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Division of Oncology, Departments of Medicine and of Genetics, Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri 63110
Received 17 December 2004/ Returned for modification 5 February 2005/ Accepted 25 February 2005
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ABSTRACT |
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INTRODUCTION |
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Based on its broad nucleic acid-binding properties, a myriad of cellular functions have been ascribed to YB-1. In mammalian cells, YB-1 has been shown to shuttle between the nuclear and cytoplasmic compartments (reference 25 and references therein). Within the nucleus, YB-1 interacts directly with other DNA- and pre-mRNA-binding proteins such as p53, AP2, CTCF, hnRNP K, and SRp30c (for a complete list, see a recent review [20]). To date, YB-1 has been implicated in nuclear activities such as chromatin remodeling, transcriptional regulation, DNA repair, and pre-mRNA splicing/transport (11). YB-1 is also abundant in the cytoplasm, where it is a major structural component of messenger ribonucleoprotein complexes and may act as an mRNA chaperone (6, 9) and a regulator of translation (reference 1 and references therein). Thus, YB-1 has been proposed to function as a multifunctional coordinator for the control of gene expression in both the nucleus and the cytoplasm (20, 34).
YB-1 is broadly expressed throughout development, and its expression level closely correlates with the cell proliferation state. YB-1 is abundantly expressed in early chicken and rat embryos, and its level decreases steadily during development (15, 19). High levels of YB-1 are also detected in vivo in actively proliferating adult tissues such as the colorectal epithelial glands (29) and regenerating liver tissue following chemical-induced damage (15) or hepatectomy (19). YB-1 is induced in various cell types in response to mitogenic stimuli, such as cytokine-stimulated T cells (27), serum-activated fibroblasts (19), and agonist-stimulated endothelial cells (31). Furthermore, increased nuclear and/or cytoplasmic expression of YB-1 has frequently been detected in a wide range of human cancers, including breast, ovarian, thyroid, and colorectal cancers, osteosarcomas, and synovial sarcomas (reviewed in reference 20). Similar results have also been described for experimental systems such as mouse and rabbit cancers (reviewed in reference 21). Importantly, an association of elevated levels of YB-1 and tumor progression has been reported for melanoma and also for lung, squamous cell, and prostate cancers (21). These clinical observations have suggested that disregulated expression of YB-1 may be associated with unfavorable clinical outcomes. However, it remains unclear whether YB-1 overexpression is causally related to the malignant phenotype or is simply a "marker" associated with rapid cell growth. Furthermore, the normal physiological role of YB-1 has yet to be defined, since knockout mice have been difficult to generate (28).
To better understand the physiological functions of YB-1 in vivo, we created homozygous mice with a true null mutation in the YB-1 gene. An analysis of YB-1-deficient animals revealed that YB-1 is required for the normal development of multiple embryonic organ systems and for perinatal survival. YB-1 plays an important role in cellular stress responses and in the prevention of premature senescence in cultured primary cells. YB-1 is therefore essential for early mammalian development and important for cellular responses to a variety of stresses.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For determination of the rate of protein synthesis, cells were washed twice with labeling medium (MP Biochemicals) and then incubated in the same medium containing 30 to 100 µCi/ml Trans[35S] label (MP Biomedicals) for 30 min at 37°C. To inhibit protein synthesis, we allowed the labeling reaction to proceed in the presence of 50 µg/ml cycloheximide (Sigma). For determination of the amount of 35S label incorporation, the samples were harvested, total cell numbers were counted, and then the samples were treated with trichloroacetic acid (TCA). The radioactivity present in the TCA precipitates was measured by liquid scintillation counting and normalized to the cell number.
The SA-ß-Gal activity at pH 6.0 was assayed by flow cytometry using an ImaGene Green C12FDG lacZ gene expression kit as described by the manufacturer (Molecular Probes), with small modifications. Briefly, harvested cells were fixed for 3 min with 3% formaldehyde at room temperature, washed with phosphate-buffered saline, stained for 1 h with the SA-ß-Gal stain solution described by Dimri et al. (7), using C12FDG in place of X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), and immediately analyzed with a FACScan cytometer.
