overexpression ofmousep140subunit ofreplication ...cgd.aacrjournals.org/cgi/reprint/7/3/319.pdf ·...

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Vol. 7, 319-326, March 1996 Cell Growth & Differentiation 319 Overexpression of Mouse p140 Subunit of Replication Factor C Accelerates Cellular Proliferation’ S. Jaharul Haque,2 Heiko van der Kuip, Aseem Kumar, Wafter E Aulitzky, Michael N. Rutherford,3 Christoph Huber, Thomas Fischer, and Bryan R. G. Williams Department of Cancer Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 [5. J. H., A. K., M. N. A., B. R. G. W.], and Department of Hematology, Medical Clinic Ill, The Johannes-Gutenberg-University School of Medicine, Langenbeckstr. 1 , 55131 Mamnz, Germany [H. v. d. K., W. E. A., C. H., T. F.] Abstract Screening of a murine cDNA expression library with an IFN-stimulated response element (ISRE), as a recognition site DNA probe, resulted in the isolation of a cDNA encoding a polypeptide of 1145 amino acids designated ISRE-binding factor-I . This was subsequently shown to be identical to the M 140,000 subunit of replication factor C (RFC). RFC is required, along with the proliferating cell nuclear antigen and DNA polymerase &, for the synthesis of the leading strand during DNA replication. RFC exhibits a structure-specffic DNA-binding activity that has been localized to its Mr 140,000 subunit (p140). Sequence- specific binding of this polypeptide to the ISRE occurs only with low affinity. Based on DNA-binding activity of the truncated RFC-p140 encoded by the partial cDNA isolated, the DNA-binding domain of this polypeptide was mapped to a region encoded by amino acids 366 to 540. Transfection of NIH 3T3 cells with an expression plasmid containing murine RFC-p140 driven by cytomegalovirus early promoter led to the establishment of stable cell lines that expressed a 2.5- to 3.0-fold higher RFC-p140 protein level in comparison with control cells. The stable clones exhibited significantly accelerated cell proliferation, indicating that RFC-p140 is the limiting subunit of an active RFC complex in normal cells. Received 9/6/95; revised 1 1/28/95; accepted 12/20/95. 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 in accordance with 18 U.S.C. Section 1734 solely to mdi- cate this fact. 1 This work was partially supported by Grant POi-CA62220 from the National ‘Cancer Institute, NIH, and Grant 405/1-4 from the German Research Council (Deutsche Forschungsgemelnschaft). 2 To whom requests for reprints should be addressed, at Department of Cancer Biology, Acorn NN1-i2, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 445-6622; Fax: (216) 445-6269. 3 Present address: Department of Pathology, Kingston General Hospital, 37 Stuart Street, Kingston, Ontario, Canada. Introduction In eukaryotic cells, DNA replication occurs only during the S phase of the cell cycle by a multitude of highly controlled mechanisms (1 , 2). A faithful duplication of DNA is essential for correct maintenance of genetic information. Since double- stranded DNA genomes contain anti-par cDNA strands, replication in eukaryotes occurs by a semicontinuous mecha- nism; one DNA strand is replicated continuously at the replka- tion fork and is called the leading strand, whereas the other is replicated discontinuously and is called the lagging strand (re- viewed in Refs. 3 and 4). This process occurs asymmetrically and involves two different members of the eukaryotic DNA polymerase family, the polymerase a and the polymerase 6. At the leading strand, polymerase has to be highly processive, whereas polymerase a at the lagging strand must be able to bind and retract from DNA frequently after each Okazald frag- ment (3, 4). In addition, effective replication requires PCNA,4 RFA, and RFC as auxiliary proteins (5-7). RFC is composed of five polypeptide subunits of Mr 140,000, Mr 40’000’ Mr 38’000’ Mr 37,000, and M, 36,000 (810). TO9eth& with PCNA and possibly RFA, RFC is a component of the protein complex responsible for synthesis of the leading strand (7, 1 1-14). By virtue of its Mr 140000 subunit, RFC binds specifically to the primer templatejunction (5, 15). AlP is bound by the M, 40’000 subunit of RFC (12). Recently, the mouse and human cDNAs encoding the Mr 140,000 subunit of RFC have been cloned (14, 16-17). It has been shown that replication factors are tightly regulated by the cell cycle; RFA from both human and yeast cells is phosphorylated by a member of the cdc2 kinase family at the time when the DNA replication apparatus is activated in the cell cycle (18, 19). This suggests that at the G,-S transition, a cell cycle-regulated protein kinase phosphorylates RFA and possi- bly other replication factors, leading directly to initiation of DNA replication (20). In addition, the amount of RFA protein ex- pressed appears to be regulated by the cell cycle (21). Exper- iments investigating human RFA and PCNA expression pre- sented evidence that these replication factors are overproduced in transformed (kidney cell line 293) and in tumor cells (HeLa cell line) (21). In yeast, the expression of replication factors has been found to be coordinated with the cell cycle (22). The biological consequence of experimentally induced deregulation of replication factorexpression in mammalian cells is not known. We have used overexpression of a mouse RFC-p140 cDNA in NIH 3T3 cellsto study the effect ofthis protoln on proliferation 4 The abbreviations used are: PCNA, proliferating cell nuclear antigen; RFA and RFC, replication factors A and C; ISRE, IFN-stimulated response element; EMSA, electrophoretic mobility shift assay; IBF-i , IFN-stimu- lated response element binding factor 1; CMV, cytomegalovirus; IPTG, isopropyfthio--D-gaIactoside; GST, glutathione S-transferase.

