The present invention relates to a protein that is interacting with the minichromosome maintenance complex in eukaryotes. More specifically, the invention relates to the use of a protein, interacting with the minichromosome complex, and the gene encoding this protein in diagnosis, prognosis and treatment of cancer.
All eukaryotic cells replicate their nuclear DNA in a conserved manner, whereby the parent molecule is unwound and each DNA strand becomes the template for nascent DNA synthesis. Coordination of DNA replication with growth and development is essential for survival, as mistakes made during DNA replication can result in apoptosis, growth defects, or oncogenesis. Because of its essential role during development, it is not surprising to see that the molecular machinery controlling DNA replication is highly conserved among organisms. Despite the many years of evolution that separate animals from plants, both organisms use the same E2F/DP pathway to regulate entry into S phase by controlling transcriptional induction of genes required for cell cycle progression and DNA replication. The genome of Arabidopsis contains six E2Fs (E2Fa, E2Fb, E2Fc, E2Fd/DEL2, E2Fe/DEL1, and E2Ff/DEL3) and two DPs (DPa and DPb) (Inzé and De Veylder, 2006). Three E2F proteins (E2Fa-c) bind DNA through the consensus E2F binding site by forming heterodimers with DP proteins. Both E2Fa and E2Fb operate as transcriptional activators, whereas E2Fc functions as a repressor (De Veylder et al., 2002; del Pozo et al., 2002). The remaining Arabidopsis E2Fs (E2Fd/DEL2, E2Fe/DEL1 and E2Ff/DEL3) contain duplicated DNA-binding domains, allowing binding to consensus E2F sites as a monomer (Kosugi and Ohashi, 2002; Ramirez-Parra et al., 2004; Vlieghe et al., 2005).
Both in mammals and Arabidopsis, numerous E2F target genes have been identified using microarrays, chromatin immunoprecipitations, and in silico analyses (Ramirez-Parra et al., 2003; Dimova and Dyson, 2005; Vandepoele et al., 2005). These genes encode proteins active during DNA replication, mitosis, DNA checkpoint control, apoptosis, or differentiation. Remarkably, almost every gene encoding for a protein involved in licensing for DNA replication is transcriptionally controlled by E2F transcription factors, such as the origin recognition complex (ORCs), CDC6, minichromosome maintenance complex (MCMs), and CDT1 genes (Vandepoele et al., 2005). Licensing for DNA replication in eukaryotes is initiated by the formation of the pre-replicative complex (pre-RC) at replication origins (Gillespie et al., 2001; Bell and Dutta, 2002; Diffley and Labib, 2002). First, ORC proteins bind to DNA during the early G1-to-S phase of the cell division cycle. Then, CDC6 binds to these ORC-DNA sites, an event that is followed quickly by binding of CDT1. Finally, replication origins are licensed by loading the MCM complex to form a pre-RC. The MCM complex is a heterohexamer composed of MCM2 to 7 and is likely a component of the helicase that unwinds DNA during replication (Tye and Sawyer, 2000; Labib and Diffley, 2001; Forsburg, 2004).
Once the formation of the pre-RC has been completed by the loading of MCMs, the DNA is primed for replication through the action of two conserved protein kinases, cyclin-dependent protein kinase (CDK) and Cdc7-Dbf4 (Dbf4-dependent kinase, DDK), resulting into the recruitment of additional replication factors to form the pre-initiation complex (pre-IC) (Kamimura et al., 2001; Masumoto et al., 2002; Takayama et al., 2003; Kanemaki et al., 2006). Loading of the pre-IC onto the origins activates the MCM helicases and recruitment of DNA polymerases, resulting into the initiation of DNA synthesis (Zou and Stillman, 2000). After origin firing, some of the initiation factors move with replication forks to support the elongation step of DNA synthesis (Aparicio et al., 1997; Takayama et al., 2003; Gambus et al., 2006; Kanemaki et al., 2006). Members of the MCM complex, particularly MCM2, have been described as markers for screening, surveillance and prognosis of cancer (Giaginis et al., 2008).
By studying E2F target genes in Arabidopsis, we have identified ETG1 (At2g40550) as a novel E2F target gene, being directly controlled by the E2Fa and E2Fb transcription factors. ETG1 null mutants display a slower cell cycle progression. Genetic analysis and transcriptional up-regulation of the PARP2, WEE1 and RAD51 genes indicated that this cell cycle delay originates from the activation of the DNA replication checkpoint. ETG1 is demonstrated to associate with the DNA replication complex, suggesting that the activation of the DNA replication checkpoint in ETG1-deficient plants originates from impaired DNA replication. Surprisingly, the absence of a functional ETG1 allele in a wee1 or atr mutant background has a profound impact on plant development, illustrating that the DNA replication checkpoint aids to the survival of ETG1-deficient plants.
