12-O-Tetradecanoylphorbol-13-acetate and UV Radiation-inducedNucleoside Diphosphate Protein Kinase B Mediates NeoplasticTransformation of Epidermal Cells*

Received for publication, October 1, 2003, and in revised form, November 14, 2003Published, JBC Papers in Press, November 17, 2003, DOI 10.1074/jbc.M310820200

Sung-Jen Wei‡§, Carol S. Trempus‡, Robin C. Ali‡, Laura A. Hansen¶, and Raymond W. Tennant‡

From the ‡National Center for Toxicogenomics, NIEHS, National Institutes of Health, Research Triangle Park,North Carolina 27709 and the ¶Department of Biomedical Sciences, Creighton University School of Medicine,Omaha, Nebraska 68178

The molecular changes associated with early skin car-cinogenesis are largely unknown. We have previouslyidentified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate(TPA) in mouse skin keratinocyte progenitor cells (Wei,S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., andTennant, R. W. (2003) J. Biol. Chem. 278, 1758–1768).Here, we show an induction of a nucleoside diphosphateprotein kinase B (NDPK-B) gene in response to TPA orUV radiation (UVR). TPA or UVR significantly inducedthe expression of NDPK-B both in vivo hyperplasticmouse skin and in vitro mouse JB6 Cl 41-5a epidermalcells. Indeed, this gene was also up-regulated in TPA orUVR-mediated skin tumors including papillomas, spin-dle cell tumors, and squamous cell carcinomas, relativeto adjacent normal skins. Functional studies by consti-tutive expression of nm23-M2/NDPK-B in TPA suscepti-ble JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneo-plastic epidermal cell lines showed a remarkable genedosage-dependent increase in foci-forming activity, aswell as an enhancement in the efficiency of neoplastictransformation of these cells in soft agar but no effect onproliferation in monolayer cultures. Interestingly, sta-ble transfection of the nm23-M2/NDPK-B delRGD orG106A mutant gene in JB6 Cl 41-5a cells selectively ab-rogated NDPK-B-induced cellular transformation, im-plicating a possible Arg105-Gly106-Asp107 regulatory rolein early skin carcinogenesis.

Mouse skin carcinogenesis is a complex multistage processthat progresses through distinct stages of initiation, promotion,and progression to malignancy (1, 2). The molecular changesassociated with the early stages of skin tumor formation haveyet to be determined. Tg�AC mice, which carry the codingregion of the v-Ha-ras oncogene fused to a fetal �-globin genepromoter (3), are considered to be genetically initiated andhave a higher sensitivity to promotional stimuli includingTPA1 (3) and full thickness wounding (4), or carcinogens suchas UV radiation (UVR) (5) and 7,12-dimethylbenz[a]anthra-

cene (6). These features establish the in vivo Tg�AC mousemodel as a valuable tool to study the early stages of skincarcinogenesis.

In an earlier study with a combination of fluorescence-acti-vated cell sorting, switching mechanism at the 5�-end of RNAtemplates cDNA amplification, and mouse cDNA array tech-nology, we identified 11 genes whose expression changed sig-nificantly in �6� CD34� keratinocytes harvested from TPA-treated mice relative to cells from untreated mice. Nine genes,including galectin-7, nm23-M2/NDPK-B, cytoskeletal epider-mal keratin 14, deleted in split hand/split foot gene 1 (Dss1),DNA double strand break repair RAD21 homolog, transcriptiontermination factor 1, thymosin �4, calpactin I light chain, and40 S ribosomal protein SA, were up-regulated, and two genes,apolipoprotein E precursor and acidic keratin complex 1 gene15, were down-regulated by TPA (7). Dss1, a gene associatedwith a heterogeneous limb developmental disorder called splithand/split foot malformation (8), has recently been identified asa novel TPA-inducible gene expressed in keratinocyte progen-itor cells, with possible involvement in early skin tumorigene-sis (7). This novel approach was highly effective in the in vivoidentification of TPA-inducible effector genes that might leadto neoplastic transformation. The protein kinase nm23-M2/NDPK-B was another one of nine TPA-up-regulated genes andwas selected for further characterization.

Nm23 is a large family of structurally and functionally con-served proteins consisting of 4–6 identical subunits of 17–20kDa each, known also as nucleoside diphosphate kinases (ND-PKs; EC 2.7.4.6) (9). NDPKs were originally identified as es-sential housekeeping enzymes required for the synthesis ofnucleoside triphosphates by catalyzing the transfer of the�-phosphoryl groups from nucleoside triphosphates to nucleo-side diphosphates via a phosphohistidine 118 enzyme interme-diate, and they play a role in maintaining intracellular nucle-otide concentrations (10). Altered expression of NDPK is alsoreportedly involved in many cellular processes, including onco-genesis (11, 12), cellular proliferation (13), differentiation (14,15), motility (16), development (17), DNA repair (18), and apo-ptosis (19). In Escherichia coli, NDPK functions as a mutatorgene and is not essential for viability (20). In Dictyosteliumdiscoideum, the membrane possesses cAMP surface receptor-stimulated NDPK that leads to the activation of G-proteins andphospholipase C (21). Recent data also indicate that NDPK-Bcan form complexes with G�� dimers and contributes to Gprotein activation (22, 23). In yeast, an NDPK knockout strain

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

§ To whom correspondence should be addressed: National Center forToxicogenomics, NIEHS, National Institutes of Health, Bldg. 101, Rm.F-149, M.D. F1-05, P.O. Box 12233, Research Triangle Park, NC 27709.Tel.: 919-316-4660; Fax: 919-541-1460; E-mail: wei2@niehs.nih.gov.

1 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; FBS, fetal bovine serum; mAb, monoclonal antibody; MEM,minimal essential medium; NDPK, nucleoside diphosphate kinase;

pAb, polyclonal antibody; RGD, Arg-Gly-Asp; RIPA, radioimmunopre-cipitation assay; RT, reverse transcription; UVR, UV radiation; WT,wild type; NS, normal skin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 7, Issue of February 13, pp. 5993–6004, 2004Printed in U.S.A.

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of Saccharomyces pombe exhibits developmental abnormalities(24), whereas the homologous abnormal wing discs (awd) infruit fly Drosophila melanogaster is required for late larvaldevelopment (25). Thus far, there are four (Nm23-M1, Nm23-M2, Nm23-M3, and Nm23-M4) (26–28) and two (Nm23-R1 andNm23-R2) (29, 30) isoforms of NDPKs described in mouse andin rat, respectively. To date, eight distinct human NDPK genes,designated nm23-H1 to nm23-H8, have also been identified.Human nm23-H1 and nm23-H2 are 88% homologous and areclosely linked on chromosome 17q21–22 near the Brca1 genelocus (31), and both have been implicated in the metastasis(32–34) and pathogenesis of tumors (11, 35). DR-nm23 (nm23-H3), located on chromosome 16, is 70% identical to nm23-H1and nm23-H2 and may play a role in normal hematopoiesis andin the induction of apoptosis (19). nm23-H5 was specificallyexpressed in testis and found to encode a protein devoid of NDPkinase activity (36). Nm23-H6 is a mitochondrial NDPK andaffects cytokinesis (37).

Nm23-H2, known as Nm23-M2 or NDPK-B (38), is a basicprotein recently identified as the human PuF factor, a tran-scriptional activator of the c-myc proto-oncogene (39, 40). Mu-tational analysis has identified residues and domains ofNm23-H2 that are involved in DNA binding, implicating a rolein the regulation of genes important to cell proliferation, dif-ferentiation, and cancer development (40, 41). In colorectalcarcinomas, nm23-H2 is among the most abundant overex-pressed transcript, suggesting that nm23-H2 helps maintainthe malignant phenotype in these tumors (35). Recently, nm23-M2/NDPK-B was also identified as a novel potential diseaselocus that was involved in mouse leukemic transformation (42).

