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Originally published In Press as doi:10.1074/jbc.M413017200 on January 5, 2005

J. Biol. Chem., Vol. 280, Issue 11, 10572-10577, March 18, 2005
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Gene Trap Mutagenesis-based Forward Genetic Approach Reveals That the Tumor Suppressor OVCA1 Is a Component of the Biosynthetic Pathway of Diphthamide on Elongation Factor 2*

Yoshitaka Nobukuni{ddagger}§, Kenji Kohno¶, and Kiyoshi Miyagawa{ddagger}

From the {ddagger}Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan and the Department of Molecular and Cell Genetics, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0101, Japan

Received for publication, November 17, 2004 , and in revised form, December 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
OVCA1 is a tumor suppressor isolated from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers (1, 2). It has been shown that expression of OVCA1 is reduced in tumors and that exogenous expression of OVCA1 inhibited growth of ovarian cancer cells (3). Furthermore, a study using Ovca1 gene knock-out mice clearly showed that OVCA1 regulates cell proliferation, embryonic development, and tumorigenesis (4). Even though the biological or cell biological functions have been elucidated, the biochemical function of OVCA1 has not been ascertained.

Diphthamide is a unique post-translationally modified histidine residue found only on translational elongation factor 2 (EF-2),1 which catalyzes the translocation of peptidyl tRNA from the ribosome A site to the P site during peptide chain elongation. Diphthamide has been found in all eukaryote and archaebacteria, however not in eubacteria. The diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) inactivate EF-2 by ADP-ribosylating the diphthamide (5, 6). The biosynthesis of diphthamide is one of the most complex post-translational modifications, and by genetic complementation analyses it has been shown that five different genes in yeast (7) and at least three genes in CHO cells (8) are involved in diphthamide synthesis. To date, three genes responsible for diphthamide formation have been elucidated in yeast and human (9–11). The biochemical function of diphthamide as a target of bacterial ADP-ribosylating toxins has been well characterized; however, its physiological role in cells has remained to be clarified.

Bacterial toxins are useful and valuable tools for investigating cell functions; important knowledge concerning cell functions has been obtained by analyses of mutants of established cell lines. Indeed, much of knowledge concerning the mechanisms of toxicity of DT has been elucidated by the study of toxin-resistant mutants (7, 8, 12–18). Although many of the DT-resistant mutants have been isolated, to clarify the genes involved in DT sensitive and/or resistant phenotype was time consuming and sometimes difficult work, especially in mammalian cells.

To increase the efficiency of insertional mutagenesis in mammalian cells, retrovirus gene trap vectors have been developed (19, 20). Gene traps are based on the integration of a reporter gene lacking a promoter into the genome and its expression from a tagged endogenous promoter. When a gene trap vector integrates into expressed genes, insertional mutants can be easily selected by selectable phenotype conferred by the gene trap vector. It is possible to increase the proportion of cells with virus-induced mutations to two to three orders of magnitude higher than in cells containing unselected proviruses by retroviral gene trap selection (21). So far, mutants having a variety of phenotypes have been isolated from CHO cells. This is because CHO cells are hypodiploid and functionally hemizygous at a number of loci (22, 23). For that reason it was expected that a single gene trap insertional mutagenetic event might result in loss of gene functions in CHO cells.

To identify the obligate genes involved in DT sensitivity and/or resistance, including diphthamide biosynthesis, we screened mutants resistant to DT from a random gene trap insertional mutants library of CHO cells. Recently, we have been able to isolate a novel mutant resistant to DT and ETA in which a mutant Ovca1 gene was disrupted by gene trap mutagenesis. Here we show genetic and biochemical evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on EF-2, the target of bacterial ADP-ribosylating toxins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture—CHO-K1 cells were obtained from the American Type Culture Collection. CHO-K1 cells and mutants were maintained in Dulbecco's modified Eagle's medium supplemented with 8% fetal calf serum, 2 mM glutamine, and antibiotics (penicillin and streptomycin).

