以具基因特異性之蛋白體學方法研究訊息傳導

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行政院國家科學委員會專題研究計畫成果報告

以具基因特異性之蛋白體學方法研究訊息傳導

計畫類別：個別型計畫計畫編號： NSC92-3112-B-002-003- 執行期間： 92 年 05 月 01 日至 93 年 04 月 30 日執行單位：國立臺灣大學醫學院臨床醫學研究所計畫主持人：周祖述報告類型：完整報告報告附件：出席國際會議研究心得報告及發表論文處理方式：本計畫可公開查詢

中華民國 93 年 9 月 4 日

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1) In aim1, Dr. Jou propose to use the dual regulatory expressing system (Ecdysone and tetracycline inducible system) …….using constitutively active Rac1 and dominant negative IkB as a study pair. This aim is well thought through, and several alternative approaches were described. One minor comment is that the limitation of 2-D gel is not discussed, and the new ICAT and /or 2-D LC system is not mentioned as alternative approaches.

We have set up a collaboration with Dr. Chung-Lin Liao in Academia Sinica using ICAT as an alternative approach to 2DGE for identifying candidates in relaying the signaling from Rac1 to NFkB (please see the following section).

2) In aim 2, Dr. Jou proposed to construct stable cell lines,……… However, it is hard to know whether these candidates are biologically relevant. This is an intrinsic problem for all proteomic approaches.

With those candidate genes identified by microarray assay, we would then use quantitative RT-PCR and Western blotting analysis to confirm the roles they play in Rho family proteins signaling.

3) and 4) In aim 3, Dr. Jou proposed to perform proteomic approaches to discover target molecules linking RhoA family small GTPases after differentially expressing RhoA/Rac1/Cdc42 mutant genes…... With very good controls and carefully designed experiments, he should be able to produce very interesting results. In aim 4, Dr. Jou proposed to apply these systems to study medically important issues. ……..

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2. Specific Aims

Our primary goal, which has not been modified so far, is to generate double inducible gene expression system in a simplistic and efficient way, and apply this system for dissecting signalling transduction pathways involving Ras related small GTPases using experimental approaches, such as DNA microarray and proteomic studies, based upon comparison of global differences in gene expression profiles.

Specific Aim I. Using the dual regulatory expressing system (Ecdysone and tetracycline inducible

system) already set up in HEK293 cell lines to demonstrate the feasibility of conjugating this double regulated expression system with proteomic studies for dissecting complex signalling transduction pathways; using constitutively active Rac1 and dominant negative IkB as a study pair.

We would like to catagorize the downstream signaling molecules of Rac1, according to the global gene expression patterns, into three groups:

Group X- molecules downstream of Rac1and upstream of NFkB Group Y- molecules downstream of NFkB and further downstream of Rac1

Group Z- molecules downstream of Rac1and unrelated to NFkB signaling pathway

Specific Aim II. Construct stable cell lines, using inducible expression systems set up in MDCK

cells, expressing candidate genes identified during the experimental approaches aimed at specific aim I to confirm the finding and also explore the relevant biological meaning

Specific Aim III. Performing proteomic approaches to discover target molecules linking RhoA

family small GTPases after differentially expressing RhoA/Rac1/Cdc42 mutant genes in either dual regulatory HEK293 or MDCK cells.

Specific Aim IV. Apply the achievement that would be made to characterize genes involved in

hepatocellular oncogensis after successfully carrying out the researches mentioned for specific aim I to III.

Specific Aim IV(A). Construct dual regulatory cell lines in differentiated rat liver cell line,

WB, and transformed hepatic cell line, such as HepG2, Hep3B, and Huh7.

Specific Aim IV(B). Generate stable cell lines expressing viral genes or other

oncogenesis-related candidate genes in cell line set up under Specific Aim IV (A), and then apply comparative proteomic studies to dissect the signalling pathways leading to hepato-oncogenesis .

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3. Progress Summary

To pursue

specific aim 1

of our proposal, we had used the following three different approaches in the past 8 months:

(a) Two-dimensional gel electrophoresis (2-DGE) analysis- both (1a) conventional and

(1b) DIGE (Difference Gel Electrophoresis), which is using the commercially available CyDye system (Amersham/Pharmacia) to differentially label different protein samples, to resolve our samples in gel.

(b) Isotope-Coded Affinity Tag (ICAT) technique- a collaboration between Dr. Chung-Lin Liao's group in the Institute of Chemistry in the Academia Sinica.

(c) DNA Microarray analysis

Before we present our data in the later section, we would like to first define our experimental settings and use E1~E4 as the abbreviations to facilitate communication.

The lysates or mRNAs from HEK293 cells differentially expressing either constitutive Rac1 (Rac1V12) or dominant negative IkB(IkB-DN) mutant were collected in the following settings:

Experimental condition 1 (E1)- neither Rac1V12 nor IkB-DN was expressed (Basal condition) Experimental condition 2 (E2)- Only Rac1V12 was induced (all group X, Y, and Z protein

expressions should be elicited)

Experimental condition 4 (E4)- Both Rac1V12 and IkB-DN were expressed (group Y proteins

should return to near basal condition, group Z should not be affected, while group X proteins might be further changed)

Experimental condition (E3) was not performed, because it was not related to our goal, in which only IkB-DN was expressed, and therefore group Y proteins might be down-regulated.

Two-dimensional gel electrophoresis (2-DGE) analysis

While we were using the conventional 2DGE to resolve the protein lysates from experimental condition 1, 2, and 4 (E1, E2, and E4), we met some problems at first including inadequate sonication of the cell pellets or inadequate use of protease inhibitors, just to name a few of them. We eventually solved out what would be the most optimal condition to get the proteins well separated on 2D gel and stained by Sybro Ruby staining for protein quantification (Fig. 1).

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In the meantime, we also tried the DIGE system advocated by Amersham/Pharmacia. Although we could see many differentially labeled spots on the gels, but many are just artifacts caused by so-called reflection errors (the single arrows in Fig. 2A, 2B, and 2C), which are characterized by the double-vision like, vertically overlapped images. We noticed they were happening no matter which combination of protein samples were subjected for the DIGE analysis. There were also some other types of differentially labeled spots visible on the gel, which were well separated and horizontally arranged (the double arrows in Fig. 2A, 2B, and 2C). Initially, we thought these might be the ones which we sought to pursue. However, we found they were still happening without any correlation to the ways how protein samples were combined; i.e., no matter whether Rac1V12 or dominant negative IkB was induced or not, they were there. Furthermore, we also found if we expressed different pair of transgenes, such as Rac1V12 and RhoAV14, they were still there with the same kind of labeling pattern (data not shown). We were puzzled by this finding and decided to isolate the protein spots for mass spectrometrical analysis to make sure the identities of those mysterious proteins.

Due to the intrinsic problems associated with DIGE, we decided to take conventional approach for 2DGE despite it is tedious and challenging to get three gels (E1, E2, and E4 on three separate gels) run at the same condition. Nevertheless, we did catagorize the differential expression pattern under these three different conditions (E1, E2, and E4) into group X, Y, and Z patterns. The examples are given in Fig. 3.

We have so far identified 7, 7, and 5 spots with group X, Y, and Z expression patterns, respectively, and would be analyzed by MS them when we get enough proteins.

Isotope-Coded Affinity Tag (ICAT) technique

Collaborating with Dr. Chung-Lin Liao in the Institute of Chemistry, Academia Sinica (Genomics/Proteomics Center), we also had our protein samples undergo ICAT approach. Dr. Liao developed a new ICAT reagent, which labels the NH2- group, instead of SH-group, of proteins. This results in higher chance of labeling in proteins containing few cysteines. After trypsinization of the protein samples prepared in E1, E2, and E4 conditions, the peptides were differentially labeled with hydrogen (D0, light) or deuterium (D4, heavy) containing reagents, and undergoing multiple dimensional liquid chromatography (MDLC) and MS analysis. Initial protein identification revealed about one thousand proteins in the samples, and further manual MS spectrum inspection, which took totally three weeks' effort because there was no commercially available software could reliably substitute this part of work, confirmed about 200 peptide pairs between E1/E2 and E2/E4 experimental conditions. A selective list of proteins and their quantification ratios in E2/E1 and E4/E2 conditions are shown in Table 1 and Table 2.

