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[Cancer Research 63, 242-249, January 2003]
© 2003 American Association for Cancer Research

Tumor Biology

Overexpression of the Autocrine Motility Factor/Phosphoglucose Isomerase Induces Transformation and Survival of NIH-3T3 Fibroblasts1

Soichi Tsutsumi, Victor Hogan, Ivan R. Nabi and Avraham Raz2

Tumor Progression and Metastasis, Karmanos Cancer Institute, Department of Pathology, Wayne State University, Medical School, Detroit, Michigan 48201 [S. T., V. H., A. R.], and Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec H3C 3J7, Canada [I. R. N.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autocrine motility factor (AMF)/phosphoglucose isomerase (PGI) is a ubiquitous cytosolic enzyme and is produced as a leaderless secretory protein, released from cells via a nonclassical pathway. AMF/PGI acts extracellularly as a potent mitogen/cytokine (CXXC, chemokine). Increased expression of AMF/PGI and its receptor/CXXC-R has been found in a wide spectrum of malignancies, and is associated with cancer progression and metastasis. To directly elucidate the functional role of AMF/PGI on cell motility and neoplastic transformation, we stably transfected AMF/PGI cDNA into NIH-3T3 cells. Ectopic overexpression of AMF/PGI results in its secretion and activation via a constitutive autocrine activation loop that renders the cells highly motile, acquiring a transformed phenotype in vitro and tumorigenicity in vivo. The transformed phenotype of AMF/PGI-transfected cells leads in part resistance to induction of apoptosis induced by serum starvation, through the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway and down-regulation of caveolin-1 expression. Overexpression of this housekeeping gene induces resistance to apoptosis and neoplastic transformation, and, thus, AMF/PGI represents a novel class of oncogenic protein.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PGI3 (EC 5.3.19) is a ubiquitous cytosolic enzyme that plays a key role in both glycolysis and gluconeogenesis pathways (1) . On secretion PGI acts as a potent mitogen/cytokine i.e., tumor AMF, neuroleukin, and maturation factor (2, 3, 4) , and therefore represents a unique example of a "moonlighting protein" that exhibits multiple cellular functions (5) . PGI/AMF/neuroleukin/MF is an orphan CXXC chemokine, and its cognate receptor is a unique Mr 78,000 seven-transmembrane glycoprotein (gp78/AMFR/CXC2CR-1; Ref. 6 ). Overexpression of AMF/PGI and its receptor (AMFR) have been found in a wide spectrum of malignancies, and is associated with migration-dependent processes during cancer progression and metastasis (7, 8, 9, 10, 11) . Whereas AMF/PGI is specifically found in the conditioned medium of transformed cells as well as in the serum and urine of cancer patients (12, 13, 14, 15, 16, 17) , it has yet to be directly demonstrated that AMF/PGI overexpression and secretion are direct determinants of tumor progression.

Exogenous AMF/PGI stimulates the growth and motility of 3T3 fibroblasts (18) , and expression of AMFR mRNA has been detected in various normal mouse tissues (6) . In the brain, AMFR expression is associated with cerebellar and hippocampal neurons, and both AMF/PGI and AMFR expression is associated with memory and learning (19 , 20) . AMF/PGI has also been reported to induce the maturation of cells of the immune system (4 , 21) . This suggests that AMF/PGI not only stimulates tumor cell motility in an autocrine manner, but that it also acts as a paracrine factor on normal cells. Therefore, the AMF/PGI/neuroleukin/MF cytokine exhibits multifunctional growth factor-like activity.

A number of growth factors, such as IGF-I, hepatocyte growth factor, PDGF, FGF, and nerve growth factor have been reported to induce cell transformation and to promote cell survival (22, 23, 24, 25, 26) . Both cellular proliferation as well as tumor cell survival are crucial for malignant progression. It was shown that preventing programmed cell death by tumor cells frequently evolve the expression of secreted growth factors that induce survival signaling pathways. Here, we show that overexpression of AMF/PGI in NIH-3T3 fibroblasts results in its secretion and the induction, via autocrine activation, of enhanced cell proliferation, transformation, and tumorigenicity in nude mice. Caveolae are vesicular invaginations of the plasma membrane that participate in cellular transport processes and signal transduction-related events (27) . Caveolin is a principal component of caveolae membranes in vivo (28) . Caveolin-1 is down-regulated or absent in oncogene (v-abl, bcr-abl, Ha-ras)-transformed NIH 3T3 cells (29) , as well as in human cancer cells (30) . Transformation of NIH-3T3 cells by AMF/PGI leads to reductions in cellular levels of caveolin and caveolin-1. More particularly, forced expression of AMF/PGI offers significant protection from apoptosis mediated by PI3K/Akt signaling pathways. Overexpression and secretion of this housekeeping gene is a novel and specific determinant of cell survival that contributes to tumor progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
LY294002, PI3K inhibitor, and PD98059, MAPK kinase inhibitor were from Calbiochem (La Jolla, CA). The following antibodies were used: anti-Akt, anti-phospho-Akt (Ser473; New England Biolabs, Beverly, MA); anti-phospho-MAPK (Promega, Madison, WI); anti-caveolin-1 and anti-CD31 (Transduction Laboratories, Lexington, KY); and anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-AMF and anti-AMFR were described previously (17 , 31) . Anti-AMF IgG and preimmune IgG were purified from serum using ImmunoPure (G)IgG according to the manufacturer’s instructions (Pierce, Rockford, IL).

