Cyclin G1 Has Growth Inhibitory Activity Linked to the ARF-Mdm2-P53 and Prb Tumor Suppressor Pathways

Vol. 1, 195–206, January 2003 Molecular Cancer Research 195

Cyclin G1 Has Growth Inhibitory Activity Linked to the ARF-Mdm2-p53 and pRb Tumor Suppressor Pathways

Lili Zhao,1 Tina Samuels,1 Sarah Winckler,1 Chandrashekhar Korgaonkar,1 Van Tompkins,1 Mary C. Horne,1,2 and Dawn E. Quelle1,2

1Department of Pharmacology and 2The Molecular Biology Graduate Program, College of Medicine, The University of Iowa, Iowa City, IA

Abstract growth. This is primarily accomplished by Mdm2, a p53- Cyclin G1 is a p53-responsive gene that is induced in responsive gene product that acts in a negative autoregulatory alternative reading frame (ARF)-arrested cells, yet its feedback loop to inactivate p53 (5). Mdm2 is an E3 ubiquitin role in growth control is unclear. We tested its effects ligase that blocks p53 function through direct binding, on growth and involvement in the ARF-Mdm2-p53 ubiquitination, and promotion of p53 nuclear export into tumor suppressor pathway. We show that cyclin G1 cytoplasmic proteasomes (5). interacts with ARF, Mdm2, and p53 in vitro and in vivo. ARF is an alternative reading frame product derived from

At high levels, cyclin G1 induces a G1-phase arrest in the INK4a/ARF tumor suppressor locus on chromosome 9p21 mammalian cells that coincides with p53 activation. (6, 7). It is the second most commonly inactivated gene in Conversely, lower levels of cyclin G1 lack intrinsic human cancer (8), and it blocks cellular transformation in growth inhibitory effects yet potentiate ARF-mediated response to activated oncogenes, such as Ras or Myc (9). As growth arrest. Notably, cyclin G1 is down-regulated by with mice lacking p53, specific disruption of ARF results in Mdm2 through proteasome-mediated degradation. spontaneous tumor development (10, 11). p53 is the major These data suggest that cyclin G1 is a positive effector of ARF-mediated growth inhibition, and ARF activates feedback regulator of p53 whose expression is p53 by antagonizing Mdm2 (7, 9). Until recently, it was restrained by Mdm2. Interestingly, growth inhibition by generally accepted that ARF neutralized Mdm2 activity by cyclin G1 does not require p53 but instead exhibits sequestering it within nucleoli, thereby allowing p53 to partial retinoblastoma protein (pRb) dependence. These accumulate in the nucleoplasm and induce expression of findings reveal that cyclin G1 has growth inhibitory growth inhibitory genes. However, recent studies showed that activity that is mechanistically linked to ARF-p53 and ARF can inhibit growth without relocalizing endogenous pRb tumor suppressor pathways. Mdm2 to nucleoli (12–14). Moreover, regions within the amino terminus of ARF were identified that were dispensable for Mdm2 binding and relocalization, but essential for its Introduction activation of p53 and inhibition of growth (14). Those findings suggested that other factors besides Mdm2 contribute to p53- Inactivation of the p53 tumor suppressor gene is the most dependent growth suppression by ARF. Consistent with that frequent genetic event in human cancers (1). p53 is a notion is the existence of multiple ARF signaling pathways. For checkpoint regulator that maintains genomic stability in the instance, once p53 is activated, both p21-dependent and p21- face of environmental and intracellular stresses, including independent pathways can contribute to the G and G arrest hypoxia, DNA damage, and oncogene activation (2). Normally, 1 2 elicited by ARF. p21Cip1 is a p53-responsive gene and potent p53 is a short-lived nuclear protein, but stress signals rapidly inhibitor of cyclin-dependent kinases (Cdks) that blocks stabilize and activate p53 through post-transcriptional mecha- phosphorylation of the retinoblastoma tumor suppressor nisms, such as phosphorylation and acetylation (3). Activated protein, pRb (15). The consequent accumulation of active, p53 suppresses growth by transactivating genes that trigger hypophosphorylated pRb arrests cells in G phase and prevents growth arrest or apoptosis (4). Once cellular damage is 1 S-phase entry. Although p21 is the primary downstream repaired, p53 must be down-regulated to allow normal cell effector of ARF-mediated cell cycle arrest, a p21-independent pathway also exists that exerts a distinct biphasic growth arrest (16, 17). In addition, ARF can induce a delayed G1-phase growth arrest in cells lacking both p53 and Mdm2 (18). Received 7/5/02; revised 11/1/02; accepted 12/13/02. Importantly, regulators of the p21- and p53/Mdm2-independent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in pathways have yet to be identified. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We previously showed that cyclin G1 is induced by ARF in Grant support: D.E.Q. from the American Cancer Society (RSG-98-254-04- both p21-positive and p21-negative cells (14, 17). Cyclin G1 is MGO) and NIH (RO1 CA90367), and by a grant from the NIH to M.C.H. (RO1 GM56900). a transcriptional target of p53 that contains two p53 binding Requests for reprints: Dawn E. Quelle, Department of Pharmacology, College of sites within its promoter, and its up-regulation coincides with Medicine, The University of Iowa, 51 Newton Road, Iowa City, IA 52242. Phone: (319) 353-5749; Fax: (319) 335-8930. E-mail: [email protected] activation of p53 by various DNA-damaging agents (19–23). Copyright D 2003 American Association for Cancer Research. Cyclin G1 is also induced by transforming growth factor h