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So far, we haven't identified a protein significantly increasing in E2 vs. E1 conditions, and also in E4 vs. E2 conditions. Our hypothesis for this phenomenon would be discussed later after we presented the data from our microarray analysis.

DNA Microarray analysis

We initially performed microarray assays at our own lab using the protocol and DNA arrays from the Third Core Laboratory in National Taiwan University Hospital, which is also sponsored by the

National Research Program in Genomic Medicine. However, we got inconsistent data which

were difficult to analyze. Later, we shifted to glass type DNA array using fluorescent probe instead of alkaline phosphatase based colorimetric probe. We think we have finally got much more reliable data (Table 3A-C) because we could identify Rac1, the transgene regulated by the tetracycline inducible system, gene up-regulated in experimental condition E2 vs. E1, and the Rac1 mRNA level is the same between experimental condition E2 and E4 (group Z expression pattern in Table 3C). Furthermore, after we used several algorithms to analyze the expression pattern we got, we could get pretty nice clustering of the gene expression patterns (Fig. 4A and 4B).

There are many interesting genes and expression patterns from the microarray assay. Taking the genes classified as group X expression pattern for examples, ARRH (RhoH) is an atypical Rho family GTPase, which doesn't have endogenous GTPase activity and is demonstrated to have a dominant negative effect of the endogenous RhoA protein. It has been previously hypothesized that Rac1 has an antagoisitic effect on RhoA, but the detailed mechanism has been elusive. If RhoH is activated by Rac1, which might be the reason why Rac1 activation might downregulate RhoA activity.

Another gene belonging to group X expression pattern, FNBP1 (formin binding protein 1), has been demonstrated to be a Cdc42 interacting partner and a GEF for Cdc42. This protein may act as a link between cdc42 signaling and regulation of the actin cytoskeleton. If FNBP1 is transcriptionally regulated by Rac1, this could provide a mechanistic basis for the interaction between Rac1 and Cdc42 on actin cytoskeleton.

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Mismatch between the data from microarray and ICAT assays, and the possible solutions to reconcile these differences in the future

Although we are satisfied with the result coming out of microarray assay, the protein expression patterns between the result from microarray do not match with each other. Our explanation is that we overload the MS capacity with a protein sample of very high complexity. The ICAT reagent we used is different from the conventional one in that the former doesn't contain biotin modification and we therefore do not enrich the ICAT labeled tryptic peptides with streptavidin beads. Basically, all the proteins, after trypsin digestion, are entering the MDLC-MS analysis, and it is very likely that the MS spectra of low abundance proteins are masked by those of high abundance proteins. If the expression products of those genes identified by microarray are low in abundance, it is very likely they could be missed from our current ICAT assay.

To solve this problem, we are planning to fractionate our protein samples first into membranous, microsomal, cytosolic, nuclear, and cytoskeletal compartments, followed by reverse phase LC separation of the protein samples of each sub-fractionation before going to the ICAT procedure. Preliminary data from Dr. Liao's group show this might be helpful in decreasing the complexity of proteins going into the ICAT analysis.

In the meantime, we are going to isolate the protein complex by immunoprecipitating either the myc-epitope tagged Rac1V12 or the inhibitor of nuclear factor kappa B (IkB) under experimental conditions differentially expressing constitutively active Rac1V12 or dominant negative IkB. We hypothesize the associated signaling molecules to either Rac1V12 IkB would be different when the signaling pathways are manipulated by Rac1 and IkB mutant proteins. This might sound a desperate approach, but could be easily tested.

Specific Aim II. Construct stable cell lines, using inducible expression systems set up in MDCK

cells, expressing candidate genes identified during the experimental approaches aimed at specific aim I to confirm the finding and also explore the relevant biological meaning

This part of the project awaits the identities of those candidate genes being disclosed after experiments related to Specific Aim I would be completed.

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Specific Aim III. Performing proteomic approaches to discover target molecules linking RhoA

family small GTPases after differentially expressing RhoA/Rac1/Cdc42 mutant genes in either dual regulatory HEK293 or MDCK cells.

To search for possible connections between different members of Rho family GTPases, we have focused in the past one year on the connections between Cdc42 and Rac1. We had then demonstrated a signaling cascade ignaling cascade Cdc42 → Rac1 → PI3K in modulating

detachment induced apoptosis (anoikis). This part of work has been written up as a submitted manuscript, and now the manuscript has been revised and re-evaluated by Experimental Cell

Research (please refer to the manuscript attached in appendix 2).

Specific Aim IV(A). Construct dual regulatory cell lines in differentiated rat liver cell line, WB,

and transformed hepatic cell line, such as HepG2, Hep3B, and Huh7.

We have so far generated Tetracycline inducible expression system in three of the above three cell lines, using the strategy we have recently published (Am J Physiol Cell Physiol 2003; 285:C711-719). Taking the Huh7 cell line as example, after three rounds of positive FACS based selection and one round of negative selection, we have enriched inducible clones with an inducibility of about 100 folds.

While we were generating the dual regulatory expression system, we accidentally found doxycycline, previously thought to be an inert chemical compound, might differentially regulate the expression level of reverse type tetracycline regulated tranactivator (rtTA). We studied the mechanistic basis of this phenomenon and wrote up a manuscript which had been submitted to

Journal of Gene Medicine (please appendix 3).

For HepG2 and Hep3B, after similar approaches, we have inducible cell populations with inducibility of about 60 and 80 folds.

As for the differentiated WB cell line, we would conduct similar selection scheme very soon. Once we finish establishing tetraycline controlled expression system in all these four cell lines, we would set up ecdysone inducible expression system in them to complete the making of dual regulatory expression systems in four cell lines.

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Table 1

Proteins identified to be up-regulated in E2 vs E1 condition by ICAT analysis

Mascot No.

Accession No. Protein Name E2/E1

23 gi|4885375 H1 histone family, member 2; histone H1d [Homo sapiens] 1.541

373 gi|27498927 similar to CG5882-PA [Homo sapiens] 1.474

422 gi|4506583 replication protein A1, 70kDa; replication protein A1 (70kD) [Homo sapiens]

1.429

396 gi|37538778 similar to dJ753D5.2 (novel protein similar to RPS17 (40S ribosomal protein S17)) [Homo sapiens]

1.426

186 gi|444021 sub2.3 [Homo sapiens] 1.409

947 gi|6424942 ALG-2 interacting protein 1 [Homo sapiens] 1.397

506 gi|243123 cytochrome c oxidase subunit VIIa='liver-type' isoform {EC 1.9.3.1} [human, skeletal muscle, Peptid

1.394

360 gi|4506681 ribosomal protein S11; 40S ribosomal protein S11 [Homo sapiens] 1.383

559 gi|9022435 ribosomal protein S3; RPS3 [Homo sapiens] 1.378

233 gi|6841566 HSPC173 [Homo sapiens] 1.370

448 gi|4757900 calreticulin precursor; Sicca syndrome antigen A (autoantigen Ro; calreticulin); autoantigen Ro [Ho

1.370

474 gi|5032087 splicing factor 3a, subunit 1, 120kDa; pre-mRNA splicing factor SF3a (120 kDa subunit) [Homo sapien