Retroviral Gene Trap Insertional Mutagenesis—The retroviral gene trap vector, ROSA{beta}geo (20), was used to construct the random gene trap insertional mutants library of CHO cells. To infect the ROSA{beta}geo to CHO cells, pseudo-retrovirus of ROSA{beta}geo was produced using a pantropic retroviral expression system (Clontech) according to the manufacturer's instruction. The pROSA{beta}geo (20) and pVSV-G plasmid constructs were co-transfected to GP293 cells with Lipofectamine (Invitrogen). After 48-h incubation, virus-containing supernatant was harvested, passed through a 0.22-µm filter, and stored at -80 °C until use. For gene trap mutagenesis, CHO-K1 cells were seeded in multiple dishes at 1 x 106 cells per 100-mm dish. After overnight incubation, the medium was replaced with a virus-containing medium. After an additional 48 h, the cells from each dish were re-seeded at 1 x 106 cells per 100-mm dish, and gene trapped mutagenized cells were selected by G418 (200 µg/ml) for 10–14 days. Typically we could get 1–2 x 103 G418-resistant colonies per dish. Cells from ~1 x 106 independent colonies were combined and stored as the random gene trap insertional mutants library of CHO cells.

Toxins—DT and fragment A of DT were purified by DEAE-cellulose column chromatography (14). ETA was purchased from List Biological Laboratories.

Selection and Cloning of Diphtheria Toxin-resistant Cells—Toxinresistant CHO mutants were isolated from the gene trap insertional mutants library. Inocula of 106 gene trap mutagenized CHO cells were incubated for 6–10 h before the addition of 1 µg/ml of DT. After about 2 weeks of selection, colonies were isolated with cloning rings, and each isolated clone was cultured in DT-free medium.

Cytotoxicity Assay with MTT—Toxin-induced cytotoxicity was evaluated by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Cells were seeded in 96-microwell plates at a density of 4 x 103/well in Dulbecco's modified Eagle's medium with 8% fetal calf serum. After 16-h incubation, the medium was replaced and cells were exposed to serial dilutions of DT or ETA (0–1000 ng/ml) for 48 h. Then 10 µl of 0.4% MTT reagent and 0.1 M sodium succinate were added to each well. After 90-min incubation, 150 µl of Me2SO were added to dissolve the purple formazan precipitate. Formazan dye was measured spectrophotometrically (570–650 nm) using the MAXline Microplate Reader (Molecular Devices, Sunnyvale, CA) (11, 24).

Southern Blot Analysis—Genomic DNA was isolated from CHO cells using PUREGENE DNA isolation kits (Gentra), digested with EcoRI, separated by electrophoresis on 0.8% agarose gel, and transferred to a nylon membrane. A 0.8-kbp PCR fragment of the Neor gene, amplified with primers 5'-AACCATGGGATCGGCCATTGAACA-3' and 5'-AGGATCCGCGAAGAACTCGTCAAGAAGGC-3' from pROSA{beta}geo, was radiolabeled with [{alpha}-32P]dCTP (3000 Ci/mmol) by random priming. The membrane was hybridized with this probe, washed, and then autoradiographed.

5'-Rapid Amplification of cDNA Ends (RACE), Cloning, and Sequence—5'-RACE analyses were conducted using the 5'-Full RACE core set (Takara Biomedicals) with total RNAs from DTR44 cells following the manufacturer's instructions. Total RNAs were prepared from mutant cells with Sepasol RNA I (Nacalai Tesque). Single strand cDNAs were prepared from the total RNAs (~1 µg of total RNA for each sample) with {beta}-galactosidase-specific 5'-phosphorylated RT primer, 5'-ATGCGCTCAGGTCAAATTCA-3' and avian myeloblastosis virus reverse transcriptase. After the degradation reaction of the hybridized RNAs by RNase H, cDNAs were circularized and/or concatemerized by using the 5'-Full RACE core set following the manufacturer's instructions. For PCR amplification of the trapped sequence, the following {beta}-galactosidase-specific primers sets were used: 5'-GTTGATGAAAGCTGGCTACA-3'/5'-GTGCTGCAAGGCGATTAAGT-3' (for the first PCR) and 5'-TGATGGCGTTAACTTGGCGT-3'/5'-TTCCCAGTCACGACGTTGTA-3' (for nested PCR). Amplification products were subcloned to pGEM-T easy vector (Promega) and sequenced by using ABI PRISMTM 377 and 310 sequencing machines (Applied Biosystems). Homology of the trapped sequences was searched by the NCBI BLAST program.