Cell Culture and Transfection.
NIH-3T3 cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin at 37°C and 5% CO2. The full-length human AMF/PGI cDNA was generated by PCR amplification (17) . The PCR product was ligated into a mammalian expression vector pcDNA3.1zeo (+; Invitrogen, Carlsbad, CA). Parental NIH-3T3 cells were transfected with AMF/PGI cDNA using LipofectAMINE according to the manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD). Isolation of single clones of the stable transfectants were accomplished by adding Zeocin (Invitrogen) to the culture medium at 750 µg/ml.

Northern Blot Analysis.
Total cellular RNA was isolated using Trizol reagent (Invitrogen), and 20 µg of RNA were analyzed via Northern hybridization using 32P-labeled AMF/PGI cDNA probe (17) .

Western Blot Analysis.
Cell lysates were separated by SDS-PAGE and analyzed by Western blotting as described previously (17) .

Anchorage-independent Growth and Tumorigenicity.
Assays were performed in six-well dishes coated with 1% SeaPlaque agarose (BMA, Rockland, ME) dissolved in DMEM supplemented with 10% FBS. One-thousand cells uniformly suspended in 0.5% agarose/DMEM were overlaid on the bottom layer. Cells were fed DMEM, 10% FBS, and 100 µg/ml Zeocin every other day. To confirm the role of the AMF/PGI in the tumor cell growth in soft agar, cells were seeded in soft agar containing the anti-AMF IgG or preimmune IgG. Colonies developed in the agar suspension were examined 3 weeks after seeding, and the number of colonies counted and photographed using phase-contrast photomicrography. Colonies measuring >=0.1 mm in diameter were scored. Results were expressed as the percentage of colonies formed per total number of seeded cells. Each experiment was performed in triplicate.

Female athymic nude mice were housed under specific pathogen-free conditions and used at 6 weeks of age. To investigate tumorigenicity in nude mice, each mouse was inoculated on both flanks with 1 x 106 cells in 0.1 ml of PBS per site. After xenografts became visible, the sizes of xenografts were determined every 3 days by externally measuring tumors in two dimensions. The volume (V) of the xenograft was calculated by the following equation: V = (L x W2) x 0.5, where L is the length and W is the width. The mean values and the SDs of the tumor volumes were calculated. Five mice were inoculated at both sides of the flank with each type of cells in two separate experiments.

Histopathological and Immunohistochemical Study.
The tumors were fixed in 10% phosphate-buffered formalin, and paraffin-embedded 4 µm-thick sections were prepared. Slides were stained with H&E according to standard laboratory protocols. Immunohistochemical study was performed using Vectastain Elite ABC kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA).

Invasion Assay.
The invasive activity of cells was assayed in Transwell cell culture chambers (Corning Costar Co., Cambridge, MA). Polycarbonate filters with 8.0-µm pore size were coated with Matrigel (Collaborative Biomedical Products, Bedford, MA; 1 mg/ml) to form a matrix barrier. Cells were resuspended to a concentration of 1 x 106/ml in DMEM with 0.5% FBS. The cell suspension (100 µl) was added to the upper compartment of the chamber, and incubated with DMEM with 10% FBS in the lower compartment for 24 h at 37°C. The filters were fixed with 4% paraformaldehyde and stained with Hema 3 (Fisher Scientific, Pittsburgh, PA). The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had invaded through Matrigel and the filter to the lower surface were counted. Each assay was performed in triplicate.

Cell Proliferation Assays.
Cell proliferation assays were performed by seeding the AMF/PGI or empty vector-transfected cells at a density of 1 x 105 cells/well in six-well plates. Cells were fed DMEM with 10% FBS every other day, and the number of cells in the wells were manually counted with a hemocytometer. For the serum dependence assay, cells were seeded at a density 1 x 105 cells/well in DMEM with 10% FBS. The next day, the medium was changed to serum-free DMEM and maintained in DMEM without FBS. In some experiments, anti-AMF IgG or preimmune IgG was incubated in the medium.