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Page 1: Overexpression ofMousep140Subunit ofReplication ...cgd.aacrjournals.org/cgi/reprint/7/3/319.pdf · motifs,suchaszincfingers,helix-loop-helix, helix-turn-helix, or leucinezipper(25)

Vol. 7, 319-326, March 1996 Cell Growth & Differentiation 319

Overexpression of Mouse p140 Subunit of ReplicationFactor C Accelerates Cellular Proliferation’

S. Jaharul Haque,2 Heiko van der Kuip,Aseem Kumar, Wafter E Aulitzky,Michael N. Rutherford,3 Christoph Huber,Thomas Fischer, and Bryan R. G. WilliamsDepartment of Cancer Biology, Research Institute, Cleveland ClinicFoundation, Cleveland, Ohio 44195 [5. J. H., A. K., M. N. A.,B. R. G. W.], and Department of Hematology, Medical Clinic Ill, TheJohannes-Gutenberg-University School of Medicine, Langenbeckstr.1 , 55131 Mamnz, Germany [H. v. d. K., W. E. A., C. H., T. F.]

AbstractScreening of a murine cDNA expression library with anIFN-stimulated response element (ISRE), as arecognition site DNA probe, resulted in the isolation ofa cDNA encoding a polypeptide of 1145 amino acidsdesignated ISRE-binding factor-I . This wassubsequently shown to be identical to the M� 140,000subunit of replication factor C (RFC). RFC is required,along with the proliferating cell nuclear antigen andDNA polymerase &, for the synthesis of the leadingstrand during DNA replication. RFC exhibits astructure-specffic DNA-binding activity that has beenlocalized to its Mr 140,000 subunit (p140). Sequence-specific binding of this polypeptide to the ISRE occursonly with low affinity. Based on DNA-binding activity ofthe truncated RFC-p140 encoded by the partial cDNAisolated, the DNA-binding domain of this polypeptidewas mapped to a region encoded by amino acids 366to 540. Transfection of NIH 3T3 cells with anexpression plasmid containing murine RFC-p140 drivenby cytomegalovirus early promoter led to theestablishment of stable cell lines that expressed a 2.5-to 3.0-fold higher RFC-p140 protein level in comparisonwith control cells. The stable clones exhibitedsignificantly accelerated cell proliferation, indicatingthat RFC-p140 is the limiting subunit of an active RFCcomplex in normal cells.

Received 9/6/95; revised 1 1/28/95; accepted 12/20/95.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was partially supported by Grant POi-CA62220 from theNational ‘Cancer Institute, NIH, and Grant 405/1-4 from the GermanResearch Council (Deutsche Forschungsgemelnschaft).2 To whom requests for reprints should be addressed, at Department ofCancer Biology, Acorn NN1-i2, Research Institute, Cleveland ClinicFoundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216)445-6622; Fax: (216) 445-6269.3 Present address: Department of Pathology, Kingston General Hospital,37 Stuart Street, Kingston, Ontario, Canada.

IntroductionIn eukaryotic cells, DNA replication occurs only during the Sphase of the cell cycle by a multitude of highly controlledmechanisms (1 , 2). A faithful duplication of DNA is essential forcorrect maintenance of genetic information. Since double-stranded DNA genomes contain anti-par� cDNA strands,replication in eukaryotes occurs by a semicontinuous mecha-nism; one DNA strand is replicated continuously at the replk�a-

tion fork and is called the leading strand, whereas the other isreplicated discontinuously and is called the lagging strand (re-

viewed in Refs. 3 and 4). This process occurs asymmetricallyand involves two different members of the eukaryotic DNApolymerase family, the polymerase a and the polymerase 6. Atthe leading strand, polymerase � has to be highly processive,whereas polymerase a at the lagging strand must be able to

bind and retract from DNA frequently after each Okazald frag-ment (3, 4). In addition, effective replication requires PCNA,4RFA, and RFC as auxiliary proteins (5-7). RFC is composed offive polypeptide subunits of Mr 140,000, Mr 40’000’ Mr 38’000’Mr 37,000, and M, 36,000 (810). TO9eth& with PCNA andpossibly RFA, RFC is a component of the protein complexresponsible for synthesis of the leading strand (7, 1 1-14). Byvirtue of its Mr 140�000 subunit, RFC binds specifically to theprimer templatejunction (5, 15). AlP is bound by the M, 40’000subunit of RFC (12). Recently, the mouse and human cDNAsencoding the Mr 140,000 subunit of RFC have been cloned (14,16-17).