Antibodies and Western analysis. We generated rabbit antisera against a mouse YB-1 peptide (QPREDGNEEDKEN; residues 252 to 264) and an MSY4 peptide (NRMQAGEIGEMKDGV; residues 249 to 263). Additional primary antibodies used were anti-actin (C-20; Santa Cruz), anti-p16Ink4a (M-156; Santa Cruz), anti-p21Cip1 (Ab-6; Oncogene), anti-Mdm2 (SMP-149; Santa Cruz), anti-p53 (Ab-7; Oncogene), and anti-green fluorescent protein (anti-GFP) (fl-1; Santa Cruz). Western blotting was performed according to a standard procedure (18) or as recommended by the suppliers, and proteins were detected by enhanced chemiluminescence using the ECL detection system (Amersham).
Lentivirus production and infection and small interfering RNA (siRNA) infection. A YB-1-GFP fusion cDNA was created by cloning the human YB-1 cDNA into HindIII and BamHI sites of pEGFP-N1 (Clontech) and subcloning this vector into a lentiviral transfer vector. To infect MEFs with the virus, we incubated 2 x 105 log-phase cells in media containing different virus titers, and we analyzed the efficiency of infection by determining the percentage of GFP+ cells with a FACScan cytometer at 2 days postinfection.
The sense sequence of the siRNA specific for p16Ink4a was 5'-GGAGUCCGCUGCAGACAGAdAdT-3', which is encoded by exon 1 of the gene. The sense sequence of the siRNA for a knockdown p21Cip1 mRNA was 5'-AACGGUGGAACUUUGACUUCG-3' and was previously described (38). A nonspecific control duplex with 47% GC content and UU overhangs which lacks homology with known gene targets (Dharmacon) was used as a nontargeting siRNA control. siRNA transfection was performed as previously described (38).
Microarray and DIGE analysis.
Total RNAs were isolated from passage 3 MEFs by use of an RNeasy kit (QIAGEN). Microarray probes were synthesized by using individual RNA samples and were hybridized with Affymetrix mouse MU74Av2 GeneChips at the Siteman Cancer Center Multiplexed Gene Analysis Core. The data set was analyzed with the Significance Analysis of Microarrays (SAM) program to identify transcripts that were differentially displayed among YB-1+/+, YB-1+/–, and YB-1–/– MEFs. Specifically, a two-class unpaired permutation test using a twofold-change threshold was used to determine significantly changed transcripts. A statistic tuning parameter (
value) was used to adjust the stringency of analysis (i.e., the predicted median false discovery rate [up to 0.140]). Whole-cell lysates were prepared from passage 3 MEFs for two-dimensional (2-D) differential in-gel electrophoresis (DIGE) analysis as previously described (5).
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RESULTS |
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Embryos homozygous for the YB-1 targeted mutation were recovered from YB-1+/– intercross matings (Fig. 1B and 2A). To measure the transcriptional output from the mutant allele, we performed real-time quantitative RT-PCR analysis of total RNAs derived from cells of individual embryos. Two primer sets (one downstream and one upstream of the deleted exon 3) gave similar results (Fig. 1C and data not shown). No YB-1 mRNA was detected in YB-1–/– cells. YB-1+/– samples showed an approximately 50% reduction in the levels of YB-1 mRNA (P < 0.001). Western blot analysis using an antibody against a peptide C-terminal to the CSD revealed the complete absence of the YB-1 protein in E13.5 YB-1–/– embryos (Fig. 1D). The steady-state mRNA and protein levels of the MSY4 gene (a YB-1 paralogue coexpressed with YB-1 in developing embryos) were not altered in YB-1+/– or YB-1–/– embryos (Fig. 1C and D).
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Next, we sought to determine when the mortality of YB-1–/– mice occurs. YB-1+/– females used for intercross matings were sacrificed at various gestational stages, genotypes of individual embryos were determined by Southern blotting and PCR analysis, and the results are summarized in Fig. 2A. At embryonic day 13.5 (E13.5), 4 of 39 homozygous embryos were nonviable, as indicated by partial embryonic resorption or a lack of cardiac activity and reflex motion. By E15.5, the fraction of nonsurviving YB-1–/– embryos was 
27% (4/15). The majority of homozygous embryos (14/18) were still alive near the end of gestation (E18.5). In contrast, on postnatal day 1, the frequency of homozygous neonates recovered was dramatically underrepresented. Except for one newborn, none of the YB-1–/– mutants was alive at this stage. This observation supports the hypothesis that most YB-1–/– embryos that survived to term died shortly after birth and were cannibalized.