Induction of Apoptosis by MAPK or/and PI3K Inhibition.
Cells (1 x 106) were seeded in six-well plates. The next day, medium was replaced with serum-free medium containing the inhibitors or vehicle controls. PD98059 and LY294002 were reconstituted in DMSO. Cells were treated with PD98059 (50 µM) for 24 h in serum-free condition. Treatment with LY294002 (50 µM) was carried out for 4 h, the medium was changed to serum-free medium without inhibitor, and cells were incubated for 24 h in serum-free conditions. Cells were harvested and the percentage of cell survival determined by trypan blue dye exclusion. Subsequently, DNAs were isolated, separated in 1.8% agarose gel, and visualized by ethidium bromide staining.

Statistical Analysis.
Statistical analyses were determined using a Student’s t test. P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive Overexpression of AMF/PGI in NIH-3T3 Transfectants.
An expression vector containing the Zeocin-resistance gene and the AMF/PGI cDNA was transfected into the NIH-3T3 cells. Individual Zeocin-resistant cell clones were isolated, and stable AMF/PGI protein-expressing clones were verified by Western blot analysis. Three clones exhibiting high level expression of AMF/PGI were selected. The ratios of AMF/PGI expression was detected by densitometry and were 6.6 (clone 1), 6.5 (clone 2), and 5.3 (clone 3) compared with the empty vector-transfected control (Fig. 1A)Citation . The expression levels of AMF/PGI mRNA were determined by Northern blot analysis using full-length AMF/PGI cDNA as a probe. The three selected clones expressed high levels of AMF/PGI mRNA, whereas empty vector-transfected cells expressed AMF/PGI at the same level as that of untransfected NIH-3T3 cells. Measurement of the expression of AMF/PGI mRNA revealed ratios of 2.3 (clone 1), 2.6 (clone 2), and 2.1 (clone 3), relative to empty vector-transfected cells (Fig. 1B)Citation . To investigate the secretion levels of AMF/PGI, subconfluent cells were incubated with serum-free DMEM for 24 h and 50 µg of protein from conditioned medium analyzed by Western blotting (17) . AMF/PGI secretion was restricted to the transfectants, and could not be detected in the conditioned medium of the parental and vector-only transfected NIH-3T3 cells (Fig. 1A)Citation . Next, we questioned whether the expression of the ligand was associated with the elevated level of its receptor, and aliquots of cell lysates were subjected to quantitative Western blot analysis using anti-AMFR antibody. No differences in expression of AMFR were observed between AMF/PGI-transfectants and vector-only transfected cells (Fig. 1A)Citation . The experiment was performed in triplicate using different protein preparations.



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  		Fig. 1. Expression of AMF/PGI in transfected clones. A, Western blot analysis. Equalized protein samples were subjected to reducing 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membrane with cell lysate proteins were probed with anti-AMF antibody (top row) or anti-AMFR antibody (second row). The secretion of AMF/PGI was analyzed by Western blotting of 50 µg/protein from conditioned medium (third row) and probed with anti-AMF antibody. The bottom row was probed with antiactin antibody as a control. The experiment was performed in triplicate using different protein preparations. Note that AMF/PGI-transfected cells produced and secreted high levels of AMF/PGI but not AMFR. B, Northern blot analysis. Twenty µg of RNA were electrophoresed through a 1% denaturing formaldehyde-agarose gel, transferred to nylon membrane, and hybridized to the 32P-labeled AMF/PGI cDNA (top row). 32P-labeled ß-actin was used as a loading control (bottom row). Lane 1, parental NIH-3T3; Lane 2, vector-only transfected NIH-3T3; Lanes 3–5, AMF/PGI-transfected clone 1, 2, and 3, respectively.

 
AMF/PGI Overexpression Is Transforming and Induces Tumorigenesis.
AMF/PGI transfection induced cellular morphological changes including a fusiform appearance and colony formation typical of cell transformation (Fig. 2, c and c’)Citation . Almost complete reversion into the flat phenotype (Fig. 2, d and d’)Citation was seen in AMF/PGI-transfected NIH-3T3 cells after administration of the anti-AMF IgG, but not by preimmune serum (data not shown), suggesting that the morphological change was caused by the overexpression of AMF/PGI. The morphology of the parental NIH-3T3 cells (Fig. 2, a and a’)Citation was similar to that of the vector-only transfected cells (Fig. 2, b and b’)Citation . The morphology of parental NIH-3T3 cells treated with anti-AMF IgG (Fig. 2, e and e’)Citation was almost the same as that of NIH-3T3 cells without anti-AMF IgG (Fig. 2, a and a’)Citation . In contrast with parental and vector-only transfected NIH-3T3 cells, AMF/PGI-transfected NIH-3T3 cells acquired the ability of anchorage-independent growth in soft agar (not shown). Furthermore, anti-AMF IgG antibody but not preimmune IgG suppressed the colony formation of AMF/PGI-transfected cells when included in the soft agar medium confirming a role for secreted protein in the enhancement of anchorage-independent growth of cells by AMF/PGI overexpression.