The biological functions of Nm23-M2/NDPK-B in cellulartransformation still remain unknown. To gain insights into thecontribution of this protein to early skin carcinogenesis, wecharacterized and studied the biological properties of Nm23-M2/NDPK-B. In this study, our data showed that Nm23-M2/NDPK-B was significantly induced either in vivo animal mod-els or in vitro cell cultures by TPA or UVR and appeared tohave a critical role in mediating neoplastic transformation ofepidermal cells in the early stages of skin carcinogenesis. Usingsite-directed mutagenesis analysis, we further identified anRGD consensus sequence domain site in Nm23-M2/NDPK-Bthat was involved in the potentiation of cellular transformationactivity. Nm23-M2/NDPK-B represents an attractive candi-date mediator of TPA- or UVR-induced tumor promotion.

EXPERIMENTAL PROCEDURES

Reagents

Nm23-M2/NDPK-B rat monoclonal antibodies (mAb) were obtainedfrom United States Biological (Swampscott, MA). V5 tag mouse mAbwas from Invitrogen; �-tubulin mouse mAb was from Zymed Laborato-ries Inc. (San Francisco, CA); actin rabbit polyclonal antibody (pAb) wasfrom Sigma. Horseradish peroxidase-conjugated secondary antibodieswere from Amersham Biosciences. Nm23-H2/NDPK-B mouse mAb(233.1) was a kind gift from Dr. Michel Veron (Institute Pasteur, Paris,France). Uridine 5�-[�-35S]thiotriphosphate triethylammonium salt(SP6/T7 grade) (�800 Ci/mmol) was purchased from Amersham Bio-sciences. Restriction enzymes, including BamHI, XbaI, EcoRI, and Hin-dIII, were obtained from New England Biolabs (Beverly, MA). Theprimers used in this study were purchased from Proligos Corp. (LaJolla, CA). Noble agar was purchased from Difco. The tumor promoterTPA was from Sigma.

Cell Cultures

TPA-susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b mouseepidermal cell clonal variants were from the American Type CultureCollection (Manassas, VA) and grown at 37 °C in a 95% air plus 5% CO2

atmosphere in Eagle’s minimal essential medium (MEM) supplementedwith 5% heat-inactivated fetal bovine serum (FBS) containing 2 mM

glutamine, 100 units/ml penicillin, and 100 �g/ml streptomycin sulfate(Invitrogen). Mouse fibroblast Rat-1, human keratinocyte HaCaT (gen-

erously provided by Dr. Norbert Fusenig, German Cancer ResearchCenter, Heidelberg, Germany), and human epidermoid carcinoma cellA431 were cultured in Dulbecco’s modified Eagle’s medium containing10% FBS. Tg�AC43 (a TPA-induced Tg�AC squamous cell carcinoma cellline) and FVB/N217 (an FVB/N carcinoma cell line) (43) were culturedin RPMI1640/Dulbecco’s modified Eagle’s medium (1:1) with 20% FBS.NIH/3T3 cells were maintained as described previously (44). Cell linesused in this study were free of mycoplasma infection.

Animals and Treatments

8–10-week-old female homozygous Tg�AC mice were obtained fromthe Taconic Laboratory of Animals and Services (Germantown, NY).Animal studies were carried out in compliance with the National Insti-tutes of Health Guidelines for Humane Care and Use of LaboratoryAnimals. The dorsal skin surface of groups of four homozygous femaleTg�AC mice were dosed twice weekly for 2 weeks with 5 �g of TPA in 200�l of acetone. Untreated control mice were sacrificed on day 1 (desig-nated as NS). Four dosing protocols were used as follows. Mice weredosed on day 1 and sacrificed on day 5 (designated as 1TPA); mice weredosed on days 1 and 5 and sacrificed on day 8 (designated as 2TPA);mice were dosed on days 1, 5, and 8 and sacrificed on day 12 (designatedas 3TPA); and mice were dosed on days 1, 5, 8, and 12 and sacrificedrespectively at 48 h (designated as 4TPA), 7 days (designated as4TPA � 7 days), 14 days (designated as 4TPA � 14 days), and 21 days(designated as 4TPA � 21 days) after the last dose. TPA-inducedpapillomas and malignant tumors (spindle cell tumors and squamouscell carcinomas) were identified, removed, and characterized as de-scribed previously (6). Tg�AC mice were irradiated with combination of30–40% UVA and 60–70% UVB, as described previously (5). Afterthree exposures to UVA/UVB with 8.67 kJ/m2 per exposure, total cu-mulative dose of 26 kJ/m2, skin tissues (designated as UV1–UV4) werecollected 24 h after the last exposure. Some animals were held forpapilloma development (designated as UVP1–UVP6), and tumors werecollected and stored at �80 °C.

Cloning and Mutagenesis

The full-length mouse nm23-M2/NDPK-B cDNA was generated byreverse transcription (RT) and PCR amplification using total RNAisolated from untreated (normal) skins harvested from Tg�AC mice.Because the nucleotide similarity (83%) and amino acid identity (88%)between nm23-M1/NDPK-A and nm23-M2/NDPK-B are relatively high(27), mouse nm23-M2/NDPK-B-specific primers were designed to ex-clude the possibility of cross-reactivity with mouse nm23-M1/NDPK-A.The specific primers used for PCR were as follows: forward (5�-CACCAT GGC CAA CCT CGA GCG TA-3�) and reverse (5�-CTC GTA CACCCA GTC ATG GGC ACA A-3�) for full-length mouse nm23-M2/NDPK-B cDNA cloning; forward (1053123) (5�-CAC CG105G TGG CCATGA AGT TCC TT123-3�) and reverse (3213297) (5�-A321TC CCC ACGGAT GGT GCC TGG TTT T297-3�) for in situ hybridization assay. Theamplified cDNA was cloned directly into pcDNA3.1D/V5-His-TOPOc

mammalian expression vector (Invitrogen). For the in situ hybridiza-tion assay, a construct expressing 217 bp of mouse nm23-M2/NDPK-Bsense or antisense RNA probe was generated as follows.pcDNA3.1D/nm23-M2/NDPK-B (1053321)-V5-His was digested withBamHI and XbaI and the mouse nm23-M2/NDPK-B cDNA fragment(1053321) was cloned into T3/T7-U19 plasmid (Ambion, Austin, TX).The deletion or point substitution mutations were introduced intomouse nm23-M2/NDPK-B cDNA by the ExSiteTM PCR-based site-directed mutagenesis kit, as described in the manufacturer’s instruc-tions (Stratagene, La Jolla, CA). The mutagenic primers were 24–29bases long and had a greater than 50% GC content. The oligonucleo-tides used to generate the different mouse Nm23-M2/NDPK-B mutantswere as follows (base changes underlined): delRGD (RGD consensussequence domain), 5�-TTC TGC ATT CAA GTT GGC AGG AAC-3�(forward) and 5�-pGAT GGT GCC TGG TTT TGA ATC AGC-3� (re-verse); G106A (RGD consensus sequence domain), 5�-CAC CAT CCGTGC GGA TTT CTG CAT-3� (forward) and 5�-pCCT GGT TTT GAATCA GCT GGA TTG GTC TC-3� (reverse); S122P (phosphoryl transferactivity site), 5�-AGT GAT CCG GTG GAG AGT GCT GAG AAA-3�(forward) and 5�-pGCC ATG AAT GAT GTT CCT GCC AAC TTG-3�(reverse); H118F (kinase activity site), 5�-CAT CAT TTT TGG CAG TGATTC AGT GGA GAG TGC-3� (forward) and 5�-pTTC CTG CCA ACT TGAATG CAG AAA TCC CC-3� (reverse). DNA sequences were verified usingan automated Applied Biosystems sequencer and the BigDyeTM Termi-nator Kit (PerkinElmer Life Sciences). Plasmid DNAs were purified usingpurification kits from Qiagen (Stanford Valencia, CA) and wereendotoxin-free when used for transfection into mammalian cells.