PCR Analyses of Chimera RNA and Genome DNA—Total RNAs were reverse transcribed and amplified by using an RNA PCR kit (Takara Biomedicals). Genome DNAs were prepared from CHO cells using PUREGENE DNA isolation kits (Gentra). For PCR amplification of the cDNA and genome DNA, the following Ovca1 and {beta}-galactosidase-specific primers sets were used: 5'-CGTTCCTCCAGCGCTGCCTT-3' (P1)/5'-GTGCTGCAAGGCGATTAAGT-3' (P3) (for the first PCR) and 5'-TCCAGCGCTGCCTTTTTGGT-3' (P2)/5'-TTCCCAGTCACGACGTTGTA-3' (P4) (for nested PCR) (see Fig. 2, B and C).



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  		FIG. 2.
The tumor suppressor Ovca1 gene was disrupted by gene trap insertional mutagenesis in DTR44 mutant. A, the nucleotides sequences (DDBJ accession number AB194396 [GenBank] ) trapped by ROSA{beta}geo in DTR44 cells were elucidated by 5'-RACE and sequence analyses of DTR44 cDNA. BLAST homology search revealed that the first exon of Ovca1 gene was trapped in DTR44 mutant cells. Homologies of the nucleotides and the deduced amino acids of mouse, Chinese hamster (CHO cells), and human OVCA1 in this region are shown. B, Ovca1-{beta}geo chimera RNA produced by gene trap event was confirmed in DTR44 mutant cells by RT-PCR as described under "Experimental Procedures." The arrow indicates the 211-bp RT-PCR (nested PCR) product amplified using primers P2 and P4 from Ovca1-{beta}geo chimera-RNA in DTR44 cells. C, schematic representation of gene trap event in the Ovca1 gene of DTR44 cells. Gene trap retrovirus was integrated in the first intron of Ovca1 gene. PCR-sequence analyses of the DTR44 genomic DNA with the primers corresponding to Ovca1 and {beta}geo sequences (P1/P3 and P2/P4) clarified that one of the gene trap retrovirus was integrated in the first intron, ~1kb downstream of exon 1, of the Ovca1 gene (data not shown).

 
cDNA Expression Experiments—The mouse Ovca1 (Ovca1 (m)), human DPH2L2 (DPH2L2 (h)), and mouse dph2l2 (dph2l2 (m)) cDNA expression vectors were constructed as described below. First, the cDNAs used in the expression experiments were RT-PCR amplified from the mouse kidney and human fibroblast mRNAs. The mRNAs were reverse transcribed with oligo dT primer and avian myeloblastosis virus reverse transcriptase. The mouse Ovca1 cDNA fragments were amplified using 5'-primers for without tag Ovca1 (5'-ATGCTAGCGTGATGGCGGCGCTGGTTGTGT-3' (the NheI site is underlined, and the start codon is in boldface)) or for with FLAG-tagged Ovca1 (5'-ATGCTAGCATGGCGGCGCTGGTTGTGTC-3' (the NheI site is underlined and the start codon is in boldface)) and a 3'-primer (5'-ATGGATCCCCCTGCTGCTCTGGCCCTCTCA-3' (the BamHI site is underlined)). The amplified cDNA fragments were then digested by NheI and BamHI and cloned into the NheI and BamHI sites of pIREShyg3 (Clontech) or pFLAG-IREShyg3. The pFLAG-IREShyg3 plasmid was constructed as follows. Oligonucleotides, 5'-AGCTTAAGTCCACCATGGATTACAAGGATGACGAC-3' (the AflII site is underlined, and the start codon is in boldface) and 5'-TAGCTAGCGATCTTATCGTCGTCATCCTTGTAATC-3' (the NheI site is underlined) were annealed, purified by PAGE, digested by AflII and NheI, and cloned into AflII and NheI sites of pIREShyg3. The mouse dph2l2 cDNA fragment was amplified using a 5'-primer (5'-ATGCTAGCAAGCTGCGCCCAATGGAGTCTA-3' (the NheI site is underlined and the start codon is in boldface)) and a 3'-primer (5'-CTGATATCCAGACTTATCACTCTGGCTC-3': the EcoRV site is underlined)). The amplified cDNA fragment was digested by NheI and EcoRV and cloned into the NheI and EcoRV sites of pIRESHyg3. The human DPH2L2 cDNA fragment was amplified using a 5'-primer (5'-CATGGATCCCAAGCTGTGCCTCATGGAGTC-3' (the BamHI site is underlined and the start codon is in boldface) and a 3'-primer (5'-GATCTCGAGCACATGGTATCAGCCGCTTCC-3' (the XhoI site is underlined)). The amplified cDNA fragment was digested by BamHI and XhoI and cloned into the BamHI and XhoI sites of pcDNA3.1/Hygro (+) (Invitrogen). These expression plasmid constructs were confirmed by restriction enzyme map and sequencing and were transfected into CHO cells using Lipofectamine reagent (Invitrogen). Stably transfected cells were selected by hygromycin B (400 µg/ml) for 10–14 days. Hygromycin-resistant colonies were isolated and analyzed.