Even more surprisingly, knock down mutants of the human ETG1 homologue (C10orf119) resulted in a similar phenotype, indicating that the gene is involved in the development of mammalian tumors. This is specifically surprising, as Sakwe et al. (2007) indicated that C10orf119 (MCM-BP) binds to the MCM complex, but only in absence of the most relevant tumor marker MCM2.
A first aspect of the invention is the use of ETG1 or an ETG1 ortholog for the diagnosis and/or prognosis of cancer. An ortholog, as used here, means a sequence with a similar, preferably an identical, function as the reference protein, and a detectable homology (expressed as percentage identity) with the reference sequence. ETG1 orthologs include, but are not limited to, rice (Oryza sativa; Os01g0166800), human (C10orf119), mouse (1110007A13Rik), Xenopus (CAJ81286), Drosophila (CG3430) and fission yeast (SPAC1687.04) orthologs. Preferably, the ETG1 ortholog is the human ortholog C10orf119 (accession NP—079110). The human ETG1 gene is located on chromosomal position 10q26.11. This particular region shows high frequency of loss of heterozygosity in human meningiomas and colorectal cancers (Mihaila et al., 2003; Karoui et al., 2004). This type of loss of heterozygosity is generally regarded as a hallmark for the localization of a tumor suppressor.
The “use” as meant herein is any use of the nucleic acid or protein and may be, as a non-limiting example, the genomic DNA for the detection of mutation, the mRNA or derived cDNA for the analysis of the expression, or the protein for the analysis of translated protein. Methods for mutation and snp analysis, expression analysis and detection and quantification of protein are known to the person skilled in the art. Abnormal chromosome content is the most common characteristic of human solid tumors. Preferably, the cancer is a cancer originating from a chromatin cohesion defect. Even more preferably, the cancer is selected from the group consisting of Seminoma, Colon carcinoma, Cervical cancer, Acute Myeloid carcinoma, Wilson tumor, Oligodendroglioma, Renal Carcinoma, Prostate carcinoma and Breast carcinoma.
Another aspect of the invention is the use of an ETG1 ortholog to treat cancer. Preferably, the use is the modulation of the expression. “Modulation,” as used herein, may be under- or over-expression. A knock down may be realized by, as a non-limiting example, the expression of RNAi. Alternatively, the level of protein may be modulated by a specific interaction, such as the binding of an antibody. Preferably, the ETG1 ortholog is the human ortholog C10orf119. One preferred embodiment is a modulation whereby modulation is a down-regulation in the cancers selected from the group consisting of Seminoma, Colon carcinoma, Acute Myeloid carcinoma, Wilson tumor, and Oligodendroglioma. Another preferred embodiment is a modulation whereby modulation is an up-regulation in the cancers selected from the group consisting of Renal Carcinoma, Prostate carcinoma and Breast carcinoma.
Still another aspect of the invention is the use of ETG1 or an ETG1 ortholog to screen compounds interfering with the interaction of ETG1 or an ETG1 ortholog with the MCM complex. Preferably, the compound is a compound that interferes with the interaction of ETG1 or an ETG1 ortholog with MCM2, MCM3, MCM4, MCM5, MCM6 and/or MCM7. “Interfering,” as used herein, can be either positive or negative, making the interaction stronger or disturbing the interaction. Methods to study protein-protein interactions (and the effect on compounds on those interactions) are known to the person skilled in the art and include, but are not limited to, two yeast hybrid studies, mappit (Tavernier et al., 2002) and reverse mappit (Lemmens et al., 2006).
Still another aspect of the invention is the use of a compound interfering with the interaction of ETG1 or an ETG1 ortholog with the MCM complex, isolated according to the invention, to treat cancer.
Previously, we identified 70 conserved plant E2F target genes (Vandepoele et al., 2005). This list holds 40 known regulators of DNA replication and chromatin dynamics, as well as 21 genes with unknown function. To identify among the latter novel S phase regulatory genes, we performed a phenotypic screening using T-DNA insertion lines obtained from the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress). One of these T-DNA insertion mutants showed an endoreduplication phenotype. We designated this T-DNA insertion mutant as E2F target gene 1 (etg1; At2g40550).
To address the role of ETG1 in plant growth and development, we analyzed the effect of loss of function of ETG1 in detail using two independent T-DNA insertion lines. Plants were grown under long-day conditions (16 hours/light, 8 hours/darkness) at 22° C. on 0.5×MS agar plate (Valvekens et al., 1988). The etg1-1 (SALK—071046) and etg1-2 (SALK—145460) alleles were found in the Salk Institute Genomic Analysis Laboratory engine (http://signal.salk.edu/cgi-bin/tdnaexpress). These seeds were acquired from the ABRC.