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  		Fig. 2. Morphological changes in growth of AMF/PGI-transfected NIH-3T3 cells. Single clones of transfected NIH-3T3 cells were selected by Zeocin. The morphology of the parental NIH-3T3 cells (a and a’) did not differ significantly from that of the empty vector-transfected cells (b and b’). However, morphological changes (fusiform appearance) and colony formation typical of the transforming growth were observed in the AMF/PGI transformants (c and c’: clone 1). Morphological changes of AMF/PGI-transfected cells were inhibited by anti-AMF IgG (50 µg/ml; d and d’). Parental NIH-3T3 cells treated with anti-AMF IgG are shown. e and e’, All cells are shown at x40 (left column) and x100 (right column) magnification.

 
Secretion of AMF/PGI was selectively detected in the conditioned medium of ras-transformed but not untransformed NIH-3T3 cells (12) , and, a ras and abl transformation of NIH-3T3 cells was shown to be associated with significant reduced expression of caveolin (29 , 32) . As seen in Fig. 3Citation , Western blot analysis of the expression of caveolin in parental, vector-only transfected, and three independent AMF/PGI-transfected NIH-3T3 cell clones revealed that the level of caveolin and caveolin-1 in AMF/PGI-transfected cell clones was reduced by approximately 30–40% relative to parental NIH-3T3 cells confirming the above reports, and supporting the function of AMF/PGI is a transforming protein. To deduce other aspects of transformation affected by AMF/PGI overexpression, we tested the invasive capacity by the cells in Matrigel using Transwell culture plates assay. The invasive potential was 2–2.5 times greater in AMF/PGI-transfected cells compared with parental or vector-only transfected NIH-3T3 cells at 24 h (Fig. 4A)Citation . Invasive capacity was specifically inhibited by anti-AMF IgG (Fig. 4B)Citation . The ability of anti-AMF antibody to revert the morphological changes, and the enhanced growth, invasivity, and anchorage-independent growth of AMF/PGI-transfected NIH-3T3 cells indicates that these cellular changes are because of constitutive autocrine activation of the cells by secreted protein.



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  		Fig. 3. Caveolin-1 expression in AMF/PGI-transfected NIH-3T3 cells. Cell lysates from parental, vector-only transfected, and three independent AMF/PGI-transfected NIH-3T3 cells were subjected to Western blot analysis with antibodies to caveolin and caveolin-1. The amount of caveolin and caveolin-1 protein in AMF/PGI-transfected cells reduced 60–70% of levels found in parental NIH-3T3 cells by densitometric tracing analysis; bars, ±SD.

 


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  		Fig. 4. Invasive ability of AMF/PGI-transfected cells. A, Two-hundred µl of a single-cell suspension (1 x 106 cells/ml) of cells were placed in the upper wells of individual Transwell inserts containing 8-µm pore polycarbonate membranes precoated with Matrigel. Cells were allowed to invade for 24 h at 37°C, and then they were fixed and stained with Hema-3. Cells on the upper surface were removed with a cotton swab, and the cells that migrated to the lower side of the membrane were mounted onto a microscope slide and counted under a light microscope at x200 magnification. In vitro invasive ability was significantly greater in AMF/PGI-transfected cells when compared with parental and vector-only transfected NIH-3T3 cells at 24 h. B, invasive capacity was inhibited by anti-AMF IgG (50 µg/ml). Data shown are the mean values from three triplicate experiments for each group; bars, ±SE. *, significant difference when compared with control (P < 005).

 
AMF/PGI-transfected NIH-3T3 cells were analyzed for their ability to form tumors after s.c. injection into female nude mice. Whereas parental and vector-only transfected NIH-3T3 cells failed to produce tumors after injection into mice, AMF/PGI-transfected cells were highly tumorigenic, forming detectable tumors within 5–7 days after inoculating with 1 x 106 cells (Fig. 5A)Citation . Histology of the implanted tumors revealed a cellular malignancy consisting of bundles of spindle-shaped cells with a storiform growth pattern (Fig. 5BCitation , panel a), and included fusiform tumor cells that exhibited markedly pleomorphic hyperchromatic nuclei and mitosis (Fig. 5BCitation , panel b). Histological examination identified the tumors as fibrosarcomas. Immunohistochemical staining for antimouse CD31 were performed to establish tumor angiogenesis and, indeed, many microvessels were detected in the growing tumors (Fig. 5BCitation , panel c).