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Cell Transfections

Cells were transfected with empty vector pcDNA3.1, wild type nm23-M2/NDPK-B (WT) or mutant types nm23-M2/NDPK-B including del-RGD, G106A, S122P, and H118F plasmid DNAs using LipofectAMINEPLUSTM reagents (Invitrogen). Transfected cells were cultured for atleast 2 weeks in medium containing 400 �g/ml of geneticin (G418)(Invitrogen). Cells were analyzed by immunoblot to confirm the expres-sion of mouse NDPK-B.

RT-PCR

Single-stranded cDNA was prepared from total RNA using the Molo-ney murine leukemia virus reverse transcriptase SuperScript II (In-vitrogen) with oligo(dT) primer and used as a template for PCR. PCRprimers for mouse nm23-M2/NDPK-B were as described above. Theforward and reverse primers of �2-microglobulin gene (217 bp in size),used as an internal control, are 5�-GAC TGG TCT TTC TAT ATC CTGG-3� and 5�-CTT TCT GCG TGC ATA AAT TG-3�, respectively. PCRcycling was as follows: denaturation (94 °C, 45 s), annealing (58 °C,45 s), and extension (72 °C, 2 min) for 30 cycles. The reaction wascarried out in a PerkinElmer-9600 thermal cycler, and PCR productswere analyzed using 2% agarose gels. DNA was quantified using Quan-tity One software version 4.0 (Bio-Rad).

In Situ Hybridization

An in situ hybridization assay was performed as previously described(45). Briefly, skin tissues were removed from Tg�AC mice treated oruntreated with multiple doses of TPA and fixed overnight in 10%neutral buffered formalin. Tissues were paraffin-embedded, and sec-tions (6 �m) were cut onto SuperFrost Plus microscope slides (Daigger,Vernon Hills, IL). The sections were deparaffinized and rehydrated bysuccessive washes in xylene and graded alcohols to 2� SSC, and then2 � 106 cpm of [�-35S]UTP-labeled mouse nm23-M2/NDPK-B sense orantisense riboprobes was applied to slides. Riboprobes were preparedfrom T7/T3-U19/nm23-M2/NDPK-B (1053321) plasmid linearized withEcoRI (antisense) or HindIII (sense) using an in vitro T7 or T3 Ribo-probe kit (Promega, Madison, WI). Following 40 °C overnight hybrid-ization, tissues were washed in 2� SSC plus 50% formamide at 40 °Cand then in 2� SSC, 1� SSC, 0.5� SSC, and 0.5� SSC for 30 min eachwash at room temperature. To remove unbound probe, tissues wereincubated with 20 �l of RNase (10 mg/ml). After several washes, theslides were dehydrated in graded alcohols and completely air-dried. Theslides were then dipped into NTB-3 autoradiographic emulsion (East-man Kodak Co.), exposed for 10 days at room temperature in the dark,dried in a light-tight container, and developed in Kodak D19 developerand fixer. The sections were counterstained with hematoxylin, coveredwith coverslips, and photographed under dark-field illumination (modelBX51, Olympus Optical Co., Tokyo, Japan).

Immunoblot Analysis

Cells were washed with ice-cold phosphate-buffered saline and lysedin ice-cold modified radioimmunoprecipitation (RIPA) buffer consistingof 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1 mM

EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 �g/ml each aprotinin,leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF. Cell suspen-sions were gently rocked on an orbital shaker in a cold room for 15 minto lyse cells. Lysates were centrifuged at 14,000 � g for 15 min at 4 °C.The skin tissues or tumors were homogenized and sonicated in ice-coldRIPA buffer and ultracentrifuged at 100,000 � g for 1 h at 4 °C. Proteinconcentration was determined by Bradford assay (Bio-Rad). Proteinswere separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes(Amersham Biosciences). Membranes were stained with primary anti-bodies, detected using horseradish peroxidase-conjugated secondaryantibodies (1:3000) (Amersham Biosciences) and enhanced chemilumi-nescence (Amersham Biosciences). Membranes were stripped and re-hybridized with anti-�-tubulin mouse mAb (1:1000) or anti-actin rabbitpAb (1:1000) as a control to confirm equal loading. Protein quantitationwas determined by ImageQuant software version 5.1 (Amersham Bio-sciences), and relative quantity is shown below the panels of Figs. 3B,4–6, and 8.

Transformation Assays

Foci-forming Activity—Cells were seeded overnight at a density of1 � 106 cells in 10-cm plates. Cells were transfected with 2 or 4 �g ofpcDNA3.1D/nm23-M2/NDPK-B or empty vector and selected in me-dium containing G418 for 14–21 days. Foci were fixed with methanol/acetic acid (v/v � 1:3), stained with 0.4% crystal violet (Sigma) (meth-

anol/acetic acid), and counted as described previously (46).Characterization of Cell Growth—Growth curves were generated as

described previously (46). In brief, 2 � 103 cells were grown as describedabove. The medium was changed every 3–4 days. Cell number wascounted in triplicate on a hemocytometer every other day for 14 days.

Anchorage-independent Growth Assay—Colony formation in softagar was assayed as described previously (46). In a 60-mm tissueculture dish, 1 � 104 cells were resuspended in 0.33% Noble agar inEagle’s MEM with 10% FBS and layered over 5 ml of 0.5% agar inEagle’s MEM with 10% FBS. Cells were grown at 37 °C in 95% air plus5% CO2, and colonies with more than eight cells were counted andphotographed 18 days postseeding.

RESULTS

nm23-M2/NDPK-B Expression Is Induced following TPATreatment—Our previous microarray data indicated thatmouse nm23-M2/NDPK-B was increased 5.4-fold in integrin�6

� CD34� keratinocytes isolated from TPA-treated Tg�ACmice skins (7). We have reported previously that keratinocytesexpressing �6 and CD34 surface markers represent a subpopu-lation of follicle-derived cells exhibiting properties of progenitorcells (47). This up-regulation was confirmed by relative RT-PCR analysis that revealed a 3.5-fold increase in integrin �6

�

CD34� keratinocytes isolated from TPA-treated Tg�AC mice,relative to integrin �6

� CD34� keratinocytes from untreatedmice (Fig. 1). The trend of nm23-M2/NDPK-B overexpressionwas in agreement with our previous microarray experiment (7).To define the specificity of tissue distribution of nm23-M2/

FIG. 1. nm23-M2/NDPK-B was up-regulated by TPA in integrin�6

� CD34� keratinocytes. TPA-treated Tg�AC mouse skin keratino-cytes carrying the cell surface markers integrin �6 and CD34 wereisolated by fluorescence-activated cell sorting (47). Control cells wereharvested from animals not treated with TPA. RNase-free DNase I-treated total RNAs from TPA-treated (TPA(�)�6�CD34�) or -untreatedintegrin �6

� CD34� keratinocytes (TPA(�)�6�CD34�) were assayed byRT-PCR, as described under “Experimental Procedures.” The productswere subjected to 2% agarose gels. Mouse nm23-M2/NDPK-B plasmidDNA was used as a positive control (PC) and showed a band of 456 bpin size. The �2-microglobulin gene (217 bp in size) was used as aninternal control. DNA was quantified using Quantity One softwareversion 4.0 (Bio-Rad).

