (28) was

equal to that using pulse labeling techniques (29). DRB hasbeen used successfully to determine the half-lives of relativelyshort-lived mRNAs (28), such as those encoding regulatoryenzymes (30).

In the presence of DRB, PKA mRNAs exhibit different ratesof decay (20). Using actinomycin D, Knutsen et al. (31) demonstrated subunit-specific differences in PKA mRNA decayfollowing stimulation and removal of cAMP from cultures ofSertoli cells. In our system, most PKA mRNAs exhibited longerhalf-lives in neoplastic E9 cells than in normal CIO cells, whilethe 1.8-kilobase Ria mRNA species and ß-actinmRNA wereequally stable in both cell lines. Degradation rates of relativelystable mRNAs cannot accurately be determined using DRB dueto cytotoxicity during extended drug exposure (28). AU-richsequences in the 3'-nontranslated regions of mRNAs confer

instability by as yet unknown mechanisms (Ref. 23; reviewedin Refs. 21 and 32). The instability motif, AUUU, appears 34times in the 3.0-kilobase mRNA for human Ria, while only 2of these sequences are found in the shorter 1.5-kilobase RiamRNA (33). Assuming homology between mouse and humanRia mRNAs, this may account for the greater stability of the1.8-kilobase Ria mRNA.

Treatment of CIO and E9 cells with Chx alone increased thelevels of most mRNAs examined, especially the Ria mRNAs.The apparent induction of Ria mRNAs by Chx is consistentwith the finding that cAMP-mediated induction of PKA subunitmRNAs in Sertoli cells is regulated by different mechanisms(34). Synthesis of Ria mRNA induced by cAMP analogues inthat system does not require protein synthesis, while that of Caand Rila does. Thus, the increases in Ria mRNA that occurwhen protein synthesis is inhibited by Chx may represent RiamRNA accumulation due to ongoing transcription. Chx maynot stabilize Ria mRNA to the same extent in neoplastic E9cells because the basal Ria transcription rate is most likely

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C10 E9

Control DRB Chx DRB+Chx Control DRB Chx DRB+Chx

Fig. 1. Effect of eycloheximide on PKAmRNA stability in normal CIO and neoplasticE9 cells. CIO and E9 cells were placed in freshmedia alone (Control) or with media containing one or more of the following additions:100 »IMconcentration of the transcriptionalinhibitor, DRB, or 10 MM(CIO) or 1 MM(E9)concentrations of the translational inhibitor,Chx, or both, for 0-12 h. Since 10 MMChxwas cytotoxic to E9 cells, we used 1 MMChxwhich produced the same magnitude of increase in all messages, including (J-actin, asthat found in CIO cells treated with 10 MMChx. Total mRNA was isolated every 3 h andanalyzed by Northern blotting as described in"Materials and Methods."

Ccx 2.4kb

Rllot 6.0kb

3.2kb3.0kb vwwv

Rio

1.8kb

0 12 369 12 369 12 369 12 0 12 369 12 369 12 369 12

Time (hrs) Time (hrs)

lower than in CIO cells (20). Analogous to lung epithelial cells,Sertoli cells exhibited a 2-3-fold increase in the levels of RI«and Ca mRNA following treatment with Chx alone, with onlyminor effects on the other PKA subunit mRNAs (31, 34). Thismay reflect an increased stabilization of mRNAs on polysomes,the site of Chx inhibition. Indeed, mRNA instability is tightlycoupled to translation (35). Stabilization of Ria mRNA in CIO

Ia

Control DRB Chx DRB+Chx

3 5Time (hours)

3 6Time (hours)

.EE

« '.(hours) Time (hours)

I 6 ITime (hours)

Time (hrs) O 12 3 69 12 369 12 36 9 12

3 6 9 12 CTime (hours)

Fig. 2. Comparison of Chx effects on DRB-treated normal CIO and neoplasticE9 cells. CIO and E9 cells were exposed to DRB in the presence (O) or absence(•)of Chx for 0-12 h. Total mRNA was isolated every 3 h and analyzed byNorthern blotting, as described in "Materials and Methods." Data are expressedas the percentage of untreated control mRNA remaining at each 3-h time interval,as determined by densitometry of bands on autoradiograms. A-C, Ca, Rila, andRio mRNA decay in CIO cells, respectively; D-F, Ca, RIIo, and Ria mRNAdecay in E9 cells, respectively.

