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Proc Natl Acad Sci U S A. Feb 27, 2007; 104(9): 3490–3495.
Published online Feb 20, 2007. doi:  10.1073/pnas.0610163104
PMCID: PMC1805544
Medical Sciences

c-Myc-mediated genomic instability proceeds via a megakaryocytic endomitosis pathway involving Gp1bα

Abstract

Genomic instability (GI) is essential for the initiation and evolution of many cancers and often precedes frank neoplastic conversion. Although GI can occur at several levels, the most conspicuous examples involve gains or losses of entire chromosomes (aneuploidy), the antecedent of which may be whole genome duplication (tetraploidy). Through largely undefined mechanisms, the c-Myc oncoprotein and its downstream target, MTMC1, promote tetraploidy and other forms of GI. In myeloid cells, c-Myc and MTMC1 also regulate a common, small subset of c-Myc target genes including GP1Bα, which encodes a subunit of the von Willebrand's factor receptor complex that mediates platelet adhesion and aggregation. Gp1bα also participates in megakaryocyte endomitosis, a form of controlled and precise whole-genome amplification. In this article, we show that both c-Myc and MTMC1 strongly up-regulate Gp1bα concurrent with their promotion of tetraploidy. shRNA-mediated inhibition of Gp1bα prevents tetraploidy by both c-Myc and MTMC1, whereas Gp1bα overexpression alone is sufficient to induce tetraploidy in established and primary cells. Once acquired, tetraploidy persists in most cases examined. Our results indicate that chromosome-level GI, induced by c-Myc overexpression, proceeds by means of the sequential up-regulation of MTMC1 and Gp1bα and further suggest that the pathways leading to megakaryocytic endomitosis and c-Myc-induced tetraploidy are mechanistically linked by their reliance on Gp1bα.

Keywords: aneuploidy, chromosomal instability, endoreduplication, MTMC1, tetraploidy

Genomic instability (GI) is critical for the development and evolution of most, if not all, naturally occurring cancers because it provides the means for a single cell to accumulate the multiple mutations needed to become transformed during an individual's lifetime (15). Ongoing GI also explains the acquisition of cancer-specific traits that tend to emerge later in the course of the disease, such as the ability to metastasize and to develop therapy resistance (15). A crucial role for GI is also supported by observations in humans and animal models showing that inherited defects in DNA repair, chronic inflammatory lesions that generate high local levels of genotoxic reactive oxygen species, and other DNA-damaging stresses are associated with an increased risk of cancer (59).

Although GI can occur at several levels, the most striking examples, and thus the first to be recognized, involve changes in whole chromosomal number, a condition termed aneuploidy (10). In the case of tumor cells, it has been proposed that aneuploidy is preceded by a doubling of the normal chromosome complement to produce transient tetraploidy (3, 11, 12), which eventually leads to other chromosomal anomalies, such as gains, losses, breaks, and translocations, with ultimate benefit to the emerging tumor cell population (1113). In contrast, tetraploidy and other polyploid states are common features of certain nontransformed cells (14). In the most well known example, bone marrow megakaryocytes, the precursors of blood platelets, undergo successive rounds of DNA replication (endomitosis), resulting in the generation of extremely large cells with a DNA content ranging from 8N to 128N (14). Lesser degrees of polyploidy are also observed in hepatocytes, myocytes, and vascular smooth muscle cells, particularly during aging (3, 14). What distinguishes these forms of “controlled” polyploidy from those associated with cancer and whether they share any mechanistic relationships remain largely unknown. Defining the means by which the induction, recognition, and stabilization of GI occur are clearly of importance because they could conceivably point the way to novel and rational means by which tumor cell initiation and progression could be controlled.

Deregulation of the c-Myc oncoprotein occurs frequently in human cancers (15) and can lead to GI at several levels (1619). Most notably, the oncoprotein promotes the acquisition of a tetraploid or pseudotetraploid state after mitotic spindle disruption or inactivation of the p53 tumor suppressor (16, 17). However, the precise pathways through which this occurs have not been clearly defined.

c-Myc functions as a general transcription factor in all proliferating cells and thus controls the expression of literally hundreds of target genes (20, 21). Among these, we have identified MTMC1 as an especially important direct positive target for c-Myc (22). Evidence for this includes the finding that MTMC1 overexpression can recapitulate multiple c-Myc functions, such as the ability to transform, promote apoptosis, alter morphology, inhibit hematopoietic differentiation, and promote tetraploidy (22). In most cases, these effects are c-Myc-independent, thus providing further evidence that MTMC1 functions downstream of c-Myc (23).