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VANDPK-B, a panel of tissues from normal Tg�AC mice wasexamined by immunoblot analysis using a rat monoclonal an-tibody specific to mouse Nm23-M2/NDPK-B, with no cross-reactivity with mouse Nm23-M1/NDPK-A molecule (27). Asshown in Fig. 2, the specific signal was detected under reducingconditions as a band of 17.5-kDa in size, which was seen invarying intensities in heart, brain, kidney, liver, skin, stomach,spleen, and small intestine. Nm23-M2/NDPK-B was expressedat higher levels in heart and kidney. In liver, spleen, stomach,small intestine, brain, and skin the expression levels of nm23-M2/NDPK-B were very low, whereas Nm23-M2/NDPK-B wasbarely detectable in lung and ovary. To determine the localiza-tion of nm23-M2/NDPK-B message in mouse skin tissues, insitu hybridization was employed. Although some nonspecificbackground was apparent in the outermost cornified cell enve-lope, the signal primarily localized to the stratified squamousepithelial regions (indicated by the arrows in Fig. 3) with an-tisense but not sense [�-35S]UTP-labeled mouse nm23-M2/NDPK-B mRNA. Some expression was evident in hair follicles,especially in the third dose of TPA-treated skins in Fig. 3A (XI).Signal was not detectable in the dermis, adipose, and muscletissues (Fig. 3A). However, there was no detectable nm23-M2/NDPK-B mRNA message in normal skins (Fig. 3A, II), relativeto the sense probe control (Fig. 3A, III). Total protein lysates ofskins harvested from Tg�AC mice treated with various doses ofTPA were used for immunoblot analysis. These doses induceextensive hyperplasia (see Fig. 3A; IV, VII, and X). Fig. 3Brevealed an induction of Nm23-M2/NDPK-B in mouse skinhomogenates following TPA treatment. Untreated mouse skinwas used as a negative control and expressed very low levels ofNm23-M2/NDPK-B (Fig. 3B, lane 1). Mouse nm23-M2/NDPK-B expression was induced 2.5-fold following one dose ofTPA (5 �g), with a maximal induction of about 8–10-fold be-tween the second and the fourth dose of 5-�g TPA treatment(Fig. 3B). Nm23-M2/NDPK-B increased with hyperplasia andwas maintained at high levels (3.8-fold) even 21 days after thelast of four doses of TPA (Fig. 3B). In contrast, Nm23-M1/NDPK-A was not significantly up-regulated in TPA-inducedhyperplastic skin tissues (data not shown).

TPA-induced Expression of nm23-M2/NDPK-B in CulturedTPA-susceptible Epidermal Cells—To investigate whether TPAis able to induce mouse nm23-M2/NDPK-B expression, TPA-susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b pre-neoplastic epidermal cell lines were employed. JB6 Cl 41-5a

cells were grown in 5% FBS/Eagle’s MEM with 0, 0.1, 1, 10, or100 ng/ml TPA. The cells were harvested 8 h post-treatment,and whole-cell lysates were prepared for immunoblot analysis.Fig. 4A showed a 1.8-fold induction of mouse Nm23-M2/NDPK-B, beginning at 0.1 ng/ml TPA, and maximal inductionof 2.5–3.2-fold with 1–100 ng/ml TPA. A kinetic analysis ofNm23-M2/NDPK-B expression in JB6 Cl 41-5a cells was con-ducted at 0, 10, and 30 min and 1, 2, 4, 8, 12, and 18 h followingtreatment with 10 ng/ml TPA (Fig. 4B). This showed thatmouse Nm23-M2/NDPK-B was induced 2.2-fold 10 min afterTPA treatment and reached a maximal 3.0-fold induction 8 hpost-treatment. Nm23-M2/NDPK-B expression then appearedto decrease from 12 to 18 h post-treatment (Fig. 4B). Interest-ingly, we did not find that mouse Nm23-M2/NDPK-B was in-duced in TPA-resistant JB6 Cl 30-7b cell line but rathershowed a remarkable decrease 10 min after treatment with 10ng/ml TPA (Fig. 4C).

Overexpression of nm23-M2/NDPK-B in Skin Tumor CellLines and Skin Tumors—In vivo and in vitro studies showedthat TPA was able to induce an increase in nm23-M2/NDPK-Bgene expression in Tg�AC mice keratinocytes, in hyperplasticTg�AC mice skins, and in TPA-susceptible mouse JB6 Cl 41-5aepidermal cells. The gene expression of nm23-M2/NDPK-B wasalso examined by immunoblot analysis in the mouse skin tu-mor cell lines as well as in TPA-induced skin tumors. Theprotein level of Nm23-M2/NDPK-B was found to respectivelyincrease 10- and 2.8-fold in two mouse skin tumor cell lines,Tg�AC43 and FVB/N217 (relative to TPA-untreated normalkeratinocytes isolated from Tg�AC mice) (TPA(�)KCs) (Fig.5A). In addition, NDPK-B expression was also increased in onehuman epidermoid carcinoma cell line A431 (5.0-fold), whencompared with human HaCaT keratinocytes (Fig. 5A). Thekeratinocytes isolated from the skins of Tg�AC mice treatedwith four doses of TPA (TPA(�)KCs) were used as a positivecontrol and showed a 3.5-fold induction (Fig. 5A, lane 2). Nm23-M2/NDPK-B protein was higher in TPA-mediated Tg�AC miceskin tumors, including 15 papillomas (3.1 � 1.0-fold) and threemalignant tumors (3.7 � 1.8-fold) (one spindle cell tumor (5.8-fold) and two squamous cell carcinomas (2.7- and 2.6-fold)),than in adjacent normal skins (1.0 � 0.3-fold) (Fig. 5B).

The nm23-M2/NDPK-B Gene Is Induced by UVR—To exam-ine whether nm23-M2/NDPK-B is induced in response to UVRin vivo, we irradiated Tg�AC mice with a combination of UVAand UVB (30–40% UVA and 60–70% UVB). After three UVA/UVB exposures, the skin was found to be extensively hyper-plastic, keratinized, and inflamed (data not shown). The UV-exposed tissues were collected 24 h after the last exposure andsnap frozen in liquid nitrogen for preparation of total proteinlysates. Immunoblot analysis revealed that Nm23-M2/NDPK-B was induced by 7.2–10-fold in these four mouse skintissues following UV exposure (designated as UV1–UV4), rel-ative to the low level expression found in nonexposed skin(designated as NS) (Fig. 6A). To further investigate if thisinduction persistently occurred in UVR-mediated skin tumors,six papillomas were examined. Our result indicated thatNm23-M2/NDPK-B was increased 7–10-fold in UVR-mediatedskin papillomas (designated as UVP1–UVP6), relative to un-treated normal Tg�AC mice skins (NS) (Fig. 6B).