Western Blot Analysis—CHO cells grown in 24-well plates were incubated with or without 1 µg/ml DT at 37 °C for 1 h, and the cells were washed and lysed by 100 µl/ml radioimmune precipitation assay buffer. Cell lysates were separated by native PAGE or SDS-PAGE and transferred to a nylon membrane (Hybond-P, Amersham Biosciences). Membranes were blocked in appropriate blocking buffers and incubated with goat anti EF-2 antibody (sc-13004, Santa Cruz Biotechnology) followed by peroxidase-conjugated anti-goat antibody. Reactive bands were detected by enhanced chemiluminescence (Amersham Biosciences).

In Vitro ADP-ribosylating Assay—CHO cell lysates preparation and ADP-ribosylation reaction of EF-2 were performed as described previously (15, 16). Cell lysates were incubated with [adenylate-14C]NAD (Amersham Biosciences, 248 mCi/mmol, catalog no. CFA 497) in the absence or presence of DT fragment A. The amount of ADP-ribosylated EF-2 was assessed by counting the radioactivity incorporated to the acid insoluble fraction in a liquid scintillation counter (Aloka, LSC-3500). ADP ribosylation of EF-2 was confirmed by SDS-PAGE followed by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of the Diphtheria Toxin-resistant Mutants—The random gene trap insertional mutants library of CHO cells was made by infecting the ROSA{beta}geo (20) followed by growth in G418 as described under "Experimental Procedures." By combining the cells from ~1 x 106 independent G418-resistant colonies, we constructed a library of mutants. The gene trap mutagenized cells were inoculated at 20 x 105 cells per 100-mm culture dish and selected with DT. Colonies, ~10 per dish, were observed 10–14 days later. A total of 24 clones were picked randomly from the 1 x 106 mutant library cells (Fig. 1A). One of these mutants, DTR44, was completely resistant to DT and ETA (Fig. 1, C and D). Southern blot analysis with a Neor gene fragment as a probe showed multiple insertions (>8 copies) in the DTR44 genome (Fig. 1B).



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  		FIG. 1.
DTR44, obtained by retroviral gene trap insertional mutagenesis, is a multiple toxin-resistant mutant. A, schematic representation of the construction of the random gene trap insertional mutants library of CHO cells and the isolation of the DT-resistant mutants. B, Southern blot analysis of the retroviral insertions in CHO cells. Genomic DNAs were digested with EcoRI, separated on 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a 32P-labeled PCR-amplified Neor gene fragment. The structure of ROSA{beta}geo provirus (20) is also shown schematically. LTR, long terminal repeat; SA, splice acceptor; {beta}geo, {beta}-galactosidase-Neor fusion gene; pA, polyadenylation signal. Cytotoxicity of DT (C) and ETA (D) to CHO cells. CHO cells were incubated with various concentrations of DT or ETA. After 48-h exposure to toxins, cell viability was determined by MTT assay as described under "Experimental Procedures."

 
Identification of the Disrupted Gene in DTR44 Cells—To identify the gene responsible for multiple toxin resistance in DTR44, we amplified the trapped sequences with a gene trap vector, ROSA{beta}geo, using 5'-RACE. A major PCR product was amplified from the DTR44 cDNA (data not shown) and 128-bp trapped sequences (DDBJ accession number AB194396 [GenBank] ) were clarified by sequencing. BLAST search revealed that 76 bp (number 53 to 128) of these 128-bp trapped sequences strongly matched those of the first exon of the mouse (25) and human OVCA1 genes (Fig. 2A). The amino acids sequence of Chinese hamster OVCA1 protein in this region completely matched with that of mouse OVCA1, and 85% of these amino acids are identical to those of human OVCA1 protein (Fig. 2A).