We have previously identified 47 MTMC1-regulated genes, many if not all of which are also c-Myc targets (24). Based on these findings, we have proposed that this small, overlapping gene subset plays a disproportionate role in promoting c-Myc phenotypes (24, 25). GP1bα, which encodes an integral subunit of the von Willebrand's factor receptor (26, 27), and whose promoter contains three c-Myc binding sites, is among the most highly up-regulated genes of this group (24). In addition to Gp1bα's established function in platelet adhesion, aggregation, and activation (26, 27), roles in megakaryocytic differentiation, apoptosis, morphogenesis, proliferation, and endomitosis have recently emerged (2830). Because c-Myc and MTMC1 overexpression can lead to tetraploidy (16, 17, 22), we have asked whether this might be a consequence of Gp1bα deregulation by these two oncoproteins. Indeed, the results presented here indicate that GpIbα is necessary to promote tetraploidy arising from c-Myc or MTMC1 overexpression, that it is sufficient for this process in cells which do not otherwise overexpress c-Myc or MTMC1, and that tetraploidy, once acquired, is maintained in the majority of cases. The additional finding that Gp1bα is more extensively expressed than previously appreciated raises the possibility that its influence on ploidy might be widespread. Taken together, our results establish a clear and heretofore unrecognized connection among c-Myc, MTMC1, and a pathway previously thought to be the exclusive domain of endomitotic megakaryocytes. Furthermore, they define a potential mechanism by which deregulated c-Myc can perturb chromosome number and lead to GI.

Results

Gp1bα Is Necessary for the Promotion of Tetraploidy by c-Myc and MT-MC1.

In keeping with previous transcriptional profiling results (24), Fig. 1 A and B confirms that Gp1bα mRNA and protein are induced in murine myeloid cells that overexpress c-Myc or its direct downstream target gene MTMC1. To determine the functional consequences of this up-regulation, a short hairpin RNA (shRNA)-based approach was used to stably suppress endogenous Gp1bα protein in these and control cells by >90% (Fig. 1C). Each cell line was then again assessed for its ability to become tetraploid or pseudotetraploid after exposure to the mitotic spindle poison colcemid (17, 22). As previously demonstrated (16, 17, 22), control cells completely arrested in G2/M (4N DNA content) after this treatment whereas the majority of 32D-c-Myc and 32D-MTMT1 cells proceeded through S-phase, without an intervening mitosis, and became tetraploid (Fig. 2). In contrast, tetraploid populations were absent in 32D-c-Myc or 32D-MTMC1 cells after knockdown of Gp1bα.

Fig. 1.
Gp1bα transcripts and protein in murine myeloid cells. 32D murine myeloid cells stably expressing c-Myc (32D-c-Myc) or MTMC1 (32D-MTMC1) or control cells (32D-neo) have been described (17, 22, 24). (A) Real-time qRT-PCRs were performed by using ...
Fig. 2.
Cell cycle analyses of myeloid cell lines. Isolated nuclei of log-phase or colcemid-treated cell lines from Fig. 1C were stained with propidium iodide and subjected to cell cycle analysis by flow cytometry (17, 22). (A–C) Control cell lines expressing ...

To determine whether the observed loss of tetraploidy in c-Myc- and MTMC1-overexpressing cells was a specific consequence of Gp1bα knockdown, each of the above cell lines was engineered to express human Gp1α. Because the human Gp1bα transcript is mismatched to the shRNA sequences in the above murine cells, human Gp1bα protein expression was readily achieved (Fig. 3A). Under these circumstances, the cells were once again able to become tetraploid after colcemid-mediated mitotic arrest (Fig. 3 B–D). We conclude that the initial loss of c-Myc- and MTMC1-mediated tetraploidy was specifically attributable to the down-regulation of endogenous Gp1bα.

Fig. 3.
Overexpression of human Gp1bα restores the ability of c-Myc and MTMC1 to induce tetraploidy in Gp1bα knockdown cells. (A) The Gp1bα shRNA cell lines shown in the first three lanes of Fig. 1C were stably transfected with a linearized ...

