Characterization of Messenger RNA for Fructose 1,6-Bisphosphate Aldolase a Isozyme of Rat Ascites Hepatoma AH 7974 Cells

Home , Aldolase B, Aldolase C

[CANCER RESEARCH 39, 502-506, February 1979] 0008-5472/79/0039-0000$02.00 Characterization of Messenger RNA for Fructose 1,6-Bisphosphate Aldolase A Isozyme of Rat Ascites Hepatoma AH 7974 Cells

Shigeru Sakiyama,1 Namiko Akiba, and Shinji Fujimura

Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2, Nitona-cho, Chiba 280, Japan

ABSTRACT control when mRNA was assayed by its ability to synthesize tt-fetoprotein in vitro (14). The messenger activity for fructose 1,6-bisphosphate In a previous paper, we reported the synthesis of aldolase aldolase (EC4.1.2.13) (aldolase) A isozyme has been char A subunit in vitro under the direction of both polysomes acterized in the polysome- or the messenger RNA-directed, and poly(A)-containing RNA of rat ascites hepatoma AH protein-synthesizing system using the pH 5 fraction of rat 7974 cells which show the nearly complete shift of isozymic liver or wheat germ extracts, respectively. The subunit of pattern from aldolase B to aldolase A (27). Meanwhile, aldolase A synthesized in vitro was detected by immunopre- Lebherz and Doyle (16) have shown the synthesis of a cipitation with anti-aldolase A antibody raised in chickens functional aldolase A subunit in vitro by polysomal RNA of followed by sodium dodecyl sulfate-polyacrylamide gel chicken muscle. Thus far, there is very little knowledge electrophoresis. The synthesis of the enzyme depended on about the metabolism of mRNA for isozymes appearing in the addition of polysomes or polyadenylate-containing RNA tumor cells. Therefore, the isolation and characterization of of rat ascites hepatoma AH 7974 cells which show a com the mRNA's coding for isozyme molecules is a prerequisite plete shift of aldolase isozyme to type A, whereas poly- step and will afford a basis for investigations of the control somes of adult rat liver were inactive. The messenger mechanisms of gene expression in tumor cells. activity for aldolase A was present exclusively on free In the present report, we describe the further characteri polysomes but absent on membrane-bound polysomes and zation of aldolase A mRNA of AH 7974 cells mainly concern in the soluble supernatant fraction of AH 7974 cells. The ing the subcellular localization of this mRNA and its size size of aldolase A messenger RNA determined by formam- estimation. ide-containing sucrose density gradient centrifugation was approximately 5.8 x 10s dallons corresponding to 1650 MATERIALS AND METHODS nucleotides. Taking into account the number of amino acid residues in the aldolase A subunit, approximately 400 nu Chemicals. ATP, GTP, creatine phosphate, and creatine cleotides correspond to the noncoding region of aldolase A phosphokinase were obtained from Sigma Chemical Co., messenger RNA. St. Louis, Mo. Poly(U)-Sepharose was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Formamide, obtained from E. Merck, Darmstadt, Germany, was vacuum INTRODUCTION distilled at 3 mm Hg (b.p. 78e) by K. Takahashi, Division of The resurgence of fetal-type isozymes of various enzymes Chemotherapy Chiba Cancer Center Research Institute. such as aldolase,2 pyruvate kinase, and hexokinase in [4,5-3H]Leucine, [U-'4C]leucine, and Protosol were pur tumors, especially in hepatomas, has been well reviewed chased from New England Nuclear, Boston, Mass. Wheat (29, 35). Three distinct isozymes of aldolase, which are germ was a gift from Nisshin Milling Co., Ltd., Chiba, different immunologically, were characterized electropho- Japan. retically regarding their substrate specificities (23). Aldolase Cells. Rat ascites hepatoma AH 7974 cells, originally A, muscle type, appears in various hepatomas at the ex induced by 4-dimethylaminoazobenzene by Yoshida (36) pense of aldolase B, adult liver type, while aldolase C, brain were supplied from Sasaki Research Institute, Tokyo, and type, is expressed only in some fast-growing hepatomas were maintained by serial transplantation via i.p. injection (20, 28). Very little is known, however, about the mechanism of ascites fluid to male Donryu rats weighing 250 g. Animals which might operate in switching the isozymic pattern at were given food and water ad libitum. About 1 week after the molecular level from the adult type to the fetal type and the transplantation of AH 7974 cells, rats were killed by wee versa in tumor tissues and in developing fetal tissues, cervical dislocation and cells were harvested and washed respectively. with 35 HIM Tris-HCI (pH 7.6)-0.14 M NaCI until the cells In considering the relationship between fetal-type iso became largely free from RBC. The livers of male Donryu zymes and fetal proteins, it should be noted that the rats weighing 250 g were perfused with the above buffer synthesis of Â«-fetoprotein in the mouse liver during its and served as the source of adult liver. developing stage was found to be under transcriptional Preparation of Polysomes. Total polysomes were pre pared from adult rat liver or AH 7974 cells according to the 1 To whom requests for reprints should be addressed. method of Falvey and Staehelin (8), except for the addition 2 The abbreviations used are: aldolase, fructose 1,6-bisphosphate aldol of Nonidet P-40 as described previously in the case of AH ase (EC4.1.2.13); poly(A), polyadenylate; poly(U). polyuridylate; mRNP, mes senger ribonucleoprotein. 7974 cells (27). Received July 31, 1978; accepted October 27. 1978. Free and membrane-bound polysomes of AH 7974 cells

