Modulation of p53 and MDM2 activity by novel interaction with Ras ...

Oncogene (2007) 26, 4209–4215

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ORIGINAL ARTICLE

Modulation of p53 and MDM2 activity by novel interaction with Ras-GAP binding proteins (G3BP) MM Kim1,3, D Wiederschain1,3,4, D Kennedy2, E Hansen1 and Z-M Yuan1 1 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USA and 2School of Biomolecular and Biomedical Sciences, Griffith University, Nathan, Queensland, Australia

Inactivation of the p53 tumor suppressor pathway is a critical step in human tumorigenesis. In addition to mutations, p53 can be functionally silenced through its increased degradation, inhibition of its transcriptional activity and/or its inappropriate subcellular localization. Using a proteomic approach, we have found that members of the Ras network of proteins, Ras-GTPase activating protein-SH3-domain-binding proteins 1 and 2 (G3BP1 and 2), bind to p53 in vitro and in vivo. Our data show that expression of G3BPs leads to the redistribution of p53 from the nucleus to the cytoplasm. The G3BP2 isoform additionally associated with murine double minute 2 (MDM2), a negative regulator of p53. G3BP2 expression resulted in significant reduction in MDM2-mediated p53 ubiquitylation and degradation. Interestingly, MDM2 was also stabilized in G3BP2-expressing cells and its ability to ubiquitylate itself was compromised. Accordingly, short hairpin RNA (shRNA)-mediated knockdown of G3BP2 caused a reduction in MDM2 protein levels. Furthermore, expression of shRNA targeting either G3BP1 or G3BP2 in human cancer cell lines resulted in marked upregulation of p53 levels and activity. Our results suggest that both G3BP isoforms may act as negative regulators of p53. Oncogene (2007) 26, 4209–4215; doi:10.1038/sj.onc.1210212; published online 5 February 2007 Keywords: G3BP; P53; MDM2

Introduction Ras-GTPase-activating protein-SH3-domain-binding proteins 1 and 2 (G3BP1 and 2) are close structural homologues that share 59% identity at the primary sequence level (Kennedy et al., 2001). These proteins directly associate with the SH3 domain of GTPaseactivating protein (GAP), which functions as an inhibitor of Ras (Tocque et al., 1997). However, Correspondence: Associate Professor Z-M Yuan, Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA. E-mail: [email protected] 3 These authors contributed equally to this work. 4 Current address: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA. Received 23 September 2006; revised 6 November 2006; accepted 6 November 2006; published online 5 February 2007

functional consequences of G3BP binding to Ras GAP have not been fully elucidated. Drosophila homologue of G3BP1 Rasputin (rin) has been proposed to function as an effector of Ras signalling independent of the Raf/ MAPK cascades (Pazman et al., 2000). In mammalian cells, G3BP1 has been found to be involved in a variety of growth-related signalling pathways. Upregulation of G3BP1 was noted in PTEN-null human cells, whereas overexpression of PTEN suppressed G3BP1 expression. Furthermore, the negative effect of PTEN expression on G3BP1 protein levels was recapitulated by direct inhibition of PI3 kinase activity (Huang et al., 2005). G3BP1 levels have also been shown to increase in response to heregulin in human Her2positive breast cancer cells (Barnes et al., 2002). Although the insights into the role of G3BPs in mitogenic signalling remain scarce, the ability of G3BP1 to regulate levels of certain mRNAs has been better characterized. G3BP1 can bind to the 30 -UTR of c-myc mRNA in a phosphorylation-dependent manner and increase its degradation in vitro (Gallouzi et al., 1998). Yet G3BP1 has no inherent endonuclease activity, and can in some instances stabilize mRNAs, as happens in the case of Tau, an essential neuronal development regulator (Atlas et al., 2004). Further evidence that G3BP1 can control the abundance of certain mRNAs was obtained when it was found in stress granules (Tourriere et al., 2003). Stress granules are sites of abortive translation initiation complexes that contain translational factors, small ribosomal subunits as well as untranslated mRNAs (Tourriere et al., 2003; Kedersha et al., 2005). It is not presently known whether G3BP2 can bind mRNA or regulate its stability. Recent epidemiological findings revealed that both G3BP isoforms are often overexpressed in human cancers. G3BP1 overexpression was detected in human head and neck tumors and cell lines as well as in human lung, prostate, colon, thyroid and breast cancer cell lines (Liu et al., 2001; Barnes et al., 2002; French et al., 2002). However, G3BP contributions to the tumorigenic phenotype have not been defined yet. Loss of the p53 tumor suppressor function, on the other hand, is fully recognized as a central event in the development of most human cancers. p53 is a transcription factor that controls expression of a number of genes implicated in cell cycle arrest, DNA repair and apoptosis in response to a variety of external and