Overexpression of Mouse nm23-M2/NDPK-B Enhances Neo-plastic Transformation in Preneoplastic Mouse EpidermalCells—Mouse nm23-M2/NDPK-B was constitutively expressedin preneoplastic epidermal cells to determine whether in-creased expression of mouse nm23-M2/NDPK-B potentiallyplays a role in skin tumorigenesis. One plasmid construct,pcDNA3.1D/nm23-M2/NDPK-B-V5-His, was prepared to con-stitutively express mouse nm23-M2/NDPK-B. Immunoblot

FIG. 2. Nm23-M2/NDPK-B was expressed in a variety of normalTg�AC mouse tissues. Tissue samples of the heart (H), brain (B),kidney (K), liver (Li), lung (Lu), ovary (O), skin (Sk), stomach (St),spleen (Sp), and small intestine (SI) were homogenized and extractedfor preparation of total protein lysates in modified RIPA buffer, asdescribed under “Experimental Procedures.” Sixty micrograms of totalprotein lysates were separated on a 15% SDS-PAGE, transferred to thepolyvinylidene difluoride membrane, and then probed with a specificanti-mouse Nm23-M2/NDPK-B rat mAb (1:200). The primary antibodywas detected using horseradish peroxidase-conjugated anti-rat IgG sec-ondary antibody at 1:3000 and enhanced chemiluminescence reagentkit. The specific signal was developed with Amersham BiosciencesHyperfilmTM ECL. Membranes were stripped and rehybridized withanti-actin rabbit pAb (1:1000). The arrow indicates the molecular massof mouse Nm23-M2/NDPK-B in size as 17.5 kDa (top panel). Actin (42kDa) served as an internal control and attested that equivalentamounts of protein were loaded in each lane (bottom panel).

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FIG. 3. Overexpression of nm23-M2/NDPK-B in early TPA-induced hyperplastic mouse skin tissues. A, in situ hybridization. To detect theexpression of nm23-M2/NDPK-B, in situ hybridization assay was performed on paraffin-embedded sections of TPA-untreated (Normal Skin; I, II, and III)or TPA-treated Tg�AC mouse skin tissues including one dose of 5 �g of TPA-treated skins (1TPA; IV, V, and VI), two doses of 5 �g of TPA-treated skins (2TPA;VII, VIII, and IX), and three doses of 5 �g TPA-treated skins (3TPA; X, XI, and XII), using a specific nm23-M2/NDPK-B (1053321) sense (S; III, VI, IX, andXII) or antisense (AS; II, V, VIII, and XI) riboprobe. The silver grains indicate the signals in probe hybridization, and slides were counterstained withhematoxylin and eosin (I, IV, VII, and X). The arrows indicate the localization of the nm23-M2/NDPK-B gene expression. Photographs were taken underlight field (I, IV, VII, and X) and dark field (II, III, V, VI, VIII, IX, XI, and XII) conditions. The white line (basal layer) indicates the border between epidermisand dermis. E, epidermis; D, dermis; A, adipose tissue; M, muscle tissue; BL, basal layer; HF, hair follicle. Original magnification was �100. B, immunoblotanalysis. The multiple doses of 5 �g of TPA-treated Tg�AC mouse skin tissues were homogenized and extracted for preparation of total protein lysates inmodified RIPA buffer. Mouse tissue total protein lysates (60 �g) were subjected to immunoblotting and probed with anti-mouse Nm23-M2/NDPK-B rat mAb(1:200) (top panel) or anti-actin rabbit pAb (1:1000) (bottom panel). Protein quantitation was determined by ImageQuant software version 5.1 (AmershamBiosciences), and relative quantity is shown under the top panel.

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analysis confirmed that mouse Nm23-M2/NDPK-B protein wasexpressed successfully and efficiently in NIH/3T3, Rat-1, JB6Cl 30-7b, and JB6 Cl 41-5a cells (Fig. 7A).

Constitutive expression of Ras family proteins and otheroncogenic proteins increase focus-forming capability and de-crease growth contact inhibition of normal untransformed cells(46). Our results showed that constitutive expression of mousenm23-M2/NDPK-B increases foci formation 3.8- (2 �g of DNA)to 4.5-fold (4 �g of DNA) and 4.0- (2 �g of DNA) to 5.3-fold (4 �gof DNA) in mouse JB6 Cl 30-7b and JB6 Cl 41-5a epidermal celllines, respectively (Fig. 7B). In addition, the increase in foci-forming activity appears to have gene dosage-dependent effects(Fig. 7B). However, expression of mouse nm23-M2/NDPK-B didnot change the foci-forming properties in fibroblasts such asNIH/3T3 and Rat-1 cells. These results demonstrate that

nm23-M2/NDPK-B alters normal contact inhibition in mouseepidermal cell lines, suggesting that nm23-M2/NDPK-B mayhave some oncogenic properties.

Transformed cells have a growth advantage in monolayerculture and acquire capacity for anchorage-independentgrowth (46). The effects of mouse nm23-M2/NDPK-B expres-sion on these growth characteristics were measured in NIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a cells (Fig. 7C). Thecells transfected with vector only or mouse nm23-M2/NDPK-Bwere grown for 48 h and then selected for at least 10 days withmedium containing 400 �g/ml of antibiotic neomycin analogG418. Stable cell clones expressing vector only or mouse nm23-M2/NDPK-B were seeded at a cell density of 2 � 103 in a 6-welltissue culture plate to assay for cell growth. Constitutive ex-pression of mouse nm23-M2/NDPK-B was not correlated withincreased growth rate in epidermal cells such as JB6 Cl 30-7band JB6 Cl 41-5a. On the contrary, our results showed thatclones overexpressing mouse nm23-M2/NDPK-B in NIH/3T3and Rat-1 fibroblasts grew at a significantly slower rate thancontrol vector-only cells (Fig. 7C). Furthermore, stable clones

FIG. 4. In vitro dose response and kinetics of TPA-inducedexpression of nm23-M2/NDPK-B in epidermal cells. JB6 Cl 41-5a(A and B) or JB6 Cl 30-7b epidermal cells (C) were treated with theindicated TPA concentrations or exposed to 10 ng/ml TPA at indicatedtime points. Cells were harvested, and whole cell lysates were extractedusing modified RIPA buffer. Sixty micrograms of whole-cell lysateswere analyzed by immunoblotting for the detection of mouse Nm23-M2/NDPK-B (top panel). �-Tubulin (50 kDa) (lower panel) served as aninternal control.

FIG. 5. Elevated NDPK-B expression in skin tumor cell linesand Tg�AC neoplasms. A, skin tumor cell lines. Two mouse skin tumorcell lines, FVB/N217 and Tg�AC43, and one human epidermoid carci-noma cell line A431 were examined for NDPK-B gene expression byimmunoblotting. HaCaT keratinocytes and TPA-untreated normal ke-ratinocytes [TPA(�)KCs] (isolated from Tg�AC mice) were used as neg-ative controls for humans and mice, respectively. TPA-treated Tg�ACmouse keratinocytes (TPA(�)KCs) served as a positive control to indi-cate the position of NDPK-B. KCs, keratinocytes. B, TPA-mediated skintumors in Tg�AC mice. Total protein lysates were prepared from sevennormal skin tissues of Tg�AC mice (NS1–NS7) and tumors, including 15papillomas (P1–P15) and three malignancies (M1–M3) with one spindlecell tumor (M1) and two squamous cell carcinomas (M2 and M3). Alltotal protein lysate (60 �g) immunoblots were probed with anti-mouseNm23-M2/NDPK-B rat mAb (1:200) or anti-human Nm23-H2/NDPK-Bmouse mAb (1:1000), anti-actin rabbit pAb (1:1000), or anti-�-tubulinmouse mAb (1:1000).

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expressing vector only or mouse nm23-M2/NDPK-B wereseeded at 1 � 104 in a 60-mm soft agar tissue culture plate toassay for anchorage-independent growth. Colony formation ef-ficiency increased �3.3- and 4.0-fold when mouse nm23-M2/NDPK-B was expressed in JB6 Cl 41-5a and JB6 Cl 30-7b cells,respectively (Fig. 7D). However, colony forming efficiency didnot increase in mouse nm23-M2/NDPK-B-transfected fibro-blasts such as NIH/3T3 and Rat-1 cells (Fig. 7D). Backgroundcolony formation was higher in JB6 Cl 41-5a cells than in JB6Cl 30-7b cells.