1.336

471 gi|36142 ribosomal protein homologous to yeast S24 [Homo sapiens] 1.335

230 gi|478813 nonhistone chromosomal protein HMG-1 - human 1.318

183 gi|14250148 Ribosomal protein L3 [Homo sapiens] 1.313

8 gi|1136741 KIAA0002 [Homo sapiens] 1.298

623 gi|5901922 CDC37 homolog [Homo sapiens] 1.293

122 gi|2136253 TCP1 ring complex protein TRiC5 - human 1.281

194 gi|1203969 filamin [Homo sapiens] 1.278

46 gi|4507677 tumor rejection antigen (gp96) 1; Tumor rejection antigen-1 (gp96); glucose regulated protein, 94 k

1.276

28 gi|28614 aldolase A [Homo sapiens] 1.273

295 gi|178663 medium tumor antigen-associated 61-kD protein 1.272

84 gi|7433799 fatty-acid synthase (EC 2.3.1.85) (version 2) - human 1.267

75 gi|115206 C-1-tetrahydrofolate synthase, cytoplasmic (C1-THF synthase) [Includes: Methylenetetrahydrofolate d

1.262

192 gi|423123 tpr protein - human 1.250

141 gi|553640 ribosomal protein S13 [Homo sapiens] 1.249

539 gi|20987810 MRPS27 protein [Homo sapiens] 1.214

18 gi|5174735 tubulin, beta, 2 [Homo sapiens] 1.213

147 gi|5174447 guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1; protein homologous to ch

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Table 2

Proteins identified to be up-regulated in E4 vs E2 condition by ICAT analysis

Mascot No.

Accession No. Protein Name 4H/2L

ratio 102 gi|37542903 similar to 60 kDa heat shock protein, mitochondrial precursor (Hsp60)

(60 kDa chaperonin) (CPN60) (

8.29

350 gi|37538336 KIAA1856 protein [Homo sapiens] 2.99

1090 gi|14591909 ribosomal protein L5; 60S ribosomal protein L5 [Homo sapiens] 1.33 59 gi|4758756 nucleosome assembly protein 1-like 1; HSP22-like protein interacting

protein; NAP-1 related protein

1.31 469 gi|2135244 chromosome segregation protein smc1 [similarity] - human 1.28

62 gi|6005854 repressor of estrogen receptor activity; B-cell associated protein [Homo sapiens]

1.22 71 gi|7433799 fatty-acid synthase (EC 2.3.1.85) (version 2) - human 1.21 589 gi|4884564 vitamin D3 receptor interacting protein [Homo sapiens] 1.21

41 gi|180555 creatine kinase-B 1.19

93 gi|105294 alternative splicing factor ASF-2 - human 1.18

142 gi|20521660 KIAA0788 protein [Homo sapiens] 1.14

186 gi|438069 thiol-specific antioxidant protein [Homo sapiens] 1.14 177 gi|2559008 chaperonin containing t-complex polypeptide 1, delta subunit;

CCT-delta [Homo sapiens]

1.10 79 gi|4503297 DEAH (Asp-Glu-Ala-His) box polypeptide 9 isoform 1; DEAD/H

(Asp-Glu-Ala-Asp/His) box polypeptide 9

1.08 90 gi|4506607 ribosomal protein L18; 60S ribosomal protein L18 [Homo sapiens] 1.07 513 gi|8923579 hypothetical protein FLJ20625 [Homo sapiens] 1.07

805 gi|4759196 symplekin [Homo sapiens] 1.06

841 gi|25136577 ELYS transcription factor-like protein TMBS62 [Homo sapiens] 1.04 96 gi|4506671 ribosomal protein P2; 60S acidic ribosomal protein P2; acidic

ribosomal phosphoprotein P2 [Homo sap

1.04 249 gi|4506691 ribosomal protein S16; 40S ribosomal protein S16 [Homo sapiens] 1.02 74 gi|9802306 DNA-binding protein TAXREB107 [Homo sapiens] 1.00

170 gi|5031635 cofilin 1 (non-muscle) [Homo sapiens] 1.00

49 gi|337424 poly(ADP-ribose) synthetase 0.99

8 gi|7106439 tubulin, beta 5 [Mus musculus] 0.99

254 gi|135538 T-complex protein 1, alpha subunit (TCP-1-alpha) (CCT-alpha) 0.99

313 gi|136066 TRIOSEPHOSPHATE ISOMERASE (TIM) 0.99

220 gi|4506209 proteasome 26S ATPase subunit 2; proteasome 26S subunit, ATPase, 2; mammalian suppressor of sgv-1 o

0.98 305 gi|1431788 Chain A, Cyclophilin A Complexed With Cyclosporin A (Nmr, 22

Structures)

0.98 456 gi|36142 ribosomal protein homologous to yeast S24 [Homo sapiens] 0.98 283 gi|18848326 Similar to GDP dissociation inhibitor 2 [Homo sapiens] 0.97

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Table 3A. Genes displaying group X expression pattern (expression ratio under E2/E1 experimental conditions is more than 2, while expression ratio under E4/E2 experimental conditions is more than 1.45) by DNA microarray assay

CLID NAME E2-E1 E4-E2

IMAGE:704274 transcriptional regulating protein 132 20.549 2.540

IMAGE:1306637 tumor protein D52 2.382 2.227

IMAGE:1285720 formin binding protein 1 2.133 1.925

IMAGE:454475 phosphorylase kinase, alpha 2 (liver) 2.035 1.557 IMAGE:302591 ras homolog gene family, member H 8.846 1.491 IMAGE:1307809 Similar to interferon-gamma receptor alpha chain 2.208 1.467

Table 3B. Genes displaying group Y expression pattern (expression ratio under E2/E1 experimental conditions is more than 2, while expression ratio under E4/E2 experimental conditions is less than 0.5) by DNA microarray assay

IMAGE:809901 collagen, type XV, alpha 1 19.213 0.032

IMAGE:789369 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein

8.462 0.083 IMAGE:196992 aldo-keto reductase family 1, member C1 8.369 0.077 IMAGE:1309018 uncharacterized hypothalamus protein HCDASE 7.863 0.062 IMAGE:430038 FYN binding protein (FYB-120/130) 6.797 0.141 IMAGE:669310 mitogen-activated protein kinase-activated protein

kinase 5

6.440 0.259

IMAGE:712341 ribonuclease 6 precursor 5.996 0.128

IMAGE:323181 fibroblast activation protein, alpha 5.856 0.241

IMAGE:21655 5'-nucleotidase, ecto (CD73) 5.680 0.181

IMAGE:223176 MAX dimerization protein 1 5.389 0.206

IMAGE:2477598 secretory leukocyte protease inhibitor 5.216 0.499 IMAGE:1271368 SH3-domain binding protein 1 4.803 0.368

IMAGE:784928 sushi-repeat protein 4.721 0.372

IMAGE:461727 phenylalanine hydroxylase 4.624 0.151

IMAGE:1302646 chromosome 13 open reading frame 9 4.579 0.363 IMAGE:814251 signaling lymphocytic activation molecule family

member 1

4.569 0.460 IMAGE:753346 aminolevulinate, delta-, synthase 2 4.341 0.477 IMAGE:1240974 Rho GDP dissociation inhibitor (GDI) beta 4.284 0.360

IMAGE:2448778 cathepsin F 4.067 0.265

IMAGE:2508044 haptoglobin 4.059 0.241

IMAGE:1287536 zinc finger protein, subfamily 1A, 1 (Ikaros) 4.006 0.250 IMAGE:1470151 GRB2-associated binding protein 2 3.926 0.168

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Table 3B. Continued

IMAGE:74537 alpha-fetoprotein 3.880 0.208

IMAGE:204541 asialoglycoprotein receptor 1 3.853 0.266 IMAGE:685022 diacylglycerol kinase, epsilon 64kDa 3.829 0.395