Regulation of p53 levels and activity by G3BPs MM Kim et al

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internal stimuli. Mutations in the TP53 gene that interfere with its transactivating function are found in a large proportion of human cancers (Vogelstein et al., 2000). Alternatively, p53 can be functionally silenced through its increased degradation, inhibition of its transcriptional activity and/or altered subcellular localization (Vogelstein et al., 2000). In breast cancers for example, the TP53 gene is mutated in only 25% of tumors, suggesting that p53 functional inactivation is likely to play an important role in the pathogenesis of this disease (Molinari et al., 2000; Vogelstein et al., 2000). Although p53 is critical to cellular response to stress, its levels are tightly regulated under physiological conditions through rapid protein turnover (Vogelstein et al., 2000). A key regulator of p53 levels and activity is its own downstream transcriptional target, the murine double minute 2 (MDM2). MDM2 acts as an E3 ubiquitin ligase that targets p53 for proteasomal degradation (Vogelstein et al., 2000). In addition, MDM2 inhibits p53 by either binding its N-terminus and thus blocking the access of basal transcriptional machinery or by promoting translocation of p53 from the nucleus to the cytoplasm (Geyer et al., 2000; Vogelstein et al., 2000). In this paper, we demonstrate a novel interaction between G3BPs and p53. Furthermore, we report here that G3BP2, but not G3BP1, can also bind to MDM2 and inhibit its ability to target both p53 and itself for degradation. We also show that upon short hairpin RNA (shRNA)-mediated knockdown of G3BPs, p53 transcriptional activity is increased, thus suggesting that G3BPs function as negative modulators of p53 function. Results We have previously shown that the C-terminus of p53 associates with factors that negatively affect p53 transcriptional activity (Wiederschain et al., 2001). To identify such p53CT-bound inhibitors, we incubated whole cell lysates of TP53-null human lung adenocarcinoma H1299 cells with either glutathione S-transferase (GST) only or GST-p53CT and used mass spectrometry analysis to identify bound proteins. Multiple polypeptides spanning the sequence of G3BP1 and G3BP2 were detected in the GST-p53CT eluates, but not in the GST only fraction (data not shown). To confirm G3BP binding to p53CT, cell lysates of 293T cells expressing Flag-tagged G3BPs were incubated with GST proteins containing either full-length p53, p53DCT or p53CT. Western blot analysis of bound proteins indicated that both G3BP1 and 2 associate with full-length p53 and p53CT, but not with the p53DCT mutant (Figure 1a). These results confirmed that p53CT is both necessary and sufficient for G3BP binding. We then sought to examine which domain of G3BPs is responsible for the physical interaction with p53. N- and C-terminal deletions of G3BP1 and G3BP2 were generated and utilized in GST-p53 pull-down assay. As shown in Figure 1b, the C-termini of both G3BP isoforms were necessary and sufficient for binding to p53. Oncogene

