O-Fucose and Fringe-modified NOTCH1 extracellular domain fragments as decoys to release niche-lodged hematopoietic progenitor cells.

Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor-ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch-ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor-ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11-13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11-13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11-13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11-13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11-13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.

Using Apache Spark on genome assembly for scalable overlap-graph reduction.

BACKGROUND: De novo genome assembly is a technique that builds the genome of a specimen using overlaps of genomic fragments without additional work with reference sequence. Sequence fragments (called reads) are assembled as contigs and scaffolds by the overlaps. The quality of the de novo assembly depends on the length and continuity of the assembly. To enable faster and more accurate assembly of species, existing sequencing techniques have been proposed, for example, high-throughput next-generation sequencing and long-reads-producing third-generation sequencing. However, these techniques require a large amounts of computer memory when very huge-size overlap graphs are resolved. Also, it is challenging for parallel computation. RESULTS: To address the limitations, we propose an innovative algorithmic approach, called Scalable Overlap-graph Reduction Algorithms (SORA). SORA is an algorithm package that performs string graph reduction algorithms by Apache Spark. The SORA's implementations are designed to execute de novo genome assembly on either a single machine or a distributed computing platform. SORA efficiently compacts the number of edges on enormous graphing paths by adapting scalable features of graph processing libraries provided by Apache Spark, GraphX and GraphFrames. CONCLUSIONS: We shared the algorithms and the experimental results at our project website, https://github.com/BioHPC/SORA . We evaluated SORA with the human genome samples. First, it processed a nearly one billion edge graph on a distributed cloud cluster. Second, it processed mid-to-small size graphs on a single workstation within a short time frame. Overall, SORA achieved the linear-scaling simulations for the increased computing instances.

The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-ß1-induced growth inhibition by targeting Smad3 linker region.

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-ß1 (TGF-ß1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-ß1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-ß1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-ß1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-ß1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

Amyloid-ß precursor protein promotes cell proliferation and motility of advanced breast cancer.

BACKGROUND: Amyloid-ß precursor protein (APP) is a highly conserved single transmembrane protein that has been linked to Alzheimer disease. Recently, the increased expression of APP in multiple types of cancers has been reported where it has significant correlation with the cancer cell proliferation. However, the function of APP in the pathogenesis of breast cancer has not previously been determined. In this study, we studied the pathological role of APP in breast cancer and revealed its potential mechanism. METHODS: The expression level of APP in multiple breast cancer cell lines was measured by Western blot analysis and the breast cancer tissue microarray was utilized to analyze the expression pattern of APP in human patient specimens. To interrogate the functional role of APP in cell growth and apoptosis, the effect of APP knockdown in MDA-MB-231 cells were analyzed. Specifically, multiple signal transduction pathways and functional alterations linked to cell survival and motility were examined in in vivo animal model as well as in vitro cell culture with the manipulation of APP expression. RESULTS: We found that the expression of APP is increased in mouse and human breast cancer cell lines, especially in the cell line possessing higher metastatic potential. Moreover, the analysis of human breast cancer tissues revealed a significant correlation between the level of APP and tumor development. Knockdown of APP (APP-kd) in breast cancer cells caused the retardation of cell growth in vitro and in vivo, with both the induction of p27(kip1) and caspase-3-mediated apoptosis. APP-kd cells also had higher sensitivity to treatment of chemotherapeutic agents, TRAIL and 5-FU. Such anti-tumorigenic effects shown in the APP-kd cells partially came from reduced pro-survival AKT activation in response to IGF-1, leading to activation of key signaling regulators for cell growth, survival, and pro-apoptotic events such as GSK3-ß and FOXO1. Notably, knock-down of APP in metastatic breast cancer cells limited cell migration and invasion ability upon stimulation of IGF-1. CONCLUSION: The present data strongly suggest that the increase of APP expression is causally linked to tumorigenicity as well as invasion of aggressive breast cancer and, therefore, the targeting of APP may be an effective therapy for breast cancer.

TRAF6 mediates IL-1ß/LPS-induced suppression of TGF-ß signaling through its interaction with the type III TGF-ß receptor.

Transforming growth factor-ß1 (TGF-ß1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1ß must overcome the anti-inflammatory effects of TGF-ß to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1ß or Lipopolysaccharide (LPS) suppresses TGF-ß-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1ß signaling, mediates this suppressive effect through interaction with the type III TGF-ß receptor (TßRIII), which is TGF-ß-dependent and requires type I TGF-ß receptor (TßRI) kinase activity. TßRI phosphorylates TßRIII at residue S829, which promotes the TRAF6/TßRIII interaction and consequent sequestration of TßRIII from the TßRII/TßRI complex. Our data indicate that IL-1ß enhances the pro-inflammatory response by suppressing TGF-ß signaling through TRAF6-mediated sequestration of TßRIII, which may be an important contributor to the early stages of tumor progression.