IMAGE:712278 c-fos 3.769 0.245

IMAGE:428083 basic transcription factor 2 3.630 0.368 IMAGE:487418 filamin A, alpha (actin binding protein 280) 3.620 0.274 IMAGE:2028959 dopa decarboxylase (aromatic L-amino acid decarboxylase) 3.615 0.371 IMAGE:1335809 hypoxia-inducible factor 1, alpha subunit 3.470 0.394 IMAGE:243174 serine (or cysteine) proteinase inhibitor, clade F (alpha-2

antiplasmin member 2

3.425 0.392

IMAGE:2353365 deoxyribonuclease I-like 1 3.371 0.290

IMAGE:704815 Inhibitor of DNA binding 2 3.274 0.239

IMAGE:70692 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin)

3.182 0.247 IMAGE:284022 Rho guanine nucleotide exchange factor (GEF) 10 3.141 0.423 IMAGE:565319 mal, T-cell differentiation protein 2 3.076 0.301

IMAGE:324492 matrix metalloproteinase 3 3.072 0.433

IMAGE:612576 3-oxoacid CoA transferase 3.055 0.402

IMAGE:2449395 aldo-keto reductase family 1, member C2 3.044 0.310 IMAGE:825645 activation-induced cytidine deaminase 3.013 0.392

IMAGE:1534853 centaurin, beta 1 3.008 0.394

IMAGE:325145 pentaxin-related gene, rapidly induced by IL-1 beta 2.932 0.456 IMAGE:2449786 tumor necrosis factor receptor superfamily, member 18 2.882 0.393

IMAGE:50604 **androgen-induced 1 2.834 0.425

IMAGE:1241180 zinc finger protein, subfamily 1A, 1 (Ikaros) 2.670 0.299

IMAGE:1334310 CGI-109 protein 2.661 0.332

IMAGE:50503 integrin, beta 2 2.650 0.232

IMAGE:840844 heat shock 70kDa protein 5 2.623 0.264 IMAGE:1656636 UDP glycosyltransferase 2 family, polypeptide B4 2.583 0.312

IMAGE:704084 centaurin, delta 1 2.567 0.341

IMAGE:487429 collagen, type VI, alpha 1 2.491 0.271

IMAGE:212188 apolipoprotein H (beta-2-glycoprotein I) 2.485 0.283

IMAGE:1306275 mutL homolog 3 (E. coli) 2.224 0.328

IMAGE:2298080 kynureninase (L-kynurenine hydrolase) 2.213 0.345 IMAGE:1681489 serine (or cysteine) proteinase inhibitor, clade E (nexin,

plasminogen activator inhibitor type 1), member 1

2.210 0.326

IMAGE:31093 cadherin 13, H-cadherin (heart) 2.155 0.205 IMAGE:1241157 tripartite motif-containing 22 2.151 0.338

IMAGE:668182 zinc finger protein 193 2.120 0.326

IMAGE:768443 microsomal glutathione S-transferase 1 2.104 0.326 IMAGE:2458975 inhibin, beta A (activin A, activin AB alpha polypeptide) 2.089 0.227

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Table 3C. Genes displaying group Z expression pattern (expression ratio under E2/E1 experimental conditions is more than 2, while expression ratio under E4/E2 experimental conditions is between 0.5 and 1.45) by DNA microarray assay

IMAGE:1369976 ecotropic viral integration site 2A 9.547 0.564

IMAGE:143523 collagen, type V, alpha 1 5.049 1.438

LCP:138 methionine-tRNA synthetase 4.800 1.217

IMAGE:2117981 47-kD autosomal chronic granulomatous disease protein 4.332 1.250 IMAGE:703774 **mitogen-activated protein kinase 8 interacting protein 3 4.332 0.699

IMAGE:1338277 RAC1 4.190 0.812

IMAGE:2491247 solute carrier family 21 (organic anion transporter), member 9

3.761 0.692 IMAGE:827132 ras-related C3 botulinum toxin substrate 2 3.489 1.426 IMAGE:781105 AHA1, activator of heat shock 90kDa protein ATPase

homolog 2 (yeast)

3.437 1.154

IMAGE:825590 xylosyltransferase 3.401 1.301

IMAGE:1307643 phosphatidylinositol transfer protein, membrane-associated 1

3.265 0.529

IMAGE:712279 kelch-like 6 (Drosophila 3.211 0.641

LCP:1 deoxynucleotidyltransferase, terminal 3.180 1.053 IMAGE:284263 myelin-associated oligodendrocyte basic protein 3.134 0.777

IMAGE:1019777 butyrylcholinesterase 3.130 0.851

IMAGE:704154 epidermodysplasia verruciformis 1 3.065 1.113

IMAGE:563574 FSHD region gene 1 2.986 0.527

IMAGE:701572 NADP-dependent retinol dehydrogenase/reductase 2.963 0.923 IMAGE:1184934 purinergic receptor P2Y, G-protein coupled, 8 2.914 0.723 IMAGE:683257 EST from selenoprotein P promoter region 2.866 0.954 IMAGE:358842 runt-related transcription factor 3 2.805 1.171 IMAGE:685210 T-cell activation GTPase activating protein 2.572 1.306

IMAGE:127636 **zuotin related factor 1 2.530 1.281

IMAGE:686274 peptidylprolyl isomerase (cyclophilin)-like 2 2.519 1.163

IMAGE:1543346 transketolase-like 1 2.474 1.221

IMAGE:701018 transcription factor Dp-2 (E2F dimerization partner 2) 2.302 1.340 IMAGE:1306711 ceroid-lipofuscinosis, neuronal 6, late infantile, variant 2.299 1.324 IMAGE:260200 myeloid cell nuclear differentiation antigen 2.201 1.401 IMAGE:80915 succinate dehydrogenase complex, subunit A 2.124 1.437 IMAGE:1286006 centrosomal kinesin-like protein 2.108 1.442 IMAGE:1670870 unc-93 homolog B1 (C. elegans) 2.062 1.429

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Figure 1

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Figure 2A

E1 lysate- neither Rac1V12 nor IkB-DN was expressed, labeled in red

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Figure 2B

E2 lysate- Only Rac1V12 was induced, labeled in red

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Figure 2C

E1 lysate- neither Rac1V12 nor IkB-DN was expressed, labeled in red

E4 lysate- Both Rac1V12 and IkB-DN were expressed, labeled in green

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patterns; the left upper quadrant in each composite image shows the quantified intensities of the index spots as a histogram in the order of E1, E2, and E4. The gels were stained with Sybro Ruby stain, scanned by Typhoon 9200 (Amersham/Pharmacia) and quantified by using the PDQuest7.1.1 software (Bio-Rad).

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Figure 4A

Hierarchical clustering result of group X (red), Y (green) , and Z (blue) genes.

Figure 4B

Non-hierarchical clustering result of group X (yellow), Y (blue), and Z (red) genes.

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4. Projected Timeline & Brief Summary of Plans for Next Year

Specific Aim I. (From 2004-01-01 to 2004-4-30)

Using the combined approaches by conventional 2DGE, ICAT, and microarray assay to dissect the complex signalling transduction pathways linking constitutively active Rac1 and nuclear factor kappa B (NFkB), we would eventually track down the possible candidates.

As we found the complexity of our protein samples might be too high for the ICAT assay, we plain to sub-fractionate the protein samples according to their distribution in different cellular compartment, and the fractionated protein samples would be further separated by reverse phase liquid chromatography. We would use quantitative real-time RT-PCR and western blotting analysis to confirm the expression patterns of several candidate genes we identified by microarray, notably those involved in Rho family protein signalling, such as RhoH and FNBP1(formin binding protein 1). We also plan to summarize our microarray data in a small methodology paper.