(TGF-h), BMP-4, p63, and p73, and is often detected in cells and tissues lacking p53, indicating that it can be regulated through p53-independent pathways (21–26). Notably, there is no proven Cdk partner for cyclin G1. It has been found to interact with pRb, cyclin G1-associated kinase (GAK), Cdk5, and the regulatory BV and catalytic C subunits of protein phosphatase 2A (PP2A) (27–32). The physiological impor- tance of those associations has not been determined, although there is suggestive new data concerning PP2A. Okamoto et al. FIGURE 1. Cyclin G1 is induced by ARF in a p53-dependent manner. (32) recently reported that cyclin G1 recruits PP2A to NIH 3T3 cells and tko MEFs were infected with vector (V ) or ARF (A) retroviruses, whereas Narf cells were treated with (+) or without (À) IPTG. dephosphorylate Mdm2 and thereby regulate p53. Western blotting was performed to measure the expression of ARF, p53, Apparently conflicting roles have been assigned to cyclin G1 and cyclin G1 in whole cell lysates (50 Ag per lane) from the indicated cells. in growth control. Some reports indicate that cyclin G1 promotes growth based on observations that its overexpression enhances the growth of certain cancer cell lines, whereas pyranoside (IPTG)-inducible ARF (39). Treatment with IPTG introduction of antisense constructs suppresses their growth for 2 days resulted in complete growth arrest (data not shown) (29, 33–35). Conversely, it has been suggested that cyclin G1 that coincided with induction of ARF, stabilization of p53, and may have growth inhibitory activities. This is based on up-regulation of cyclin G1 (Fig. 1). These data indicate that substantial yet largely correlative data showing that cyclin G1 ARF-mediated induction of cyclin G1 is commonly observed and requires p53. expression is high in differentiated tissues and in G2-phase- arrested hepatocytes, is induced by DNA-damaging agents, and The p53-dependent up-regulation of cyclin G1 in ARF- is up-regulated during TGF-h- or BMP4-mediated growth arrested cells suggested that it might have intrinsic growth arrest (23, 24, 36, 37). Moreover, it was recently shown that inhibitory activity. This was supported by preliminary cyclin G1À/À mouse embryo fibroblasts (MEFs) are partially experiments showing that ectopically expressed cyclin G1 deficient in an irradiation-induced G -M-phase checkpoint (38). induced a G1-phase arrest in Chinese hamster ovary (CHO) 2 1 Others found that overexpression of cyclin G1 had no effect on and 293 cells. To further test that idea, green fluorescent the growth properties of mouse fibroblasts, yet it sensitized protein (GFP)-tagged cyclin G1 plasmids were expressed by those cells to tumor necrosis factor a (TNFa)-mediated transient transfection in U2OS and NIH 3T3 cells. Cells apoptosis (19, 23). expressing GFP or GFP-cyclin G1 (GFP-G1) were collected The data presented here implicate cyclin G1 as a regulator by fluorescence-activated cell sorting, and the DNA content within the ARF-Mdm2-p53 and pRb tumor suppressor path- of each population was measured by staining with Hoescht ways. Moreover, our findings suggest that cyclin G1 has dye or propidium iodide (PI). Fig. 2 shows that cells intrinsic growth inhibitory activity that is dependent on the expressing high levels of GFP remained in cycle, similar to magnitude of its expression. That observation may help to GFP-negative cells isolated from the same populations. In explain, at least in part, why there are conflicting reports contrast, cells expressing GFP-G1 were dramatically arrested concerning its role in growth control. in the G1 phase of the cell cycle. In experiments using live U2OS cells stained with Hoescht, cells were simultaneously stained with PI to identify dead cells within the population. Results No increase in cell death was observed in the GFP-G1- Cyclin G1 Has Intrinsic Growth Inhibitory Activity positive cells versus those expressing GFP (data not shown), We previously demonstrated that cyclin G1 protein indicating that in this system, cyclin G1 does not initiate expression is induced concomitantly with p53 activation in apoptosis. ARF-arrested mouse fibroblasts (14, 17). Given that ARF can inhibit growth independent of p53 and that cyclin G1 is Cyclin G1 Potentiates ARF-Mediated Growth Arrest regulated by multiple transcription factors, we tested whether To examine the significance of cyclin G1 up-regulation by up-regulation of cyclin G1 by ARF is p53 dependent. Cyclin ARF, we tested its contribution to ARF-mediated growth arrest G1 protein levels were assayed following introduction of in NIH 3T3 cells. Mouse fibroblasts were chosen for these ARF into cells expressing or lacking p53. Mouse NIH 3T3 studies because we routinely achieve nearly complete intro- fibroblasts (INK4a/ARF-null, wild-type p53) and triple- duction of our genes of interest into the population via knockout (tko) MEFs lacking p53, Mdm2, and ARF were retroviral-mediated infection (14, 17). As controls, retroviruses infected with retroviruses encoding mouse ARF or empty encoding cyclin G1, empty vector, or mouse ARF were vector control. ARF causes a rapid p53-dependent G1- and individually transduced into the ARF-null cells. Western G2-phase growth arrest in 3T3 cells versus a delayed G1- blotting confirmed expression of the exogenous hemaglutinin phase block in tko cells (14, 18). Immunoblots showed (HA)-tagged cyclin G1 (Fig. 3A), although levels achieved by equivalent expression of ARF in both populations, yet cyclin retroviral infection were approximately 5-fold lower than that G1 expression and induction was only evident in NIH 3T3 cells (Fig. 1). For comparison, we also examined cyclin G1 expression in human Narf cells, a derivative of U2OS osteosarcoma cells that express isopropyl-1-thio-h-D-galacto- 1S. Winckler and M.C. Horne, unpublished observations.

divided at a slightly faster rate (1.2-fold, P < 0.01) than vector control cells (Table 1). This finding was in accordance with studies showing that cyclin G1 modestly enhanced colony formation of human diploid fibroblasts (29). It seems most likely that an inability to achieve high levels of cyclin G1 expression in the retroviral system accounts for its lack of growth inhibitory activity. Indeed, a correlation between growth arrest and high-level expression of cyclin G1 was observed (Fig. 3D). NIH 3T3 cells that were arrested, either by transfection of GFP-G1 or introduction of ARF, expressed high levels of tagged or endogenous cyclin G1, respectively (Fig. 3D). Conversely, cells infected with cyclin G1 retroviruses