To confirm the in vivo association of G3BPs with p53, Flag-tagged G3BP1 and G3BP2 were co-expressed with myc-tagged p53 in H1299 cells. Western blot analysis following anti-Flag immunoprecipitation indicated that p53 was present in both G3BP1 and G3BP2 protein complexes (Figure 1c). Using human breast adenocarcinoma MCF7 cells that express wild-type p53, we set out to examine the interaction of endogenously expressed p53 and G3BPs. As under normal conditions p53 is expressed at low levels, MCF7 cells were irradiated to increase p53 abundance. Both G3BP isoforms, as well as MDM2 that served as a positive control, were detected in the p53 complex following anti-p53 immunoprecipitation (Figure 1d). Collectively, these data show that G3BPs interact directly with p53 both in vitro and in vivo. To investigate whether G3BPs and p53 co-localize in the cell, we created fluorescence-tagged constructs of p53, G3BP1 and G3BP2 to track their localization. G3BPs are generally thought to reside in the cytoplasm, however, both G3BPs are capable of shuttling into the nucleus in a cell cycle-dependent manner (Guitard et al., 2001; French et al., 2002). Although p53 is a mostly nuclear protein, a small fraction of it is detected in the cytoplasm under normal conditions and can be further increased by certain negative regulators of the p53 function. From the biochemical data, we predicted that a subpopulation of p53 would colocalize with G3BPs. When expressed alone, GFP-p53 was found almost exclusively in the nucleus, whereas G3BPs were mostly cytoplasmically localized. Coexpression of G3BP1 or 2 with p53 resulted in a significant increase in p53 cytoplasmic fraction and co-localization with G3BPs (Figure 1e). Changes in p53 subcellular distribution caused by G3BP expression prompted us to examine whether these proteins can interact with MDM2, a negative regulator of p53 that can increase its nuclear export (Geyer et al., 2000; Gu et al., 2002). To this end, lysates of 293 T cells transiently transfected with either full-length MDM2, MDM2 lacking its zinc and RING finger domains (MDM2DRFD, amino acid (aa) 1–301) or MDM2 RING and zinc finger domain (MDM2RFD, aa 302– 491) were incubated with GST-G3BP1 or GST-G3BP2. Western blot analysis with the antibody that specifically recognizes MDM2RFD indicated that G3BP2, but not G3BP1, bound to both wild-type MDM2 and its RFD only mutant (Figure 2a). The in vivo association of fulllength MDM2 and G3BP2 was further confirmed by anti-MDM2 immunoprecipitation and Western blot analysis (data not shown). The RING finger domain of MDM2 is essential to its function as an E3 ubiquitin ligase. To determine whether G3BP2 binding to the RFD could affect MDM2’s ability to target p53 for degradation, p53 was co-expressed with MDM2 and G3BP2 in DKOs. G3BP2 expression resulted in nearly complete rescue of p53 from the degradation by MDM2. When the cells were treated with the proteasome inhibitor MG132 before harvesting, ubiquitylated high-molecular-weight p53 species were reduced in G3BP2-expressing cells,


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Figure 1 G3BPs are novel p53-binding proteins. (a) Flag-G3BP1, Flag-G3BP2 were expressed in 293T cells as indicated. Cell lysates were incubated with GST only, GST-p53 (aa 1–393), GST-p53DCT (aa 1–320), GST-p53 CT (aa 320–393). Bound proteins were analysed by SDS-PAGE and Western blot with the indicated antibodies. (b) Lysates of 293T cells transfected with either fulllength G3BPs or their deletion mutants were incubated with GST-p53, beads were washed, boiled in SDS-PAGE loading dye to release bound proteins and analysed by anti-Flag antibody. Summary of the binding data is presented on the right. (c) Flag-G3BP1, Flag-G3BP2 and myc-p53 were co-expressed in 293T cells as indicated. Cell lysates were incubated with anti-Flag (M2) agarose beads or anti-mouse IgG agarose beads as a negative control. Bound proteins were analysed by SDS-PAGE and Western blot with the indicated antibodies. (d) Endogenous p53 was immunoprecipitated from X-ray-irradiated (5 Gy) MCF7 cells using anti-p53 agarose-conjugated antibody. p53-associated proteins were detected by Western blot. (e) RFP-G3BP1, RFP-G3BP2 and GFP-p53 were expressed in Saos-2 cells as shown. Subcellular localization of proteins was assessed by fluorescent confocal microscopy.