To screen for homozygous insertion alleles, the following primer pairs were designed: 5′-AGACCAAGATGGTCAGAGGATC-3′ (SEQ ID NO:1) and 5′-ACTGGAACACAGTAAAGCAAGC-3′ (SEQ ID NO:2) for etg1-1, and 5′-AAATTAACCGGAATGGGTTTG-3′ (SEQ ID NO:3) and 5′-ATGACTCAGATTGATGCCTGG-3′ (SEQ ID NO:4) for etg1-2. The T-DNA was inserted in the first intron (etg1-1; SALK—071046) or last exon (etg1-2; SALK—145460) of the ETG1 gene (
At the stage of bolting, younger leaves showed a slightly elongated and serrated leaf phenotype (
To study the effect of loss of ETG1 function on cell cycle progression in more detail, we performed a leaf kinematic growth analysis. Kinematic analyses of leaf growth was performed as described by Boudolf et al. (2004) and De Veylder et al. (2001). Leaf growth of etg1 and Col-0 was analyzed on five plants from 5 to 22 DAS by measuring the total leaf blade area of all cells from the abaxial epidermis drawn with a drawing tube attached to the microscope, the total number of cells. The average cell area was determined from the number and total area of drawn cells, and the total number of cells per leaf was calculated by dividing the leaf area by the average cell area (averaged between the apical and basal positions).
Finally, the average cell division rate for the whole leaf was determined as the slope of the log 2-transformed number of cells per leaf, which was done using five-point differentiation formulas (Erickson, 1976). Subsequently, seedlings were fixed in 100% ethanol overnight, replaced by lactic acid for microscopy. When wild-type and etg1-1 were compared, significant differences were observed (
Until nine days after sowing, the average cell division rate for the whole leaf, calculated on the basis of the increase of cell numbers over time, were constantly lower in the etg1-1 mutant than wild-type leaves (
To pinpoint the cell cycle arrest point, we measured the ratio of 4C/2C cells by flow cytometry using eight-day-old leaves. As at this time point, leaf cells of both genotypes are dividing (
As a confirmation, the expression levels of a number of cell cycle marker genes were analyzed by real-time RT-PCR. RNA was extracted from Arabidopsis tissues with RNEASY® Plant Mini Kit (Qiagen). First-strand cDNA was prepared from total RNA with the Superscript™ III First-Strand Synthesis System (Invitrogen) and according to the manufacturer's instructions. Quantitative PCR was performed with the LIGHTCYCLER® 480 SYBR Green I Master (Roche) with 100 nM primers and 0.1 μg of RT reaction product. Reactions were run and analyzed on the LIGHTCYCLER® 480 Real-Time PCR System (Roche) according to the manufacturer's instructions. All quantifications were normalized to ACTIN2 cDNA fragments amplified under the same conditions. Quantitative reactions were done in triplicate and averaged. Primers used were 5′-GGCTCCTCTTAACCCAAAGGC-3′ (SEQ ID NO:5) and 5′-CACACCATCACCAGAATCCAGC-3′ (SEQ ID NO: 6) for ACTIN2, 5′-TTGCAACCAGGCACCTTGAA-3′ (SEQ ID NO:7) and 5′-CAAATCGGCGGGCATTATGT-3′ (SEQ ID NO:8) for ETG1, 5′-TGGTGCTGGACATTTCAGTCGG-3′ (SEQ ID NO:9) and 5′-CAAGAGCTTGCACTTCCATCATAG-3′ (SEQ ID NO:10) for WEE1, 5′-CGAGGAAGGATCTCTTGCAG-3′ (SEQ ID NO:11) and 5′-GCACTAGTGAACCCCAGAGG-3′ (SEQ ID NO:12) for RAD51, 5′-CTCAAAATCCCACGCTTCTTGTGG-3′ (SEQ ID NO:13) and 5′-CACGTCTACTACCTTTGGTTTCCC-3′ (SEQ ID NO:14) for CYCB1;1, and 5′-CTCGAGATGGACGAAGAAGG-3′ (SEQ ID NO:15) and 5′-CGACGCAGAGTAATCGAACA-3′ (SEQ ID NO:16) for CDKB1;1. A substantial increase in the transcript level of the G2-to-M-phase-specific cyclin CYCB1;1 and CDKB1;1 genes were seen in the etg1-1 mutant (
The ETG1 gene was originally identified by microarray analysis, comparing the transcriptome of wild-type Arabidopsis plants with that of plants ectopically expressing the heterodimeric E2Fa-DPa transcription, showing a strong up-regulation of ETG1 into the latter (Vandepoele et al., 2005). This induction was confirmed by quantitative real-time PCR analysis (
To address whether ETG1 is an E2F target gene in vivo, we performed chromatin immunoprecipitation (ChIP) experiments. Immunoprecipitations were performed using anti-E2Fa, -E2Fb, -E2Fc, and DEL1 antibodies. ChIP was performed according to Bowler et al. (2004) with few modifications. Briefly, 1 g of eight-day-old seedlings was harvested, rinsed in ddH2O, and cross-linked in 1% formaldehyde for ten minutes. Cross-linking was stopped by addition of glycine to a final concentration of 0.125 M. Tissue was grinded and chromatin extracted. The chromatin solution was sonicated using a Branson 1200 sonifier. After preclearing, 10 μl of the appropriate antibodies was added to the chromatin solution and incubated overnight at 4° C. After collection of the immunoprecipitate with protein A agarose beads, beads were washed and immunocomplexes eluted. Cross-linking was reversed by incubation at 65° C. overnight. Proteinase K digestion was followed by phenol/chloroform extraction and ethanol precipitation. Recovered DNA was used in 25 cycle PCR reaction.