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Aldolase A mRNA of Rat Ascites Hepatoma were prepared as described by Adelman et al. (1). The mined by averaging the radioactivity on both sides of the polysomes recovered from 16 g of cells were 368 and 180 peak on the gel. A)61)unitsfor free and membrane-bound polysomes, respec Sucrose DensIty Gradient Centrifugatlon.Total poly tively. somal ANA (400 lLg) of AH 7974 cells was dissolved in 3 mM Crude polysomes and the postmicrosomal supemnatant of Tnis-HCI (pH 7.6), 3 mM EDTA, and 70% fonmamide and heat AH 7974 cells were prepared according to the method treated for 3 mm at 7@ (12). Linear 5 to 20% sucrose described by Zahninger et a!. (38). gradients were made in the above buffer. Following centnif RNA Extraction.RNA'swereisolatedfromtotalor crude ugation in a Beckman SW 27 motorat 26,000 rpm for 72 hn, polysomes or from the postmicrosomal supennatant frac the gradient was fractionated from the top of the tube. Each tion of AH 7974 cells by phenol-chlonoform-isoamyl alcohol fraction (0.4 ml) was adjusted to 0.2 M with respect to NaCI, at pH 9.0 (3). Poly(A)-containing RNA was obtained by an and 2 volumes of ethanol were added. After the RNA was affinity column chromatography on poly(U)-Sepharose as washed twice with 2 volumes of ethanol in the presence of described previously (27). RNA concentration was deter 0.2 M NaCI, it was finally dissolved in 15 pi of H@O,and 10- mined on the assumption that 1 mg/mI equals 25 A@ nm. @.tIaliquots were used for the in vitro assay for protein In Vitro Protein Synthesis. Polysome-dimected protein synthesis. synthesis was carried out as described in a previous paper (27). The incubation mixture (1 ml) contained per ml: 30 RESULTS @.tmolTnis-HCI(pH 7.6), 70 @molmonovalent cations (KCI plus NH4CI), 4 @molmagnesium acetate, 1 @moIATP,0.4 Since aldolase A is known to be entirely absent in adult @moIGTP,20 p.mol creatine phosphate, 100 @gcreatine nat liver, studies were carried out to investigate whether this phosphokinase, 1 @moIdithiothreitol, 40 nmol 19 amino absence is associated with that of messenger activity at the acids, 1 pCi [U-14Cjleucine (300 @.tCi/@mol),0.2ml of pH 5 polysomal level. Total polysomes prepared from adult rat fraction prepared from adult rat liven and polysomes as liven were compared with those from AH 7974 cells for the indicated. The incubation was carried out at 25Â°for60 mm. ability to synthesize aldolase A in vitro. The results shown ANA-dimected protein synthesis was carried out as de in Chart 1 and Table 1 clearly demonstrate that there is no scnibed in a previous paper (27), except that the concentra detectable messenger activity for aldolase A on rat liver tions of some ingredients were changed. The incubation polysomes in spite of the presence of a significant amount mixture (0.2 ml) contained per ml: 20 @.tmolN-2-hydroxy of total messenger activity. When ANA extracted from ethylpiperazine-N'-2-ethanesulfonic acid (pH 7.6), 98 @mol