Specific Aim II. (From 2004-5-01 to 2004-08-31)

Construct stable cell lines, using inducible expression systems set up in MDCK cells, expressing candidate genes identified during the experimental approaches aimed at specific aim I to confirm the finding and also explore the relevant biological meaning

Specific Aim III. (From 2004-01-01 to 2004-12-31)

Given the experience we obtained in the past 8 months or so, we think we could start using the combined proteomic and microarray approach to discover target molecules linking Rho family small GTPases after differentially expressing RhoA/Rac1/Cdc42 mutant genes in either dual regulatory HEK293 or MDCK cells. Since we have already written up a manuscript, which is under second revision after being sent to Experimental Cell Research, describing the signaling cascade Cdc42 → Rac1 → PI3K in modulating detachment-induced apoptosis (anoikis) (please

refer to the manuscript in appendix 2). We would propably start from the constitutively active Cdc42 and dominant negative Rac1 double mutant cells first.

Specific Aim IV. (From 2004-01-01 to 2004-06-30)

Specific Aim IV(A). Finish constructing dual regulatory cell lines in differentiated rat liver

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5. Personnel

Summarize the

personnel involved in the project during the grant period

. List

the personnel in accordance to the following categories: (1) senior investigators,

including visitors; (2) postdoctoral fellows; (3) graduate students; (4) technicians or

research assistants. Specify for each individual the period of involvement and the

percentage commitment of effort.

Name In Chinese In English Position Title Education Degree % of personal effort on this project Job Description or Responsibilities

周祖述

鄭子琳

鄭心遠

蔡舒聿

林育誼

Tzuu-Shuh Jou Tzu-Ling Cheng Hsin-Yuan Cheng Su-Yu Tsai Yu-Yi Lin Assistant Professor (Principal Investigator) 1st year technician 1st year technician 1st year technician 3rd Year Resident in internal medicine MD;Ph.D Master degree Master degree Master degree M.D. 50% (with another NSC sponsored project at hand) 100% 100% 100% 50%

Coordinating the works and planning the experiments associated with this project Writing proposals,

manuscripts, and rebuttals for the publication

DNA plasmid constrtuction, Cell Culturing, and

Making stable clones

. cell biology and biochemical assay; carrying the project related to the submitted paper No-1

cell biology and biochemical assay; carrying the project related to the submitted paper No-2

Protein extraction, Running 2D gel

FACS sorting

Microarray assay and data analysis

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6. Publications and/or Patents

6a. Publications

List the title and complete references (author(s), journal or book, year, page number)

of all publications

directly resulting from studies supported by the project (i.e.,

with citation of this grant in the acknowledgement section)

. List the publications

for the project in accordance to the following categories: (1) manuscripts published

and accepted for publications; (2) manuscripts submitted; and (3) conference

proceedings. Provide one copy of each publication not previously reported to the

National Science Council in the Appendix.

1.

Lai JF, Juang SH, Hung YM, Cheng HY, Cheng TL, Mostov KE, Jou TS.

Appendix 1

Lai JF, Juang SH, Hung YM, Cheng HY, Cheng TL, Mostov KE, Jou TS. An ecdysone

and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling. Am J Physiol Cell Physiol. 2003 Sep;285(3):C711-9. Epub 2003

May 07 (To save pages, please log onto

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２３

Appendix 2 (Submitted to Experimental Cell Research, revised and

resubmitted).

Regulation of Anoikis by Cdc42 and Rac1

Tzu-Ling Cheng, Marc Symons*, Tzuu-Shuh Jou§

Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine

No. 7, Chung-Shan S. Road Taipei, 100 Taiwan

* Center for Oncology and Cell Biology, North Shore-LIJ Research Institute, 350 Community Drive, 11030, Manhasset, NY, USA

§To whom reprint should be addressed. Tel: 8862-23123456 ext.7258

Fax: 8862-23709820

E-mail: [email protected]

Manuscript Information: 41 pages of text, and 8 figures in the paper.

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ABSTRACT

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily

members contribute significantly to this process. Anchorage-independent growth and the ability to

avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this

study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin Darby

canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 could stimulates the

ERK, JNK and p38 MAPK pathways in suspension condition; however, inhibition of these

signalling does not affect Cdc42-stimulated cell survival. However, we demonstrated that

iInhibition of phosphatidylinositol 3-kinase (PI3K) pathway however abolishesd the protective

effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also

showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by

Rac1. The consequence of Rac1 activation initiates aWe also provide evidence for a positive

regulatory feedback loop between involving Rac1 and PI3K. In addition, we show that the

survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by

Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin

cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results

suggest indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also

propose conclude that this survival function depends on a positive feedback loop involving Rac1

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INTRODUCTION

The interaction between cells and the surrounding matrix is a major determinant of cellular

behavior, modulating gene expression, cell growth and differentiation, cell migration, and overall

tissue architecture [1]. Anchorage-dependent survival is also an important consequence of

cell-matrix interaction [2]. Epithelial cells, endothelial cells, and muscle cells undergo

programmed cell death when they are deprived of the contact with extracellular matrix [3].

Apoptosis induced by disruption of the interaction between epithelial cells and extracellular

matrix has been termed as "anoikis", which means homelessness in Greek [4]. Anoikis plays an

essential role in regulating tissue homeostasis in normal epithelial tissues. When keratinocytes

and colonic epithelial cells migrate from the dividing basal layer toward the outer lining layer [5],

the cells lose the ability to divide and eventually exofoliate from the monolayer. Anoikis also

regulates involution of mammary glands [6], and is an important step in the first cavitation of

embryogenesis [7]. Anoikis also modulates many pathological conditions. An important

characteristic of transformed cells is the loss of anchorage-dependent growth control, thereby

disrupting an essential surveillance mechanism that prevents cells from colonizing elsewhere

when they are detached from their normal residence [8][9]. The capability of escaping anoikis

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Initially identified as major players relaying the signalling from lipid and growth factor

components in serum to actin cytoskeleton organization, Rho family GTPases have been

demonstrated to regulate a large number of biological processes in response to cell-cell and

cell-substratum adhesion [10, 11]. Rho family members regulate distinct actin cytoskeleton-based

structures; namely, Cdc42 induces filopodia, Rac1 stimulates lamellipodia formation, while RhoA

regulates the formation of stress fibers and focal adhesions [12-14]. There is considerable cross

talk between members of the Rho family, the details of which appear to depend on the cell type and

observation conditions [15]. Notably however, in most circumstances, Cdc42 appears to act

upstream of Rac1 [14].

Rho family GTPases play an important role in cell transformation [16]. Expression of

constitutively active (hydrolysis-defective) Rac1 in Rat1 fibroblasts induces serum- and

anchorage-independent growth and is tumorigenic in nude mice [17]. Cdc42 regulates

anchorage-independent growth and dominant negative Cdc42 N17 inhibits Ras focus formation

and anchorage-independent growth [18]. We have also shown that constitutively active Rac1

protects MDCK cells from anoikis, while dominant negative Rac1 potentiates anoikis in MDCK

cells [19]. In this paper, we address the role of Cdc42 in the regulation of anoikis, and examine its

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Experimental procedures

Plasmids

Coding sequences expressing constitutively active and dominant negative Cdc42 were

amplified by PCR using pCMVneoMYC-Cdc42V12 and pCMVneoMYC-Cdc42N17 (gifts of Dr.