As shown in
The regulation of the ETG1 promoter activity through its E2F consensus sites was further analyzed using transgenic plants expressing the β-glucuronidase (GUS) reporter gene under control of the ETG1 promoter. To define the contribution of each of the E2F binding sites, we deleted either one (ΔI or ΔII) or both (ΔI,II) of the E2F elements. The ETG1 promoter sequence was amplified from Arabidopsis genomic DNA by PCR with the FP-ETG1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATATGAAAACCTAATTCCTCTG-3′ (SEQ ID NO:17)) and RP-ETG1 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCG GTCAGACAATCGTAAGCTGGT-3′ (SEQ ID NO:18)) primers. Each E2F element in the ETG1 promoter were mutated by two-step overlap extension PCR with FP-ETG1, RPETG1 primers and promoter-specific primers, FPΔI-ETG1 (5′-ATGGATAATGAACCTAGGAGATATG-3′ (SEQ ID NO:19)) and RPΔI-ETG1 (5′-CTCCTAGGTTCATTATCCATGCCCATTC-3′ (SEQ ID NO:20)) for mutation of the first E2F element (I), and FPΔII-ETG1 (5′-AGGAGATATGGGCCCAACTATACACACTTG-3′ (SEQ ID NO:21)) and RPΔII-ETG1 (5′-TAGTTGGGCCCATATCTCCTAGGTT-3′ (SEQ ID NO:22)) for mutation of the second E2F element (II), respectively (
Each PCR fragment was cloned into pDONR201 entry vector by BP recombination reaction and subsequently transferred into the pKGWFS7 destination vector (Karimi et al., 2002) by LR recombination reaction, resulting in a transcriptional fusion between the ETG1 promoter and the eGFP-GUS fusion gene. All constructs were transferred into the Agrobacterium tumefaciens C58C1RifR strain harboring the plasmid pMP90. The obtained Agrobacterium strains were used to generate stably transformed Arabidopsis with the floral dip transformation method (Clough and Bent, 1998). Seedlings were stained on multiwell plates (Falcon 3043; Becton Dickinson). GUS assays were performed as described by Beeckman and Engler (1994). Samples mounted in lactic acid were observed and photographed with a differential interference contrast microscope (Leica).
More than five independent transgenic lines were analyzed per gene construct, all showing identical results. In six-day-old seedlings, high levels of ETG1 expression were observed in the shoot apical and root meristem (
The ETG1 gene encodes for a protein of 589 amino acid residues (At2g40550; genbank accession AAY25444). ETG1 is a singleton in Arabidopsis. When searching for similar proteins by sequence comparison, no identification was found with any other functional annotated protein, neither a specific amino acid domain could be identified with the exception for a putative nuclear localization signal, PFKKMKV (SEQ ID NO:23) (amino-acids 184-190), suggesting that ETG1 resides in the nucleus. To investigate the subcellular localization of ETG1, an ETG1:eGFP (GFP for enhanced green fluorescent protein) fusion protein was transiently expressed in tobacco leaf epidermal cells. ETG1:eGFP fluorescence was observed in the nucleus only, illustrating that ETG1 is a nuclear protein (
ETG1 orthologous proteins were found in rice (Oryza sativa; Os01g0166800), human (C10orf119), mouse (1110007A13Rik), Xenopus (CAJ81286), Drosophila (CG3430) and fission yeast (SPAC1687.04) (Supplemental
Patterns of coexpression can reveal networks of functionally related genes and provide deeper understanding of processes requiring multiple gene products (Stuart et al., 2003; Wei et al., 2006). To predict ETG1 function, we searched for genes coexpressed with ETG1 using the ATTED-II coexpression database (Obayashi et al., 2007). This search revealed that ETG1 is highly coexpressed with genes encoding DNA replication proteins, such as minichromosome maintenance family proteins (MCM2, 3, 4, 5 and 7), proliferating cell nuclear antigen proteins (PCNA1 and 2), DNA primase small subunit protein, and DNA polymerase alpha subunits (Table 1). Moreover, when searching for proteins interacting with orthologous ETG1 proteins, using the BioGRID protein interactions database (http://www.thebiogrid.org/index.php), we found that the Drosophila ortholog CG3430 interacted with MCM5 protein. An identical interaction between the Arabidopsis ETG1 and MCM5 (At2g07690) proteins was demonstrated using the yeast two-hybrid system (
The ETG1-MCM5 protein-protein interaction was confirmed in planta by BiFC experiments (Bracha-Drori et al., 2004; Walter et al., 2004). The full-length open reading frames of ETG1 and MCM5 with/without stop codon were amplified by PCR using F-ETG1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAGGACCAGCTTACGATT-3′ (SEQ ID NO:24)) and R-ETG1 (5′-GGGGACCACTTTGTACAAGAAAGCTGGG TCTTACTTGAGCCTCTCCTTTCTA-3′ (SEQ ID NO:25)) primers, and F-MCM5 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCAGGATGGGACGAAGGAG-3′ (SEQ ID NO:26)) and R-MCM5 (5′-GGGGACCACTTTGTACAAGAAAGCTG GGTCCTCAAGCTTTGCGGACAATAGAA-3′ (SEQ ID NO:27)) primers, respectively, and cloned into pDONR201 ENTRY vector by BP recombination reaction according to the manufacturer's instructions (Invitrogen).