Gp1bα Overexpression Is Sufficient for the Induction of Tetrapoidy in Multiple Cell Types.

The foregoing studies established that Gp1bα is necessary for the induction of tetraploidy in 32D-c-Myc and 32D-MTMC1 cells and sufficient for the induction of tetraploidy in the parental cells, which do not normally overexpress either protein. To determine whether it is also sufficient in other cell types, epitope-tagged Gp1bα was overexpressed through retroviral transduction in several immortalized or primary cell types in which c-Myc is regulated normally and MTMC1 expression is either low or undetectable [Fig. 4A and supporting information (SI) Fig. 6)]. As seen in Fig. 4B, colcemid treatment of control cells resulted in the expected, albeit variable, G2/M arrest in all cases. In contrast, similar treatment of all Gp1bα-overexpressing cells developed tetraploidy to varying degrees. Although primary human foreskin fibroblasts (HFFs) also became tetraploid after colcemid treatment, the extent to which this occurred was less than that seen in any of the established cell lines, thus suggesting that the genomes of primary cells are less susceptible to the overexpression of Gp1bα.

Fig. 4.
Enforced expression of Gp1bα is sufficient for the induction of tetraploidy in various cell types. Stable expression of Gp1bα in the indicated immortalized or primary cells was accomplished through transduction with a bicistronic LXSN ...

Two exceptions to the absolute reliance on colcemid were seen in the cases of NIH 3T3 murine and BJ-T/T/R human fibroblasts (31), both of which spontaneously developed tetraploidy after transduction. Repeat immunoblotting with an anti-Gp1bα mAb showed that, in all cell lines, the levels of ectopically expressed protein were only 2- to 3-fold higher than endogenous levels (SI Fig. 7), thus indicating that tetraploidy induction does not require excessive Gp1bα expression. Cell-surface localization of Gp1bα and/or its proper glycosylation does, however, appear to be important, because tetraploidy was abrogated when colcemid-treated cells were concurrently exposed to brefeldin A, a potent inhibitor of protein secretion at the level of the endoplasmic reticulum and Golgi complex (SI Fig. 8). Finally, we have demonstrated that tetraploidy persists subsequent to the removal of colcemid in immortalized cells but disappears within several days in primary cells (Fig. 5). We have also shown that tetraploids are larger than their control counterparts and contain approximately twice as many chromosomes (SI Fig. 9).

Fig. 5.
Persistence of tetraploidy after removal of colecemid. The indicated Gp1bα-transduced diploid cell lines in log-phase growth (A, D, and G) were exposed to colcemid for 16–27 h as described in the legends to Figs. 11 ...

It has been proposed that the promotion of tetraploidy by deregulated c-Myc is a consequence of its ability to drive unscheduled DNA synthesis without an intervening mitosis (16, 17). To determine whether GpIbα-mediated tetraploidy operates through a mechanism that relies on endogenous c-Myc, we expressed GpIbα in c-Myc-null fibroblasts (32) (SI Fig. 6). Because of the extremely slow growth rate of these cells, it was quite difficult to achieve a complete G2/M arrest, even when colcemid exposure times were extended to 4 d. Nonetheless, a significant fraction of the cells were rendered tetraploid after this treatment. From these results, we conclude that Gp1bα-mediated induction of tetraploidy is at least partially c-Myc-independent.

Widespread Expression of Gp1bα.

The expression of Gp1bα by 32D myeloid cells is perhaps not surprising, given that they share a common lineage with bone marrow megakaryocytes (27, 29). GpIbα has also been detected in normal endothelial cells, dermal dendrocytes, MCF7 breast cancer cells, and primary breast cancers but not in normal mammary tissue (3336). To explore the possibility that ploidy might normally be under the control of Gp1bα in other cell types, we examined a variety of tissues and cell lines for the expression of Gp1bα mRNA and protein. As shown in SI Fig. 10, both transcripts and protein for Gp1bα were identified at varying levels in virtually all of the tested specimens. Thus, we conclude that Gp1bα is more widely expressed than was previously assumed.