Arie Abo and Matt Hart, Onyx Pharmaceuticals) as a template respectively. The PCR primers

were designed so that the amplified products were tagged with a 5' EcoRI and a 3' XhoI site. The

PCR products were cloned into the EcoRI and XhoI restricted pCMV-Tag2B (Stratagene) to tag a

FLAG epitope at their amino terminals, and resulted in two intermediate plasmids,

pCF-Cdc42V12 and pCF-Cdc42N17. The FLAG-tagged Cdc42V12 coding sequence was

released from pCF- Cdc42V12 by NotI and XhoI digestion, and cloned into similarly restricted

vector, pIND(SP1), to generate pISF-Cdc42V12. By a similar strategy, pISF-Cdc42N17 was also

made. pISF-Cdc42V12 and pISF-Cdc42N17 were confirmed to express FLAG-tagged

constitutively active or dominant negative Cdc42 in an ecdysone responsive manner by

immunoblotting and immunofluorescence after they were transiently transfected into MDCK and

HEK293 cell clones which had been stably transfected with heterodimeric ecdysone receptors

system [20]. pVgRXR (Invitrogen) is used to express the heterodimeric ecdysone receptors and

confer zeocin (Invitrogen) resistance during drug selection. All the engineered plasmids were

made according to standard molecular biological techniques and were confirmed by DNA

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Stable cell lines construction

Madin-Darby canine kidney (MDCK) cells were grown in DMEM containing 10% fetal

bovine serum at 37oC in a humidified atmosphere containing 5% CO2. Transfection was

performed using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's

instruction. Our ultimate goal is to establish dual regulatory cell lines expressing different small

GTPase genes independently under tetracycline and ecdysone inducible systems. Therefore, we

started with a well characterized MDCK cell line expressing dominant negative Rac1 (Rac1N17)

gene under tetracycline repressible system [21, 22], and established ecdysone inducible expression

system by zeocin selection and a FACS enrichment strategy [20] in this cell line which we named

Rac1N17.1. We co-transfected 6 x 105 Rac1N17.1 cells in a p-35 well with 3.8 µg

pISF-Cdc42V12 or pISF-Cdc42N17 respectively and 0.2 µg pUB-Bsd (Invitrogen), which carried

blasticidin-S selection marker. Six hours after transfection, the cells were trypsinized and splitted

onto 6 p-100 wells with the addition of 20 ng/ml of doxycycline to suppress the expression of the

originally established (stably integrated) Rac1N17 transgene. The day after transfection, cell

culture medium was replete with 10 µg/ml of blasticidin-S for selection of stably transfected

clones. Individual candidate clones expressing FLAG-tagged Cdc42V12 or Cdc42N17 were

identified by immunofluorescence and Western blotting using anti-FLAG antibody (M2) from

Sigma. In parallel, empty pIND(SP1) vector was cotransfected and selected in the same way to

establish mock transfectant control (used to generate data described in Figure 1C). To induce the

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concentration of ponasterone (Stratagene), and an aliquot of cells were added with the same

volume of vehicle (alcohol) as a control. To establish MDCK cell line expressing constitutively

active PI3K mutant in the background of tetracycline repressible Rac1N17 transgene, we followed

the same transfection and selection protocol as described above except we co-transfected 6 x 105

Rac1N17.1 cells in a p-35 well with 3.8 µg plasmid expressing CMV promoter driven PI3K

constitutively active mutant (p110*) and 0.2 µg pVgRXR (Invitrogen), which carried zeocin

selection marker.

Antibodies and inhibitors

Immunoblotting was performed with one of the following primary antibodies: rabbit

anti-Cdc42 and anti-p110 catalytic domain of PI3K (Santa Cruz), rabbit anti-total ERK p42/44,

rabbit anti-phosphorylated ERK p42/44 (T202/Y204), rabbit anti-total and anti- phosphorylated

JNK, rabbit anti-total and anti- phosphorylated JNK, anti- phosphorylated GSK-3 (the above

antibodies were from Cell Signaling Technology), mouse anti Rac1 (UBI), anti-myc (purified

from 9E10 hybridoma) or anti-FLAG tag (M2, Sigma). The secondary antibody used for Western

blotting analyis was either an anti-rabbit or anti-mouse HRP-linked antibody at 1:3000 dilution

(Amersham). The following small molecule inhibitors were dissolved in DMSO and used for

dissecting Cdc42V12 mediated survival signalling pathway taken by suspended cells: MEK

inhibitor U0126 (Promega), JNK inhibitor SP600125 (Tocris), p38 inhibitor SB203580, PI3K

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used, the same volume of DMSO was also added to the control wells.

Western blot analysis

Adherent or suspended cells were washed in PBS, and cell extracts were prepared by lysing

cells in boiling SDS sample buffer (2% SDS, 150 mM NaCl, 62.5 mM Tris-HCl (pH 6.8), 10%

glycerol, 50 mM dithiothreitol, 0.01% bromophenol blue). The protein samples were separated by

SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell) and the

membranes were blocked in 5% non-fat milk in PBST (0.1% Tween-20 in PBS) followed by

immunoblotting analysis. Blots were developed with ECL reagent (Amersham-Pharmacia Biotech)

for Western blotting analysis of total lysate or SuperSignal West Femto substrate (Pierce) for

GST-PAK pull-down assay.

P38 and PI 3-kinase activity assay

Immobilized phospho-p38 (Thr180/Tyr182) monoclonal antibody (Cell Signaling

Technology) and immobilized phospho-Akt (Ser473) monoclonal antibody (1G1) (Cell Signaling

Technology) were used to determine p38 and Akt activities of MDCK cells in suspension.

Extracts were made from 6 x106 cells that were induced to express transgenes for 24 hours under

adherent condition and further cultured in suspension for 2 hours. The cells were then extracted

using lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5

mM sodium pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin)

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overnight and for 3 hours at 4oC respectively. Immunoprecipitates of activated Akt were washed

twice with lysis buffer and twice with kinase buffer (25 mM Tris pH 7.5, 5 mM β-glycerol

phosphsate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). Then kinase reactions were carried

out at 30oC for 30 minutes in the presence of 200 µM ATP and 1 µg of GSK-3 to assay PKB

activity. Reactions were terminated by adding 2X SDS sample buffer and the boiled samples were

loaded onto a SDS-PAGE gel. Immunoprecipitates of activated p38 were washed three times with

lysis buffer and 2X SDS sample buffer was added before loading onto a SDS-PAGE gel. Western

blotting analysis was processed as described above and incubated with rabbit anti-phosphorylated

GSK-3 or anti-phosphorylated p38 antibody to assess the activities of Akt and p38 respectively.

p38 activity assay

Immobilized phospho-p38 (Thr180/Tyr182) monoclonal antibody (Cell Signaling

Technology) was used to determine p38 activities of MDCK cells in suspension. Extracts were

made from 6 x106 cells that were induced to express transgenes for 24 hours under adherent

condition and further cultured in suspension for 2 hours. The cells were then extracted using lysis

buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5 mM sodium

pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin) supplied by

the manufacturer. Immunoprecipitation of activated p38 was carried out overnight at 4oC.

Immunoprecipitates of activated p38 were washed three times with lysis buffer and 2X SDS

sample buffer was added before loading onto a SDS-PAGE gel. Western blotting analysis was

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assess the activities of p38.

Akt activity assay

Immobilized phospho-Akt (Ser473) monoclonal antibody (1G1) (Cell Signaling

Technology) was used to determine Akt activities of MDCK cells in suspension. Extracts were

made from 6 x106 cells that were induced to express transgenes for 24 hours under adherent

condition and further cultured in suspension for 2 hours. The cells were then extracted using lysis

buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5 mM sodium

pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin) supplied by

the manufacturer. Immunoprecipitation of activated Akt were carried out for 3 hours at 4oC.

Immunoprecipitates of activated Akt were washed twice with lysis buffer and twice with kinase

buffer (25 mM Tris pH 7.5, 5 mM β-glycerol phosphsate, 2 mM DTT, 0.1 mM Na3VO4, and 10

mM MgCl2). Then kinase reactions were carried out at 30oC for 30 minutes in the presence of 200 µM ATP and 1 µg of GSK-3. Reactions were terminated by adding 2X SDS sample buffer and the boiled samples were loaded onto a SDS-PAGE gel. Western blotting analysis was processed as

described above and incubated with rabbit anti-phosphorylated GSK-3 to assess the activities of

Akt.