It has been shown that replication factors are tightly regulatedby the cell cycle; RFA from both human and yeast cells isphosphorylated by a member of the cdc2 kinase family at thetime when the DNA replication apparatus is activated in the cellcycle (18, 19). This suggests that at the G,-S transition, a cellcycle-regulated protein kinase phosphorylates RFA and possi-

bly other replication factors, leading directly to initiation of DNAreplication (20). In addition, the amount of RFA protein ex-pressed appears to be regulated by the cell cycle (21). Exper-iments investigating human RFA and PCNA expression pre-sented evidence that these replication factors areoverproduced in transformed (kidney cell line 293) and in tumor

cells (HeLa cell line) (21). In yeast, the expression of replication

factors has been found to be coordinated with the cell cycle(22). The biological consequence of experimentally inducedderegulation of replication factorexpression in mammalian cellsis not known.

We have used overexpression of a mouse RFC-p140 cDNAin NIH 3T3 cellsto study the effect ofthis protoln on proliferation

4 The abbreviations used are: PCNA, proliferating cell nuclear antigen;RFA and RFC, replication factors A and C; ISRE, IFN-stimulated responseelement; EMSA, electrophoretic mobility shift assay; IBF-i , IFN-stimu-lated response element binding factor 1 ; CMV, cytomegalovirus; IPTG,isopropyfthio-�-D-gaIactoside; GST, glutathione S-transferase.

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320 AFC-p140 Overexpression Increases Cell Proliferation

and cell growth. A cDNA clone encoding the M� 140�000 sub-unit of mouse RFC was isolated by screening a Agtl 1 library

with an oligonucleotide duplex containing the ISRE of the mu-rine 2’,S’-adenylate synthetase gene (23). Fusion proteins of

munne RFC-p140 were expressed in bacteria and used to raise

polyclonal antibody that specifically recognizes this polypep-tide. The expression pattern of munne RFC-p140 gene wasanalyzed in Northern blot experiments. Cell proliferation assaysand �l-lJthymidine incorporation measurements in stable trans-

fectants of munne RFC-pl 40 demonstrate that overexpressionsignificantly enhances cell proliferation.

ResuftsIsolation of the Large Subunit of Murine RFC. To isolateISRE-binding proteins, we screened a munne cDNA expressionlibrary using a trimer of a 29-bp ISRE derived from the munne2’,S’-adenylate synthetase gene (23) as a recognition site DNAprobe. We isolated a recombinant Agtl 1 clone that encodes aMr 140,000 �-galactosidase fusion protein (Fig. 1A). This fusionprotein was found to bind a monomer ISRE (29-bp) oligonucle-otide duplex on a Southwestern blot (Fig. 1B). The cDNA insertof the clone was 526-bp long, and its nucleotide sequence wasopen in one reading frame encoding 175 amino acids. Partiallypurified p-galactosidase fusion protein exhibited an ISRE-bind-ing activity in solution, as measured by EMSA (Fig. 1C). How-ever, this binding was competed by relatively high concentra-tions of unlabeled oligonucleotide probe, indicating a low

affinity DNA-protein interaction.Using this partial cDNA, we screened the same and other

cDNAlibraries and isolated several overlapping clones(data notshown). The restriction maps and nucleotide sequence analy-

585 of these clones allowed us to construct a composite cDNAof 361 5-bp length.5 Northern blot analysis revealed that thiscDNA hybridizes with two mRNA species of sizes 4.0 and 4.8

kb. Thus, the composite clone is not a full-length cDNA, but it

contains the full-length coding sequence of 1 145 amino acids(calculated molecular weight, Mr 127,446). We described thisprotein as an ISRE-binding factor, IBF-1 (24). While character-

izing the DNA-binding activity of IBF-1 , a homology search inthe Genbank revealed that IBF-1 is almost 100% homologousto the Mr 140,000 large subunit of the munne RFC (RFC-p140)