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Fig. 3. Northern blot of the effect of DRB and Chx on (3-actin mRNAexpression. E9 cells were exposed to DRB in the presence or absence of Chx for0-12 h and total mRNA was isolated every 3 h and analyzed by Northern blotting.The /3-actin message was very stable over the 12-h time course of DRB treatment.Chx alone slightly increased this message. Decay of/?-actin mRNA in the presenceof DRB was unaffected by the addition of Chx, as determined by densitometricanalysis of autoradiograms.

cells treated with both DRB and Chx resembles that increaseseen with Chx alone (Fig. 1). In contrast to the general increasesin mRNA levels in Chx-treated CIO and E9 cells, severalmRNAs, including those encoding tubulin (36) and histone(37), are selectively stabilized by Chx. Rapid degradation ofmany labile growth factor mRNAs including c-myc requiresprotein synthesis (21).

When Chx is added to DRB-treated E9 cells, only the Coand RHa mRNAs are markedly destabilized and decay at ratessimilar to those in CIO cells. This is one of the few examplesof Chx-mediated destabilization of an mRNA species and thefirst report of a difference in this effect between normal andneoplastic cells. Addition of Chx to cAMP-treated Sertoli cellsstrongly stabilized the mRNAs for RUßand Rila and increasedthe steady state levels of Ria and Ca (31). Perhaps a proteinfactor(s) with a relatively short half-life selectively stabilizes

Rila and Ca mRNAs in neoplastic E9 cells. Since differentialPKA mRNA stability was observed in the presence of DRBalone, the mRNA which encodes this putative EC-specific factormust also be comparatively stable. E9 cells may preferentiallystabilize the mRNAs which encode PKA II to help compensatefor their nearly complete loss of PKA 1(18). Transferrin receptor mRNA is stabilized during iron starvation by a M, 90,000cytoplasmic protein which binds to five iron response elementslocated in the 3'-untranslated region of the message (38).

Eukaryotic mRNA stability is regulated in part by the poly(A)binding protein, which binds to poly(A) tails of mature Iran-

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scripts, thereby protecting them from degradation by nucleases(reviewed in Ref. 35). Other fra/is-acting factors interact withdestabilizing AREs located in the 3'-untranslated regions ofshort-lived messages, such as those encoding several oncogenesand lymphokines (35). These AREs are also found within thetranscribed portion of many labile messages encoding immediate early gene products and promote rapid removal of thepoly(A) tail as a first step in mRNA decay (39). Indeed, little isknown about the regulation of PKA subunit mRNA stability,other than the recent finding that cAMP can stabilize themRNA for RII0 in actinomycin D-treated Sertoli cells (31).cAMP had no effect on PKA subunit mRNA decay rates inCIO and E9 cells.'

The sequence determinants that govern PKA mRNA stabilityare completely uncharacterized, but AU-rich regions may beinvolved (33). Perhaps a protein which interacts with stabilization elements present in both Ca and Rila mRNA is overex-pressed in neoplastic E9 cells. If so, then the stability of othermRNAs containing analogous elements is likely to be affected,inasmuch as common mechanisms are thought to mediatemRNA-degradative pathways (21). This type of change, inwhich several mRNAs encoding important regulatory proteinsare preferentially stabilized, could provide neoplastic cells withtheir characteristic growth advantage. Thus, inappropriateexpression of a mRNA-regulatory factor or class of factorscould help to maintain the oncogenic state. In c-wyc-inducedmouse monocytic tumors, granulocyte-macrophage-colony-stimulating factor mRNA, which contains AREs, is selectivelystabilized by a tumor-specific frans-acting factor (25). Granu-locyte-macrophage-colony-stimulating factor production bythese monocytic tumor cells may represent an oncogenic lesionpromoting autonomous growth (40).