Full-length open reading frames of ETG1 and MCM5 were transferred into the pH7FWG2 destination vector (Karimi et al., 2002) by LR recombination reaction, resulting in ETG1:EGFP and MCM5:EYFP fusion proteins, respectively. BiFC assay was performed as described by Walter et al. (2004). The coding region of ETG1 was amplified with 5′-GCCACTAGTGGATCCATGGGAGGACCAGCTTACGATT-3′ (SEQ ID NO:28) and 5′-AGCGGTACCCTCGAGGTACTTGAGCCTCTCCTTTCTA-3′ (SEQ ID NO:29) primers, and cloned via BamHI-XhoI into the plasmid pUC-SPYNE (Walter et al., 2004), resulting in the plasmid expressed as the ETG1:YFPN fusion protein; the coding region of MCM5 was also obtained with 5′-TGGCGCGCCACTAGTATGTCAGGATGGGACGAAGGAG-3′ (SEQ ID NO:30) and 5′-ACCCTCGAGGTCGACGTAAGCTTTGCGGACAATAGAA-3′ (SEQ ID NO:31) primers, and cloned via SpeI-SalI into the plasmid pUC-SPYCE (Walter et al., 2004) to give rise to the plasmid expressed as the MCM5:YFPC fusion protein.
The Agrobacterium strains containing both BiFC constructs were co-infiltrated into tobacco leaves. Epidermal cell layer of tobacco leaves were assayed for fluorescence using confocal laser scanning microscope (Zeiss) two to four days after infiltration. After transfection, YFP fluorescence was observed in the nuclei of cells transfected with the ETG1-YFPN and MCM5-YFPC fusion proteins, demonstrating that the ETG1 protein interacted with MCM5 in the plant nucleus (
To identify additional ETG1-associated proteins, tandem affinity purification (TAP) in combination with MALDI-TOF-TOF-MS based protein identification was performed. TAP experiments were done according to Van Leene et al. (2007). In short, the ETG1-coding sequence was cloned by recombination into the pKNTAP vector generating a Pro35S:TAP-ETG1 cassette (pKCETG1TAP). Arabidopsis cell suspension cultures were stably transformed by Agrobacterium-mediated co-cultivation with pKNETG1TAP. Transformed Arabidopsis cells were selected and transferred to liquid medium for upscaling. Expression levels of TAP-tagged proteins were checked by protein blotting with an anti-CBP antibody. In a first round of affinity purification, protein extracts of 15 g plant material were incubated with an IgG resin. Bound complexes were released and eluted from the resin by tag cleavage with TEV protease.
In a second affinity step on a calmodulin agarose column, co-eluting non-interacting proteins and the TEV protease were removed with the flow-through. Finally, both the ETG1 bait and interacting proteins were eluted from the calmodulin agarose via EGTA-mediated removal of calcium. Eluted proteins were separated on 4-12% NuPAGE gels, excised and analyzed by Maldi-TOF/TOF MS as described (Van Leene et al., 2007). To increase the stringency of the data set, contaminating proteins due to experimental background as determined by Van Leene et al. (2007) were systematically subtracted from the lists of co-purified proteins. Next to MCM5, we identified five interacting proteins, including other components of the MCM complex, being MCM2 (At1g44900), MCM3 (At5g46280), MCM4 (At2g16440), MCM6 (At5g44635) and MCM1 (At4g02060) (Table 2).
Combined with the subcellular localization results, these data indicated that ETG1 assembles into the replisome complex. Therefore, ETG1 depletion is expected to affect the efficiency of DNA replication. To test this hypothesis, a bromodeoxyuridine (BrdU) pulse-labeling experiment was performed. Three-day-old seedlings grown on MS agar plates were incubated in the labeling solution containing 10 μM bromodeoxyuridine (BrdU) (Roche) at room temperature for various time points, and the genomic DNA was extracted with a DNEASY® Plant Mini Kit (QIAGEN). The amounts of BrdU were determined by ELISA using Anti-BrdU-POD antibody (Roche). Three biological and two technical replicates were used at each time point for ELISA. Fifty microliters of the extracted DNA (0.2 μg/ml) was placed in each well. The ELISA procedure was chiefly that of 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit III (Roche). In wild-type, the level of BrdU incorporation into cells gradually increased to a saturation level by three hours (
1All proteins were detected in the two independent TAP experiments.