polysomes of adult rat liver was investigated for aldolase A potassium acetate, 3.0 @moImagnesium acetate, 1 @mol messenger activity in extracts derived from wheat germ, no ATP, 0.1 @molGTP, 8 @tmoIcreatine phosphate, 6 @g activity was detected (data not shown). creatine phosphokinase, 2 @moldithiothmeitol, 20 nmol 19 It has been widely believed that mRNA can exist in various amino acids, 96 @gwheat germ tANA, 320 @Ci[4,5- forms as regards intracellular sites, i.e., polysome bound 3H]Ieucine (50 mCi/@.tmoI), 0.3 ml wheat germ extracts (10, 17, 32, 33, 37, 38) or as free mRNP particles in the prepared as described previously (19), and RNA as mdi cytoplasm (4, 5, 38). The former can be further divided into cated. The incubation was carried out at 30Â°for60 mm. membrane-bound and free polysomes for the synthesis of ProductAnalysis.In eitherthe polysome-orthe mANA proteins for export and nonsecneted proteins, respectively. directed system, 1 @lof the supemnatant obtained after centnifugation of the reaction mixture at 180,000 x g for 60 mm was spotted on filter papers, and total messenger activity was measured after tnichlomoacetic acid wash (27). albuminaldolas.A chymotrypsinogen The details of the method of identification of one of the in II vitro products with aldolase A were described in a previous 300 paper (27). Briefly, after incubation, the reaction mixture was centrifuged at 180,000 x g for 60 mm, and the super natant containing released polypeptides was fractionated by ammonium sulfate precipitation between 40- and 60% E 150 saturation. After dialysis and the addition of 14 @gof aldolase A purified from AH 7974 cells as a carrier, aldolase A was precipitated by chicken anti-aldolase A y-globulin. Immunoprecipitates formed were analyzed by electropho 60 nesis on sodium dodecyl sulfate-polyacrylamide gels (7.5%) 0 20 40 Slice Number (34). The gels were then sliced, solubilized with Pnotosol, Chart 1. Electrophoretic profiles of in vitro products synthesized by AH and then counted with a counting efficiency of 35 and 60% 7974 cells and rat liver total polysomes. @Iypeptidessynthesized and for [3H]- and [â€˜4C]leucine-Iabeledproducts, respectively. released were partially purified for aldalase A by ammonium sulfate fraction The values for the samples on filter papers were 10 and ation and then precipitated by anti-aldolase A chicken y-globulin as de scribed under â€œMaterialsandMethods.â€•Sodium dodecyl sulfate-polyacryl 60%, respectively. amide gel (7.5%) electrophoresis was carried out according to the method of The messenger activity for aldolase A was quantitated by Weber and Osbarn (34). Electropharetic migration is from left to right. Arrows, relative distance of migration of aldolase A subunit as well as marker adding the radioactivity which appeared in the region of the proteins. Polypeptides synthesized by 32.0 A2,,,/ml ofAH 7974 polysomes (â€¢) subunit molecule of aldolase A minus background deter and 36 A@,,/mlof rat liver polysomes (0).