The existence of the Ovca1-{beta}geo chimera RNA resulting from gene trap mutagenesis was confirmed by RT-PCR of DTR44 mRNA (Fig. 2B). Although multiple insertions of the gene trap vector into DTR44 genome were ascertained by Southern blot (Fig. 1B), PCR sequence analysis of the DTR44 genomic DNA with the primers correspond to Ovca1, and {beta}geo sequences clarified that one of the gene trap retrovirus was integrated within the first intron, ~1kb downstream of exon 1, of the Ovca1 gene (Fig. 2C).

Disruption of the Ovca1 Gene Renders a Toxin-resistant Phenotype—To define the role of OVCA1 in DTR44, Ovca1 cDNA expression plasmids were constructed and transfected into DTR44 cells; stable transformants were then established by selecting with hygromycin B (400 µg/ml). Stable transfectant colonies were picked and evaluated for sensitivity to the toxins. The majority of clones arising from DTR44 cells transfected with Ovca1 cDNA regained sensitivity to DT and ETA (Fig. 3A). In contrast, in cells transfected with the empty vector or DPH2L2, proteins that have higher similarity to DPH2 than OVCA1 (26), expression vector remained toxinresistant (Fig. 3A).



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  		FIG. 3.
Expression of OVCA1 restores the sensitivity of DTR44 mutant cells to bacterial ADP-ribosylating toxins. A, effect of OVCA1 or DPH2L2 expression on sensitivity to toxins. The expression plasmids of the indicated genes, mouse Ovca1 (OVCA1 (m)), FLAG-tagged mouse Ovca1 (FLAG-OVCA1 (m)), mouse dph2l2 (DPH2L2 (m)), and human DPH2L2 (DPH2L2 (h)) were transfected to DTR44 mutant cells, and stably transformed cells were established through growth for 10–14 days in hygromycin B (400 µg/ml). The cell colonies were isolated and subcultured to 48-well plates and then treated with DT (1 µg/ml) or ETA (1 µg/ml) for 96 h. The numbers of toxin-sensitive mutants (DTs, DT-sensitive; ETAs, ETA-sensitive) versus those that tested the sensitivities to toxins were determined by microscopic observation. B, RT-PCR analysis of the Ovca1-{beta}geo chimera RNA. Ovca1-{beta}geo chimera RNA was examined in CHO cells as in Fig. 2B. R1 to R4 are DTR44 cells transfected with mouse OVCA1 expression constructs and confirmed the recovery of sensitivity to toxins. R1 and R2 clones were used for further analyses. Cytotoxicities of DT (C) or ETA (D) to CHO cells were determined by MTT assay as in Fig. 1. DTR44-EV is a stably transformed DTR44 cells transfected with an empty vector, pIREShyg3.

 
Cytotoxicities of DT or ETA to DTR44 cells and the clones of DTR44 transfected with OVCA1 expression constructs were further analyzed by MTT assay. Clones R1 and R2, DTR44 stable transformants of mouse Ovca1 cDNA expression vector, regained their parental sensitivity and were fully sensitive to DT and ETA (Fig. 3, C and D).

The presence of Ovca1-{beta}geo chimera RNA in these transformants (Fig. 3B) clearly showed that the recovery of toxin sensitivity was not caused by the deletion of the gene trap vector integrated in the Ovca1 gene. Taken together, we concluded that OVCA1 was required for the process involved in sensitivity to DT and ETA.

OVCA1 Is Required for Diphthamide Biosynthesis—DTR44 cells showed the characteristics of the DTRII phenotype, that is, multiple toxin resistances and a resistant to high concentrations of toxins. DTRII mutants have shown that DT sensitivity was affected at the level of EF-2 (8, 12, 13, 15–18).

To determine whether DTR44 cells are altered in their susceptibility of EF-2 to ADP-ribosylation, CHO cell extracts were assayed for transfer of radiolabeling from NAD+ to EF-2. Lysates were incubated with [adenylate-14C]NAD in the absence or presence of DT fragment A. The amount of ADP-ribosylated EF-2 was assessed by counting the radioactivity incorporated to the acid insoluble fraction with a liquid scintillation counter (Fig. 4A). ADP ribosylation of EF-2 was confirmed by SDS-PAGE followed by autoradiography. (Fig. 4B).