(16). The central 950 amino acids of IBF-1/RFC-p140 (aminoacids 101 to 1050) were expressed in bacteria as a hexahisti-dine-tagged fusion protein of an approximate molecular weight

of M, 1 15,000 and was partially purified by nickel-agarose af-finity chromatography. This bacterially expressed protein wasfound to bind to the ISRE oligonucleotide duplex on a South-western blot (data not shown). It also binds to the ISRE insolution, as measured by EMSA, and the DNA-r�rotein complex

was supershifted by specific antiserum raised against thispolypeptide (Fig. 1D). This histidine-tagged fusion protein, how-ever, was found to bind other unrelated oligonucleotide probes(data not shown). Nonspecific interaction of RFC-p140 withDNA has also been shown by others (16). It is worth mentioningthat the IBF-1 cDNA clone5 we isolated contains an extra 14

5 The GenBank Accession number of the IBF-1/AFC-p140 sequence isU07157.

amino acids at position 187. This is not a cloning artifact sincethis sequence is present in more than one ovetlapping inde-pendent clone. The DNA-binding domain of RFCp-140/IBF-1was mapped to a central region of the polypeptide, rangingfrom amino acid 366 to 540, based on EMSA using: (a) a-ga-lactosidase fusion protein (Fig. 1 , B and C); and (b) staphylo-coccal protein A fusion protein (data not shown). The DNA-binding domain of this protein does not contain any definedmotifs, such as zinc fingers, helix-loop-helix, helix-turn-helix, orleucine zipper (25).

Establishment of Stable Transfectant Cell LinesExpressing RFC-p140 Protein. A pUC19-based eukaryoticexpression vector driven by the CMV early promoter ex-pressing pRFC-1 40/IBF-1 was transfected into NIH 3T3cells, along with a neomycin resistance-mediating plasmidas described (26). The expression vector lacking the RFC-p1 40/IBF-1 insert was used as a control. Positive stabletransfectant clones were identified by Northern blot analysis,and from 36 clones screened, 7 were found to expressexogenous RFC-pl 40/IBF-1 (data not shown). Steady-statelevels of the RFC-pl 40 mRNA in the stable transfectantclones are shown in Fig. 2. In control cells transfected withvector lacking the p1 40-RFC insert (clones 344-1 9 and 344-15) and in cells transfected with p140-RFC cDNA, two mo-lecular species of RFC-pl 40/IBF-1 mRNA were detected(Fig. 2). Positive transfectant clones were identified by over-expression of the faster-migrating mRNA species corre-sponding to an approximate size of 4.0 kb (Fig. 2: clones34-302-15, -17, and -5). The highest levels of RFC-p140mRNA expression were observed in clones 34-302-5 and 34-302-1 7, and these were subjected to further analyses.

Protein expression of RFC-p140 was assessed usingWestern blot analysis. A comparative Western blot analysisof RFC-pl 40 from whole-cell extracts of 1 0 x 106 cells each,

by using the polyclonal antiserum specifically recognizing themurine RFC-p140, revealed a single band with an apparentmolecular mass of 140 kD (Fig. 3A). The specificity of thedescribed band was examined by parallel incubation withpreimmune serum (Fig. 3B). Positive stable transfectantclones 34-302-5 and 34-302-1 7 revealed high levels of ex-pression of RFC-pl 40 protein (Fig. 3A, Lanes 3 and 4) ascompared with the endogenous RFC-p140 protein level ofcontrol cells (Fig. 3A, Lanes 1 and 2). Equal loading wasverified by Ponceau staining of the membrane prior to anti-body probing (data not shown). Quantitative analysis usingdensitometnc scanning demonstrated a 2.5- to 3.0-fold in-crease in the intensity of the M, 140,000 band in positiveclones. There was no obvious difference in the apparentmolecular mass of exogenous RFC-pl 40 in comparison withthat of endogenous protein. Thus the positive transfectantclones exhibit markedly increased levels of RFC-pl 40 pro-tein expression compared with the control NIH 3T3 cells.

RFC-p140 Expression Correlates with Cell Prolifera-tion. To determine the possible effects of RFC-pl 40 over-expression on cellular proliferation, the growth rates of ex-ogenous RFC-pl 40-expressing clones and control cloneswere compared. Control clones 344-1 9 and 344-4 presentednearly identical cell numbers when proliferation was followedfor 7 days (Fig. 4). During this time period, cell numbers

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1�1

,t, .� , . � . : �

t�_ � J*._J-

IP

140

120

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ow...�

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Cell Growth & Differentiation 321