FEBRUARY 1979 503

Table 1 containing RNA prepared from total polysomes of AH 7974 Messenger activity for aldolase A on total polysomes of AH 7974 cells to synthesize aldolase A in the wheat germ system. cells and rat liver The synthesis of aldolase A was saturated at the concentra released A tion of 100 j.@g/ml.In this experiment, leucine incorporation Sourceof polypeptides synthesized into aldolase A represented about 0.02% of the total incon polysomesPolysomes (dpm x 10@'Aldolase (dpm)bAH7974ceIIs32.06.5738Ratadded (A@/ml)Total ponation into released polypeptides. This number is appnox imately one-fifth of that obtained from experiments using liver36.04.233Rat liver72.05.038a polysomes (Table 1; Ref. 27). Since a number of mRNA's are known to carry the Determinedfrom the radioactivity after tnichloraacetic acid wash. nontranslatable nucleotides to a certain degree (12, 21, 24), b Calculated by summing the radioactivity which appeared in the the molecular weight of aldolase A mRNA was measured by aldolase A region on sodium dodecyl sulfate-polyacrylamide gel formamide-contamning sucrose density gradient centnifuga electrophoretic patternsas shown in Chart 1. tion. As shown in Chart 3, aldolase A mRNA appeared as a

Table 3 Table 2 Distribution of poly(A)-containing RNA and its messenger activity Distribution of aldolase A messenger activity between free and for aldolase A between crude polysomal and postmicrosomal membrane-boundcellsPolysomes polysomes of AH 7974 fractions of AH 7974 cells Total released AldalaseA RNAyieldIn vitroproteinsynthesisTotal Sourceof poly- added(A@/ polypeptides synthesized (dpm)bFreesomes ml) (dpm x 10@)â€• re leasedpoly @ 546Membrane 66.0 5 75a bound 66.0 3 Aldal ARNATotalPoly(A)-pep ase Determined from the radioactivity after tnichloroacetic acid ex contain RNAtidessynthe wash. RNA x b Calculated by summing the radioactivity which appeared in the (dpmfCrudeSource of RNAtracteif(mg)ing (/Lg)added(@@g/ml)(dpmi0'/@sized aldolaseA region after electropharesisan sodium dodecyl sulfate polyacrylamide gel as described under â€œMaterialsand Methods.â€• 3.2590Postmicrasomal7.0136757.30fractionpolysames22.5607801

Therefore, we performed the following experiments to see a Extracted from 15 g (wet weight) of AH 7974 cells. which subcellular fraction is enriched with mRNA for aldol b Determined from the radioactivity after tnichloroacetic acid ase A. Since the above method for the isolation of total wash. polysomes from AH 7974 cells included the use of deoxy C Calculated by summing the radioactivity which appeared in the aldolase A region after electrophoresis an sodium dodecyl sulfate cholate which solubilizes membrane, hence membrane polyacrylamide gel as described under â€œMaterialsand Methods.â€• bound polysomes, a method without the use of the deter gent should be introduced, and the method of Adelman et a!. (1) used in the present experiment was found to be most 0 suitable for the preparation of an active subfraction of polysomes of AH 7974 cells. Table 2 indicates that the 3 messenger activity for aldolase A is located exclusively on 0 free polysomes, although overall messenger activity was 20 E within the same range between these 2 polysomal subfnac tions. It is conceivable that a trace amount of the messenger activity for aldolase A present on membrane-bound poly @ somes might be due to the contamination of this fraction by 10 free polysomes. 10 As a next step, we investigated whether mRNA for aldol a ase A is present as free mRNP particles in the cytoplasm. E AH 7974 cells were fractionated into crude polysomes and 9 cell sap according to the method of Zahninger et a!. (38), and ANA extracted from each fraction was assayed for 6 the aldolase A messenger activity in the wheat germ extract after partial purification of RNA by passage through a 0 50 100 poly(U)-Sepharose column. There was no detectable mes RNA (pg/mi) senger activity for the synthesis of aldolase A in RNA obtained from the supemnatant fraction (Table 3). The me Chart 2. Synthesis of total released polypeptides and aldolase A by poly(A)-containing RNA extracted from total polysomes of AH 7974 cells. suits shown in Table 3 also suggest the presence of a Protein-synthesizing assay using extracts from wheat germ was carried out significant quantity of poly(A)-containing RNA which pos with varying amounts of RNA. One @.tIofthe supernatant after centrifugation sesses messenger activity in the supemnatant fraction as at 180,000 x g far 60 mm was processed for the measurement of total messenger activity ( 0). The rest of the incubation mixture served for the shown previously (37, 38). measurement of aldolase A synthesized as described under â€˜â€˜Materialsand Chart 2 indicates the dose-response curve of poly(A) Methodsâ€•(â€¢).

504 CANCER RESEARCH VOL. 39

crosomal supennatant fraction suggests that there is no regulatory mechanism at this step for the synthesis of . aldolase A. This evidence, taken together with the above 3 finding that there is no messenger activity for aldolase A on polysomes of adult matliver, could imply that the expression : ofaldolaseAgeneisunderatranscriptionalcontrol.This t: speculation is based on the assumption that the messenger

.â€˜ activity observed in vitro protein-synthesizing systems me flects the quantity of mRNA as reported previously (6, 25).. U Although it is difficult to make a direct comparison ,< between values of translation efficiency observed at the I, 4 C different laboratories, the fact that the incorporation of

V U, leucine into aldolase A represents only 0.02% of the total a 3 E incorporation by poly(A)-containing RNA shows a signifi