FIGURE 2. High levels of cyclin G1 induce G1-phase growth arrest. continued to proliferate and expressed relatively low levels of Representative histograms showing cell cycle distributions of sorted GFP- exogenous cyclin G1. Thus, the magnitude of cyclin G1 positive (+) and GFP-negative (À) U2OS and NIH 3T3 cells following transfection with GFP or GFP-cyclin G1 (GFP-G1) plasmids. The expression correlated with its effects on cell growth. percentage of cells in S phase for each population is denoted. Previous studies showed that cyclin G1 was unable to induce apoptosis when overexpressed in NIH 3T3 cells, but it potentiated cell death induced by TNFa (23). To test whether induced by ARF. Flow cytometric analyses revealed no exogenous cyclin G1 could enhance ARF-induced growth significant effect of cyclin G1 on 3T3 cell growth, similar to arrest, NIH 3T3 cells were first infected with retroviruses vector control cells, whereas ARF induced a complete G1- and encoding empty vector or cyclin G1, followed by a second G2-phase growth arrest (Fig. 3B and C). round of infection with ARF retroviruses. Although this method The lack of growth arrest by cyclin G1 in these cells was resulted in essentially complete infection with ARF (at least surprising given its ability to block U2OS and NIH 3T3 cell 96% of cells in each population expressed ARF, as determined growth in transfection experiments (Fig. 2). However, it was by immunofluorescence), reduced expression of ARF was consistent with earlier studies showing that cyclin G1 was routinely achieved. Consequently, a less robust arrest and up- unable to initiate growth arrest or apoptosis when overex- regulation of p53 by ARF was observed (Fig. 3A and B). Under pressed in mouse fibroblasts (19). Interestingly, cell counts these conditions, the addition of exogenous cyclin G1 taken 2 days after infection showed that cyclin G1 expressors consistently potentiated the G1-phase growth arrest exerted by

FIGURE 3. Cyclin G1 enhances ARF-mediated growth arrest. A. Equivalent amounts of total cellular protein from NIH 3T3 cells infected with the indicated retroviruses were analyzed by Western blotting for expression of ARF, cyclin G1 (HA-tagged form indicated by asterisk), p53, Mdm2, and B23 (loading control). B. Cell cycle distributions of infected NIH 3T3 cells from a representative experi- ment, in which V/A and G1/A represent sequential infections of vector or cyclin G1 plus ARF viruses. C. The relative percentage S phase for cells expressing the indicated viruses relative to vector control was calculated from three independent experiments. Asterisk, statistically significant difference between G1/A and V/A samples, as determined by a paired, two-tailed Student’s t test analysis (P = 0.008); bars, SD. D. Cyclin G1 immunoblot assessing expression levels in an equivalent number of NIH 3T3 cells infected with vector, ARF, or HA-cyclin G1 retroviruses compared to NIH 3T3 cells transfected with GFP or GFP- G1. Transfected cells were analyzed (not sorted) by fluorescence-activated cell sorting, and 33% expressed GFP-G1. Asterisks indicate endogenous cyclin G1 (*), HA-cyclin G1 (**), and GFP-G1 (***).

ARF (Fig. 3C). Although the effect was modest, it was highly GST. GST-G1 proteins containing NH2-terminal residues 1–24 reproducible and statistically significant when subjected to a or 1–57 of cyclin G1 were incapable of binding to either ARF, Student’s t test analysis (P = 0.008). Identical results were p53, or Mdm2 (Table 2). Relatively weak binding was observed obtained in BrdUrd-incorporation assays, in which 40% of vec/ between those proteins and residues 1–187 of cyclin G1, a ARF-infected cells incorporated BrdUrd compared to only 30% construct that contains the conserved cyclin box (24, 41). In of G1/ARF-infected cells. contrast, GST-G11 – 217 exhibited strong association with ARF The above results indicated that cyclin G1 can contribute to and optimal binding to p53 and Mdm2 when compared to the ARF-mediated growth arrest. It is notable that the enhanced binding ability of wild-type cyclin G11 – 294. Thus, residues arrest of G1/ARF-expressing cells did not coincide with 187–217 are required for efficient binding of ARF, p53, and increased p53 stabilization (Fig. 3A) or transcriptional Mdm2. That region comprises the first three a helices of the so- activation in reporter assays (negative data not shown). The called ‘‘box repeat,’’ a COOH-terminal region of five a helices slight increase in Mdm2 expression observed in G1 + ARF- containing structural similarity to the cyclin box (41). versus Vec + ARF-infected cells was not reproducible in To determine whether cyclin G1 formed complexes with multiple experiments (see Fig. 3A), and we have no evidence Mdm2, ARF, and p53 in vivo, immunoprecipitation (IP)-Western for up-regulation of another p53 target, p21, in these cells (data blot analyses were performed from U2OS cells expressing not shown). Therefore, the effects of exogenous cyclin G1 GFP-G1 with each of the individual proteins in U2OS cells appeared to be independent of p53, as one would expect given (Fig. 4B and C). GFP control did not associate with either that its expression is normally downstream of p53. Hdm2, p53, or ARF. In contrast, GFP-G1 efficiently associated with ectopically expressed Hdm2 and p53 (Fig. 4B). It also Cyclin G1 Associates with ARF, Mdm2, and p53 coprecipitated with endogenous Hdm2 in p53 immunoprecipi- Previous studies showed that cyclin G1 is a nuclear protein, tations from cells overexpressing p53, suggesting that a trimeric ARF is nucleolar, and p53 and human Mdm2 (Hdm2) reside complex between G1-Hdm2-p53 can be formed. By compar- within the nucleoplasm (22, 36, 40). Therefore, we tested ison, cyclin G1 appeared to interact weakly with ARF relative whether cyclin G1 might physically bind to those regulators. to the association between ARF and Hdm2 (Fig. 4C). In vitro binding assays were performed using glutathione NIH 3T3 cells infected with ARF retroviruses were then used S-transferase (GST)-tagged cyclin G1 mixed with recombinant to determine if endogenous cyclin G1, p53, and Mdm2 form ARF, p53, or Mdm2 produced in Sf9 insect cells (Fig. 4A). complexes in ARF-arrested cells. As expected from earlier GST-cyclin G2 and GST-PP2AC (the C subunit of PP2A) were studies (39, 42), protein complexes between ARF, Mdm2, and included for comparison. Cyclin G2 shares significant homol- p53 were observed in the arrested cells (Fig. 4D). Two different ogy with cyclin G1, and the BV and C subunits of PP2A are antibodies efficiently precipitated cyclin G1 and coprecipitated a known to associate with both G cyclins (24, 30–32). We found small amount of p53, yet failed to coprecipitate ARF or Mdm2. that GST-cyclin G1 bound to ARF, p53, and Mdm2, although Conversely, low levels of cyclin G1 were detectably precipitated the interaction with Mdm2 was reproducibly more efficient in by antisera to Mdm2 and p53 compared to IgG control, repeated assays (Fig. 4A and data not shown). Interestingly, p53 suggesting that a small percentage of cyclin G1 is associated associated equally well with cyclin G2 and more strongly with with those regulators during ARF-mediated growth arrest. PP2AC than it did with cyclin G1. The interaction between ARF and Mdm2 with cyclin G2 was weak in vitro. Likewise, their ability to complex with PP2AC was limited but modestly Cyclin G1 Is Relocalized by ARF and Hdm2 stronger given the relatively low levels of GST-PP2AC in the Given that cyclin G1 forms complexes with Hdm2, ARF, reactions. Thus, cyclin G1 is able to associate independently and p53 in vivo, we tested whether its localization was altered with ARF, p53, and Mdm2 in vitro, and p53 can also interact by those proteins in U2OS cells (Fig. 5). As expected, GFP was with cyclin G2 and PP2AC. expressed in both the cytoplasm and nucleus, and its local- To define the region(s) of cyclin G1 that interact with ARF, ization was not altered by expression of ARF, p53, or Hdm2, or p53, and Mdm2, similar in vitro binding studies were performed vice versa. GFP-G1 was distributed throughout the entire with COOH-terminal deletion mutants of cyclin G1 fused with nucleus, including the nucleoli, when expressed alone in U2OS cells. In contrast, GFP-G1 localization was altered by exogenous ARF and Hdm2. GFP-G1 became exclusively nucleolar in nearly 60% of transfected cells co-expressing Table 1. Low Levels of Exogenous Cyclin G1 Modestly Promote Cell Growth in NIH 3T3 Fibroblasts ARF, whereas Hdm2 retained GFP-G1 in the nucleoplasm in 65% of the population (Fig. 5). To assess the specificity of the Retrovirus Relative Cell Number ARF effect, GFP-G1 was co-expressed with an ARF mutant, D1-62, that localizes to the nucleoplasm and lacks growth Vector 1.0 F 0 inhibitory activity (40, 43). GFP-G1 localization was unaffected ARF 0.35 F 0.09 by D1-62. Importantly, expression levels were key determinants Cyclin G1 1.21 F 0.02 (P < 0.01) of cyclin G1 localization because lower levels of ARF or Hdm2 Note: Cells were infected with the indicated retroviruses and cell counts were were less efficient at relocalizing GFP-G1 (data not shown). taken 2 days later. Each value represents the mean F SD from three independent Overall, colocalization and/or relocalization of cyclin G1 with experiments. The probability (P) value, which indicates that cyclin G1-infected samples are statistically distinct from vector controls, was determined using a p53, Hdm2, and ARF supports the notion that in vivo paired, two-tailed Student’s t test analysis. complexes exist between those proteins.