A. 1 2 3 4 B. 1234

Fig. 1 . A, Coomassie blue stainof p-galactosidase/RFC-p140protein in SDS-polyacrylamidegel. Partially enriched fractionsof lysogen extracts derived fromIPTG-induced (Lane 1) and unin-duced (Lane 2) culture of recom-binant clone and IPTG-induced(Lane 3) and uninduced (Lane 4)culture of Agti 1 control clonewere resolved on a 7.5% SDS-polyacrylamide gel. The gel wasstained with Coomassie blue. Ar-rowheads, Mr 1 40,000 recombi-nant protein and Mr 120,000/3-galactosidase protein. B,Southwestern blot analysis of�-galactosidase-RFC-pi40 fu-sion protein. Partially enrichedfractions of �3-galactosidase-AFC-p140 fusion protein (Lane1, IPTG induced; Lane 2, unin-duced) and crude lysogen ex-tract containing f3-galactosi-dase-RFC-pl 40 fusion protein(Lane 3, IPTG induced; Lane 4,uninduced) were probed with anend-labeled monomer ISRE oIl-gonucleotide duplex. : Arrow-head, the induced fusion protein.C, EMSA of f3-galactosidase-RFC-p140 fusion protein. Fivehundred ng of the partially en-riched fusion protein and 0.2 ngof end-labeled ISRE probe wereused in each reaction. Competi-tions were carried with 100-,200-, 500-, 1000-, and 1500-foldmolar excess of cold ISRE oligo-nucleotide duplex in Lanes 2-6.D, EMSA of hexahistidine-tagged RFC-p140. Twenty-fiveng of purified protein and 0.2 ngof end-labeled ISRE probe wereused in each reaction. Preim-mune serum (0.2 �.tl) was addedbefore and after the addition ofprobe to binding reactionsloaded in Lanes 1 and 2, respec-tively, and 0.2 �tl anti-RFC-p140serum was added to binding re-actions before and after the ad-dition of probe and loaded inLanes 3 and 4, respectively.*, supershifted DNA-proteincomplex.

increased from 6,000 cells plated on day 0 to 63,875 and68,750 cells, respectively, on day 7. In contrast, clones 34-

302-1 7 and 34-302-5 exhibited a significant accelerated

growth rate revealing 163,750 cells and 208,125 cells afterincubation for 7 days. This experiment was repeated three

times, and very similar results were obtained. In positive

transfectant clones, proliferation appeared to correlate with

the expressed level of RFC-pl 40 protein. Clone 34-302-5

consistently exhibited a slightly higher growth rate in corn-

parison with the clone 34-302-1 7 and also revealed the

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plasmid

Clone number I 17 1 5 I

RF-C 140

I1........j....:6 � 8 � 9 �

34-302

10 � 12 � 13 � 15

344

� 22 123 19 � 15

CHO-B

a

A B

344 I 34�3O2�Jplasmid

Clone number

RF-Cp140 -.

19 � 4 � 5 1 17 � marker(kDa)

-200.0

97.4

4-. 69.0

a- 46.0

.-. 30.0

322 RFC-pl 40 Overexpression Increases Cell Proliferation

Fig. 3. Western blot analysis ofAFC-p140 from positive stabletransfectant clones (34-302-5and 34-302-i 7) and controls(344-19 and 344-4). Whole-cellextracts of 1 0 x 106 cells eachwere subjected to SDS-PAGE(7.5% gels) and blotted onto ni-trocellulose membranes. ForWestern blotting, strips were in-cubated with RF-pl 40-specificantiserum (A) or with preimmuneserum (B) to reveal the extent ofnonspecific binding. Molecularweight markers are indicated.

Fig. 2. Northern blot analysis ofp1 40-RFC-specific mRNA levelsof stable clones transfected witha p1 40-RFC expression vector(plasmid 34-302) and with vectorlacking p140-AFC insert (plas-mid 344). Size-fractionated totalcellular ANA (20 �g) from stabletransfectant clones was blottedand hybridized to the indicated32P-labeled cDNA probes.

higher expression level of RFC-p140 protein, as judged by

Western blot analysis. Similar data were obtained by using

[3H]thymidine incorporation assays of proliferating cells fol-

lowed for 5 days. Positive transfectant clone 34-302-5

showed significantly enhanced incorporation of [3H]thymi-dine in comparison with the control clone on day 5 (Fig. 5).We noted that the initial rate of [3H]thymidine incorporation

on day 2 is very similar between RFC-p140-overexpressing

clones and control clones (Fig. 5). Therefore, the difference in

the total amount of thymidine uptake on day 5 is due to the

difference in the number of cells in the plate (Fig. 4), although

the experiment was started with the same number of cells in

each plate. Cell cycle analyses of RFC-pl 40-overexpressing

clones and control clones indicated that overexpression of

the large subunit of RFC resulted in a higher percentage of

cells in S phase and a reduced percentage of cells in G1 (Fig.

6). Taken together, these data clearly indicate that RFC-

p1 40-overexpressing cells exhibited a differential growth

pattern compared with the control cells.Cells overexpressing RFC-p140 did not exhibit obvious

morphological changes. The stable expression of RFC-pl 40

did not result in anchorage-independent growth, as judged

by the soft agar assays. Stable transfectant clones and wild-

type NIH 3T3 cells were seeded out in soft agar at a densityof 7000 cells/mI, as described in “Materials and Methods.”