An Increased Release of nm23-M2/NDPK-B from Cells follow-ing Exposure to TPA or UVR—Next we examined whetherNm23-M2/NDPK-B is released from the cells and is affected bystresses such as TPA and UVR. For these studies, TPA-suscep-tible mouse epidermal cell line JB6 Cl 41-5a and human kera-tinocyte cell line HaCaT, which is routinely used in UVR stud-ies, were employed. Prior to TPA or UV exposure, subconfluentJB6 Cl 41-5a and HaCaT cell monolayers were starved inserum-free medium for at least 12 h. As seen in Fig. 8, Nm23-M2/NDPK-B was detected in serum-free conditioned mediacollected from untreated 18-h cultures of JB6 Cl 41-5a andHaCaT cells. At this time, cells were observed to still be viable,as ascertained by examination of trypan blue dye exclusionusing an inverted microscope (data not shown). The resultindicates that Nm23-M2/NDPK-B is released from the cells.More interestingly, the release of Nm23-M2/NDPK-B into themedium was enhanced about 2.5–3-fold in JB6 Cl 41-5a (Fig.

8A) and HaCaT cells (Fig. 8B) 18 h following exposure to TPA(10 ng/ml), UVA (10 J/cm2) or UVB (10 mJ/cm2). The increasedrelease of Nm23-M2/NDPK-B may explain why the expressionof NDPK-B in JB6 Cl 41-5a cells was significantly down-regu-lated 12–18 h after treatment with 10 ng/ml of TPA (Fig. 4B).

Deletion of Arg105-Gly106-Asp107 Consensus Sequence Domainor Point Substitution Gly106 with Ala in Mouse Nm23-M2/NDPK-B Selectively Abrogates Cellular Transformation—To dis-sect and determine which amino acid residues of Nm23-M2/NDPK-B might be involved in cellular transformation, weemployed site-directed mutagenesis to generate mutants for de-letion and point substitution and examined anchorage-inde-pendent growth using a standard in vitro soft agar assay. Themutants include Arg105-Gly106-Asp107 deletion (delRGD), Gly106

substitution with Ala (G106A), Ser122 substitution with Pro(S122P), and His118 instead of Phe (H118F). We stably trans-fected the TPA-susceptible mouse preneoplastic epidermal cellline JB6 Cl 41-5a with vector only, wild type (WT), delRGD,G106A, H118F, or S122P nm23-M2/NDPK-B gene and assayedtheir activity for cellular transformation. Immunoblot analysisshowed that the nm23-M2/NDPK-B gene constructs were effi-ciently expressed and released into the serum-free conditionedmedia collected from the different JB6 Cl 41-5a stable cell clones,when probed with anti-V5 tag mouse monoclonal antibody (Fig.9A). The Nm23-M2/NDPK-B (delRGD) mutant protein had asignificant shift in electrophoretic gel mobility with a higher ratethan the other proteins, whereas the mobility of mutant proteinssuch as Nm23-M2/NDPK-B (G106A), Nm23-M2/NDPK-B(S122P), and Nm23-M2/NDPK-B (H118F) were not changed (Fig.9A). The stable clones expressing vector only, WT, delRGD,G106A, H118F, and S122P nm23-M2/NDPK-B genes wereseeded separately at 1 � 104 in a 60-mm soft agar tissue cultureplate to assay for anchorage-independent growth. The colonywith more than eight cells was counted 18 days postseeding. Asshown in Fig. 9, B and C, colony formation efficiency was reduced�7.2- and 3.5-fold relative to nm23-M2/NDPK-B (WT), whennm23-M2/NDPK-B (delRGD) and nm23-M2/NDPK-B (G106A)were expressed in JB6 Cl 41-5a cells, respectively. Overexpres-sion of nm23-M2/NDPK-B (S122P) and nm23-M2/NDPK-B(H118F) did not significantly reduce the efficiency of cellulartransformation.

DISCUSSION

In this study, we further characterize the biological functionsof the NDPK-B gene product using the in vitro JB6 mouseepidermal clonal genetic variant cell system that has proven tobe valuable in studying tumor promoter-dependent biologicalevents occurring during preneoplastic progression (48). In thisway, we first demonstrate that NDPK-B is a potentially impor-tant TPA- or UVR-responsive gene required for neoplastictransformation in epidermal cells.

Our RT-PCR data showed a significant up-regulation inmouse nm23-M2/NDPK-B gene expression in integrin �6

�

CD34� keratinocytes following TPA treatment (Fig. 1). Thisinduction is closely associated with the TPA- and UVR-inducedpromotion stage of skin carcinogenesis in mice (Figs. 3 and 6A).Immunoblot analysis also showed an in vitro TPA-inducedincrease in Nm23-M2/NDPK-B protein level in JB6 Cl 41-5apreneoplastic epidermal cells (Fig. 4, A and B). In addition, adramatic increase in gene expression of NDPK-B occurs con-sistently in skin tumor cell lines (Fig. 5A) and in TPA- orUVR-mediated mouse skin tumors as well (Figs. 5B and 6B).These results clearly suggest that nm23-M2/NDPK-B is a novelTPA- or UVR-responsive gene that may be a useful marker forearly skin tumorigenesis.

Although our results indicate a close correlation betweennm23-M2/NDPK-B gene expression and the tumor promotion

FIG. 6. Induction of nm23-M2/NDPK-B by UVR. A, Nm23-M2/NDPK-B increase in UVR-mediated hyperplastic skins. Tg�AC mouseskins were irradiated with a combination of UVA and UVB (30–40%UVA and 60–70% UVB). After three exposures to UVA/UVB with 8.67kJ/m2 per exposure (total 26 kJ/m2), the skin tissues were collected 24 hafter the last exposure and snap frozen in liquid nitrogen. UV1–UV4,UV-exposed skin tissues from mouse 1–4. B, nm23-M2/NDPK-B over-expression in UVR-induced papilloma in Tg�AC mice. Tg�AC mice wereirradiated with a combination of 30–40% UVA and 60–70% UVB. Afterthree exposures to UVA/UVB with 8.67 kJ/m2 per exposure (total of 26kJ/m2), the skin tumors were collected and snap frozen in liquid nitro-gen. NS, normal skin; UVP1–UVP6, UV-induced papillomas frommouse 1–6. Sixty micrograms of the total protein lysates were subjectedto a 10–20% SDS-PAGE and then probed with anti-mouse Nm23-M2/NDPK-B rat mAb (1:200) or anti-actin rabbit pAb (1:1000).