Discussion

Chromosomal instability and other forms of GI occur in the vast majority of human cancers and provide an explanation for the rapid and ongoing evolution of tumor phenotypes, such as the ability to metastasize and develop resistance to various therapeutic modalities (13, 5, 7). Indeed, considerable evidence favors the idea that GI is an early or even inciting event that permits the subsequent stable accumulation of an otherwise unattainable number of mutations required for the development of a fully transformed cell during an organism's life span (1, 2, 7). Consistent with this idea is the presence of aneuploidy and/or allelic imbalances in a variety of preneoplastic lesions such as cervical dysplasia, ulcerative colitis, and Barrett's esophagus (3741). In a more recent study, mammary epithelial cells derived from p53−/− mice, and subsequently rendered tetraploid by treatment with a mitotic spindle poison, were shown to be tumorigenic after exposure to a chemical carcinogen and implantation into nude mice (42). Such findings support the idea that tetraploidy, and concurrent centrosome duplication, lead to bipolar mitoses and subsequent abnormal chromosome partitioning and/or breakage (3, 11, 12, 42, 43). During subsequent in vivo growth, various subpopulations of such genetically unstable cells would be selected as a consequence of their having acquired the characteristics needed to allow their sustained survival and expansion (1, 2, 7, 11, 12, 44).

The findings reported here and elsewhere have identified a previously unrecognized pathway by which both c-Myc and its downstream target, MTMC1 can promote GI. This work was predicated on earlier observations that MTMC1 possesses the unique ability to recapitulate multiple c-Myc phenotypes, including the induction of tetraploidy (2224). Unlike the case of c-Myc, however, whose tetraploid-promoting properties require that cells first be exposed to a mitotic spindle poison, or that p53 be rendered inactive (16, 17), the induction of tetraploidy by MTMC1 is independent of such interventions. Instead, it arises spontaneously and in a stochastic manner in longer-term cultures of MTMC1-overexpressing cells (22, 23). Previous gene expression profiling had indicated that MTMC1 deregulates a small subset of c-Myc targets, thus suggesting that these may represent a “privileged” group with a disproportionate role in mediating c-Myc phenotypes (24, 25). Because Gp1bα is one of the most highly up-regulated targets for both of these oncoproteins, and because it has previously been shown to regulate the DNA content of megakaryocytes (30), we reasoned that it might be involved in c-Myc and MTMC1-mediated tetraploidy induction. Indeed, the results presented here indicate that Gp1bα is both necessary and sufficient for this property.

Three distinct cellular outcomes were observed in response to Gp1bα overexpression (Figs. 3 and and44 and SI Fig. 6). The first involved the acquisition of a tetraploid genome arising only as a consequence of prior exposure to colcemid. This response was seen in the majority of established cell lines examined and remained a stable property for at least 2 wk upon resumption of logarithmic growth after drug removal. Similar behavior has been previously observed in established cell lines that overexpress c-Myc (16, 17). In the second case, seen in primary human foreskin fibroblasts (HFFs), a small tetraploid population arose only after colcemid treatment and was quickly supplanted by a diploid population upon removal of the drug. Finally, in the third case, which included two cell lines (NIH 3T3 and BJ-T/T/R), stable tetraploidy arose rapidly and in the absence of prior mitotic blockage.

Consideration of the latter two groups may provide some insights into the requirements for and constraints on Gp1bα-mediated GI. Thus, in contrast to the situation with virtually all established cell lines, primary HFFs possess intact p53 and pRb tumor-suppressor pathways, both of which have been implicated in a G1 checkpoint that denies cells with a tetraploid genome the opportunity for continued proliferation (11, 12, 4548). In the studies reported here, only a small fraction of HFFs underwent an additional aberrant S-phase and developed tetraploidy, and those that did were quickly eliminated upon the removal of colcemid, most likely through p53-dependent proliferative arrest and/or apoptosis. In contrast, both p53 and Rb proteins have been inactivated in BJ-T/T/R-immortalized human primary fibroblasts as a result of their expressing SV40-T antigen (31). Such cells might be expected to be more susceptible to the acquisition of tetraploidy under the influence of Gp1bα overexpression, given the loss of these two major checkpoints.

That Gp1bα-mediated GI, once acquired, remains a stable property for at least 2 wk (Fig. 5) is consistent with previous findings that constitutive c-Myc expression is necessary for both the induction and maintenance of aneuploidy in Rat1a fibroblasts (16, 17, 49). Whether GI can be maintained for longer periods, and whether it requires continuous Gp1bα expression, await confirmation. Given the heterogeneity of the responses we have thus far observed, however, it seems likely that the answers to these questions will depend on the intactness of the G2/M and other checkpoints and the degree to which they are incapacitated in different cell types.