After 3 weeks, no colony formation was detected (data not

shown). Since anchorage-independent growth frequently ac-

companies malignant transformation, our data suggest that

overexpression of RFC-pl 40 protein does not lead to atransformed phenotype.

DiscussionWe have fortuitously cloned a cDNA encoding the large

subunit of mouse RFC from an expression library by using an

ISRE as a recognition site DNA probe. The DNA-bindingdomain of RFC-p140 was mapped to the central region of

this protein, ranging from amino acid 366 to 540, as revealed

by EMSA analyses using the ISRE as probe. However, the

significance ofthis finding for the functional role of RFC-p140

in vivo is unknown. DNA-binding activity of RFC-p140 hasbeen reported already by Luckow et a!. (16).

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2

Tlme,d

300�

T

200

.0Ez

C.)

Fig. 4. Analysis of cellular proliferation of positive transfectant clones(3T3 34-302-17 and 3T3 34-302-5) and of control clones (3T3 344-19 and3T3 344-4). Cell numbers were determined at the indicated times using ahemocytometer. One representative experiment of a total of three wasshown here.

400 . . .

I ±

I ,

0 � �4�:�\

Fig. 5. �H]Thymidine incorporation assay of clones 344-19 (control) and34-302-5 (AFC-pl 40 overexpressing). Cells were incubated in triplicates,and the rate of DNA synthesis was monitored at times indicated asdescribed in “Materials and Methods.”

This report describes several novel aspects of the func-

tional relationship of RFC-pl 40 expression and control of cellproliferation and growth. The data presented here show that

overexpression of the p1 40 subunit of RFC results in anaccelerated cell proliferation. With a CMV early promoter-

driven murine cDNA that encodes a M� 140,000 subunit of

RFC, we carried out stable transfection of NIH 3T3 fibro-blasts. Positive transfectant clones revealed a significant

100

�Th

Cell Growth & Differentiation 323

acceleration in cellular growth, which closely correlated with

the 2.5- to 3.0-fold higher RFC-p140 protein expression ob-

served in Western blot analysis. In positive transfectantclones, no evidence that the exogenously expressed RFC-

p140 differs from the endogenous form could be detected.

This indicates that there are no gross structural differences

between the endogenously expressed RFC-p140 protein

and the protein expressed by the cDNA clone in fibroblasts.A contribution of the amount of replication factor expressed

to the control of DNA replication and cellular proliferation

was already suggested by analysis of RFA expression within

the cell cycle and in transformed and malignant cells (21).

Because RFA expression data demonstrate that phosphor-ylation of replication factors at the G1-S-phase transition isthe crucial step in activation of DNA replication, it seems

8 conceivable that an up-regulated protein level facilitates thisreaction by simply increasing the concentration of the en-

zyme substrate. This hypothesis is supported by the cellcycle analyses, which are consistent with an accelerated

G1-S transition. However, the precise mechanisms by which

the overexpression of RFC-pl 40 results in an increased pro-

liferation rate remain to be defined. RFC-pl 40-overexpress-ing clones may provide a powerful tool to evaluate the con-tribution of replication factors to the control of the cell cycle.

An inherent potential problem in expression studies is thedifficulty in distinguishing between the effects of deregulated

expression and overexpression per Se. A close correlation ofacceleration in cellular proliferation with the expressed pro-

tein level suggests that RFC-p140 is the limiting subunit of

RFC and that high levels of a functional RFC multiproteincomplex promote DNA replication and cellular proliferation.This suggestion is indirectly supported by recent data dem-

onstrating that the distribution of RFC-p140 changes signif-icantly during the cell cycle, with a peak of abundance in the

nuclei of cells in G1 (27). Similarly, in vitro, it could be shown

that efficacy of replication of SV4O DNA correlates well with

the amount of RFC activity added to the DNA replicationassay (7). DNA replication is a highly complex process in-volving the participation of a number of enzymes and acces-

sory factors in which the multi-subunit protein RFC functionsto recognize the primed template and load a second acces-

soy protein PCNA to the complex in the presence of ATP;

this complex binds polymerase �, resulting in efficient chain

elongation (1 3, 28-30). Thus, the ordered recruitment of

different proteins to replication complex assembly seems to

exclude the possibility of causing inhibition of DNA replica-

tion by sequestration of an essential component by an over-

produced factor like RFC-pl 40. Whether deregulation of

RFC-pl 40 can be detected in malignant disease, e.g.,

chronic myelogenous leukemia, in which deregulation of cell

cycle events is believed to be of pathogenic significance, is

a subject for additional studies.