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FIG. 7. Constitutive expression of mouse nm23-M2/NDPK-B remarkably enhances neoplastic transformation in JB6 Cl 41-5aepidermal cells. A, immunoblot analysis. Sixty micrograms of total protein lysates from parental (P), pcDNA3.1 vector only (V), or nm23-M2/NDPK-B-transfected (N) cell lines including NIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a were separated on 15% SDS-PAGE. The membranes

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stage (induced by TPA or UVR), the specific functions of nm23-M2/NDPK-B in this process, such as proliferation, differentia-tion, or neoplastic transformation, remains unknown. Earlierreports identified NDPK-B as a differentiation-inhibitory fac-tor, which is able to inhibit the differentiation of several hema-topoietic cell lines in vitro, where the inhibition was independ-ent of the phosphotranferase activity, as demonstrated withNDPK-B mutants lacking the enzymatic activity (14, 49).Moreover, several hematopoietic tumor cell lines stained posi-tive for NDPK-B in flow cytometric analysis (50), and up-regulation of the human NDPK-B gene was also observed innormal lymphocytes induced to proliferate with phytohemag-glutinin (51). Down-regulation of the NDPK-B and c-myc geneswas also reported in 1,25-dihydroxyvitamin D3-induced differ-entiation (52). These results suggest that NDPK-B may play acritical role in the inhibition of differentiation or the promotionof proliferation of these cells. As seen in Fig. 7, we found thatnm23-M2/NDPK-B was efficiently expressed in NIH/3T3,Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a (Fig. 7A). The elevatedexpression of mouse nm23-M2/NDPK-B in genetically modifiedNIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a stable clones,which were transfected with pcDNA3.1D/nm23-M2/NDPK-Bmammalian expression vector and selected with G418 for atleast 10 days, does not significantly promote cell proliferationunder standard in vitro tissue culture conditions (Fig. 7C) butappears to markedly increase foci-forming activity in epider-mal cell lines (e.g. JB6 Cl 30-7b and JB6 Cl 41-5a) in a genedosage-dependent manner but not in fibroblast cell lines (e.g.NIH/3T3 and Rat-1) (Fig. 7B). Notably, we found the rate of cellgrowth to be selectively inhibited in nm23-M2/NDPK-B-over-expressing mouse fibroblasts, NIH/3T3 and Rat-1. Our dataconsistently showed a decrease of proliferation rate from 100%down to about 1% (NIH/3T3) or 50% (Rat-1) and implicatedthat more than 50% of the growth inhibition observed in fibro-blasts was mediated by activation of Nm23-M2/NDPK-B-initi-ated signaling pathways (Fig. 7C), presumably suggesting thatNDPK-B plays an important role in the maintenance of integ-rity of skin tissues. More importantly, overexpression of mousenm23-M2/NDPK-B increased colony-forming efficiency in JB6Cl 41-5a and JB6 Cl 30-7b cells but not in NIH/3T3 and Rat-1cells using anchorage-independent growth assay (Fig. 7D).Thus, the ability of nm23-M2/NDPK-B to regulate cellular

transformation may be specific for epithelial cells.NDPKs have previously been reported to localize at different

subcellular localizations, such as nucleus (53), cytoplasm (54),and mitochondria (37), with low levels in the plasma mem-brane (50). Previous studies have already revealed that inhib-itory factor was purified from the conditioned medium of themouse myeloid cell line M1, although NDPKs have no signalpeptide for their secretion (49, 55). Anzinger et al. (56) recentlyfound secretion of NDPK-B by cells isolated from humanbreast, colon, pancreas, and lung tumors. In addition, Willemset al. (57, 58) also demonstrated that extracellular NDPK mod-ulated normal hematopoietic cell differentiation. Our data haveshown NDPK-B release into the serum-free media of mouseJB6 Cl 41-5a epidermal cells and an about 2.5–3.0-fold increasein release levels 18 h following TPA treatment (Fig. 8). Allpreviously identified NDPKs, except that in bacteria, share aspecific tripeptide Arg-Gly-Asp (RGD) domain (27). To furtherdetermine whether the RGD domain of NDPK-B protein isrequired for the enhancement of neoplastic transformation inepidermal cells, site-directed mutagenesis was employed tocreate mutants for deletion or point substitution. Immunoblotanalysis showed that Nm23-M2/NDPK-B proteins were re-leased into the serum-free conditioned media collected fromJB6 Cl 41-5a individual stable clones, which were transfectedwith vector only, wild type (WT), delRGD, G106A, S122P, orH118F nm23-M2/NDPK-B gene (Fig. 9A). The elevated expres-sion of mouse nm23-M2/NDPK-B (WT) into JB6 Cl 41-5a cellsacquired the susceptibility to transformation in an in vitro softagar assay. Indeed, constitutive expression of mouse nm23-M2/NDPK-B (delRGD) or nm23-M2/NDPK-B (G106A) mutant genein JB6 Cl 41-5a cells selectively abolished NDPK-B-inducedanchorage-independent growth (Fig. 9, B and C). Thus, theenhancement of cellular transformation requires the presenceof the RGD consensus sequence domain. Previous reports indi-cated that the point substitutions of nm23-H2/NDPK-B atHis118 with Phe (H118F) and at Ser122 instead of Pro (S122P)result in a defective kinase activity and phosphoryl transferactivity, respectively (59, 60). Interestingly, overexpression ofmouse nm23-M2/NDPK-B (S122P) (lack of phosphoryl transferactivity) and nm23-M2/NDPK-B (H118F) (lack of kinase activ-ity) did not significantly reduce the efficiency of cellular trans-formation and suggests that NDPK-B kinase activity is not

were probed with anti-V5 tag mouse mAb (1:5000) (top panel) or anti-�-tubulin mouse mAb (1:1000) (bottom panel). B, mouse nm23-M2/NDPK-Boverexpression confers gene dosage effects in foci-forming activity. NIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a cells were transfected with 2or 4 �g of pcDNA3.1 or mouse nm23-M2/NDPK-B expression construct for 48 h. pcDNA3.1 vector or nm23-M2/NDPK-B-transfected cell lines wereplated at a density of 1 � 104 and then selected respectively in medium with 400 �g/ml G418 for 14–21 days. Foci were fixed, stained, and counted.C, constitutive expression of mouse nm23-M2/NDPK-B was not correlated with increased growth rate in epidermal cells. Growth curves weregenerated for NIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a cells stably expressing vector only or mouse nm23-M2/NDPK-B, as described under“Experimental Procedures.” Cells were counted in triplicate every other day for 14 days. D, elevated mouse nm23-M2/NDPK-B expression enhancestransformation efficiency in epidermal cell lines. NIH/3T3, Rat-1, JB6 Cl 30-7b, and JB6 Cl 41-5a cells were stably transfected with vector only ornm23-M2/NDPK-B plasmid DNA and seeded (1 � 104) into 0.33% soft agar over a 0.5% agar bottom layer. The colony with more than eight cellswas counted 18 days postseeding. The data represent an average of two experiments.

FIG. 8. An increased release of NDPK-B protein from cells in response to TPA or UVR. Prior to TPA or UV exposure, subconfluent mouseJB6 Cl 41-5a and human HaCaT cell monolayers were starved in serum-free medium for at least 12 h. JB6 Cl 41-5a cells (A) were treated with10 ng/ml TPA, and HaCaT keratinocytes (B) were irradiated with 10 J/cm2 UVA or 10 mJ/cm2 UVB. After 18-h cultivation, the conditioned mediawere collected, concentrated, and subjected to 15% SDS-PAGE for the detection of NDPK-B protein. The immunoblots were probed with anti-mouseNm23-M2/NDPK-B rat mAb (1:200) or anti-human Nm23-H2/NDPK-B mouse mAb (1:1000).

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required for transformation (Fig. 9, B and C). Taken together,these results point to a crucial regulatory role of the RGDconsensus sequence domain, but not the catalytic domain, inNDPK-B-mediated cellular transformation.

Although NDPK-B protein can be released into the mediumin our cell model systems, we cannot exclude the possibleeffects of NDPK-B in the nucleus. Nm23-H2/NDPK-B has re-cently been identified as the human PuF factor (40) and cantransactivate a human c-myc proto-oncogene via a functionalnuclease hypersensitive element (39). Activation of the c-mycproto-oncogene contributes to cellular transformation, mito-genesis, differentiation, and apoptosis (61–63). Recent reportsalso indicated that c-Myc promotes differentiation of humanepidermal stem cells (64), and c-myc activation in transgenicmouse epidermis results in mobilization of stem cells and dif-ferentiation of their progeny (65). Moreover, the constitutiveexpression of human c-myc has been demonstrated to depleteepidermal stem cells by reducing �1 integrin expression (66).Mutational analysis of human Nm23-H2/NDPK-B has identi-fied amino acid residues and structural domains that are in-volved in DNA binding, implicating a multifunctional role inthe regulation of genes (e.g. c-myc) important to cell prolifera-tion, differentiation, and cancer development (41, 67). Researchis in progress to further identify how NDPK-B, integrin, andc-Myc coordinately regulate cell growth, differentiation, andtransformation in epidermal stem cells following treatmentwith TPA.