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  		Fig. 5. A, transfected cells and parental NIH-3T3 cells (1 x 106) were s.c. inoculated into the back of 6-week-old female athymic nude mice. Five mice were inoculated at both sides with each type of cells in two separate experiments. Tumor formation was monitored for up to 4 weeks. The two independent experiments produced similar results. B, pathological characteristic of AMF/PGI-induced tumors in nude mice. Tumors were generated by inoculation of AMF/PGI-transfected NIH-3T3 cells as described in "Materials and Methods." Histological appearance of xenografts tumors from mice that were inoculated with AMF/PGI-transfected cells. Tumor consisted of spindle-shaped cells and storiform growth pattern like fibrosarcoma. (H&E stain; original magnification: a, x40; b, x200). Pleomorphic hyperchromatic nuclei and mitosis were observed (c, arrow). c, detection of tumor vascularization. Deparaffinized sections of AMF/PGI-transfectant tumors were incubated with antimouse CD31 antibody at 4°C overnight, followed by biotinylated rat antimouse IgG secondary antibody. Detection was carried out with an avidin-biotin complex kit from Vector Laboratories (x100).

 
AMF/PGI Overexpression Confers Protection against Apoptosis via the PI3K/Akt Pathway.
Each of the AMF/PGI-transfected cell clones grew at rates faster than those of parental and vector-only transfected NIH-3T3 cells (Fig. 6A)Citation . The increase in cell number of the AMF/PGI-transfected cell clones was 2-fold greater than the control cells after 7 days in culture. Notably, whereas serum deprivation results in rapid cell death of parental and vector-only transfected NIH-3T3 cells (95% of these cells die within 72 h), approximately 80–90% of AMF/PGI-transfected cells survive after serum withdrawal and continue proliferating in serum-free conditions (Fig. 6B)Citation . The effect of anti-AMF IgG on cell proliferation of parental and AMF/PGI-transfected NIH-3T3 cells was determined by growth curve analysis as demonstrated in Fig. 6CCitation . Anti-AMF IgG (50 µg/ml) inhibited parental, vector-only transfected, and AMF/PGI-transfected NIH3T3 cell proliferation by 19%, 21% and 33%, respectively, after 7 days of cultivation compared with control. Growth inhibition of anti-AMF IgG on AMF/PGI-transfected NIH-3T3 cells was significantly greater than that on parental and vector-only transfected NIH-3T3 cells (Fig. 6, A and C)Citation . Preimmune rabbit IgG had no effect on proliferation in cultures of these cell lines (data not shown).



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  		Fig. 6. Growth of AMF/PGI-transfected NIH-3T3 cells. Cells (1 x 105/well) were seeded into six-well plates in DMEM with 10% FBS. The next day, the medium was replaced by DMEM with or without 10% FBS. Cells were fed with fresh medium every other day, and the number of cells in the wells were manually counted with a hemocytometer daily for up to 7 days after seeding. A, AMF/PGI-transfected cells grew faster than the parental and empty vector-transfected cells in DMEM with 10% FBS. B, AMF/PGI-transfected cells survived in serum-free conditions. Serum-deprivation resulted in rapid cell death of control NIH-3T3 cells and empty vector-transfected cells. AMF/PGI-transfected cells were resistant to serum withdrawal. Approximately 80–90% of cells survived, and continued growing in serum-free conditions. C, influence of antibody to the AMF/PGI on the growth of cells in DMEM containing 10% FBS. Anti-AMF IgG (50 µg/ml) inhibited parental, vector-only transfected, and AMF/PGI-transfected NIH-3T3 cell proliferation by 19%, 21%, and 33%, respectively, after 7 days of cultivation compared with control. Growth inhibition effect of anti-AMF IgG on AMF/PGI-transfected NIH-3T3 cells was significantly greater than that on parental and vector-only transfected NIH-3T3 cells. Preimmune rabbit IgG had no effect on proliferation in cultures of these cell lines (data not shown). Each bar represents the mean of triplicate wells ± SD. The experiment was repeated twice with similar results. ({bullet}), parental NIH3T3; (x), empty vector-transfected NIH3T3; ({circ}), AMF/PGI-transfected clone 1; ({triangleup}), clone 2; ({square}), clone 3.

 
Pharmacological inhibitors were used to determine the signaling pathway(s) activated by AMF/PGI overexpression that inhibit apoptosis. Survival of the AMF/PGI-transfected cells under serum deprivation was specifically inhibited by the PI3K inhibitor LY294002 and relatively resistant to growth inhibition by PD98059 inhibition of MAPK kinase activation (Fig. 7A)Citation . Inhibition of both pathways simultaneously showed only a limited additive effect on serum starvation-induced apoptosis. Induction of oligonucleosomal DNA fragmentation, an indicator of apoptosis, occurred in parental and vector only-transfected NIH-3T3 cells after 24 h of serum withdrawal alone and when treated with PD98059 and/or LY294002 (data not shown). In contrast, DNA fragmentation could be detected in AMF/PGI-transfected NIH-3T3 cells when treated with LY290042 but not with PD987059 (Fig. 7B)Citation . Therefore, AMF/PGI-induced apoptosis protection is mainly mediated by the PI3K/PKB pathway.