FIGURE 4. Cyclin G1 interacts with Mdm2, p53, and ARF in vitro and in vivo. A. Equal amounts of Sf9 lysates containing Mdm2, p53, and ARF were mixed with the indicated GST fusion proteins, and Western blots performed to measure binding. GST fusion proteins were detected by Ponceau S staining (bottom panel). B. GFP or GFP-G1 was co-expressed with Hdm2 or p53 in U2OS cells, and cell lysates were subjected to IP- Western blot analyses. Antibodies to GFP (G), p53 (P), and Hdm2 (H) were used in the IPs. C. U2OS cells expressing GFP or GFP- G1 with ARF, or cells transfected with ARF and Hdm2, were analyzed by IP-Western blotting, as described above. Antibodies to ARF (A) were used. D. Endogenous cyclin G1 complexes were examined in ARF- arrested NIH 3T3 cells treated with MG132 for 3 h. Direct lysates (50 Ag per lane) from vector (V )- and ARF (A)-infected cells, and immunoprecipitated complexes (from 700 Ag per IP) were analyzed by Western blotting. IPs were performed with the indicated antibodies, including pAb421 conjugated to Sepharose (ap53) and two different antibodies to cyclin G1 (aG1-sc, Santa Cruz Biotechnology, Santa Cruz, CA; aG1*, polyclonal 1133).

Cyclin G1 Stabilizes and Activates p53 that GFP-ARF and GFP-G1 activated p53 transcription, with an The mechanism by which cyclin G1 potentiated ARF- average 5- to 8-fold increase above background levels observed mediated growth inhibition appeared to be p53 independent. in GFP-expressing cells (Fig. 6B). Thus, cyclin G1 is a positive However, the G1-phase arrest induced by high-level expression regulator of p53. Because U2OS and NIH 3T3 cells lack the of cyclin G1 correlated with an ability to bind Mdm2 and p53. INK4a/ARF gene, these results also demonstrate that cyclin G1 We hypothesized that an association between cyclin G1 with is able to activate p53 in the absence of ARF. Mdm2 or p53 might block the ability of Mdm2 to negatively regulate p53, thereby fostering p53 activation. Consequently, Cyclin G1 Function Does Not Require p53 Yet Shows we measured the stability and transcriptional activity of p53 in Partial Dependence on pRb U2OS and NIH 3T3 cells expressing GFP-G1 (Fig. 6). GFP- To test whether growth arrest mediated by cyclin G1 positive cells were collected by cell sorting from populations required p53 activity, GFP or GFP-G1 was expressed in mouse transfected with GFP or GFP-G1 and analyzed by Western and human cells lacking p53 (Fig. 7). As shown previously, blotting for expression of p53, Mdm2, and the respective GFP GFP expression alone had minimal effects on the cell cycle proteins (Fig. 6A). In U2OS cells, GFP-ARF was included as a distributions of each cell line tested, whereas p53-positive NIH positive control, and as anticipated, it stabilized p53 and led to 3T3 and U2OS were efficiently arrested by cyclin G1 (Fig. 7A). up-regulation of Mdm2 compared to GFP controls. Notably, Cyclin G1 also exhibited growth suppressive activity in murine GFP-G1 also caused a 2- to 5-fold increase in p53 and Mdm2 10-1 cells, an immortalized derivative of Balb 3T3 fibroblasts levels, although the magnitude of up-regulation by cyclin G1 that lacks p53 (44). In contrast, cyclin G1 failed to block was consistently less than that achieved by ARF. A similar growth in p53-null Saos-2 osteosarcoma cells. Besides lacking result was observed in NIH 3T3 cells in which p53 and Mdm2 p53, Saos-2 cells carry a homozygous deletion of RB genes expression was enhanced by GFP-G1 compared to GFP- (45), whereas 10-1 cells are pRb-positive.2 positive and GFP-negative control cells (Fig. 6A, right panel). The above data suggested that growth inhibition by cyclin G1 The increased expression of p53 indicated that it was stabilized was p53 independent but might require pRb. To test that idea by cyclin G1, whereas the up-regulation of Mdm2 suggested more directly, we established isogenic derivatives of U2OS cells that p53 was activated. stably expressing the human papilloma viral proteins, E6 or E7, To directly measure the effects of cyclin G1 on p53 activity, U2OS and NIH 3T3 stable cell lines expressing a p53 luciferase reporter construct were generated and transiently transfected with GFP, GFP-ARF, or GFP-G1. Luciferase assays revealed 2C. Korgaonkar and D.E. Quelle, unpublished observations.