indicative of impaired MDM2-mediated p53 ubiquitylation. Moreover, exogenously expressed MDM2 protein also appeared to be stabilized by G3BP2 expression (Figure 2b). To confirm and extend these observations, we coexpressed G3BP1 or G3BP2 with either p53 or MDM2 in H1299 cells. As MDM2 is a transcriptional target of p53, it was imperative to test the stability of MDM2 in a cell that lacked p53 expression. As shown in Figure 2c, MDM2, an inherently unstable protein, was stabilized by G3BP2, but not by G3BP1, by more than twofold. Expression of G3BP1 had virtually no effect on p53 stability, whereas G3BP2 coexpression resulted in significantly increased p53 protein abundance (Figure 2d). MDM2’s inherent instability stems from its capacity to self-ubiquitylate (Kawai et al., 2003). As G3BP2 overexpression suppressed MDM2-mediated p53 ubiquitylation, we wanted to know if G3BP2 could also

interfere with MDM2 auto-ubiquitylation. When MDM2 was co-expressed in H1299 cells with either G3BP1 or G3BP2, the high-molecular-weight smear detected by the anti-MDM2 antibody that typically represents ubiquitylated form of MDM2 was nearly absent when G3BP2 was coexpressed, whereas G3BP1 had no such effect (Figure 2e). To confirm these findings, we utilized shRNA to downregulate endogenous G3BPs. G3BP1 protein levels were not affected by transient transfection with the shRNA construct targeting G3BP2 and vice versa, thus confirming the specificity of knockdown (Figure 2f, upper panels). Endogenous MDM2 was significantly reduced in G3BP2 shRNA-expressing H1299 cells that lack p53 but express detectable levels of MDM2. Downregulation of G3BP1 in these cells failed to have an appreciable effect on MDM2 protein levels (Figure 2f, lower panels). Taken together, these results indicate that G3BP2 can bind to MDM2 RING domain Oncogene


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Figure 2 G3BP2 binds to MDM2 and decreases its E3 ligase activity. (a) Full-length MDM2 or its truncated mutants were expressed in 293 T cells and cell lysates were incubated with the indicated GST fusion proteins. Bound proteins were analysed by anti-MDM2 antibody (Ab-3) (upper panel) that specifically recognizes an epitope found within the MDM2RFD, or by anti-MDM2 (Ab-1) (lower panel) that binds to the N-terminus of MDM2. (b) H1299 cells were transfected with various constructs as shown and incubated with proteasome inhibitor MG132 (7.5 mg/ml) or vehicle for 6 h before harvesting. Whole cell lysates were analysed by Western blot with the indicated antibodies. (c and d) H1299 cells were transfected with either MDM2 or p53 and with G3BP expression constructs. Protein levels were analysed by Western blot and quantified using densitometry. Relative density of each protein band was calculated relative to vector control. (e) MDM2 was co-expressed in H1299 cells with either Flag-G3BP1 or Flag-G3BP2. Cell lysates were resolved by SDS-PAGE and probed with anti-MDM2 antibody (Ab-1). High-molecular weight smearing represents ubiquitylated forms of MDM2. (f) shRNA constructs targeting either G3BP1 or G3BP2 were transfected into H1299 cells and the cell lysates were subsequently analysed by Western blot using indicated antibodies.

and interfere with the ability of MDM2 to ubiquitylate itself and p53 resulting in the stabilization of both proteins. Under the conditions of long-term knockdown of either G3BP1 or G3BP2, we studied the half-life of p53 and MDM2 proteins. MCF7 cells stably expressing shRNA targeting one of the G3BP isoforms were treated with cyclohexamide and harvested at appropriate time points. The p53 half-life was extended in G3BP2 shRNA-expressing cells, but remained largely unchanged in G3BP1 shRNA-expressing cells. Steady state levels of MDM2 appeared to be increased in both G3BP1 and 2 shRNA-expressing cells, most likely due to increased p53 transcriptional activity in these cells. Oncogene

However, the half-life of MDM2 was significantly diminished only in the cells expressing G3BP2 shRNA, thereby confirming our earlier observations of G3BP2mediated inhibition of MDM2 ubiquitylation activity (Figure 3a). To test the effect of G3BP knockdown on p53 functional activity, we introduced G3BP shRNA into isogenically matched TP53/ and TP53 þ / þ HCT116 colon carcinoma cells. In HCT116 cells with intact p53, its levels were substantially increased by the G3BP2 shRNA and elevated to some extent by the expression of G3BP1 shRNA. More importantly, an increase in p53 activity, as demonstrated by the upregulation of canonical p53 downstream target cell