Gp1bα has previously been viewed as playing an important role in the adhesion, aggregation, and activation of platelets by virtue of its comprising a major subunit of the von Willebrand's factor receptor (26, 27). However, more recent studies demonstrating that the 96-aa-long intracytoplasmic domain of GpIbα interacts with and/or regulates important oncogenic signaling molecules such as PI3 kinase, c-src, Akt1, and 14-3-3ζ, are also consistent with a role in mediating chromosomal instability or in relaying potentially oncogenic signals (30, 5052). It will clearly be important in future work to delineate more precisely how the Gp1bα-mediated control of normal megakaryocytic endomitosis differs from that governing the abnormal tetraploidy associated with deregulated c-Myc and MTMC1.

Materials and Methods

Cell Culture.

Murine 32D myeloid cell lines expressing c-Myc and MTMC1 and conditions for their growth have been described (17, 22). These were maintained in RPMI medium 1640 supplemented with 10% FCS (Invitrogen, Carlsbad, CA) and 1 ng/ml recombinant murine interleukin-3 (IL-3; R & D Systems, Minneapolis, MN). All other cell lines were routinely maintained in Dulbecco's MEM containing 10% FCS, 2 mM glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. These included Rat1a fibroblasts; NIH 3T3 fibroblasts; C2C12 myoblasts; C3H10T1/2 fibroblasts; primary human BJ fibroblasts immortalized by SV40 T-antigen, telomerase, and a Ras oncogene (BJ-T/T/R cells, ref. 31); and primary HFF cells. The latter were obtained from the American Type Culture Collection (Manassas, VA).

Cell Cycle Assays.

Cell cycle analyses were performed on propidium iodide-stained nuclei by using a FACSstar flow cytometer (Becton-Dickinson Biosciences, San Jose, CA). Data were analyzed by single histogram statistics as described (17, 22). G2/M arrest was achieved by incubating cells in the presence of colcemid (25–35 ng/ml, Sigma–Aldrich, St. Louis, MO) for 16–27 h except in the case of mycKO cells, where the incubation was extended to 96 h.

Recombinant DNA Methods.

The optimal shRNA sequence for murine Gp1bα was determined by using Dharmacon software (Dharmacon, Lafayette, CO) and corresponded to the region encoding nucleotides 674–692 of murine Gp1bα cDNA (GenBank accession no. NM_010326). The double-stranded oligonucleotide, with overhanging BamHI and HindIII sites (IDT, Coralville, IA), was cloned into the pSilencer-H1-hygro vector (Ambion, Austin, TX). The sequence and orientation of the insert were confirmed by automated DNA sequencing. An expression vector for human Gp1bα in the pcDNA-Zeo vector (Invitrogen) was a kind gift from Michael Kroll (Baylor College of Medicine, Houston, TX). Stable transfections were performed by electroporation of linearized plasmid DNA, followed by selection and pooling of Zeocin-resistant clones. For some experiments, we used a previously described bicistronic LXSN-EYFP retroviral vector (23). The human Gp1bα coding sequence, minus the termination codon, was amplified with forward and reverse primers containing XhoI and SalI sites, respectively. After XhoI and SalI digestion, the fragment was cloned into the above vector so as to be in-frame with a c-Myc epitope tag in the 3′ end of the polylinker site. Retroviral vector transfections and packaging in amphotropic Phoenix-A cells, and subsequent infections, were performed as described (23).

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Michael Kroll (Baylor College of Medicine, Houston, TX) and John Sedivy (Brown University, Providence, RI) for plasmids, Bob Lakomy for cell sorting, Bill Hahn (Dana–Farber Cancer Institute Boston, MA), and Yatin Vyas (Children's Hospital of Pittsburgh) for cell lines, and Michael Long for helpful comments on the manuscript. This work was supported by National Institutes of Health Grant R01 CA078259 (to E.V.P.).

Abbreviations

GI
genomic instability
Gp1bα
glycoprotein 1b α
HFF
human foreskin fibroblasts.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610163104/DC1.

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