4 2@ cant contrast with the value obtained in the case of the a synthesis of aldolase A in vitro by chicken muscle polysomal a to RNA (approximately 1%) (16). This difference might be due 0 V to the predominant synthesis of the aldolase A subunit 4 6 10 molecule in chicken muscle, which represents about 6% of Fraction Number the total protein synthesis in vivo (16). On the other hand, the figure of 0.02% is within the same order of magnitude Chart 3. Analysis of aldolase A mRNA of AH 7974 cells by sucrose gradient as that observed in the case of translation in vitro of centrifugation . Polysomal RNA (400 @g)wascentrifuged after heat treatment in linear 5 to 20% sucrose gradients containing 3 m@Tris-HCI (pH 7.6)-3 mM tryptophan oxygenase (13) and tyrosine aminotnansferase EDTA-70% formamide in a Beckman SW27 rotor at 27,000 rpm far 72 hr. (22). The lower efficiency of translation of aldolase A by - - - -, absorbance at 260 nm; arrows, positions of 18 and 28S rRNA's. Total messenger activity (0); aldolase A messenger activity (â€¢). mRNA than by polysomes (0.1%) might be caused by partial degradation of aldolase A mRNA during RNA extraction and sharp peak at around 175, whereas the total messenger storage. activity appeared also as a sharp peak but at about 165. There always exists the technical problem of how to avoid The molecular weight of aldolase A mANA was calculated completely the aggregation of mRNA's with other mRNA's to be 5.8 x 10@from the relative migration distance of 28 or nRNA's. As observed in the present study and others (12, and 185 nRNA's. When RNA was analyzed in a similar way 18), the use of formamide in the sucrose density gradient as in the above experiment but without formamide, the enables one to make an estimation of the chain length of peak of the messenger activity for aldolase A was found at mRNA. The molecular weight of aldolase A mRNA has been the position slightly larger than 18S nRNAwith an approxi calculated at 5.8 x 10@,corresponding to approximately mate molecular weight of 7.5 x 10@(datanot shown). 1650 nucleotides. After subtracting 150 nucleotides, on the average, for the poly(A) segment, 1,500 nucleotides could DISCUSSION represent the transcribed region. Since rabbit muscle aldol ase A, the molecular weight of which (40,000) is the same Evidence from numerous studies reveals that secretory as that of aldolase A of AH 7974 cells (27), has been shown proteins are synthesized primarily on membrane-bound to be composed of 361 amino acid residues (15), the polysomes as exemplified by albumin (37), other plasma minimum length for the coding region is 1100 nucleotides. proteins (10), and amylase (17), whereas cytosol proteins Therefore, the noncoding region of aldolase A mRNA is such as anginase (32) and catalase (33) are synthesized on about 400 nucleotides in length, which is longer than that free polysomes. In certain cases, however, this distinction reported for mANA's for globin (24) or the immunoglobulin is not strict. Aldolase A can be considered, from its catalytic light chain (21), but quite comparable with that obtained for nature to be located in the cytoplasm. Recently, it has been mANA's for enzyme proteins such as tyrosine aminotrans shown that aldolase A localizes in the cytoplasm of mat fenase and tryptophan oxygenase (12). hepatoma cells from immunopenoxidase studies (11). The As a next approach, a model is required in which the predominant presence of aldolase A messenger activity on isozymic pattern can be modulated during the course of free polysomes could be related to the location of the cellular transformation by oncogenic viruses (26, 30) or of aldolase A molecule. On this point, there is an argument carcinogenesis by chemicals (7, 31). In this connection, the about the localization of aldolase A inside the cell, i.e., the study on mANA coding for aldolase B, which decreases or association with endoplasmic reticulum (9) and the pres disappears during hepatocarcinogenesis (31), will be nec ence in the soluble supemnatant fraction (2). essary to investigate the control mechanism of biosynthesis Although the precise nature of cytoplasmic mRNP parti and the degradation of both isozyme mRNA's and isozymes des still remains to be determined, a number of mANA's in tumor cells. coding for various proteins such as femnitin(38), actin (4), and myosmn (5) are present in this state. One possible REFERENCES explanation for the presence of mRNP particles could be a 1. Adelman, M. R.,. Blabel, G., and Sabatini, D. D. An Improved Cell storage function of the precursor of polysomal mANA. The Fractionation Procedure far the Preparation of Rat Liver Membrane absence of messenger activity for aldolase A in the postmi bound Ribosomes. J. Cell Biol., 56: 191-205, 1973.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Characterization of Messenger RNA for Fructose 1,6-Bisphosphate Aldolase A Isozyme of Rat Ascites Hepatoma AH 7974 Cells

Shigeru Sakiyama, Namiko Akiba and Shinji Fujimura

Cancer Res 1979;39:502-506.

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