Materials and MethodsLibrary Screening. A Agtl 1 cDNA library was prepared from poly(A)’ANA of a munne pre-B cell line (70Z/3). The primary partial cDNA encod-ing the ISRE-binding domain of RFC-p140 was cloned from this library by

using a tnmer of the 29-bp ISRE of the munne 2’,S’-adenylate synthetase

gene (5’-CCCTTCTCGGGAAATGGAAACTGAPA�TC-3’) as a recognitionsite probe (31 , 32). Overlapping clones were isolated from the same library

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A

460

400

B

I Gi: 68,9%I S: 21,8%

G2/M : 9,3%

160

60

n

480

400

320

240

160

60

0

01: 49,1%S: 37,6%

G2/M: 13,3%

(I 80 ieo 240 320 400 48

DNA Content

80 1 0 240 320 400 48

DNA Content

324 RFC-pl 40 Overexpression Increases Cell Proliferation

I..I 320

- 240

U

NIH 3T3 344 19

z

U

NIH 3T3 34-302 5

Fig. 6. Cell cycle analysis of clones 344-19 (control) and 34-302-5 (RFC-pi4O overexpressing). Propidium iodide-stained cells were analyzed f�rtheir DNAcontent in a fluorescence-activated cell sorter. The percentage of cells in different phases of the cell cycle is indicated.

and another munne cDNA expression library (a gift from Dr. John A.

Hassell, McMaster University, Hamilton, Ontario, Canada) by using the526-bp partial cDNA insert as a hybridization probe.

Expression and PurificatIon of Fusion ProteIns. The lysogens of therecombinant and nonrecombinant (control) Agtl 1 clones were isolated

according to standard procedure. The fusion protein and the f3-galacto-sidase protein were induced by IPTG, and bacterial extracts were partiallyenriched by 33% ammonium suifate fractionation. The apparent molec-ular weight of the fusion protein was determined to be Mr 1 40,000 byCoomassie blue staining of the SDS-polyacrylamide gel.

A GST-RFC-pi4O/IBF-i fusion construct was prepared by ligation of a

blunted i266-bp BamHl-EcoRI fragment to the EcoRl (blunted) site ofGST-expression vector pGEx-2T. The fusion protein was expressed inEscherichia coil (DH5-a) by IPTG induction and was purified by affinity

chromatography using glutathione-Sepharose beads (33).A BamHl cDNA fragment encoding amino acid 101 to 1050 of murine

RFC-pi4O/1BF-i was cloned in the bacterial expression vectorpQEi0 intothe BamHI site and was expressed as a hexahistidine-tagged fusionprotein of Mr 1 i5,000. The IPTG-induced insoluble fusion protein waspurified from the bacterial extract under denatunng conditions by nickel-agarose affinity chromatography, according to the manufacturer’s

(Quiagen) protocol.

Southwestern Blot Analysis. A crude lysogen extract or its partiallyenriched fraction was mixed with SOS-PAGE sample buffer[50 mp,i Tris-CI(pH 6.8), iOO mM OTT, 2% SOS, 0.1 % bromophenol blue, and 10%glycerol] and heated at 37”C for 5 mm and loaded on 7.5% SOS-poly-

acrylamide gel. Proteins were transferred onto the nitrocellulose mem-brane at 4#{176}Cusing Southwestern transfer buffer(25 m� Tns base and 190mM glycmne). The membrane was subjected to denaturation and renatur-

ation, followed by probing with an end-labeled 29-bp ISRE oligonucleo-tide duplex as described (23).

EMSA. EMSA was performed according to the protocol describedpreviously (26).

Preparation of Polyclonal Antibody. The GST-RFC-pi4O,IBF-i pro-ten from iOO ml bacterial culture was affinity purified by glutathione-Sepharose beads as described above, and the fusion protein was re-

solved in a 7.5% preparative SDS-polyacrylamide gel. The gel was brieflystained with 0.05% aqueous solution of Coomassie blue and destained inwater for 2 h. A Mr 73,000 protein band containing 200-250 �g of

GST/AFC-pi4O was excised from the gel, crushed in sterile PBS, andinjected s.c. into two rabbits. Injections were repeated five times at2-week intervals. A week after the last injection, serum was tested for itsability to detect cellular RFC-pi4O. Whole-cell extracts were prepared (26)and were resolved on a 7.5% SDS-PAGE, transferred electrophoreticallyat 4#{176}Conto a nitrocellulose membrane, incubated with immune serum,followed by 125l-Iabeled protein A [ICN; 50,000 cpm/ml in 5% milk inTBS-T (iO m�.i Tris Cl, pH 8.0, i50 mp.i NaCI, 0.1 % Tween 20)], andexposed to a X-ray film as descnbed below.