Inhibition of DNA replication in Arabidopsis results into the simultaneous induction of DNA repair genes and the cell cycle inhibitory WEE1 gene, which arrests cells in the G2 phase of the cell cycle (De Schutter et al., 2007). The decreased rate of DNA replication and observed interaction of ETG1 with replication proteins suggested that the G2-arrest noticed in ETG1-deficient plants might be the consequence of activation of the replication checkpoint. To test this hypothesis, we compared the expression levels of the RAD51 (DNA repair) and WEE1 (cell cycle checkpoint) marker genes by real-time RT-PCR in wild-type versus etg1 mutant plants. Ionizing radiation (γ-irradiation and UV) and radiomimetic drugs (HU, aphidicolin and bleomycin) are known to induce RAD51 and WEE1 expression (Chen et al., 2003; De Schutter et al., 2007). Expression of both RAD51 and WEE1 was significantly up-regulated in the etg1-1 seedlings (
Activation of the DNA stress checkpoint was confirmed by using plants that carried as transgene the poly(ADP-ribose) polymerase 2 (PARP2) and WEE1 promoter fused to GUS, being markers for DNA stress and activation of the G2 replication checkpoint, respectively (Babiychuk et al., 1998; Doucet-Chabeaud et al., 2001; De Schutter et al., 2007). As shown in
DNA replication stress caused by blocking of the replication fork is mainly sensed by the ATR kinase (Culligan et al., 2004). Previously, we have demonstrated that WEE1 is one of the main targets of the ATR signaling cascade. WEE1 transiently arrests cells in the G2 phase, allowing them to finalize DNA replication before proceeding into mitosis (De Schutter et al., 2007). When assuming that the increased cell cycle duration time observed in the etg1 mutant plants is the result of the activation of the replication checkpoint, it is expected that ETG1-deficiency should have a dramatic impact on the development of plants that are unable to arrest their cell cycle in response to DNA stress. To test this hypothesis, double mutants were constructed between etg1-1 and two DNA stress checkpoint mutants, atr-2 and wee1-1. The atr-2 and wee1-1 mutants have been described previously (Culligan et al., 2004; De Schutter et al., 2007). atr-2 and wee1-1 single mutants are hypersensitive to replication-blocking or DNA damaging drugs plants, but are viable and develop normally in the absence of exogenous DNA-stress treatments (
Scanning electron microscopy revealed severe growth suppression (
To gain more insight into cell cycle effect in etg1 mutants, we examined transcript levels of 24,000 genes by using Affymetrix ATH1 GeneChip arrays. Triplicate batches of first leaf pairs of nine-day-old wild-type and etg1 plants were harvested for total RNA preparation. The statistical analysis identified a total of 220 differentially expressed genes between wild-type and etg1 at P<0.01, among which 89% were up-regulated and 11% were down-regulated with fold change expression ranging from 1.3 to 14.8 and 0.15 to 0.75, respectively (Tables 3, 4). Interestingly, among the 196 up-regulated genes in etg1, 103 genes (52%) express with a peak in mitosis. Transcription of genes expressed specifically during mitosis is regulated by common upstream cis-acting element, called MSA (mitosis-specific activator; CAACGG). When we checked MSA element in promoter region of up-regulated genes in etg1, significant enrichment of MSA elements was detected. We also calculated the timing of maximal expression of up-regulated genes during the cell cycle. Genes expressed during M phase were clearly more strongly induced than those expressed during the S, G2, and G1 phases (
Sister chromatid cohesion is apparently established during DNA replication in S phase and maintained until anaphase onset. To assess the role of ETG1 in the establishment and maintenance of cohesion, we performed FISH analysis, as described by Schubert et al. (2008). To analyze sister chromatid cohesion in interphase nuclei, individual BACs (BAC T2P11/T7N9 and F11P17) from different positions along chromosomes 1 were hybridized to flow-sorted 4C leaf nuclei. The number of FISH signals was taken as a measure for sister chromatid separation. One FISH signal (
Arabidopsis NPK1-related protein
Arabidopsis thaumatin-like protein
The human ETG1 cDNA (hETG1) was PCR amplified from the 2961492 clone (openbiosystem) using PFU turbo (Stratagene) and the following primers 5′-CACCATGCCGTGTGGGGAGG-3 (SEQ ID NO:32) and 5′-TCTAGAAAGTTCATTTCCATTCACACATTT-3′ (SEQ ID NO:33); following the manufacturer's instruction. The PCR product was ligated in the pENTR/D/Topo vector (InVitrogen) according to the manufacturer instruction.