As shown in Fig. 2B to I, YB-1–/– embryos were generally smaller than their wild-type and YB-1+/– littermates. Embryonic growth retardation was detected in most living homozygotes at E13.5 and was increasingly prominent at later stages. By E18.5, the body weights of surviving homozygotes were 50 to 75% that of their wild-type and heterozygous littermates (Fig. 2J). In addition, about one-third of the YB-1–/– embryos (21/72) also displayed craniofacial lesions, including improper neural tube closure (Fig. 2C) and exencephaly (Fig. 2E, G, and I). No such abnormalities were observed with wild-type embryos or heterozygotes. All embryos with craniofacial defects were markedly pale, perhaps as a consequence of severe blood loss from cerebral hemorrhage (Fig. 2H and I). Of these, one-third (3/9) did not survive to E18.5, and the remainder died shortly after C-section. The majority of E18.5 YB-1–/– embryos without exencephaly (8/9) were initially alive but quickly became cyanotic. Five of eight such neonates lost viability within the first hour, whereas all wild-type (n = 21) and YB-1+/– neonates (n = 38) were viable at this point. Histological analyses of serial embryo sections revealed that the alveolar spaces in the lungs of dead E18.5 (n = 2) and P1 (n = 1) mutants were not inflated (data not shown). The other major organ systems of these animals were small but grossly normal. Taken together, these data suggest that neurological lesions, severe hemorrhage, and respiratory failure at least partially account for the observed embryonic/perinatal lethality.
Multiorgan hypoplasia in YB-1–/– embryos. We next examined the organs of YB-1-deficient embryos to define the number and size of their constituent cells. The number of cells recovered from E15.5 YB-1–/– livers was about 50% that of their wild-type and heterozygous counterparts (P < 0.01; Fig. 2K). The undergrowth defect was not due to a developmental arrest, since the onset of fetal/adult hematopoiesis occurred in E13.5 YB-1–/– livers without a noticeable delay (Z. H. Lu and T. J. Ley, unpublished data). Similarly, there were about 40% fewer cells recovered from trypsin-digested E13.5 YB-1–/– carcasses (embryos with neurological tissues and internal organs removed [data not shown]). Flow cytometric analysis revealed similar forward- versus side-scatter profiles for both fetal hepatocytes (Fig. 2L) and fibroblasts (data not shown) obtained from all embryos tested, suggesting that YB-1 deficiency does not alter the size or granularity of these cell types in vivo. We next performed terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays on serial cryo-sections of E18.5 embryos and found that the YB-1–/– tissues contained normal numbers of TUNEL+ cells relative to wild-type samples (data not shown). These data suggest that YB-1–/– embryos manifest growth retardation as a result of multiorgan hypoplasia (a defect in cellular proliferation) without detectable cellular hypotrophy (undergrowth of cell mass) or increased apoptosis.
YB-1 deficiency does not cause "global" changes in the transcriptome, proteome, or rate of protein synthesis in cells derived from developing embryos.