or empty vector. The E6 protein targets p53 for degradation, whereas E7 binds to and inactivates pRb (46–48). Cyclin G1 effectively arrested U2OS-vector and U2OS-E6 cells, support- ing the notion that p53 is not required for cyclin G1-mediated growth suppression (Fig. 7B). By comparison, cells expressing E7 were only partially arrested by cyclin G1, consistent with the data obtained in Saos-2 cells that suggested a role for pRb in cyclin G1-induced growth arrest. Identical results were obtained in BrdUrd incorporation assays using the same cell types transfected with either GFP or GFP-G1 (data not shown).

Mdm2 Targets Cyclin G1 for Degradation in the 26S Proteasome During the course of our studies, we found it difficult to achieve and maintain high levels of ectopically expressed cyclin G1. This was evident in both transfection and infection experiments in which exogenous cyclin G1 levels dramatically decreased from 30 to 72 h post-expression (data not shown). As such, we tested whether cyclin G1 was degraded by the 26S proteasome. NIH 3T3 cells were infected with retroviruses encoding vector or cyclin G1, and 2 days later, the populations were treated with the proteasome inhibitor, MG132, for increasing amounts of time. As shown in Fig. 8A, treatment of vector control cells with MG132 resulted in the stabilization of endogenous cyclin G1, p53, and Mdm2. As a target of p53, the up-regulation of endogenous cyclin G1 could be attributed to induction by stabilized p53 rather than inhibition of proteasome-mediated degradation. Therefore, we analyzed expression of HA- or GFP-tagged cyclin G1 constructs that lack p53 promoter sites. Both HA-cyclin G1 and GFP-G1 were markedly stabilized by MG132, indicating FIGURE 5. Cyclin G1 subnuclear distribution is distinctly altered by Hdm2 and ARF. U2OS cells were transfected with GFP or GFP-G1 that cyclin G1 is a target of the 26S proteasome (Fig. 8A and constructs, with or without ARF, ARF mutant D1-62, Hdm2, or p53. B). Interestingly, the stabilization of exogenous HA-cyclin G1 Immunofluorescence was used to determine the localization of GFP was maximal after 1 h treatment with MG132, and this and GFP-G1 proteins (green) versus ARF, Hdm2, and p53 (Texas Red). Relocalization of GFP-G1 by Hdm2 and ARF was quantified coincided with accelerated stabilization and up-regulation of from three independent experiments in which 100 cells or more were endogenous p53 and Mdm2 compared to vector control cells counted per condition. (Fig. 8A). Such data are consistent with the ability of cyclin G1 to stabilize and activate p53. However, the high levels of HA- cyclin G1 obtained in MG132-treated cells, which are Given that cyclin G1 interacts with Hdm2, an ubiquitin comparable to the levels of endogenous cyclin G1 induced by ligase that targets p53 for degradation via the proteasome, we ARF, did not stabilize p53 as well as ARF. This indicates that assayed whether degradation of cyclin G1 was mediated by additional events besides up-regulation of cyclin G1 contribute Hdm2. Equivalent amounts of either p53, GFP-G1, or GFP to ARF-mediated stabilization of p53. were co-expressed with increasing amounts of Hdm2 in U2OS cells, and the expression of each protein was assessed by Table 2. The COOH-Terminal ‘‘Box Repeat’’ Region of Cyclin Western blotting (Fig. 8C). As a control for Hdm2 activity and G1 Is Required for Efficient in Vitro Binding to ARF, Mdm2, to establish the validity of our assay, we showed that p53 and p53 expression was progressively reduced as Hdm2 expression was increased. Moreover, p53 was not destabilized by an Hdm2 Relative Binding mutant (Hdm2.Ala466 – 473) which is disrupted in the RING domain required for ubiquitin ligase activity (49). Identical Cyclin G1 Fusion Protein ARF p53 Mdm2 results were obtained with GFP-G1, whereas GFP stability was GST-G11–24 ÀÀ Ànot affected by Hdm2 expression. These results strongly GST-G11–57 ÀÀ Àsuggested that cyclin G1, like p53, is a target of Hdm2- 1 – 187 GST-G1 ++ + + mediated degradation. GST-G11 – 217 +++ ++++ ++++ GST-G11 – 294(wt) ++++ ++++ ++++ Discussion Note: Relative in vitro binding efficiencies of various GST-cyclin G1 fusion proteins for recombinant ARF, p53, and Mdm2 were determined from at least two A key finding of this work was that cyclin G1 over- experiments. expression caused a G1-phase growth arrest. Since its discovery

in 1994 as a transcriptional target of p53 (19), cyclin G1 has been described as either a positive or negative regulator of cell growth. For instance, stable overexpression of cyclin G1 was found to accelerate clonal expansion in primary fibroblasts (29), whereas transient overexpression of cyclin G1 in cell lines and primary hepatocytes induced apoptosis (36). Our finding that low levels of cyclin G1 lack growth inhibitory activity (and may even promote growth) while high levels suppress it may help to reconcile data suggesting opposite roles for cyclin G1 in growth control. One likely reason the growth inhibitory effects of cyclin G1 have not been widely appreciated is that its expression is markedly down-regulated by the proteasome in a

FIGURE 7. Cyclin G1-mediated growth arrest does not require p53 but exhibits partial pRb dependence. A. The relative percentage S phase for sorted cells expressing GFP (black bar) or GFP-G1 (gray bar) compared to GFP-negative cells within the same populations. Two independent experiments were performed for each cell type. B. U2OS cell lines expressing the human papilloma viral proteins, E6 or E7, or empty vector (Vec) were transfected with GFP (black bar) or GFP-G1 (gray bar). GFP- positive cells were collected by sorting and their DNA content measured by PI staining and flow cytometry. The mean percentage S phase for each sample is shown from two independent experiments.