To study the role of human hETG1, transgenic MCF7 cell cultures were generated that either overexpress or silence the hETG1 gene. MCF-7 cell lines were obtained from the American Cell Type Culture Collection (Rockville, Md.) and maintained in DMEM supplemented with 5% FCS, 5% newborn bovine serum, 2 mmol/L-glutamine, 0.4 mmol/L sodium pyruvate, 100 units/mL penicillin and 100 μg/mL streptomycin and in DMEM supplemented with 5% FCS, 2 mmol/L L-glutamine, 0.4 mmol/L sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, 6 ng/ml bovine insulin (Sigma-Aldrich, St Louis, Mo.), respectively. All recombinant lentiviruses were produced by transient transfection of HEK293T cells according to standard protocols. Briefly, 0.6 million cells of the packaging cell line HEK293T were seeded in two wells of six-well plates. After 24 hours, 3 μg of the lentiviral knock-down vector pGIZ V2HS-158067 purchased from Open-Biosystems (Huntsville, Ala.), 3 μg of the packaging plasmid pCMV-ΔR8.91 (Zufferey et al, 1997), and 1.5 μg of the envelope plasmid pMD2G-VSVG (Zufferey et al, 1997) were first ethanol-precipitated together and then transfected in the presence of chloroquine (25 μM) into the HEK293T cells using the calcium phosphate precipitation method.
Transduction of the MCF7 cells was performed in triplicate by resuspending 25,000 cells with 200 ml viral supernatant and plating them in a 96-well plate. The plate was centrifuged for 1.5 hours at 32° C. and 1500 rpm and incubated at 37° C. in a water-saturated incubator under a 5% O2/95% CO2 atmosphere. After 96 hours, the cells were trypsinized, pooled and amplified. Transduction efficiencies were determined by measuring EGFP expression using FACS analysis (Epics Altra from Beckman Coulter, Fullerton, Calif., USA). For the overproduction of hETG, we modified the pWPI (addgene ref 12254) lentiviral vector (Pham et al., 2004) to include a C-terminal tag (Myc) at the end of the gene of interest, a Tet operon sequence in front of the promoter sequence of the vector to allow conditional control of the expression cassette and finally a Gateway® cloning cassette located between the promoter and a C-terminal tag (Myc or V5His) to allow rapid transfer of the genes of interest from Gateway-compatible entry vectors. Co-transduction with a suitable lentiviral vector, pLV-tTR-KRAB-Red (Wiznerowicz and Trono, 2003), allows controlling the expression of the transgene by addition of doxycycline. Finally, the vector also bears an EGFP selection marker driven by an IRES sequence following the Gateway cassette to follow infection efficiency and eventually enrich the population by FACS sorting.
Overexpression and silencing of the hETG1 gene were confirmed by RT-PCR analysis. RNA was extracted from MCF-7 cells with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was prepared from 1 μg total RNA with the iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) according to the manufacturer's instructions. For quantitative PCR, PCR reactions were run in triplicate on a LightCycler® 480 Real-Time PCR system (Roche) using the SYBR Green I master Mix (Roche), 100 nM primers and 20 ng of cDNA according to the manufacturer's instructions The CT (threshold cycle when fluorescence intensity exceeds ten times the SD of the baseline fluorescence) values for the target amplicon and endogenous control (TBP) were determined for each sample. Quantification was performed using the comparative CT method (DDCT) Primers used were 5′-ACTCTCCACGAAATACCACTTTG-3′ (SEQ ID NO:34) and 5′-GTAGGATGTTGAGGGACTGACTCG-3′ (SEQ ID NO:35) for hETGJ and 5′-CGGCTGTTTAACTTCGCTTC-3′ (SEQ ID NO:36) and 5′-CACACGCCAAGAAACAGTGA-3′ (SEQ ID NO:37) for TBP. For both the overexpressing and silencing lines appropriate cell lines were selected. The V5-C10 overexpressing line showed an 17-fold induction of hETG1 expression levels. The 158067-Const knock-down cell line showed a depletion of 80% in transcription of hETG1.