Coordinate control of cellular R and C subunit concentrationsoccurs at the level of protein turnover (41, 42). The Chx-mediated destabilization of both Ca and RII«mRNA evokesthe intriguing possibility of coordinate regulation at the mRNAlevel as well. The question is whether these two mRNAs arepreferentially stabilized in neoplastic E9 cells via the samemechanism. Basal expression of mRNA for both Ca and Riais low in E9 cells compared CIO cells, while that of Rila issomewhat greater (19). This elevated basal expression of RilamRNA (as well as the specific stabilization of PKA II mRNAs)could represent a compensatory or homeostatic adjustment.The altered levels of PKA subunit mRNAs in neoplastic E9cells may arise from a single lesion rather than through independent events. Possible candidates for such a lesion includeK-ras oncogene activation or retinoblastoma tumor suppressorgene mutation, aberrations which have been associated withlung tumorigenesis (11, 16, 43, 44). These hypotheses arecurrently being tested in the mouse lung tumor system.

Acknowledgments

We gratefully acknowledge Dr. Jeffrey Rosen (Department of CellBiology, Baylor College of Medicine, Houston) for his helpful comments on the manuscript.

References

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

1. Beebe, S.. J. and Corbin, J. D. Cyclic nucleotide-dependent protein kinases. 28.In: P. D. Boyer (ed.). The Enzymes, Ed. 3, Vol. 17, pp. 43-111. Orlando,FL: Academic Press, Inc., 1986.

2. Gill, G. N., and Garren, L. D. Role of the receptor in the mechanism of 29.

3C. A. Lange-Carter and A. M. Malkinson, unpublished results.

action of adenosine 3':5'-cyclic monophosphate. Proc. Nati. Acad. Sci. USA,68: 786-790, 1971.0yen, O, Mykleburst, F., Scott, J. D., Hansson, V., and Jahnsen, T. Humantestis cDNA for the regulatory1 subunit RII«of cAMP-dependent proteinkinase encodes an alternate amino-terminal region. FEBS Lett., 246: 57-64,1989.Clegg, C. H., Cadd, G. G.. and McKnight, G. S. Genetic characterization ofa brain-specific form of the type I regulatory subunit of cAMP-dependentprotein kinase. Proc. Nati. Acad. Sci. USA, 85: 3703-3707, 1988.Ratoosh, S. J., Lifka, J., Hedin, L., Jahnsen, T., and Richards, J. S. Hormonalregulation of the synthesis and mRNA content of the regulatory' subunit ofcyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells. J. Biol. Chem., 262: 7306-7313, 1987.0yen, O., Sandberg, M., Eskild, W., Levy, F. O., Knutsen, G., Beebe, S.,Hansson, V., and Jahnsen, T. Differential regulation of messenger ribonucleicacids for specific subunits of cyclic adenosine 3'.5'-monophosphate (cAMP)-