Smad7 binds to the adaptors TAB2 and TAB3 to block recruitment of the kinase TAK1 to the adaptor TRAF2.

Transforming growth factor-beta1 (TGF-beta1) regulates inflammation and can inhibit activation of the transcription factor NF-kappaB in certain cell types. Here we show that the TGF-beta-induced signaling protein Smad7 bound to TAB2 and TAB3, which are adaptors that link the kinase TAK1 to 'upstream' regulators in the proinflammatory tumor necrosis factor (TNF) signaling pathway. Smad7 thereby promoted TGF-beta-mediated anti-inflammatory effects. The formation of Smad7-TAB2 and Smad7-TAB3 complexes resulted in the suppression of TNF signaling through the adaptors TRAF2, TAB2 and/or TAB3, and TAK1. Furthermore, expression of a transgene encoding Smad7 in mouse skin suppressed inflammation and NF-kappaB nuclear translocation substantially and disrupted the formation of endogenous TRAF2-TAK1-TAB2 and TRAF2-TAK1-TAB3 complexes. Thus, Smad7 is a critical mediator of TGF-beta signals that block proinflammatory TNF signals.

Smad6 negatively regulates interleukin 1-receptor-Toll-like receptor signaling through direct interaction with the adaptor Pellino-1.

Transforming growth factor-beta1 (TGF-beta1) is a potent cytokine with pleiotropic effects, including anti-inflammatory activity. Here we show that the signaling protein Smad6 bound to Pellino-1, an adaptor protein of mammalian interleukin 1 receptor (IL-1R)-associated kinase 1 (IRAK1), and thereby promoted TGF-beta-mediated anti-inflammatory effects. Smad6-Pellino-1 interaction abrogated signaling mediated by a complex of IRAK1, Pellino-1 and adaptor protein TRAF6 that formed after stimulation by IL-1beta treatment. Blockade of IRAK1-Pellino-1-TRAF6 signaling prevented degradation of the inhibitor IkappaBalpha and subsequent nuclear translocation of transcription factor NF-kappaB and thus expression of proinflammatory genes. 'Knockdown' of endogenous Smad6 expression by RNA interference reduced anti-inflammatory activity mediated by TGF-beta1 or the TGF-beta family member BMP-4. Thus Smad6 is a critical mediator of the TGF-beta-BMP pathway that mediates anti-inflammatory activity and negatively regulates IL-1R-Toll-like receptor signals.

The survival kinase Mirk/dyrk1B is activated through Rac1-MKK3 signaling.

The serine/threonine kinase Mirk/dyrk1B is activated in several solid tumors where it mediates cell survival, but the mechanism by which Mirk is activated in tumors is unknown. We now demonstrate that Mirk is activated as a kinase by signaling from Rac1 to the mitogen-activated protein kinase kinase MKK3. Rac is a Ras superfamily GTPase that, when activated, functions downstream of Ras oncoproteins to promote cell survival, transformation, and membrane ruffling. The constitutively active mutant Rac1QL activated Mirk in several cell types through MKK3, which in turn activated Mirk by phosphorylation. Dominant negative Rac1, dominant negative MKK3, and knockdown of MKK3 by RNA interference inhibited the kinase activity of co-expressed Mirk. E-cadherin ligation in confluent Madin-Darby canine kidney (MDCK) epithelial cells is known to transiently activate Rac1. Mirk was activated by endogenous Rac1 following E-cadherin ligation in confluent MDCK epithelial cells, whereas treatment of confluent MDCK cells with an Rac1 inhibitor decreased Mirk activity. Disruption of cadherin ligation by EGTA or prevention of cadherin ligation by maintenance of cells at subconfluent density blocked activation of Mirk. Engagement of cadherin molecules on subconfluent cells by an E-cadherin/Fc chimeric molecule transiently activated both Rac1 and Mirk with a similar time course. Rac activity is up-regulated in many human tumors and mediates survival signals, which enable tumor cells to evade apoptosis. This study characterizes a new anti-apoptotic signaling pathway that connects Rac1 with a novel downstream effector, Mirk kinase, which has recently been demonstrated to mediate survival in human tumors.

Up-regulation of defense enzymes is responsible for low reactive oxygen species in malignant prostate cancer cells.