In both the hETG1 overexpression and knock-down lines substantial changes in the cellular morphology were observed. These changes were characterized by the appearance of multi-nucleated and/or giant cells (
Two hundred thousand hETG1 knocked-down and control MCF-7 cells (non infected MCF-7) were plated into six-well plates and grown two days in 4 ml of MCF-7 medium. Cells were next trypsinized, centrifuged at 2000 rpm for 5 minutes re-suspended in 1 ml of FAC's buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and filtered on a 40 μm strainer (Becton Dickinson, San Jose, Calif.). Cells were incubated on ice in the dark during 15 minutes in FACS buffer supplemented with DAPI. The DNA content was analyzed in triplicate by flow cytometry. Compared to the control cultures, the knocked-down cultures are characterized by a depletion of the 2C (G1-phase) cell population, correlated with an increment in the population of cells with a DNA content equal to 4C (G2-phase) or greater (polyploidy) (
This cell cycle arrest was further demonstrated by transcriptional up-regulation of G2-M marker genes (such as Cyclin B1, Cyclin B2 and Cyclin H) (
To further investigate this issue, metaphases in MCF7 wild-type and h-ETG1 knock-down karyotypes were characterized by counting metaphase chromosomes with totally detached chromatids. Briefly, upon h-ETG1 knock-down, cells were cultivated during 2 days at 37° C. To enrich for mitotic chromosomes, subconfluent cells were treated with KaryoMax colcemid (Sigma) for five hours before harvesting. Cells were trypsinized, pelleted and resuspended in hypotonic solution (60 mM KCl) for 30 minutes at room temperature. Cells were repelleted, the hypotonic solution was removed and cells were resuspended in freshly made methanol: glacial acetic acid (3:1) added drop-wise. Two or three drops of suspended cells were applied to precleaned blood smear glass slides and finally chromosomes were counterstained with VectaShield containing DAPI (Vector Laboratories). Microscopic analysis illustrated an increment of chromosomes with totally detached metaphase chromosomes upon h-ETG1 knock-down (
cDNA synthesis on RNA samples was performed on 1.5 μg total RNA using the Iscript cDNA synthesis kit (Bio-Rad). Subsequently qPCR on the LC480 (Roche) was done for ETG1 and different reference genes (Vandesompele et al. 2002) using LCS480 Sybr Green I master kit (Roche), Fast SYBR master mix kit (Applied Biosystems) and Taqman fast univ. PCR Mastermix (Applied Biosystems). Using GeNorm (Vandesompele et al. 2002) we determined the most accurate set of reference genes for normalisation (HMBS, ACTB, HPRTI, RPL13A, SDHA, TBP and UBC). The average threshold cycle of triplicate reactions was used for all subsequent calculations using the delta Ct method. Relative ETG1 expression levels (average of ten samples with low expression set to 1) were depicted ranking low to high (
The expression of ETG1 was further compared with different clinicopathological parameters available for the different analyzed tumors. High ETG1 expression was correlated with a negative estrogen receptor (ER) status (
Early detection of cancer by screening remains an important effective method for improving cancer survival. ETG1 is tested for potential clinical practice by the generation of diagnostic antibodies. A full-length human ETG1 cDNA clone was used as a template for PCR amplification with Pfu polymerase to generate an ETG1 coding cassette. The primers used for amplification were hETG1-Inf-Fw 5′-CAAGGTACCAAGCTTAATGCCGTGTGGGGAGG-3′ (SEQ ID NO:38) and hETG1-Inf-Rv 5′-TGCGGCCGCATGCATTTAAAGTTCATTTCCAT-3′ (SEQ ID NO:39). The resulting PCR product was inserted by fusion cloning into the HindIII pLHX32 plasmid downstream of the His6 tag, to generate pLHXhETG1. Plasmid insert is controlled by DNA sequencing. Plasmid is transformed in MC 1061 bacteria containing a pICA2 plasmid. Exponentially growing E. Coli bacteria are induced overnight with 1 mM isopropyl β-D-thiogalactopyranoside at 20° C. The cells are harvested by centrifugation and the cell paste is frozen until required. Frozen MC1061 cell pellets are suspended in buffer A, comprising 20 nM Tris-HCl, pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 200 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 50 μM leupeptin and 20 μg/ml aprotinin, and are lysed by sonication or French press. Insoluble proteins are removed by centrifugation. Bacterial DNA is removed over a DEAF column equilibrated by buffer A. The flow through is applied on a Co+2 metal chelate column which is washed with buffer A for 4 to 16 hours. Low strength metal binding proteins are removed by a short washing with buffer B, consisting of 20 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 200 mM NaCl and 10 mM imidazole. His6-tagged ETG1 is eluted from the column by buffer C, containing 20 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 50 mM NaCl and 100 mM imidazole. The purity of the preparation is checked by SDS-PAGE and further purified if needed by Q-resource or monoQ columns. Purified proteins are used for immunization of rabbits for the production of polyclonal antibodies (Eurogentec). In addition purified ETG1 protein is provided to the VIB Nano-body service facility for lama immunization. Generated anti-ETG1 polyclonal antibodies and nanobodies are used for a wide range of applications like immunohistochemistry, immunomodulation, intracellular expression (intrabodies), biosensor-applications, etc. Collaborations are initiated with different pathology departments from which Tissue MicroArrays (TMAs) are utilized for high throughput molecular pathology characterization of various kinds of tumors (e.g. skin, colon, breast). TMAs containing samples with long-term and complete clinical follow-up data are available within this framework, allowing us to obtain prognostic and predictive information.
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Number | Date | Country | Kind |
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61130552 | May 2008 | US | national |
This patent application is a national phase entry under 35 U.S.C. §371 of PCT International Patent Application PCT/EP2009/056658, filed May 29, 2009, designating the United States and published in English as International Patent Publication WO 2009/144311 A1 on Dec. 3, 2009, which claims the benefit, under Article 8 of the PCT, of U.S. Provisional Patent Application No. 61/130,552, filed May 29, 2008, the entire disclosure of each of which is hereby incorporated herein by this reference.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2009/056658 | 5/29/2009 | WO | 00 | 5/19/2011 |