potentially Mdm2-dependent manner. As such, it is difficult to achieve high levels of cyclin G1 except in transient assays. Also, studies that relied on stable overexpression would naturally exclude cells arrested by cyclin G1 (23, 29, 35). A particularly novel observation was that growth inhibition by cyclin G1 coincided with stabilization and activation of p53. As depicted in the model in Fig. 9, this shows that cyclin G1 can act in a positive regulatory feedback loop to activate p53. The molecular mechanisms underlying p53 activation by cyclin G1 are presently undefined, but we speculate that Mdm2 function is somehow blocked. This could result from cyclin G1 stoichiometrically limiting the binding between p53 and Mdm2, in keeping with the finding that both proteins associate with the same COOH-terminal region of cyclin G1. FIGURE 6. Cyclin G1 stabilizes and activates p53. A. U2OS and NIH However, cyclin G1 overexpression actually enhanced the 3T3 cells were transfected with GFP, GFP-G1, or GFP-ARF plasmids, and GFP-positive cells (as well as GFP-negative cells from NIH 3T3 experi- detection of Mdm2-p53 complexes in vivo (Fig. 4 and Ref. ments) were collected by sorting. Cell lysates were electrophoresed on 32), and binding studies revealed that p53 and cyclin G1 separate gels and immunoblots performed to determine expression of p53 interact with distinct regions of Mdm2.3 Thus, Mdm2 might and GFP (upper blots) or Mdm2 (lower blots), using Stat5 as the loading control for each membrane. B. U2OS and NIH 3T3 cells stably expressing a p53 luciferase reporter construct were similarly transfected, and the relative luciferase activity within GFP-G1- or GFP-ARF-expressing cells was calculated compared to GFP controls. Data are representative of three independent experiments. 3L. Zhao and D.E. Quelle, unpublished observations.

bridge the interaction between cyclin G1 and p53 in vivo.As serves a role in p53 signaling. Indeed, a transient surge in such, introduction of cyclin G1 into Mdm2-p53 complexes cyclin G1 expression following p53 stimulation might mimic may partially disrupt Mdm2 conformation and cause reduced the high levels achieved in our transfection assays and amplify p53 ubiquitination. p53 activity. Subsequent down-regulation of cyclin G1 by the It is also conceivable that cyclin G1 promotes p53 proteasome, presumably mediated by Mdm2, would then activation via its association with PP2A. That interaction reduce cyclin G1 expression and remove its contribution to causes the dephosphorylation of Mdm2 at S166 (32), an event p53 signaling. Consistent with such a temporal response, associated with the cytoplasmic relocalization of Mdm2 and others showed that cyclin G1 expression precedes Mdm2 up- consequent stabilization of p53 in the nucleus (50). A notable regulation in response to DNA damage, and over time, cyclin complication with that notion is that cyclin G1-PP2A G1 levels decrease while Mdm2 expression increases (51, 52). complexes also promote dephosphorylation of Mdm2 at The circumstance or cellular context in which cyclin G1 T216 (32). Dephosphorylation at that residue is thought to expression is induced may also determine its impact on p53- result in p53 degradation, not stabilization, because cells dependent events. There is evidence that cyclin G1 contributes lacking cyclin G1 express elevated p53 and hyperphosphory- to DNA damage-induced checkpoints and G2-M-phase arrest lated Mdm2 at T216. Those results led Okamoto et al. (32) to in primary fibroblasts and hepatocytes (36–38). suggest that cyclin G1 can negatively regulate p53, although On the other hand, several lines of evidence support our given the opposing effects of cyclin G1-PP2A on Mdm2 observation that cyclin G1 can function independent of phosphorylation, they also speculated that cyclin G1 could p53. First, cyclin G1 is expressed in cells and tissues activate p53. Unfortunately, the biological effects of cyclin G1 lacking p53 (21–25). Second, it can sensitize cells to on p53 activity or cell growth were not tested in that study. undergo apoptosis irrespective of p53 status (23), and its Because of its ability to activate p53, we were somewhat ability to potentiate ARF-induced growth arrest did not surprised that growth inhibition by cyclin G1 did not require correspond with greater stabilization or activation of p53. p53. Several cell types lacking functional p53 were efficiently Third, if the sole function of cyclin G1 was to regulate arrested by cyclin G1. Although those results suggested that p53, cyclin G1-null animals would be expected to either p53 is not important for cyclin G1-mediated growth arrest, lack p53 function and be predisposed to cancer or possibly die they do not rule out the alternative possibility that cyclin G1 during embryogenesis due to unchecked p53 activity. Neither

FIGURE 8. Cyclin G1 degradation by the 26S proteasome is promoted by Hdm2. A. NIH 3T3 cells were infected with retroviruses encoding vector (V )or HA-cyclin G1 (marked with an asterisk), and treated with DMSO at time 0 or 50 AM MG132 for the indicated times. ARF-infected cells (A) were left untreated. Cyclin G1, p53, Mdm2, and B23 (loading control) expression was examined by immunoblotting. B. GFP-G1-transfected NIH 3T3 cells were treated for 6 h with (+) or without (À)20AM MG132. Matched images using the same confocal settings are represented. Although not shown, GFP expression was not altered by MG132 treatment. C. U2OS cells were transfected with the indicated plasmids, and the expression of p53, Hdm2, GFP, GFP-G1, and Stat5 (loading control) was determined by Western blotting.