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  		FIG. 4.
DTR44 mutant cells are defective in diphthamide formation of EF-2. CHO cell lysates were incubated with [adenylate-14C]NAD in the absence or presence of DT. A, in vitro ADP-ribosylating assay. EF-2 of DTR44 cells is resistant to ADP-ribosylation by DT. The amount of ADP ribosylated EF-2 in vitro was assessed by counting the radioactivity incorporated to the acid insoluble fraction as described previously (15, 16). R1 and R2 are DTR44 clones transfected with mouse OVCA1 expression vector. B, ADP-ribosylation of EF-2 in vitro was confirmed by SDS-PAGE followed by autoradiography. One major band (~95 kDa) corresponding to ADP-ribosylated EF-2 was observed. EF-2 of CHO, R1, and R2 cells were the ADP-ribosylatable form. The non-ribosylatable EF-2 in DTR44 cells could be converted to the ADP-ribosylatable form by transfection of cells with OVCA1 expression vector. C, Western blots analyses of the EF-2. After 1 h incubation with or without 1 µg/ml of DT at 37 °C CHO cells were lysed by radioimmune precipitation assay buffer, and cell lysates were separated by native PAGE (top) or SDS-PAGE (bottom) followed by Western blots analyses using an anti-EF-2 antibody as described under "Experimental Procedures."

 
ADP-ribosylation assay clearly showed that ADP-ribosylated acceptor activity in DTR44 cell lysate was dramatically reduced compared with that of wild type parental CHO cells (Fig. 4, A and B). Furthermore, the non-ribosylatable EF-2 from DTR44 cells restored the ADP-ribosyl acceptor activity when transfected with OVCA1 expression vector (Fig. 4, A and B). From these observations, we concluded that DTR44 cells are defective in diphthamide formation on EF-2 and that OVCA1 is required for the biosynthesis of diphthamide.

Identification of Intermediate in Diphthamide Synthesis— ADP-ribosylated, the non-ADP-ribosylated form, and biosynthetic intermediate of diphthamide on EF-2 have been shown to be easily distinguished by native PAGE followed by Western blotting using an anti-EF-2 antibody (11). Using this detection system, we examined the diphthamide biosynthetic intermediate in DTR44 cells. ADP-ribosylated EF-2 has two added negative charges compared with EF-2, and this increased negative charge could be detected as faster migration on native PAGE. The EF-2 of DTR44 migrates between the ADP-ribosylated and non-ADP-ribosylated forms (Fig. 4C). This observation further confirmed that OVCA1 is required for the biosynthesis of diphthamide on EF-2, the target site for the ADP-ribosylating bacterial protein toxins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To elucidate the molecular mechanisms underlying DT sensitivity and/or resistance, including diphthamide biosynthesis, we screened mutants resistant to DT from a library of random gene trap insertional mutants of CHO cells. CHO cells are functionally hemizygous at a number of loci (22, 23). It was expected that a single integration event of gene trap retrovirus might result in the loss of gene function in CHO cells, and we would be able to get mutants with specific phenotype by proper selection from this library of mutant cells.

DTR44 mutant cells isolated from the gene trap insertional mutants library showed a phenotype with multiple toxin resistance to DT and ETA. It was revealed that the Ovca1 gene was disrupted by gene trap mutagenesis and that the expression of OVCA1 fully recovered the sensitivity to toxins and EF-2 diphthamide formation. These genetic and biochemical data clearly show that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on EF-2.

So far the amino acid sequences of OVCA1 did not reveal any functional protein domains suggesting its biochemical function except for low level sequence similarity (~20%) (1, 2) between OVCA1 and DPH2, a yeast protein necessary for diphthamide formation (10). The forward genetic approach using gene trap mutagenesis in this study clearly demonstrated the biochemical function of OVCA1 in the formation of diphthamide in mammalian cells as described above.

The retroviral gene trap mutagenesis approach described in this study is relatively simple and straightforward. Even though multiple copies of gene trap vectors are integrated into DTR44 mutant genome (Fig. 1B), we could easily identify the obligate gene for DT-resistant phenotype in DTR44 cells by analyzing the trapped sequences in the chimera RNA produced by gene trap mutagenesis. Retroviruses can be used as insertional mutagens to isolate specific genes in mammalian cells. However, in practice, conventional retroviruses are inefficient mutagens (27). In ROSA{beta}geo, the retroviral gene trap vector used in this study, {beta}geo is flanked by an upstream 3'-splice consensus sequence (splice acceptor) and a downstream polyadenylation site to ensure its activation from integrations into introns ("intron trap"), and the gene trap events by SA{beta}geo are estimated to ~4.5 to 11.6% of integration events (20). So even if multiple integration events occurred, it was estimated that a large portion of retroviral vector integrations were not involved in the specific phenotypes.