Construction of Expression Plasmids. The composite cDNA of3615-bp murine RFC-pi4O/IBF-i was linked to a NotI linker and sub-cloned into the Nofl site of an eukaryotic expression vector containingCMV early promoter, SV4O polyadenylation, and transcriptional termina-tion signals. The resulting expression vector encoding murine RFC-pi4O

and vector lacking the RFC-pi4O insert were designated 34-302 and 344,respectively. The plasmid pSV2neo used as a selective marker was de-

scribed elsewhere (26).Cell Culture and Transfections. NIH 3T3 cells obtained from the

American Type Culture Collection (Rockville, MD) were grown in RPMIi640 (Seromed) supplemented with iO% FCS, 50 units/mI streptomycin,and 50 units/mI penicillin. For transfection experiments, cells were seeded

at a density of 2 x i0� cells in a 60-mm Petri dish. Cells were cotrans-fected with 20 � of plasmid DNA containing mouse RFC-p140 cDNA-insert and 2.5 �g of plasmid containing the selective marker (pSv2neo).Control cells were cotransfected with a plasmid lacking the RFC-pi4O

cDNA insert and pSV2neo plasmid. NIH 3T3 cells were transfected with

the indicated expression plasmids, as described previously (34). Cellswere treated for 8 h with a medium-precipitate mixture and washedseveral times with PBS. After a nonselective period of 48 h, cells weregrown in a selective medium containing 400 pg/mI of G4i8 (Boehringer).The medium was changed twice a week for 3-4 weeks, and the mono-

clonal colonies were transferred to small dishes and propagated in 6418-containing medium for analyses.

RNA Extraction and Northern BlotAnalysis. Total RNA was isolated

from cells according to the method described by Chomczynski andSacchi(35). Electrophoretic separation, RNAtransferto nykn membranes(Amersham; Hybond N), and autoradiographic identification were done bystandard techniques (36). Filters were hybridized with RFC-p140 cDNAinsert radiolabeled by random priming method.

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Cell Growth & Differentiation 325

Protein Extraction and western Blot Analysis. Whole-cell extracts

were prepared as described previously (26). The proteins were resolved ina 8% SDS-polyacrylamide gel and electrotransferred onto a nitrocellulosemembrane. The membrane was blocked in a solution (5% nonfat driedmilk, 0.02% sodium azide, and 0.02% Tween 20 in PBS) for 20 h andincubated with immune serum diluted in the blocking solution at a con-

centration of 1 :200. After extensive rinsing in PBS plus 0.02% Tween 20,the membrane was probed with 1251-Iabeled Staphylococcus aureus pro-

tein A and was exposed to DuPont Cronex 4 film at -80#{176}Cas described(37).

Determination of Cell Proliferation. For growth experiments, 6000cells of positive transfectant clones and controls were seeded in medium-sized flasks; culture medium was changed every alternate day. Cell num-bers were determined using a hemocytorneter.

�HJThymIdine Incorporation Assay. The rate of DNA synthesis wasmonitored by incubating the cells in 96-well, flat-bottomed plates (Greiner)

in triplicate with 5000 cells/well in a total volume of 200 �.d RPMI 1640

(Seromed) supplemented with 10% FCS at 37#{176}Cand 5% CO2. After theindicated time, cells were pulsed with 1 �CVweII [�H]thymidmne (DuPont)for 16 h and collected on a PHD-Cell Harvester (Dunn Labortechnik).

[�H]Thymidine incorporated by the cells was quantified by liquid scintil-

lation counting in a beta counter (Beckmann Munich).Cell Cycle Analysis. Cells were grown to semiconfluency, trypsinized,

and washed once in 10% FCS containing medium and twice in samplebuffer(0.1 % glucose in PBS). Cell fixation was carried out in 70% ethanolovernight, and fixed cells were resuspended in 1 ml sample buffer con-taming so �g/mI propidium iodide solution and 100 units/mI RNase A asdescribed (38). The stained cells were analyzed in a fluorescence-activated cell sorter (EPICS XLR; Coulter), and the percentage of cells in

different phases of the cell cycle was determined by using the Multi-Cycleprogram.

Colony Formation in Soft Agar. Cells (7000) wereadded to 1 .0 ml ofplating agar consisting of supplemented RPMI containing 0.33% agar and

poured onto 6-well plates containing a base of 0.5% agar with supple-rnented RPMI. Cells were fed after 10 days with APMI-supplernentedplating agar.

AcknowledgmentsWe thank Drs. Gregory Hannigan, Christine E. Campbell, RobertSilverman, and Ganes Sen for helpful discussions; Dr. John A. Hassell

for providing a cDNA library; and Dr. Elizabeth Sexsmith for raisingantibodies.

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