an essential role in controlling the G1-to-S phase transition (55). When hypophosphorylated, the pRb proteins sequester E2F transcription factors and block S-phase entry (56). Protein phosphatase 1 (PP1) and PP2A represent two different classes of serine/threonine phosphatases that have been implicated in a number of biological processes, including phosphorylation of the pRb proteins (57). Considerable evidence suggests that PP1 dephosphorylates pRb and activates pRb-dependent growth arrest (58–60), whereas PP2A preferentially targets p107 (61, 62). However, there is a possibility that PP2A can also act on pRb (63). Given its ability to associate with both PP2A and pRb, it is conceivable that cyclin G1 induces G1-phase arrest by promoting PP2A-mediated dephosphorylation of either pRb or p107. A potential role for PP1 still cannot be excluded, nor can the possibility that cyclin G1 binding to pRb directly blocks its FIGURE 9. Model depicting the functional relationship between cyclin phosphorylation by Cdks. At this point, additional studies are G1 with ARF, Mdm2, p53, and pRb. Cyclin G1 and Mdm2 are transcrip- warranted to define the molecular basis of the functional tional targets of p53; ARF activates p53 and induces their expression by antagonizing Mdm2, an ubiquitin ligase and negative regulator of p53. relationship between pRb and cyclin G1. Mdm2 may also down-regulate cyclin G1 activity because it enhances An important discovery during the course of these studies proteasomal degradation of cyclin G1. At high levels of expression, cyclin G1 inhibits cell growth and stimulates p53 activity. We hypothesize that was that cyclin G1 expression is limited by proteasomal cyclin G1 activates p53 by disrupting Mdm2 function (dashed line), degradation. Moreover, Mdm2 overexpression accelerated that possibly by altering its phosphorylation status via PP2A. Once activated by process, whereas a RING finger mutant of Mdm2 that lacks cyclin G1, p53 would be expected to induce expression of p21, an inhibitor of Cdks that blocks growth by suppressing pRb phosphorylation. ubiquitin ligase activity failed to promote cyclin G1 degrada- Consistent with that idea is our finding that cyclin G1 exhibits partial tion. Those findings are exciting because they represent the first dependence on pRb to suppress growth. However, our observation that demonstration of cyclin G1 regulation by the proteasome, and growth inhibition by cyclin G1 does not require p53 is inconsistent with that model. Consequently, a more direct functional link between cyclin G1 and they suggest that Mdm2 normally controls cyclin G1 expression pRb is likely, perhaps mediated through PP2A. Arrows, activating events; via ubiquitination. In fact, preliminary data from a variety of in perpendicular bars, inhibitory processes. vivo ubiquitination assays support that notion.4 The ability of Mdm2 to regulate both p53 and cyclin G1 is striking, particularly because cyclin G1 can activate p53. As such, this outcome was observed in mice lacking cyclin G1 (38). Indeed, work implies that Mdm2 can negatively regulate p53 function the lack of tumorigenesis or overt developmental defects in by promoting cyclin G1 degradation, in addition to its more cyclin G1-null mice suggests that it does not function as a tumor direct effects on p53. suppressor or essential regulator of growth. Rather, it may The significance of cyclin G1 relocalization to nucleoli in contribute to growth control in response to genotoxic stresses or cells overexpressing ARF is presently unclear. Binding studies at particular times in development, and interactions with other revealed relatively weak association between cyclin G1 and regulators, such as pRb and PP2A, may dictate its role. An ARF in vivo; therefore, the relocalization of cyclin G1 to important point when considering cyclin G1 function, however, nucleoli likely requires other factors. Although Hdm2 and p53 is the possibility that cyclin G2 may have redundant or showed more efficient interaction with cyclin G1 in vivo, both compensatory functions. That idea is bolstered by findings that remained largely or completely nucleoplasmic in cells express- cyclin G2 also inhibits cell growth (31), and it associates with ing cyclin G1 and ARF (data not shown), suggesting that they many of the same proteins, including p53, PP2A, Mdm2, and were not responsible for directing cyclin G1 to nucleoli. It ARF (data herein and Refs. 31, 32, and 52). remains to be determined whether the localization of cyclin G1 Our data showed that cyclin G1-mediated growth suppres- correlates with its effects on growth and ability to activate p53. sion was partially dependent on pRb. Cyclin G1 had no growth Various types of tumors, including osteosarcomas, and breast inhibitory activity in RB-null Saos-2 cells and only partial and prostrate cancers, express high levels of cyclin G1 (22). activity in U2OS cells expressing E7, an oncoprotein known to Because we found that high expression of cyclin G1 is growth cause the degradation and inactivation of pRb (46, 48, 53, 54). inhibitory, it is presumed that those tumor cells lack essential Earlier work showed that cyclin G1 can associate with pRb, and regulators of G1 function, such as pRb. It is also possible that that the effects of cyclin G1 on growth in RKO colon cancer mislocalization of cyclin G1 in cancer cells cancels its growth cells were lost on inactivation of pRb (29). While the authors of inhibitory effects. Others showed that cyclin G1 failed to cluster that study proposed that cyclin G1 promoted growth in a pRb- in discrete nuclear foci in response to DNA damage in dependent manner, we suggest that pRb may be required for transformed cells, but did so in normal breast epithelial cells, growth inhibition by cyclin G1. In fact, both ideas may be prompting them to postulate that ‘‘clustering’’ enabled cyclin correct. As mentioned earlier, the level of cyclin G1 expression G1 to act as a p53 effector (22). may determine its effects on growth. The noteworthy point of agreement is that pRb may be essential for cyclin G1 action. The pRb tumor suppressor protein and its closely related family members, p107 and p130, are phosphoproteins that play 4T. Samuels, S. Winckler, and M.C. Horne, unpublished observations.