Immunofluorescence mMicroscopy

The procedure for morphological studies was the same as the one published previously [21].

The mouse anti-FLAG antibody (M2, Sigma) was used at 1 ng/ml for indirect

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Anoikis induction and DNA fragmentation assays.

Mutant GTPases expressing MDCK cells were induced to express transgenes by adding the

indicated concentration of ponasterone (for ecdysone inducible transgenes) or removing

doxycycline (for tetracycline repressible transgenes) for 24 hours as a monolayer and subsequently

trypsinized and cultured in suspension on ultra low attachment plates (Costar) at a density of 5

x104 cells/ ml for 16-18 hours. Cells were then processed for assessing the level of DNA

fragmentation using the Cell Death ELISA kit (quantifying histone-associated DNA fragments)

using the protocol suggested by the manufacturer (Roche Molecular Biomedicals). Lysates

assayed were equivalent to 2.5 x104 cells. Each condition contained at least four independent

samples. Results were representative of at least three independent experiments, and shown as the

means with S.E.M.

Cell survival assay

10,000 MDCK cells expressing Cdc42V12 and control cells in 200 µl of culture medium

were maintained in suspension condition for 18 hours. Then, 40 µl of a modified MTT reagent,

MTS

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

mixed with an electron coupling reagent (phenazine ethosulfate) from Promega, was added to the

well with gentle pipetting. After incubation in the incubator at 37oC for 60 minutes, absorbance at 490 nm was recorded. In the meantime, 10,000 control cells were detached from plastic dish and

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Rac1 activation assay

The procedure of preparing GST-PAK fusion protein and processing cellular lysates to

perform the pull down assay is essentially as described before [23]. In brief, the p21-binding

domain (PBD) of p21-activated kinase 1 (PAK1) was fused with glutathione-S-transferase to make

a recombinant protein (GST-PBD). The GST-PBD fusion protein was then expressed, purified,

and coupled to glutathione-sepharose beads (Amersham-Pharmacia Biotech). One twentieth of

the clarified cell lysate was immunoblotted for the corresponding GTPase specific antibody to

confirm the presence of equal loading, while the rest of the lysate was then incubated for 60 min at

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Results

Constitutively activeRegulation of anoikis by Cdc42 suppresses, while inhibition

of Cdc42 activities enhances, MDCK cell anoikis

We choose MDCK cells, which readily undergo apoptosis when cultured in suspension, for

studying anoikis [4, 24]. In order to explore the role of Cdc42 in cell survival in anchorage

independent conditions, we generated stable MDCK cell lines that expressed constitutively active

Cdc42V12 or dominant negative Cdc42N17 under the control of an ecdysone-inducible promoter

[25]. We selected several lines for each mutant transgene, and examined the effect of regulated

expression of Cdc42V12 and Cdc42N17 on anoikis. Results were consistent among independent

clones, and were presented here with those generated from a representative clone. Addition of

ponasterone, a synthetic ecdysone analogue, induced expression of Cdc42V12 comparable to that

of endogenous Cdc42 (Fig 1A). Interestingly, the endogenous Cdc42 expression appeared to be

down-regulated by the over-expressed transgene, possibly reflecting a regulatory mechanism to

keep the activation status of Cdc42 in check (Fig. 1A).

Expression of Cdc42V12 by addition of ponasterone reduces the extent of apoptosis in

suspension conditions in a dose-dependent manner, as measured by the level of histone-associated

DNA fragments (Fig. 1B). In contrast, addition of the same concentration of ponasterone did not

have any effect on apoptosis in the parental cell line (Fig. 1B). To exclude the possibility of

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similar doses of ponasterone into the medium of wild type MDCK, and mock transfectant controls.

No significant effect of ponasterone was observed on the anoikis of these cells (Fig. 1C), arguing

for the specificity of the effect elicited by Cdc42 activation on anoikis regulation.

To confirm that Cdc42 significantly enhances the survival potential of MDCK cells in

suspension, we determined the viability of control and Cdc42V12-expressing cells after keeping

them in suspension culture for 18 hours. We used a modified MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay to quantify cell survival. Whereas only 40% of

the control cells survived in suspension conditions, up to 70% of the Cdc42V12-expressing cells

remained viable (Fig. 1D).

In contrast to the effect of Cdc42V12 on anoikis, ponasterone-regulated expression of

dominant negative Cdc42 very slightly, but reproducibly, increased apoptosis in suspension

conditions (Fig. 1ED). Inhibition of Cdc42 activity under adherent condition, however, did not

elicit any detectable difference in apoptosis in comparison to the control (Fig. 1ED). Since the

endogenous Cdc42 activity significantly declines after substratum detachment (unpublished data),

the marginal effect of Cdc42N17 on cell survival could result from the inhibition of the residual

Cdc42 activity in the suspended cells. The effects of Cdc42V12 and Cdc42N17 on the survival of

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Cdc42 survival signaling is independent of p42/44 ERK activation

To investigate the signalling mechanisms that mediate Cdc42V12- stimulated cell survival

under anchorage-independent situation, we first examined the contribution of MAPK cascades.

Cdc42 and Rac1 have been demonstrated to synergize with Raf in activating ERKs [26, 27].

Recently, Cdc42 and Rac1 were shown to enhance association of ERK2 with MEK1 through the

activity of PAK in a fibronectin-dependent manner [28]. Furthermore, several reports indicate

activated ERK prevents anoikis [29, 30]; conversely, inhibition of Cdc42 activity has been shown

to trigger anoikis in murine fibroblasts which depend on an activated ERK activity [31].

To determine the role of ERK in Cdc42-stimulated survival signaling, we examined ERK

activity in attached and suspension conditions using the ecdysone-inducible

Cdc42V12-expressing cells. In control cells, ERK activity decreased significantly after cell

detachment, and subsequently gradually returned to baseline levels (Fig. 2A). At present, we do

not know which mechanism governs the restoration of the ERK activity after cell detachment.

Expression of Cdc42V12 did not significantly enhance ERK activiation in either attachment or

suspension conditions (Fig. 2A). In line with these observations, inhibition of MEK by U0126 did

not affect Cdc42-induced protection against anoikis, although ERK activation was nearly

abolished by the inhibitor (Fig. 2B). These results indicate that ERK activity does not play a

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Cdc42 survival signaling is independent of JNK/SAPK pathway

The role of JNK in anoikis regulation remains controversial. Whereas JNK activation has

been shown to be required for the induction of anoikis in one study [32], this has been contradicted

by another study [24]. To investigate the role of JNK activation in the inhibition of anoikis caused

by Cdc42, we used SP600125, a reversible ATP-competitive inhibitor of JNK-1, -2, -3 [33].

Whereas SP600125 efficiently inhibited the Cdc42V12-induced JNK activation, indicated by the

diminished autophosphorylation of JNK (Fig. 3A) [34], the protective effect of Cdc42V12 against

anoikis was not affected (Fig. 3B). Notably, the application of SP600125 at 10 µM, which inhibits

JNK activation significantly, didn't have any effect on Cdc42V12 expression (Fig. 3A). We

therefore conclude the protective effect of Cdc42 on anoikis is independent of the JNK pathway

activation.

Cdc42 survival signaling is independent of p38

The role of p38 in anoikis regulation has also been controversial in that both pro-apoptotic

[35] and anti-apoptotic [36] effects have been reported. This controversy could reflect the

observation that distinct members of the p38 subfamily of mitogen-activated protein kinases may

have different functions in apoptosis [37], which needs further to be reconciled within different

cellular contexts [38]. Since p38 has been implicated in Cdc42-mediated survival signalling in

fibroblasts grown in suspension condition [36], we investigated the role of p38 in survival

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compound specifically inhibiting p38 at micromolar concentration [39]. Since p38 can

autophosphorylate [40, 41], we examined the extent of p38 activation with a polyclonal serum

specific for phosphorylated p38, after immunoprecipitating the phosphorylated p38 with a mouse

monoclonal antibody. In the presence of 10 µM SB203580, p38 activation was significantly

suppressed, while Cdc42V12 expression still fully protected suspended MDCK cells from anoikis

(Fig. 4A and 4B). We therefor conclude that although Cdc42V12 can stimulate p38 in the absence

of cell attachment, p38 activation is dispensable for Cdc42-mediated MDCK cell survival in these

conditions.