Since YB-1 has been proposed to act as an important transcription/mRNA stability factor for a variety of genes that could be relevant for embryonic development, we performed a microarray analysis of cells derived from individual embryos to identify genes with altered expression patterns due to the loss of YB-1. Total RNAs were isolated from primary fibroblasts which had been freshly established from E13.5 wild-type (n = 2), YB-1+/– (n = 4), or YB-1–/– (n = 4) embryos (MEFs), labeled, and hybridized with Affymetrix mouse MU74Av2 chips. The left panel of Fig. 3A shows a scatter plot of the average signal of YB-1–/– samples for each probe set on the y axis, with corresponding average wild-type signals shown on the x axis. The microarray data set was analyzed with the SAM program to identify mRNAs that were differentially displayed from MEFs derived from YB-1+/+/YB-1+/– embryos (n = 6) versus those from YB-1–/– embryos (n = 4) (Fig. 3A, right panel). Using nonstringent parameters (with a twofold-change cutoff and a relaxed false discovery rate [median = 14%; 90% percentile = 42%]), we found that YB-1 itself displayed a greatly reduced abundance (>24-fold) in YB-1–/– cells. Four mRNAs displayed a small increase in abundance (more than twofold but less than threefold) (adenylyl cyclase-associated CAP protein homolog 1, Gap junction membrane channel protein ß2, translation elongation factor 1 homolog ß2, and cardiac 
-actin), and only one other mRNA was reduced more than twofold (FMS-like tyrosine kinase 1). These results, together with the observation that the total RNA yields per cell were indistinguishable among all samples studied, strongly suggest that the transcriptome is not globally altered in E13.5 embryonic tissues deficient in YB-1.
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YB-1 has been proposed to play a role in the global regulation of mRNA translation. We therefore performed a trans-35S label incorporation assay with freshly established MEFs. The amount of protein synthesis in serum-starved or restimulated YB-1–/– MEFs was compared with that in wild-type MEFs (Fig. 3C). The protein synthesis inhibitor cycloheximide was used to verify that trans-35S label incorporation exclusively measured protein synthesis. As shown in Fig. 3D, there was no detectable difference in 35S label incorporation into proteins (on a per cell basis) between freshly prepared quiescent YB-1–/– and wild-type MEFs. In addition, YB-1–/– and wild-type MEFs showed similar increases in 35S label incorporation in response to mitogen stimulation.
YB-1–/– MEFs display an elevated sensitivity to oxidative, genotoxic, and oncogene-induced stresses. We next cultured MEFs from individual E13.5 YB-1–/– embryos to test the effect of YB-1 deficiency on cellular growth in vitro under conditions of oxidative stress (20% O2) or under "physiological" conditions (3% O2) (24). MEFs were cultured and serially passaged according to a 3T8 protocol, and their cell numbers were monitored at the end of each passage. As expected, wild-type (Fig. 4A) and YB-1+/– cells (data not shown) proliferated rapidly in early passages but showed a steady decline in the rate of cell growth in 20% O2. In contrast, YB-1–/– MEFs exhibited lower cell densities throughout the early passages and entered complete growth arrest as early as passage 4, two to three passages earlier than the wild-type cells. In 3% O2, both wild-type and YB-1–/– MEFs showed significantly prolonged proliferative life spans. As shown in Fig. 4B, a lower seeding density did not release the growth arrest of YB-1–/– cells grown in 20% O2 (passage 5), ruling out the possibility that an increased sensitivity to contact inhibition (which is reversible) was responsible. In addition, YB-1–/– MEFs also displayed an increased sensitivity to the genotoxic drugs mitomycin C and cisplatin compared with YB-1+/+ and YB-1+/– MEFs (Fig. 4C), and they had a reduced proliferative response to c-Myc overexpression (data not shown).
YB-1–/– MEFs cultured in 20% O2 senesce prematurely. To determine the effects of YB-1 deficiency under conditions of oxidative stress, we subjected exponentially growing, asynchronous passage 3 MEFs serially passaged in 20% O2 to flow cytometric analysis to define their DNA contents. The extent of apoptosis was evaluated by measuring the proportion of cells containing sub-G0/G1 DNA content (Fig. 4D) and also by assessing the number of nonadherent cells in the medium (data not shown). No excess of apoptotic cells was detected in either assay.
YB-1–/– MEFs exhibited a slightly higher percentage of cells in G0/G1, with a smaller proportion of cells in the S and G2/M phases (Fig. 4D). To more precisely define the effects of YB-1 deficiency on cell cycle progression, we synchronized MEFs by serum starvation. The cells were then stimulated to reenter the cell cycle by the addition of 10% serum. Growth factor deprivation did not cause a completely irreversible growth arrest in YB-1-deficient cells (Fig. 4E), nor did it cause apoptosis (data not shown). We next performed a BrdU pulse-labeling assay with passage 3 MEFs to assess the timing of accumulation of S-phase cells (Fig. 4F). After a lag phase of 8 to 12 h, the wild-type cultures showed a steady increase in the proportion of S-phase cells, so that by 16 h, >55% of the cells were in S phase. In contrast, only a small proportion of YB-1–/– cells entered S phase at 12 h, and the percentage of YB-1–/– cells in S phase was consistently lower than that of wild-type cells for up to 20 h. At this point, the majority of wild-type cells had exited S phase.