This study was initiated to determine if cyclin G1 is a polybrene for 18 h, and selected in 0.6 mg/ml neomycin (G418) regulator within the ARF signaling pathways. The data suggest for 2–3 weeks. Expression of E7 was confirmed by Western that it is, because it can contribute to ARF-mediated cell cycle blotting (Zymed, San Francisco, CA), whereas E6 expression arrest and has intrinsic growth inhibitory activity. Notably, the was confirmed by reduced p53 expression (measured by ability of cyclin G1 to potentiate growth arrest by ARF did not Western blotting) and diminished ability of ARF to induce involve activation of p53, suggesting that cyclin G1 functions growth arrest (data not shown). as a downstream effector of p53 in the p21-independent pathway induced by ARF. We can conclude that cyclin G1 is DNA Constructs not a participant in the p53/Mdm2-independent pathway, Expression constructs containing HA-tagged ARF and its because it is not expressed in p53-null tko cells arrested by mutant, D1-62, in pcDNA3.1, pSRa-MSV-tk-neo, or pEGFP, ARF. At present, we have not addressed whether cyclin G1 is have been described elsewhere (6, 65). DNA constructs for required for ARF-induced arrest. That seems highly unlikely, GFP-tagged cyclin G1 were prepared by polymerase chain however, because ARF can inhibit growth in the absence of p21 reaction amplification of murine cyclin G1 cDNA (forward or p53, demonstrating that it has multiple mechanisms of action primer: 5V-GCGAAGCTTGGATCCACCATGGTA- (16–18). Rather, cyclin G1 may affect the magnitude or GAAGTACTGACAACTGACTCTC-3V and reverse primer: 5V- kinetics of growth suppression by ARF and p53. GAGCCCGGGAATTCTTACAAATGGTCTCAG- In conclusion, these studies provide some explanation for GAATCGTTGG-3V). A 950-bp BamHI-SmaI cyclin G1 cDNA how cyclin G1 can exhibit differential effects on cell growth. was subcloned into pEGFP-N1 (Clontech Laboratories, Inc., We propose that the levels and timing at which it is expressed Palo Alto, CA) at BglII-SmaI sites. Cyclin G1 cDNA was largely dictate its function, possibly by modulating the further amplified from pEGFP-cyclinG1 (forward primer: regulators with which it associates. In agreement with that 5V-GCGAAGCTTGGATCCACCATGGTAGAAGTACTGA- idea, cyclin G1 affected both the accumulation and degradation CAACTGACTCTC-3V and reverse primer: 5V-GCGGAATTCT- of p53 depending on whether it was in complexes with Mdm2/ CAACTCGAGGTCGACTGACAAATGGTCTCAG- ARF or Mdm2/PP2A, respectively (52). In that regard, GAATCGT-3V). Products were subcloned into pcDNA3.1, a interesting parallels can be drawn with another p53 target, pBlueScript vector containing an HA epitope, and HA-cyclin p21, because low levels of p21 facilitate the assembly of G1 was ligated into pSRa-MSV-tk-neo. GST expression growth-promoting cyclin D/Cdk4 complexes while high levels constructs for cyclin G1, cyclin G2, and PP2A/C have been are redistributed among the Cdks and consequently inhibit described (31). Mutants of cyclin G1 in the pGex vector were growth (15). Our current understanding of cyclin G1 suggests generated by deletion of internal fragments from full-length that its different roles in growth control correlate, at least in part, cyclin G1 and vector religation, including XbaI/XhoI (GST- with differential regulation of the ARF-Mdm2-p53 pathway. G11–24), BglII/XhoI (GST-G11–57), StuI/XhoI (GST-G11 – 187), and SnaBI/XhoI (GST-G11 – 217). Materials and Methods Cell Culture and Protein Expression Analyses for Growth Arrest NIH 3T3 fibroblasts and U2OS osteosarcoma cells (both The DNA content of GFP-positive and GFP-negative cells ARF-null, p53 and Mdm2 wild type) were grown in DMEM was determined by staining live cells with Hoescht dye 33342 containing 10% fetal bovine serum, 2 mM glutamine, and 100 (Sigma Chemical Co., St. Louis, MO), exactly as described Ag/ml of penicillin and streptomycin. Two p53-null cell lines, (31). Dead cells within the populations were identified by Saos-2 and 10-1 (44), and primary MEFs lacking p53, Mdm2, staining with 5 Ag/ml PI for 5 min at room temperature. Cells and ARF (kindly provided by Gerry Zambetti, St. Jude were sorted by an Epics 753 dual laser cytometer (Beckman Children’s Research Hospital) (18), were similarly maintained. Coulter Corporation, Miami, FL). PI-positive cells and doublets Narf6 cells (kindly provided by Gordon Peters, ICRF) were were excluded to ensure that only single viable cells were used treated with 1 Ag/ml IPTG for 2 days to induce ARF for analysis of DNA content and GFP expression. Alternatively, 5 expression. Cells were treated with 20–50 AM MG132 1.5 Â 10 GFP-positive and GFP-negative cells were sorted (Calbiochem, San Diego, CA) to inhibit the proteasome. from unstained populations, stained with PI, and analyzed for Retroviruses containing HA-tagged wild-type ARF or DNA content using a FACScan (Becton Dickinson, San Jose, murine cyclin G1 were produced and infected into NIH 3T3 CA) (6). For infected cells, DNA content was determined 48 h cells, as described (6). For sequential infections, cells were first post-infection by PI staining and FACScan analysis. Final cell infected with vector or cyclin G1 viruses for 6 h, followed by cycle distributions were determined using ModFit (Verity 4 ml of ARF retrovirus overnight. The next day, fresh medium Software House, Topsham, ME) or Watson Pragmatic (FlowJo, was added and cells were collected 24 h later. Plasmid DNAs Tree Star Inc., San Carlos, CA) software. Cell cycle progression were transfected by a modified calcium phosphate precipitation into S phase was also monitored by BrdUrd incorporation (14). method (64). To select stable lines expressing human papilloma virus E6 or E7 proteins, amphotropic viruses were first Protein Interaction Analyses collected from PA317 cells expressing either the empty pLXSN Cells were lysed (1 Â 107 cells/ml) for 1 h on ice in retroviral vector, pLXSN-E6 or pLXSN-E7 (kindly provided by NP40 buffer [50 mM Tris (pH 7.5), 120 mM NaCl, 1 mM Denise Galloway, Fred Hutchison Cancer Center). U20S cells EDTA, 0.5% NP40] supplemented with 0.1 mM sodium were then infected with 10 ml of each virus containing 8 Ag/ml vanadate, 1 mM sodium fluoride, 5 Ag/ml leupeptin, and 30 AM

phenylmethylsulfonyl flouride. Lysates were sonicated (1 Â 5s reagents. We also thank Jussara Hagen, Aruni S. Arachichige Don, and Brian Haugen for technical assistance. These studies were performed with assistance pulse) and clarified by centrifugation at 12,000 rpm for 10 min from the University of Iowa Flow Cytometry Facility, the Holden Comprehensive at 4jC. Equivalent amounts of protein were immunoprecipi- Cancer Center, and core facilities of the Diabetes and Endocrinology Research tated with protein A- or G-Sepharose at 4jC using antibodies to Center at the University of Iowa. mouse ARF (6), GFP (Abcam, Cambridge, UK), Mdm2 [2A10 or Ab-1 (Oncogene Research Products, Cambridge, MA)], p53 References [DO-1 (Santa Cruz Biotechnology) or pAb421 conjugated to 1. Hainaut, P., Soussi, T., Shomer, B., Hollstein, M., Greenblatt, M., Hovig, E., Harris, C. C., and Montesano, R. 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Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2003 American Association for Cancer Research. Cyclin G1 Has Growth Inhibitory Activity Linked to the ARF-Mdm2-p53 and pRb Tumor Suppressor Pathways 1 1 D.E.Q. from the American Cancer Society (RSG-98-254-04-MGO) and NIH (RO1 CA90367), and by a grant from the NIH to M.C.H. (RO1 GM56900).

Lili Zhao, Tina Samuels, Sarah Winckler, et al.

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