dependent protein kinase by cAMP in rat Sertoli cells. Endocrinology, 122:2658-2666, 1988.0yen, O., Eskild, W., Beebe, S. J.. Hansson, V., and Jahnsen, T. Biphasicresponse to 3',5'-cyclic adenosine monophosphate (cAMP) at the messengerribonucleic acid level for a regulatory' subunit of cAMP-dependent proteinkinase. Mol. Endocrino!., 2: 1070-1075, 1988.D0skeland, S. O., Kvinnsland, S., and Ueland. P. M. Protein kinases activatedby cAMP in the genital tract of spayed mice treated with oestradiol-170. J.Reprod. Fértil.,44: 207-216, 1975.Dumont, J. E., Jauniaux. J. C., and Roger, P. P. The cyclic AMP-mediatedstimulation of cell proliferation. Trends Biochem. Sci., 14:67-71, 1989.Malkinson, A. M. The genetic basis of susceptibility to lung tumors in mice.Toxicology, 54:241-271, 1989.You, M., Candrian, U., Maronpot, R. R.. Stoner, G. D., and Anderson, M.W. Activation of the K-ras protooncogene in spontaneously occurring andchemically induced lung tumors of the strain A mouse. Proc. Nati. Acad.Sci. USA, 86: 3070-3074, 1989.Droms, K. A., Haley, B. E., and Malkinson, A. M. Decreased incorporationof the photoaffinity probe 8N3-[7-"Pl-GTP into a 45KD protein in lungtumors. Biochem. Biophys. Res. Commun., 144: 591-597, 1987.Smith, G. J., LeMesurier. S. M., De Montfort, M. L., and Lykke, A. W. J.Development and characterization of type 2 pneumocyte-related cell linesfrom normal adult mouse lung. Pathology, 16:401-405, 1984.Smith, G. J., and Lykke, A. W. J. Characterization of a neoplastic epithelialcell strain derived by dexamethasone treatment of cultured normal mousetype 2 pneumocytes. J. Pathol., 147: 165-172, 1985.Grady, H. G., and Stewart, H. L. Histogenesis of induced pulmonary tumorsin strain A mice. Am. J. Pathol., 16: 417-432, 1940.Pan, Y. H. L., Nuzum, E. O., Hanson, L. A. and Beer, D. G. Ki-ras activationand expression in transformed mouse lung cell lines. Mol. Carcinog., 3:279-286, 1990.Droms, K. A., Haley, B. E., Smith, G. J., and Malkinson, A. M. Decreased8N3-[i-"P]GTP photolabeling of Gsa in tumorigenic lung epithelial celllines: association with decreased hormone responsiveness and loss of contact-inhibited growth. Exp. Cell. Res.. 182: 330-339, 1989.Lange-Carter, C. A., Fossli, T., Jahnsen, T., and Malkinson, A. M. Decreasedexpression of the type I isozyme of cAMP-dependent protein kinase in tumorcell lines of lung epithelial origin. J. Biol. Chem., 265.-7814-7818, 1990.Lange-Carter, C. A., and Malkinson, A. M. Alterations in the cAMP signaltransduction pathway in mouse lung tumorigenesis. Exp. Lung Res., 77:341-357, 1991.Lange-Carter. C. A., and Malkinson, A. M. Altered regulation of mRNAlevels encoding the type I isozyme of cAMP-dependent protein kinase inneoplastic mouse lung epithelial cells. J. Biol. Chem., in press, 1991.Nielsen, D. A., and Shapiro, D. J. Insights into hormonal control of messenger RNA stability. Mol. Endocrino!., 4: 953-957, 1990.Shannon, J. M., Emrie, P. A., Fisher, J. H., Kuroki, Y., Jennings, S. D., andMason, R. J. Effect of a reconstituted basement membrane on expression ofsurfactant apoproteins in cultured adult rat alveolar type II cells. Am. JRespir. Cell. Mo!. Biol., 2: 183-192, 1990.Shaw, G., and Kamen, R. A conserved AU sequence from the 3' untranslatedregion of GM-CSF mRNA mediates selective mRNA degradation. Cell, 46:659-667, 1986.Piechaczyk, M., Yang, J-Q., Blanchard, J-M., Jeanteur, P., and Marcu, K.B. Posttranscriptional mechanisms are responsible for accumulation of truncated c-myc RNAs in murine plasma cell tumors. Cell, 42: 589-597, 1985.Schuler, G. D., and Cole, M. D. GM-CSF and oncogene mRNA stabilitiesare independently regulated in trans in a mouse monocytic tumor. Cell, 55:1115-1122, 1988.Laub, O., Jakobovits, E. B., and Aloni, Y. 5,6-Dichloro-l-é-ribofuranosyl-benzimidazole enhances premature termination of late transcription of simian virus 40 DNA. Proc. Nati. Acad. Sci. USA, 77: 3297-3301, 1980.Gordon, I., and Stevenson, D. Kinetics of decay in the expression of inter-feron-dependent mRNAs responsible for resistance to virus. Proc. Nati. Acad.Sci. USA, 77:452-456, 1980.Helms, S. R.. and Rottman. F. M. Characterization of an inducible promotersystem to investigate decay of stable mRNA molecules. Nucleic Acids Res.,IS: 255-259, 1990.Harpold. M. M., Wilson, M. C., and Darnell, J. E. Chinese hamster poly-adenylated messenger ribonucleic acid: relationship to nonpolyadenylatedsequences and relative conservation during messenger ribonucleic acid proc-