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  		Fig. 7. Signal pathways involved in protection against apoptosis induced by serum-deprivation. A, figure shows that on serum starvation the AMF/PGI-transfected cell-induced cell survival is decreased by LY294002, but the AMF/PGI-transfected cells were relatively resistant to growth inhibition by PD98059. Simultaneous treatment with both inhibitors showed an addictive effect. No differences were observed in vector-only transfected NIH-3T3 cells that were treated with exogenous AMF/PGI. ({square}), serum deprivation; (), serum deprivation + PD98059; (), serum deprivation + LY294002; ({blacksquare}), serum deprivation + PD98059 + LY29002. *, significant difference when compared with control (P < 005); bars, ±SD. B, induction of oligonucleosomal DNA fragmentation treated with PD98056 and LY294002 in AMF/PGI-transfected NIH-3T3 cells. Cells were cultured in serum-free condition. Treatment with PD98059 (50 µM) was carried out for 24 h and for 4 h with LY294002 (50 µM). DNA was isolated, subjected to agarose gel electrophoresis, and visualized by ethidium bromide staining. DNA fragmentation was only observed under LY294002 treatment. These results indicate that AMF/PGI overexpression-induced protection may be mainly mediated by the PI3K/PKB pathway. Lane 1, standard marker (1 kb ladder). Lanes 2, 5, and 8, AMF/PGI-transfected clone 1. Lanes 3, 6, and 9, AMF/PGI-transfected clone 2. Lanes 4, 7, and 10, AMF/PGI-transfected clone 3.

 
PKB/Akt is catalytically inactive in serum-starved primary and immortalized fibroblasts (33) . The activation of PKB/Akt results from phosphorylation of both Thr308 and Ser473 (34) . Using phosphospecific antibodies revealed that on serum deprivation the PBK/Akt phosphorylation levels were increased only in AMF/PGI-transfected cells and not in vector-only transfected or parental NIH-3T3 cells (Fig. 8A)Citation . The phosphorylation of PBK/Akt was abrogated by inhibition of PI3K with pharmacological inhibitor LY294002 demonstrating the involvement of PI3K-mediated signaling in AMF/PGI-induced PKB/Akt activation. The MAPK pathway has also been identified as an important signaling pathway activated by various growth factors (35) . Using the phospho-MAPK antibody, MAPK activation was equivalent in untransfected and AMF/PGI-transfected NIH-3T3 cells (Fig. 8B)Citation . The ability of the MAPK kinase inhibitor PD98059 to inhibit phosphorylation of MAPKs indicates that the MAPK pathway probably does not regulate AMF/PGI-mediated cell survival. In conclusion, AMF/PGI overexpression and secretion induce NIH-3T3 cell transformation, invasion, and tumorigenicity associated with apoptosis resistances via PI3K-dependent activation of PKB/Akt phosphorylation.



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  		Fig. 8. PKB/Akt is activated in AMF/PGI-transfected NIH-3T3 cells. A, Western blot analysis with phosphorylation specific antibodies using PhosphoSer473PKB/Akt antibody (top) and control with antibodies recognizing phosphorylation-independent total PKB/Akt (bottom). Lysates were obtained after serum deprivation for 24 h and after starvation plus additional treatment with LY294002 (50 µM) or PD98059 (50 µM). Lane 1, parental NIH-3T3 cells. Lane 2, vector-only transfected NIH-3T3 cells. Lane 3, vector-only transfected NIH-3T3 cells incubated with PGI. Lane 4, AMF/PGI-transfected clone 1. Lane 5, AMF/PGI-transfected clone 2. Lane 6, AMF/PGI-transfected clone 3. Lane 7, AMF/PGI-transfected clone 1 treated with LY290042. The same results were obtained when clones 2 and 3 were treated with LY290042. B, phosphorylated MAPK was detected with phosphospecific MAPK antibody. Lane 1, parental NIH-3T3 cells. Lane 2, vector-only transfected NIH-3T3 cells. Lane 3, vector-only transfected NIH-3T3 cells incubated with PGI. Lane 4, AMF/PGI-transfected clone 1. Lane 5, AMF/PGI-transfected clone 2. Lane 6, AMF/PGI-transfected clone 3. Lane 7, clone 1 treated with PD98059. The same results were obtained when clones 2 and 3 were treated with LY290042.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AMF/PGI Overexpression Is Transforming.
The transforming ability of the glycolytic enzyme AMF/PGI to induce cell transformation and tumorigenesis represents the first demonstration of the oncogenic potential of overexpression and secretion of this housekeeping protein. Expression of AMF/PGI and other glycolytic enzymes are up-regulated in response to enhanced energy requirements under hypoxic condition (36) . However, AMF/PGI is different from other glycolytic enzymes in that it acts as a moonlighting protein that also exhibits cytokine activity when secreted from cells. AMF/PGI gene expression has been shown to be developmentally regulated in brain (37) as well as induced during differentiation of osteoblast cells (38) , and to be elevated under hypoxic conditions (39 , 40) . The demonstration that AMF/PGI-overexpressing NIH-3T3 cells exhibit enhanced growth properties in vitro, anchorage-independent growth, and tumorigenicity in vivo highlights the possible role of AMF/PGI overexpression during normal cellular responses, and its contribution to cellular transformation.