Due to inefficiency of the antibody for immunohistochemicalstaining in mouse skins, we used in situ hybridization to local-ize the nm23-M2/NDPK-B mRNA message. Our findings on thelocalization of nm23-M2/NDPK-B mRNA in homozygous Tg�ACmice skin tissues following treatment with variable doses ofTPA showed a dramatically increased expression in the strat-ified squamous epithelial regions, in particular in basal layerand spinosum, and with some expression in hair follicles (Fig.3A). Indeed, the induction of nm23-M2/NDPK-B mRNA seemsto occur in a TPA dose-dependent manner, consistent withimmunoblot analysis (Fig. 3B). Unlike immunoblot analysis, insitu hybridization did not detect the nm23-M2/NDPK-B signalin normal mouse skin tissues, probably in part due to therelative insensitivity of in situ hybridization. Interestingly, ourprevious study indicates that the expression patterns of Dss1and Nm23-M2/NDPK-B were similar in respect to tissue dis-tribution and localization (7). Moreover, recently reported dataindicate that human Nm23-H2/NDPK-B binds to single-stranded oligonucleotides in a non-sequence-specific mannerbut that it exhibits strong specificity for single-stranded DNA(68). One Dss1 functional model, as shown by its 37% acidicresidue (aspartic and glutamic acid-rich domains, �21 chargeat pH 7.0) and its 13% aromatic residue content, suggests thatit mimics oligonucleotides, possibly regulating the accessibilityof a subset of the putative DNA binding sites on the helical andOB1 domains of BRCA2 (69). Recently, a Dss1/BRCA2 interac-tion was shown to be required for proficiency in DNA repair,recombination, and genome stability in the fungi Ustilago may-dis (70). Postel et al. (18) also revealed that catalysis of DNAcleavage and nucleoside triphosphate synthesis by human

FIG. 9. Deletion of Arg105-Gly106-Asp107 consensus sequence do-main or point substitution Gly106 with Ala in mouse Nm23-M2/NDPK-B selectively abrogates cellular transformation. A, immu-noblot analysis. The deletion or point substitution was introduced intomouse nm23-M2/NDPK-B cDNA by the ExSiteTM PCR-Based site-di-rected mutagenesis kit, as described under “Experimental Procedures.”These mutants include delRGD, G106A, S122P, and H118F. JB6 Cl41-5a cells were transfected with pcDNA3.1 vector only (Vector), wildtype nm23-M2/NDPK-B (WT), or mutant types nm23-M2/NDPK-B in-cluding delRGD, G106A, S122P, and H118F plasmid DNAs. To obtainstable clones, transfected cells were selected under 400 �g/ml G418 forat least 10 days. Serum-free conditioned media were collected andconcentrated from stable clones and analyzed by immunoblotting toconfirm the release of mouse NDPK-B proteins. B, anchorage-indepen-

dent growth assay. JB6 Cl 41-5a-stable cell clones were seeded at adensity of 1 � 104 into 0.33% soft agar over a 0.5% agar bottom layer.Cells were grown at 37 °C in 95% air plus 5% CO2, and a colony withmore than eight cells was counted 18 days after seeding. The datarepresent an average of two experiments. C, photographs were taken 18days postseeding under an inverted microscope. Original magnificationwas �100. I, pcDNA3.1 (Vector); II, nm23-M2/NDPK-B (WT); III, nm23-M2/NDPK-B (delRGD); IV, nm23-M2/NDPK-B (G106A); V, nm23-M2/NDPK-B (S122P); VI, nm23-M2/NDPK-B (H118F).

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Nm23-H2/NDPK-B share an active site, thus implicating aDNA repair function for the NDPK-B molecule. It raises apossibility that Dss1, in addition to binding the DNA repairprotein BRCA2 (71), could interact with the DNA repair pro-tein Nm23-M2/NDPK-B in mouse skins following exposurewith TPA. Although we have found that Dss1 binds directlywith Nm23-M2/NDPK-B in in vitro studies using TNT QuickCoupled Transcription/Translation Systems,2 further studieswill be necessary before the significance of this unique protein-protein interaction can be fully understood.

In summary, our studies provide a new insight into thebiological function of mouse Nm23-M2/NDPK-B and demon-strate that it is likely to play a unique role in mediating TPA-or UVR-induced skin carcinogenesis. NDPK-B was capable ofbeing released outside of cells in culture, and an increase inrelease was observed in response to TPA and UVR. This proteinkinase nm23-M2/NDPK-B was also induced in hyperplasticmouse skins and in TPA-mediated mouse skin tumors. In ad-dition, constitutive expression of mouse nm23-M2/NDPK-B inpreneoplastic epidermal cells not only promoted foci-formingactivity but also enhanced the process of neoplastic transfor-mation in these cells. More interestingly, stable transfection ofthe mouse nm23-M2/NDPK-B (delRGD) or nm23-M2/NDPK-B(G106A) mutant gene in JB6 Cl 41-5a cells selectively abro-gated NDPK-B-induced cellular transformation, implicating apossible RGD regulatory role in early skin carcinogenesis.

Acknowledgments—We thank Drs. John O’Bryan (Laboratory of Sig-nal Transduction, NIEHS, National Institutes of Health (NIH)) andRobert Langenbach (Laboratory of Molecular Carcinogenesis (LMC),NIEHS, NIH) for critically reading the manuscript and giving invalu-able advice. We also thank Dr. Michel Veron (Unite de RegulationEnzymatique des Activites Cellulaires, Institute Pasteur, Paris,France) for kindly providing the Nm23-H2/NDPK-B mouse monoclonalantibody (mAb 233.1); Dr. Norbert Fusenig (Division of Differentiationand Carcinogenesis in Vitro, German Cancer Research Center, Heidel-berg, Germany) for generously providing the human keratinocyte cellline HaCaT; Dr. Ronald Cannon (National Center for Toxicogenomics(NCT), NIEHS, NIH) for contributing the mouse skin tissues; AndreaMoon (NCT, NIEHS, NIH) for preparing the FVB/N217 tumor cell totalprotein lysates; John Otstot (LMC, DNA Sequencing Core Facility,NIEHS, NIH) for confirming the DNA sequences; Jian-Li Huangand Dr. Yu-Ying He (Laboratory of Pharmacology and Chemistry(LPC), NIEHS, NIH) for manipulating the UV apparatus;Drs. Alexandra Heinloth (NCT, NIEHS, NIH), Thomas Gray (LMC,NIEHS, NIH), Michelle Block (LPC, NIEHS, NIH), and Bryan Betz(NCT, NIEHS, NIH) for technical suggestions and comments; andDr. Wen K. Yang (Institute of Biomedical Sciences, Academia Sinica,Taipei, Taiwan, Republic of China) for communicating results prior topublication.

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TennantSung-Jen Wei, Carol S. Trempus, Robin C. Ali, Laura A. Hansen and Raymond W.

CellsDiphosphate Protein Kinase B Mediates Neoplastic Transformation of Epidermal

-Tetradecanoylphorbol-13-acetate and UV Radiation-induced NucleosideO12-

doi: 10.1074/jbc.M310820200 originally published online November 17, 20032004, 279:5993-6004.J. Biol. Chem. 

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