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Figure 3 Downregulation of G3BP1 or G3BP2 by shRNA leads to p53 activation. (a) MCF7 cells stably transfected with G3BP shRNA were treated with cyclohexamide (1 mg/ml) for the indicated period of time to stop de novo protein synthesis. Cell lysates were probed by Western blot with the appropriate antibodies. (b) Isogenic HCT116 cell lines with defined TP53 status were transfected with G3BP-targeting shRNA and cell lysates were analysed by Western blot. (c) MCF7 cells were transfected with G3BP-targeting shRNA and cell lysates were analysed by Western blot.

cycle inhibitor p21/Waf-1, was observed in these cells. p21/Waf-1 levels were unchanged in TP53/ HCT116 cells expressing G3BP shRNA, indicating that this is a p53-specific effect (Figure 3b). To ensure that this phenomenon was not specific to colon cancer cells, similar approach was utilized to downregulate G3BP levels in MCF7 cells that contain wild-type TP53. Expression of shRNA targeting either of the G3BP isoforms led to p53 activation and an induction of p21/ Waf-1, thereby confirming earlier observations. Discussion It is widely accepted that p53 is a critical tumor suppressor and its function is compromised in nearly all human malignancies. Although inactivating p53 mutations occur in approximately half of all cancers, wild-type p53 in the other half of human tumors can be functionally inhibited in a variety of ways. Here, we provide evidence of the direct physical and functional interaction of p53 with the components of the Ras network of proteins, G3BP1 and G3BP2.

Our results indicate that both G3BP isoforms bind to p53 C-terminus, which plays a crucial role in regulating p53 activity. The C-terminus harbors the oligomerization domain of p53 that is required for efficient DNA binding, as well as multiple sites of post-translational modifications that positively regulate p53 function (Levine, 1997; Wiederschain et al., 2001). Therefore, G3BP binding in this region might physically disrupt normal p53 oligomer assembly or obscure critical residues that are subject to post-translational modifications. Furthermore, the nuclear localization and export signals (NLS and NES, respectively) are located within the C-terminus of p53 and their accessibility could be altered by G3BP binding. Consistent with this notion, our data show that G3BP overexpression results in abnormal p53 subcellular distribution. Initially, we hypothesized that G3BPs might also be binding to MDM2, which is capable of actively exporting p53 from the nucleus. However, only G3BP2 displayed strong interaction with MDM2, and its overexpression resulted in marked stabilization of both p53 and MDM2. As MDM2 is a potent inhibitor of p53 transactivation, a pool of stabilized, yet functionally Oncogene


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inactivated, p53 is created in the cells where G3BP2 is overexpressed. Experimental evidence presented here suggests that binding of G3BP2 to the RFD of MDM2 results in its reduced E3 ligase activity. It is possible that binding of G3BP2 to MDM2 can exclude other important ubiquitin conjugating enzymes from binding. G3BPs have also been shown to interact with the ubiquitin-specific proteases (USPs), particularly with USP10 (Soncini et al., 2001). Therefore, it is plausible that G3BP2 can facilitate active de-ubiquitylation of p53 and MDM2 via recruitment of USP enzymes. In conclusion, our data show that the lack of both family members of G3BPs via shRNA can lead to the upregulation of p53 and their overexpression can lead the cytoplasmic redistribution of p53. Although they might influence p53 differently, regardless of their mechanism of action, both G3BP proteins can suppress p53 function. Interestingly, recently reported G3BP1 knockout mice succumb to early embryonic lethality owing to massive apoptosis (Zekri et al., 2005). Although this phenomenon can be partially explained by G3BP1 function in regulating the translation of certain mRNAs, it is also possible that p53 is upregulated in G3BP1/ cells. This hypothesis is consistent with our shRNA data. Given that G3BPs have been shown to be overexpressed in a number of human malignancies, reducing their protein levels and/or activity might provide a novel target to sensitize cancer cells to genotoxic stress through enhanced p53 response.