Anti-AMF antibodies abrogate the altered morphology, enhanced growth, increased invasivity, and acquisition of anchorage-independent growth of the AMF/PGI-overexpressing NIH-3T3 cells; thus, the acquired phenotype is a direct consequence of its secretion and autocrine activation of the AMFR, and probably not because of enhanced glycolytic function. The cytokine function of the enzyme was acquired over evolution because only mammalian and not bacterial or yeast PGIs express cytokine activity (41) and may represent a prosurvival mechanism to protect glycolytically active cells from premature death. Similarly, glycolysis is generally up-regulated in tumor cells, and is an essential element of tumor cell motility and pseudopodial protrusion (42 , 43) . Therefore, increased expression and secretion of AMF/PGI should contribute to tumor progression not only via increased glycolytic stimulation of tumor cell motility and invasion but also acting as a survival factor.

Mutated forms of PGI have been identified and are associated with hemolytic anemia, a consequence of decreased glycolytic activity (44 , 45) . Certain mutations were also associated with neurological deficiencies suggesting that they disrupt the extracellular cytokine function of the enzyme (42) . The fact that specific inhibitors of this enzyme activity of PGI also inhibit its cytokine activity suggests that the active site is also the receptor-binding motif (2 , 38 , 46) . This overlap of domains may explain, at least in part, why tumor-associated mutations of PGI have not yet been identified. The route of secretion of AMF/PGI, like other proteins secreted, is via the nonclassical secretory pathway yet to be identified. AMF/PGI peptide lacks a secretory signal peptide and is thought to be actively secreted via a novel alternative pathway, like FGF (25) and PDGF (47) . Whether this is a consequence of specific signals in the protein sequences or a consequence of overexpression, as shown here, remains elusive.

Down-regulation of caveolin-1 mRNA and protein expression is associated with NIH-3T3 cells transformation by a variety of activated oncogenes like v-Abl, bcr-Abl, crk1, and H-RasG12V, and caveolin-1 expression levels inversely correlated with the ability of these cells to grow in soft agar (29) . The fact that caveolin expression was also reduced in AMF/PGI-transfected cells implicates autocrine activation of the AMFR caveolin down-regulation. Caveolin, a Mr 21,000–24,000 integral membrane protein, is a principal component of caveolae membranes in vivo, and caveolae participate in vesicular trafficking events and signal transduction processes (48) . The human caveolin-1 gene is localized to a suspected tumor suppressor locus (49) but is neither mutated nor methylated in cancer cells (50) questioning whether it is a true tumor suppressor gene. Caveolin-1 gene expression is directly regulated by hyperactivation of p42/44MAPK cascade (51) . Here, we show that p42/44MAPK is not activated in response to autocrine AMF/PGI stimulation suggesting that overexpression of AMF/PGI may stimulate reduction in caveolin-1 levels via a different pathway. AMFR is internalized to the endoplasmic reticulum via a caveolin-dependent pathway that is negatively regulated by caveolin-1 expression (32 , 52) . The autocrine activation loop by AMF/PGI of its receptor may therefore occur in the caveolae, and its stimulation acts to regulate caveolae internalization and stabilization at the plasma membrane, and potentially affect caveolin expression levels.

Secreted AMF/PGI Is Antiapoptotic.
It was shown recently that AMF/PGI acts as an angiogenic factor in vivo and in vitro (46) . AMF/PGI stimulates in vitro motility of human umbilical vein endothelial cells, elicited the formation of tube-like structures mimicking angiogenesis when human umbilical vein endothelial cells were grown in three-dimensional type I-collagen gels, and specific PGI inhibitors prevented angiogenic activity and neovascularization in the mouse model. Here, staining of the developing with endothelial cell marker (antimouse CD31) identified many microvessels in AMF/PGI-transfectant tumors supporting the notion that a tumor secreted AMF/PGI may act as an angiogenic factor that acts in a paracrine manner.