After the isolation of the Ovca1 gene (1, 2), the biological functions of OVCA1 were clarified by cell biological analyses and the study of gene knock-out mice. It was speculated that the loss or haploinsufficiency of OVCA1 might be an important event in ovarian tumorigenesis from the observation that expression of OVCA1 protein in ovarian tumor tissues or cell lines was reduced (3). It was also seen that exogenous expression of OVCA1 in ovarian cancer cells causes suppression of cell growth with an increased number of cells in G1 phase of the cell cycle, suggesting that OVCA1 may play a role in the control of cell cycle/cell growth (3). Furthermore, study using knock-out mice demonstrated that OVCA1 regulates cell proliferation, embryonic development, and tumorigenesis (4).

The fact that the tumor suppressor OVCA1 is involved in diphthamide biosynthesis on EF-2 suggests the possibility that aberrations in translational regulation may be one of the molecular mechanisms underlying the tumorigenesis caused by the defect of OVCA1. So far it has been elucidated that components of the protein synthesis apparatus seem to be involved in the control of cell proliferation, and aberrations in protein synthesis are commonly encountered in established cancers (28). Furthermore, it has been demonstrated that removal of regulation of the expression of components of the translational machinery, such as elongation factor-1 {alpha}, a GTP-binding protein that catalyzes the binding of aminoacyl-transfer RNAs to the ribosome, predispose cells to become more susceptible to malignant transformation (29).

It has also been demonstrated that the activity of EF-2 kinase was markedly increased in several forms of malignancies and that inhibition of EF-2 kinase inhibited the growth of a variety of cancer cell lines (28, 30–32). Phosphorylation of EF-2 by EF-2 kinase results in a drastic inhibition of protein synthesis, and dephosphorylation of EF-2 by phosphatase restores its activity. The phosphorylation of EF-2 directly affects the elongation stage of translation, and this represents a novel mechanism of translational control (33). It is possible to speculate that the defect of OVCA1 also disturbs the translational regulation through the abnormal diphthamide formation on EF-2 and results in the cause of tumorigenesis.

The fact that OVCA1 is a component of diphthamide biosynthetic pathway may also provide an important clue for a better understanding of the biological function of diphthamide. The biosynthesis of diphthamide represents one of the most complex post-translational modifications of an amino acid known to date and is widely well conserved (5, 6), suggesting that it has real importance for biological function. However, the function and role of diphthamide in cellular physiology still remains obscure.

Thus far, the existence of endogenously ADP-ribosylated EF-2 and cellular ADP-ribosyltransferase activity has been found in a variety of animals and tissues. The enzyme transfers ADP-ribose from NAD to elongation factor 2, inactivating the factor like bacterial toxins (34–36). However, the nature of the cellular ADP-ribosyltransferase and its physiological significance remain unknown. To clarify that the effect on cell proliferation, embryonic development, and tumorigenesis observed in the Ovca1 knock-out mice (4) truly result from the defect of diphthamide, the generation of mice lacking other genes in the diphthamide biosynthesis may be helpful.

The finding that OVCA1 is a component of the diphthamide synthetic pathway will shed light for the further understanding of the function of OVCA1, molecular mechanisms underlying the tumorigenesis in the defect of OVCA1, and the physiological role of diphthamide. Furthermore, a functional genetic approach utilizing the random gene trap mutants library of CHO cells described above should become a useful strategy to identify the genes responsible for specific phenotypes.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB194396 [GenBank] .

* This work was supported by Grants-in-Aid for Scientific Research (A) 15659074 and (B) 12204091 from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the ONO Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Tel.: 81-82-257-5829; Fax: 81-82-256-7102; E-mail: nobukuni{at}hiroshima-u.ac.jp.

1 The abbreviations used are: EF-2, elongation factor 2; DT, diphtheria toxin; ETA, Pseudomonas exotoxin A; CHO, Chinese hamster ovary; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RACE, rapid amplification of cDNA ends; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Philippe Soriano for kindly providing the plasmid construct, pROSA{beta}geo, and Dr. Masahiko Nishiyama for critical comments on the MTT assay.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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