We further examined the morphology of passage 4 YB-1–/– MEFs that were serially passaged in 20% O2 and found that most cells were large and flattened, a feature which is typical of senescent fibroblasts in culture (Fig. 5A). YB-1–/– MEFs also accumulated higher levels of endogenous senescence-associated ß-galactosidase (SA-ß-gal) activity (pH 6.0) than did wild-type or YB-1+/– cells (P < 0.01) (Fig. 5B). Collectively, these results suggest that YB-1–/– MEFs have deceased proliferative potential when cultured under oxidative stress because they prematurely senesce.
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A microscopic analysis revealed that most passage 4 YB-1–/– cells expressing YB-1-GFP adopted a small and light-refractile morphology typical of proliferating cells (Fig. 5F, YB-1-GFP, –/–, arrows), similar to the morphology of wild-type cells (Fig. 5F, GFP, +/+, arrows). This morphology contrasted with the flattened, senescent phenotype displayed by nonexpressing cells (Fig. 5F, YB-1-GFP, –/–, arrowheads) and GFP+ YB-1–/– cultures (Fig. 5F, GFP, –/–, arrowheads). These results, reproduced with four independent MEF preparations from individual YB-1–/– embryos, collectively suggest that the premature senescence observed in oxidatively stressed YB-1–/– MEFs is due to the YB-1 deficiency per se and not to a "neighborhood effect" caused by the retained PGK-neo cassette.
Several regulatory pathways are altered in stressed YB-1–/– MEFs. We next examined the status of various G1 regulatory and senescence-related proteins in YB-1–/– MEFs cultured in 20% O2 for various periods of time. We could not detect consistent changes in the protein levels of p53, p19Arf, or CDK4 between YB-1–/– and wild-type/YB-1+/– cells as a function of the passage number (Fig. 6A and data not shown). p27Kip1 levels were high in all passage 2 MEFs but declined more rapidly in continuously passaged YB-1–/– cells than in wild-type or YB-1+/– cells. In contrast, the cyclin-dependent kinase inhibitor p16Ink4a and the p53 transcriptional targets p21Cip1 and Mdm2 accumulated more rapidly in YB-1–/– MEFs. The abundance of p16, p21, and Mdm2 in passage 3 YB-1–/– cells (lane 6) was significantly higher than that in passage 4 wild-type and YB-1+/– cells (lanes 7 and 8). The increased abundance of p16 and p21 was due to increased levels of their respective mRNAs (Fig. 6B), suggesting that a transcriptional mechanism(s) is responsible. To determine the relevance of p16 and p21 overexpression in mediating premature senescence, we "knocked down" the levels of these proteins in YB-1–/– MEFs by using specific siRNAs (Fig. 6C). As shown in Fig. 6D, the p16 and p21 double knockdown extended the proliferative capacity of late-passage YB-1–/– cells. The early senescent phenotype of YB-1–/– cells was not completely rescued by the double knockdown of p16 and p21, but not all cells were transfected and inhibition was incomplete. The double knockdown of p16 and p21 also enhanced the proliferation of wild-type cells, as predicted.