Materials and methods Identification of novel p53-binding proteins Using proteomic approach described previously (Li et al., 2002), we isolated proteins that specifically bind to the C-terminus of p53 (p53CT, aa 320–393). Briefly, H1299 cell lysates were incubated with either GST protein or the GSTp53CT fusion protein bound to glutathione sepharose beads (Amersham Biosciences, Piscataway, NJ, USA). Beads were washed and bound proteins were eluted using a high-salt buffer. The eluted fraction was subjected to in-solution tryptic digest, resolved by liquid chromatography and analysed by mass spectroscopy at the Harvard Medical School Proteomics Center. Cell culture and transfection MCF7, H1299, Saos-2, 293T cells (American Type Culture Collection) and MDM2/ p53/ MEFs (DKO) were maintained in minimal essential medium supplemented with 10% fetal bovine serum. HCT116 (Dr B Vogelstein, Johns Hopkins University) were maintained in McCoy’s media. Cells were transfected by the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) method according to the manufacturer’s instructions. Plasmid design p53 and MDM2 expression plasmids have been described previously (Gu et al., 2002). G3BP1 and G3BP2 constructs were created by cloning their respective cDNAs into pCDNA3.1 (Invitrogen) vector containing either an N-terminal Flag epitope Oncogene

tag or the red fluorescent protein (RFP). shRNA sequences (available upon request) targeting either G3BP1 or G3BP2 were cloned into the pBS/U6 vector generously provided by Dr Y Shi (Harvard Medical School, Boston, MA, USA). Creation of stably expressing shRNA cells shRNAs targeting G3BPs were cloned into pSuper vector (Oligoengine, Seattle, WA, USA). Retroviruses were generated by transfecting the resulting vectors into Pheonix-Amphoteric HEK 293 cells. Supernatant was harvested 48 h post-transfection and used to infect target cells in the presence of polybrene. Preparation of whole-cell extracts and Western blotting Preparation of whole cell lysates and Western blot analysis have been described previously (Gu et al., 2001). In short, cells were transfected with the appropriate plasmid DNA, harvested 24 h post-transfection, lysed, and the extracts were centrifuged to remove cell debris. Protein concentrations were determined by using the BioRad protein assay (BioRad, Hercules, CA, USA). Samples were boiled in the loading dye and resolved by sodium dodecyl sulfate–polyacryl amide gel electrophoresis (SDS—PAGE). Proteins were transferred onto nitrocellulose membranes and probed with the indicated antibodies: anti-p53 (Ab-6; Calbiochem, San Diego, CA, USA), anti-Flag (M5; Sigma, St Louis, MO, USA), anti-b-actin (AC-15; Sigma), anti-MDM2 (Ab-1, Ab-3 Oncogene), anti-G3BP1 (Transduction Labs, Franklin Lakes, NJ, USA), anti-G3BP2 (11) and anti-green fluorescent protein (GFP) (Clontech, Mountain View, CA, USA). Proteins were visualized with an enhanced chemiluminescence detection system (NEN). GST-protein binding assay GST constructs were made according to standard laboratory protocols. 293T cells were transfected as indicated, harvested 24 h later and lysed in 1% Triton X-100 lysis buffer. Cell lysates were incubated with the indicated GST fusion proteins immobilized on glutathione beads. Beads were then washed and protein complexes were liberated by boiling in SDS-PAGE loading buffer. Samples were subsequently analysed by Western blotting. Immunoprecipitation analysis Immunoprecipitations were performed as described elsewhere (Gu et al., 2002). Cell lysates were prepared in 1% Triton X100 lysis buffer and incubated with the appropriate antibodies conjugated to agarose beads. Immune complexes and whole lysates were analysed by Western blotting. Subcellular distribution assay Cells were grown on coverslips, transfected and, following 24– 48 h incubation, fixed with 4% paraformaldehyde. Slides were mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL, USA). Specimens were examined under a fluorescent microscope (Zeiss, Stuttgart, Germany). Acknowledgements We thank Dr Bert Vogelstein for his generous gift of cells used in this study, and Dr Yang Shi for the pBS/U6 plasmid. We also thank the Biological Analysis Facility Care (NIEHS 5P30ES00002) for their expertise. This work was funded by the National Institutes of Health RO1CA85679-02 to ZMY. DW is supported by the Ruth L Kirschstein National Research Service Award (CA009078). MMK is supported by National Institutes of Health T32ES007155.


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