Cdc42 mediates survival signaling in MDCK cells cultured in suspension by

activating PI3

We have shown that activated Rac1 protects MDCK cells against anoikis via activation of

PI3K [19]. To investigate whether PI3K/Akt signalling also plays a role in Cdc42-mediated

survival in suspension culture, we performed in vitro kinase assays for Akt, and observed that

Cdc42V12 significantly stimulates Akt in suspended MDCK cells (Fig. 5A). Furthermore,

LY294002, a pharmacological inhibitor of PI3K, strongly inhibited PI3K activity (Fig. 5A) as well

as the protective effect of Cdc42V12 (Fig. 5B). These results provide strong evidence for a

significant role for PI3K in the protective effect induced by Cdc42 activation in MDCK cells in

suspension.

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Since both Cdc42 and Rac1 can inhibit anoikis via activation of PI3K, we next addressed the

question whether there is a signaling cascade from Cdc42 to Rac1 in suspended MDCK cells,

similar to the one observed in fibroblasts [14]. We used a Rac1N17 and Cdc42V12 double

expressing cell line and examined whether differential expression of these two transgenes could

affect apoptosis of MDCK cells kept in suspension condition. Fig. 6A shows the morphologyies of

this particular MDCK cell line under adherent conditions. When Cdc42V12 was induced, the cells

tended to be flatter than uninduced controls (panel b and a in Fig. 6A). This Cdc42V12 cell line

also displayed other morphological features similar to previously characterized MDCK cell lines

that express constitutively active Rac1 [21] (panel f in Fig. 6A). These included the formation of

lamellipodia and macropinocytotic vesicles [21]. Immunostaining of Cdc42V12-expressing

MDCK cells showed a spectrum of morphological characteristics from cells displaying abundant

filopodia to cells with broad lamellipodia (Fig. 6B). Between these two types of cellsextremes, we

observed cells with long protrusions embedded in sheet-like structures similar to those fibroblasts

reported to be microinjected with constitutively active Cdc42 recombinant protein [14]. The

Cdc42V12 microinjected fibroblasts first developed filopodia followed by lamellipodia

progressively growing between the pre-formed filopodia and finally coalescing into a web like

structure enclosing the majoritiesy of the filopodia [14]. When Rac1N17 was induced in addition

to the expression of Cdc42V12, the cells had an aggregated and contracted morphology similar to

the one when Rac1N17 was singly induced (panels d and c in Fig. 6A and [21]). The

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viable, provided they are attached to substratum or embedded in a three dimensional collagen gel.

In addition, they can polarize marker proteins at specific membrane domains [21, 22].

The observations that induction of Cdc42V12 induction elicitings filipodia as well as

lamellipodia, a hallmark feature for Rac1 activation, and that dominant negative Rac1 expression

revertings these phenotypes, in MDCK cells prompted us to examine the possibility thatwhether

constitutively active Cdc42 could lead to activation of Rac1. GST-PAK fusion protein pull-down

assay demonstrated that Rac1 activity was higher when Cdc42V12 was induced in attached and

suspended MDCK cells (Fig. 6C). In addition, Cdc42 activated PI3K in a Rac1 dependent manner,

as indicated by the observation that dominant negative Rac1 abolished Akt activation by

Cdc42V12 (Fig. 6D). In addition, expression of Rac1N17 strongly inhibited the protective effect

of Cdc42V12 against anoikis (Fig. 6E). These results suggest that Rac1 acts downstream of Cdc42

in survival signalling in MDCK cells cultured in suspension.

PI3K-mediated protection against anoikis requires Rac1

While the survival signals stimulated by Rac1 in suspended cells clearly depend on PI3K

activity [19], there are many conditions where Rac1 functions downstream of PI3K [42-44]. To

further address this issue, we generate MDCK cell lines stably expressing constitutively active

PI3K activity in the background of a tetracycline repressible dominant negative Rac1 (Rac1N17)

(46)

４４

catalytic sub-unit and the SH2 domain from the p85 regulatory sub-unit of PI3K. This chimeric

protein was demonstrated to activate PI3K signaling pathways independent of growth factor

stimulation [45]. Two independent MDCK clones were selected and demonstrated to express

p110* by Western blotting analysis (upper panel in Fig. 7A). It should be noted that the fusion of

p110 and the SH2 domain makes p110* migrate as a 140 kDa molecule on the PAGE, and also the

rabbit antibody we used for Western Blot could not recognize the endogenous canine form of PI3K.

Inducible expression of Rac1N17 did not affect the expression level of the CMV promoter driven

p110*, while the endogenous Rac1 expression levels seemed to be down-regulated by the

expression of Rac1N17 (Fig. 7A).

When these p110* expressing MDCK cells were cultured in suspension conditions, they were

more resistant to anoikis than the mock control (Fig. 7B) which was consistent with a previous

report [46]. This protective effect of constitutively active PI3K, however, was inhibited by the

inducible expression of Rac1N17 (Fig. 7B). This result indicates Rac1 could also function

downstream of PI3K in providing survival signals against anoikis.

Cdc42- and Rac1-mediated protection against anoikis requires an intact actin

cytoskeleton

Since both Cdc42 and Rac1 play major roles in controlling the organization of the actin

(47)

４５

depends on an intact actin cytoskeleton. Latrunculin B, a potent actin polymerization inhibitor,

significantly inhibited the anoikis-protective effects of both Cdc42V12 and Rac1V12 (Fig. 87B),

suggesting an important role for Cdc42/Rac1-controlled actin polymerisation in survival

signalling by Cdc42 and Rac1. Interestingly however, the activation of PI3K by both Cdc42 and

Rac1 was not significantly affected by latrunculin B (Fig. 8A), indicating that an intact actin

cytoskeleton is largely dispensable for the Cdc42- and Rac1-induced PI3K activation.

(48)

４６

Discussion

We have examined the role of Cdc42 in apoptosis induced by cell detachment from the

substratum. Our study shows that inducible expression of Cdc42V12 significantly inhibits anoikis

in MDCK cells. Our results also strongly suggest indicate that Cdc42-induced protection against

anoikis is mediated by the activation of Rac1,. In addition, our data support a model in which Rac1

and PI3K participate inwhich then initiates a positive regulatory feedback loop involving Rac1

and PI3K to inhibit anoikis. Furthermore, our data suggest that both Cdc42 and Rac1-stimulated

cell survival depends on an intact actin cytoskeleton.

Our previous [19] and present studies demonstrate that PI3K activity is critical for Rac1V12-

and Cdc42V12-mediated protection against anoikis. These observations are in line with several

studies showing that Rac1-regulated functions, including cell survival, can be inhibited by

pharmacological inhibitors of PI3K [47-49]. In addition, similar to the results that we obtained in

MDCK cells, expression of constitutively active Rac1 in hematopoietic cells can stimulate PKB in

a PI3K-dependent fashion [50]. Interestingly however, a number of earlier reports had indicated

that Rac1 could function downstream of PI3K [42-44]. It appears therefore that PI3K may function

both upstream and downstream of Rac1 (reviewed in [51]). In several cases, this could be

demonstrated in the same type of cells [50]. Based on these observations, a positive feedback loop

between Rac1 and PI3K has been postulated to function in the establishment of front-to-back

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