6702

Research. on January 8, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

STABILIZATION OF PKA II mRNA IN NEOPLASTIC LUNG CELLS

essing. Mol. Cell. Biol., /: 188-198, 1981.30. Hargrove, J. H., and Schmidt, F. H. The role of mRNA and protein stability

in gene expression. FASEB J., 3: 2360-2370, 1989.31. Knutsen, H. K., Tasken, K. A., Eskild, W., Jahnsen, T., and Hansson, V.

Adenosine 3',5'-monophosphate-dependent stabilization of messenger ribo-nucleic acids (mRNAs) for protein kinase-A (PKA) subunits in rat Sertolicells: rapid degradation of mRNAs for PKA subunits is dependent on ongoingRNA and protein synthesis. Endocrinology, 129: 2496-2502, 1991.

32. Cleveland, D. W. Gene regulation through messenger RNA stability. Curr.Opin. Cell Biol., /: 1148-1153, 1989.

33. Sandberg, M., Skalhegg. B., and Jahnsen, T. The two mRNA forms for thetype la regulatory subunit of cAMP-dependent protein kinase from humantestis are due to the use of different polyadenylation site signals. Biochem.Biophys. Res. Commun., 167: 323-330, 1990.

34. Tasken, K. A., Knutsen, H. K., Attramadel, H., Tasken, K., Jahnsen, T..Hansson, V., and Eskild, W. Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent proteinkinases. Mol. Endocrinol., 5: 21-28, 1991.

35. Sachs, A. The role of poly(A) in the translation and stability of mRNA. Curr.Opin. Cell Biol., 2: 1092-1098, 1990.

36. Pachter, J. S., Yen, T. J., and Cleveland, D. W. Autoregulation of tubulinexpression is achieved through specific degradation of polysomal tubulinmRNAs. Cell, 51: 283-292, 1987.

37

38

Graves, R. A., Pandey, N. B., Chodchoy, N., and Marzluff, W. F. Translationis required for regulation of histone mRNA degradation. Cell, 48: 615-626,1987.Mullner, E. W., Neupert, B., and Kühn,L. C. A specific mRNA bindingfactor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell, 58: 373-382, 1989.

39. Shyu, A-B., Belasco. J. G., and Greenberg, M. E. Two distinct destabilizingelements in the c-fos message trigger deadenylation as a first step in rapidmRNA decay. Genes Dev., 5: 221-231, 1991.

40. Stocking, C., Loliger, C, Kawai, M., Suciu, S., Gough. N., and Ostertag, W.Identification of genes involved in growth autonomy of hematopoietic cellsby analysis of factor-independent mutants. Cell, 53: 869-879, 1988.

41. Weber, W., and Hilz, H. Stoichiometry of cAMP binding and limitedproteolysis of protein kinase regulatory subunits RI and RII. Biochem.Biophys. Res. Commun., 90: 1073-1081, 1979.

42. Often, A. D., and McKnight, G. S. Overexpression of the type II regulatorysubunit of the cAMP-dependent protein kinase eliminates the type I holoen-zyme in mouse cells. J. Biol. Chem., 264: 20255-20260, 1989.Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res., 49: 4682-4689, 1989.Harbour, J. W., Lai, S-L., Whang-Peng, J., Gazdar, A. F., Minna, J. D., andKaye, F. J. Abnormalities in structure and expression of the human retino-blastoma gene in SCLC. Science (Washington DC), 241: 353-357, 1988.

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Research. on January 8, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

1991;51:6699-6703. Cancer Res   Carol A. Lang-Carter and Alvin M. Malkinson  Mouse Lung Epithelial Cells

NeoplasticversusProtein Kinase Messenger RNA in Normal Differential Regulation of the Stability of Cyclic AMP-dependent

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