Reactive oxygen species (ROS) are involved in a diversity of important phenomena in the process of tumor development. To investigate the alterations of oxidative stress and their related systems in tumor progression, a variety of components in the antioxidative stress defense system were examined in prostate cancer cell lines, PC3 and LNCaP. Cell surface molecules involved in metastasis were expressed highly in PC3 cells compared with LNCaP cells, and strong invasion ability was shown in PC3 cells only. ROS level in LNCaP cells was twice higher than that in PC3 cells, although nitric oxide (NO) level was similar between the two cell lines. The content of GSH increased up to about 2-fold in PC3 compared with LNCaP. Activities of glutathione reductase, thioredoxin reductase, and glutathione S-transferase except catalase are significantly higher in PC3 cells than in LNCaP cells. Furthermore, oxidative stress-inducing agents caused down-regulation of GSH and glutathione S-transferase much more significantly in LNCaP cells than in PC3 cells. These results imply that malignant tumor cells may maintain low ROS content by preserving relatively high anti-oxidative capacity, even in the presence of stressful agents.

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway.

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.

Mirk/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts.

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21(Cip1). Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.

Serine/threonine kinase Mirk/Dyrk1B is an inhibitor of epithelial cell migration and is negatively regulated by the Met adaptor Ran-binding protein M.

Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas. In the current study, the Met adaptor protein Ran-binding protein M (RanBPM) was identified as a Mirk-binding protein by yeast two-hybrid analysis. The Mirk-RanBPM association was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation studies, and in vivo cross-linking. Met plays an important role in tumor cell invasion and cell migration. RanBPM has been reported to bind to the tyrosine kinase domain of the hepatocyte growth factor (HGF) receptor Met, enhance Met downstream signaling, and enhance HGF-induced A704 kidney carcinoma cell invasion (Wang, D., Li, Z., Messing, E. M., and Wu, G. (2002) J. Biol. Chem. 277, 36216-36222). We made a stable Mirk-inducible subline from nontransformed Mv1Lu lung epithelial cells and now demonstrate that induction of Mirk inhibited the migration of these cells in wounding experiments and inhibited their invasion through polycarbonate Transwell filters. Furthermore the ability of Mirk to inhibit Mv1Lu cell migration was attenuated when cells were exposed to HGF or to elevated levels of transiently expressed RanBPM. RanBPM inhibited the kinase activity of Mirk/Dyrk1B and Dyrk1A. In addition, RanBPM and HGF inhibited the function of Mirk as a transcriptional coactivator. Our findings suggest that Mirk plays a role in modulating cell migration through opposing the action of the Met signaling cascade adaptor protein RanBPM.

Rapid turnover of cell-cycle regulators found in Mirk/dyrk1B transfectants.

Mirk/dyrk1B is an arginine-directed protein kinase, which functions as a transcriptional activator and mediates serum-free growth of colon carcinoma cells by an unknown mechanism. We now report that turnover of the cdk inhibitor p27(kip1) and the G(1)-phase cyclin cyclin D1 is enhanced in each of 4 Mirk stable transfectants compared to vector control transfectants and Mirk kinase-inactive mutant transfectants. This enhanced turnover is proteasome-dependent and leads to lower protein levels of both p27(kip1) and cyclin D1. Lower protein levels of the cdk inhibitor p21(cip1) were also observed in the 4 Mirk stable transfectants. Mirk did not alter the activity of a p27(kip1) promoter construct or p27(kip1) mRNA levels by stable expression, indicating that the decrease in p27(kip1) protein levels was due to a posttranscriptional mechanism. These data are consistent with mirk enhancing the expression of some component common to the proteolysis of both p27(kip1) and cyclin D1.

The transcriptional activator Mirk/Dyrk1B is sequestered by p38alpha/beta MAP kinase.

Mirk/Dyrk1B protein kinase was shown in an earlier study to function as a transcriptional activator of HNF1alpha, which Mirk phosphorylates at Ser(249) within its CREB (cAMP-response element-binding protein)-binding protein (CBP) binding domain (). The MAPK kinase MKK3 was also shown to activate Mirk as a protein kinase, implicating Mirk in the biological response to certain stress agents. Another MKK3 substrate, p38MAPK, is now shown to inhibit the function of Mirk as a transcriptional activator in a kinase-independent manner. Co-immunoprecipitation experiments demonstrated that kinase-inactive p38AF, as well as wild-type p38, sequestered Mirk and prevented its association with MKK3. Only the p38alpha and p38beta isoforms, but not the gamma or delta isoforms, complexed with Mirk. p38alphaMAPK blocked Mirk activation of HNF1alpha in a dose-dependent manner, with high levels of kinase-inactive p38alphaAF completely suppressing the activity of Mirk. Size fractionation by fast protein liquid chromatography on Superdex 200 demonstrated that Mirk is not found as a monomer in vivo, but is found within 150-700 kDa subnuclear complexes, which co-migrate with the nuclear body scaffolding protein PML. Endogenous Mirk, p38, and MKK3 co-migrate within 500-700-kDa protein complexes, which accumulate when nuclear export is blocked by leptomycin B. Stable overexpression of Mirk increases the fraction of Mirk protein and p38 protein within these 500-700 kDa complexes, suggesting that the complexes act as nuclear depots for Mirk and p38. Sequestration of Mirk by p38 may occur within these subnuclear complexes. Synchronization experiments demonstrated that Mirk levels fluctuate about 10-fold within the cell cycle, while p38 levels do not, leading to the speculation that endogenous p38 could only block Mirk function when Mirk levels were low in S phase and not when Mirk levels were elevated in G(0)/G(1). These data suggest a novel cell cycle-dependent function for p38, suppression of the function of Mirk as a transcriptional activator only when cells are proliferating, and thus limiting Mirk function to growth-arrested cells.