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DISCUSSION |
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Our data revealed that YB-1 deficiency results in fully penetrant late embryonic runting and perinatal lethality. Morphological and histological analyses revealed that many YB-1–/– embryos displayed major developmental defects (neurological abnormalities, hemorrhage, and respiratory failure) that probably contributed to lethality. Growth retardation occurred in all late-stage embryos and was the result of hypoplasia in multiple organ systems. Since late-stage embryogenesis involves massive cellular proliferation that occurs over a very short period of time, the late embryonic period can be thought of as a time of considerable proliferative stress. The multiorgan hypoplasia observed in YB-1–/– embryos may therefore occur because of defective stress response signaling pathways in the constituent cells. This hypothesis is also consistent with the observation that YB-1 expression is elevated in many cell lines in response to various environmental stresses (see below). An alternative explanation for the late embryonic hypoplasia is a placental defect caused by YB-1 deficiency. This explanation seems unlikely to us, however; although YB-1–/– placentas were noted to be slightly smaller than those of wild-type controls, they did not display any of the abnormalities associated with placental failure (26). To formally rule out of this possibility, a conditional knockout approach with adult mice would be required.
Numerous in vitro studies have implicated YB-1 in important cellular functions such as protein synthesis, mRNA stabilization, cell cycle regulation, and cell survival. Two previous reports of targeted YB-1 mutations in cultured cell lines (for both, only YB-1+/– cells were established) have suggested that haploinsufficient phenotypes exist. Guntaka and colleagues showed that a chicken lymphoid DT40 cell line heterozygous for a YB-1 targeted mutation displayed major cellular defects, such as aneuploidy and severe apoptosis (33). Kuwano's group found that the disruption of one allele of the YB-1 gene in a mouse ES cell line caused an abnormal sensitivity to external cytotoxic stimuli (28). In contrast with these studies, we showed that YB-1+/– mice and MEF preparations have no detectable phenotype. The mechanisms for the discrepancies among these mutations are currently unknown, but the two mutant alleles described in the previous studies could have potentially encoded truncated proteins that may have acted as dominant-negative molecules. Regardless, it is clear that heterozygosity for our "true null" mutation of YB-1 supports all of the normal physiological functions of this protein in vivo.
Based on the observation of Evdokimova et al. (10) that immunodepletion of YB-1 results in a complete inhibition of protein synthesis in a rabbit reticulocyte lysate, we expected that a loss of YB-1 would not be compatible with cell viability. However, our studies with YB-1-deficient mice revealed that a high percentage of mouse embryos develop and survive to the late stage of embryogenesis. Furthermore, we detected "wild-type" numbers of apoptotic cells in YB-1-deficient embryos and cultured MEFs. These results strongly suggest that YB-1 is not a cell viability factor or a bona fide cell cycle regulator. We further showed that the loss of YB-1 does not cause "global" changes in the transcriptome, the proteome, or the rate of total protein synthesis in freshly isolated MEFs. These findings, while surprising, raise the possibility that other Y-box proteins may substitute for YB-1 to "rescue" these activities. Three Y-box proteins, YB-1, MSY2, and MSY4 (13, 35), have been identified in mice, and their orthologues are known from Xenopus to humans (4, 14). In mice, YB-1 and MSY4 are ubiquitously expressed throughout development. In addition to the highly conserved central CSD, all vertebrate Y-box proteins contain a short and variable amino-terminal domain and a long structurally conserved carboxyl tail consisting of four basic/aromatic islands. Y-box paralogues have been shown to possess similar biochemical properties in vitro (reference 13 and references therein). Regardless of these observations, YB-1 is required for normal late embryonic development and viability and for normal responses to cellular stresses; it clearly has nonredundant functions that are not compensated for by MSY4. YB-1 is distinct from other CSD family members because its exceptionally high degree of phylogenetic conservation occurs not only in the CSD but also in the rest of the molecule. Alternatively, YB-1 and MSY4 may perform similar functions in vivo but differ mainly in their tissue distribution patterns. For example, YB-1 mRNA levels are very high in human fetal brains, where MSY4 is minimally expressed (32). The observation that neurological development is sometimes adversely affected by YB-1 deficiency (i.e., partially penetrant neural tube defects and exencephaly) therefore suggests that YB-1 may have important functions in central nervous system development. In order to better understand the functional similarities and differences among Y-box paralogues in vivo, it will be important to compare loss-of-function models of MSY2 and MSY4 with that of YB-1.
Primary MEFs proliferate only for a finite duration in vitro before irreversibly entering into a nonproliferative state called culture shock, stasis, or stress-induced senescence. Campisi and colleagues recently showed that MEFs senesce as a result of the oxidative stress imposed by culturing in 20% O2, which is a condition used for standard tissue culture protocols (24). In addition, a variety of cytotoxic stimuli, including UV and ionizing irradiation, anticancer drug treatments, and oncogenic viral infections, have also been shown to trigger senescence both in vitro and in vivo (3). These findings have suggested that stress-related senescence may represent one of the tumor-suppressive mechanisms of somatic cells (24). Interestingly, many of the same cytotoxic stimuli that trigger senescence are also known to induce the overexpression and/or nuclear accumulation of YB-1 (2, 8, 17, 25, 30). In the present study, we found that YB-1–/– MEFs cultured in 20% O2 prematurely senesce and have a reduced proliferative response to serum stimulation. In addition, YB-1–/– MEFs are abnormally sensitive to genotoxic drugs (mitomycin C and cisplatin) and display a reduced ability to support the accelerated proliferation caused by oncogene overexpression. Our data therefore provide an important line of genetic evidence showing that YB-1 is an important component of a cellular stress response signaling pathway that is required to protect cells from a variety of stresses.
We do not yet know how YB-1 protects cells from senescence. However, late-passage YB-1–/– MEFs displayed a high percentage of cells in G0/G1 and prematurely accumulated the negative cell cycle regulators p16Ink4a and p21Cip1. These findings are consistent with the view that cell cycle arrest is an important step towards the initiation of the senescent phenotype (3). As a transcription factor, YB-1 has been described to regulate (both positively and negatively) a host of target genes involved in the stress response pathway and in cell growth (references 2, 28, and 29 and references therein). However, it remains to be established whether YB-1 plays a direct role in negatively regulating p16Ink4a and p21Cip1 transcription in oxidatively stressed MEFs. In addition, YB-1 could also directly interact with other important cytotoxic stress-related factors and modulate their biological activities. YB-1 has previously been shown to physically interact with p53 both in vitro and in vivo (23, 37). The p53/Mdm2/p19Arf pathway plays an integral role in the cellular stress response, genomic integrity surveillance, and senescence pathways. While our results do not suggest that the early senescent phenotype is caused by disregulation of p53 or p19ARF gene expression, YB-1 may still play a role in this pathway by altering p53 or p19Arf activities. Indeed, two p53 transcriptional target genes (p21Cip1 and Mdm2) are prematurely induced in YB-1–/– MEFs, suggesting that p53 activity does indeed increase with a YB-1 deficiency.
Alternatively, YB-1 has been shown to exhibit high affinities for damaged DNA (apurinic or cisplatin- or UV-modified DNA) and H-DNA structures, and it physically interacts with several DNA repair factors in genotoxically stressed cells (references 12 and 22 and references therein), raising the possibility that YB-1 may function directly in DNA damage recognition and/or processing. The loss of murine Fus/TLS, a YB-1-interacting multifunctional ribonucleoprotein, has been shown to cause chromosomal instability and perinatal death (16). Many mouse mutants lacking genes important for DNA repair pathways (e.g., Xrcc2, Lig4, Xrcc4, Ku70/80, Polß, and Xpa) also result in a premature senescence phenotype (references 3 and 24 and references therein). Recent clinical studies have also reported a strong correlation between the nuclear level of YB-1 and the cellular resistance to chemotherapy of various primary human cancers (reviewed in reference 12). We are therefore using our YB-1-deficient mice to investigate the importance of YB-1 for DNA repair, genome stability, and the genetic interaction with the p53 pathway (Fig. 7).
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ACKNOWLEDGMENTS |
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We thank Kelly Schrimpf and Mieke Hoock for expert blastocyst injections and animal husbandry. Philip Horwitz was responsible for the production of the polyclonal antisera against YB-1. We thank Charles Sherr, Martine Roussel, and Jason Weber for helpful discussions. The Embryonic Stem Cell Core, Multiplexed Gene Analysis Core, and Proteomics Core of Siteman Cancer Center all contributed to the execution of these studies. Nancy Reidelberger provided expert editorial assistance.
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FOOTNOTES |
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, this pup was small for its age but was alive 2 h after surgical birth; 