Mirk protein kinase is activated by MKK3 and functions as a transcriptional activator of HNF1alpha.

Mirk/Dyrk1B is an arginine-directed serine/threonine protein kinase that is expressed at low levels in most normal tissues but at elevated levels in many tumor cell lines and in normal skeletal muscle. Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium, but the mechanism of Mirk action is unknown. DCoHm (dimerization cofactor of hepatocyte nuclear factor 1alpha ( HNF1alpha) from muscle), a novel gene of the DCoH family with 78% amino acid identity to DCoH, was identified as a Mirk-binding protein by yeast two-hybrid analysis and cloned. Mirk co-immunoprecipitated with DCoHm and bound to DCoHm in glutathione S-transferase pull-down assays. DCoH stabilizes HNF1alpha as a dimer and enhances its transcriptional activity on the beta-fibrinogen promoter reporter, and DCoHm had similar activity. Mirk enhanced HNF1alpha transcriptional activity in a dose-dependent manner, whereas two kinase-inactive Mirk mutants and a Mirk N-terminal deletion mutant did not. Mirk, DCoHm, and HNF1alpha formed a complex. Mirk bound to a specific region within the CREB-binding protein-binding region of HNF1alpha and phosphorylated HNF1alpha at a site adjacent to the Mirk-binding region. Conversely, the HNF1alpha binding domain was located within the first five conserved kinase subdomains of Mirk. Mirk co-immunoprecipitated with the MAPK kinase MKK3, an upstream activator of p38. MKK3 enhanced Mirk kinase activity and the transcriptional activation of HNF1alpha by Mirk, suggesting that Mirk, like p38, is activated by certain environmental stress agents. The Mirk-binding protein DCoH has been shown to be selectively expressed in colon carcinomas but not in normal tissue. Mirk may function as an HNF1alpha transcriptional activator in response to an MKK3-mediated stress signal, and the selective expression of DCoH could restrict the Mirk response to carcinoma cells.

Insulin-like growth factor-I has a biphasic effect on colon carcinoma cells through transient inactivation of forkhead1, initially mitogenic, then mediating growth arrest and differentiation.

IGF-I stimulates intestinal cell differentiation after initiating a short proliferative burst, similar to its effect on muscle cell differentiation. Levels of IGF-I attainable in serum (10-20 ng/ml) induced transient growth stimulation of colon carcinoma cells, then growth arrest. When IGF-I functioned as a mitogen, it blocked differentiation. Intestinal cell differentiation occurred once cells had undergone the IGF-I-initiated growth arrest and IGF-I and butyrate acted synergistically to induce maturation markers. IGF-I induces NIH-3T3 cell proliferation and survival by activating the kinase akt, which in turn inhibits various apoptotic mediators and the forkhead family of transcription factors, which mediate expression of p27(kip1). Promoter reporter assays demonstrated that forkhead1 mediates transcription of p27(kip1) in colon carcinoma cells. The mitogenic effects of IGF-I on 4 colon carcinoma cell lines were transient because the inactivating phosphorylation of forkhead1 by akt was short-lived. This allowed transcriptional upregulation of the cdk inhibitor p27(kip1), with a resulting growth arrest. In contrast, in NIH-3T3 cells treated in parallel with identical IGF-I levels, forkhead phosphorylation levels were sustained; thus, no increase in p27(kip1) levels was seen and cells continued to proliferate. Intestinal epithelial cells in vivo undergo a limited number of divisions, then growth arrest and completion of their maturation. IGFs found in intestinal tissue may control the timing of this process. In addition, colon cancers may have developed strategies to overcome IGF-I-mediated growth arrest. Earlier (Kansra et al., Int J Cancer 2000;87:373-8), we found that levels of IGFBP-3 were elevated at least 2-fold in 70% of resected colon cancers compared with adjacent normal tissue. In the current study, growth inhibition by IGF-I and IGF-II was blocked by concurrent addition of IGFBP-3, implying that colon cancers with elevated IGFBP-3 levels would be selected for in vivo because they could bind and inactivate high serum IGF-I levels and continue to proliferate.