Human basic FGF (FGF-2) is a prototype member of the large family of heparin-binding growth factor genes that regulates various biological processes, such as proliferation, differentiation, migration, angiogenesis, and survival in different cells (25) . Similar to PGI, FGF-2 is expressed in a broad range of tumor cells; its expression and its release is modified during tumor progression (53) . FGF-2 also induces endothelial cell proliferation, migration, and angiogenesis in vitro, and regulates the expression of several molecules that mediate angiogenesis (25) . Overexpression of FGF-2 in NIH-3T3 fibroblasts modulates Bcl-2 (54) and interleukin 6 levels (55) leading to inhibition of apoptosis through alternate molecular mechanisms. The Mr 18,000 low molecular weight isoform (LMW FGF-2) is cytosolic and like AMF/PGI is also secreted via an endoplasmic reticulum-Golgi-independent pathway; therefore, this isoform acts as a paracrine/autocrine factor. In contrast, the high molecular weight isoforms of FGF-2 (HMW FGF-2) contain nuclear localization sequence-like signals responsible for nuclear targeting (25) . The selective expression of either LMW or HMW FGF-2 forms induces NIH-3T3 cell transformation as measured by enhanced saturation density and growth in soft agar (56) . Therefore, AMF/PGI and FGF are both angiogenic factors secreted via the nonclassical pathway that exhibit similar biological functions. Like FGF-2, PGI transfectants possess an enhanced proliferative potential under serum-free conditions and antiapoptotic ability. The data indicate that AMF/PGI protects cells from apoptosis, and that the specific PI3K inhibitor LY294002 sensitizes cells to apoptosis and abolishes protection. Inhibition of apoptosis by AMF/PGI is associated with kinase activity of the antiapoptotic mediator Akt. The PI3K/Akt pathway is activated in response to many stimuli resulting in an antiapoptotic effect (57) . On the basis of this, Akt was determined to be an antiapoptotic regulating protein through which various survival signals suppress cell death (58) . The activation of the PI3K/Akt pathway is required for cell spreading and matrix-induced cell survival, and integrin-mediated adhesion activates Akt in a PI3K-dependent manner (59) .

MAPK was shown to be involved in the protection of cells from apoptosis induced by growth factor withdrawal (60) . IGF-I is capable of preventing apoptosis by activation of multiple signal transduction pathways including PI3K and MAPK pathways. These two pathways appear to interact with each other and to a certain extent may replace each other (22) . FGF-2 stimulated DNA synthesis and induced a sustained phosphorylation of p42/44MAPK in rat mammary fibroblasts (61) . In contrast, use of PD98059 showed that MAPK appeared to have a lesser effect on AMF/PGI-mediated survival than PI3K/Akt. The selective mediation of tumor cell survival by AMF/PGI via the PI3K/Akt pathway is consistent with previous reports that AMF/PGI stimulated a 300% increase in inositol trisphosphate after 90 min of exposure (62) . Activation of survival mediator Akt involves the binding of PI3K that generates inositol lipids to Akt via pleckstrin homology domain (63) .

In conclusion, overexpression and consequent secretion of AMF/PGI constitutively activates its receptor via an autocrine loop that induces cellular transformation, tumorigenicity, and apoptosis survival through the activation of the PI3K/Akt signaling pathway. The protection of cells from apoptosis by this ubiquitous glycolytic enzyme suggests that the alternate cytokine function of this moonlighting protein may have been acquired to protect active glycolytic cells from cell death under hypoxic condition of growing tumors.


   FOOTNOTES
 
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grant CA-51714 (to A. R.) and by Grant MT-15132 from the Canadian Institutes for Health Research (to I. R. N.). Back

2 To whom requests for reprints should be addressed, at Tumor Progression and Metastasis, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: (313) 833-0960; Fax: (313) 831-7518; E-mail: raza{at}karmanos.org Back

3 The abbreviations used are: PGI, phosphoglucose isomerase; AMF, autocrine motility factor; AMFR, autocrine motility factor receptor; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PI3K, phosphatidylinositol 3'-kinase; MAPK, mitogen-activated protein kinase; FBS, fetal bovine serum; PKB, protein kinase B. Back

Received 8/ 6/02. Accepted 10/29/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Harrison R. A. The detection of hexokinase, glucosephosphate isomerase and phosphoglucomutase activities in polyacrylamide gels after electrophoresis: a novel method using immobilized glucose 6-phosphate dehydrogenase. Anal. Biochem., 61: 500-507, 1974.[Medline]
  2. Watanabe H., Takehana K., Date M., Shinozaki T., Raz A. Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res., 56: 2960-2963, 1996.[Abstract/Free Full Text]
  3. Chaput M., Claes V., Portetelle D., Cludts I., Cravador A., Burny A., Gras H., Tartar A. The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase. Nature (Lond.), 332: 454-455, 1988.[Medline]
  4. Xu W., Seiter K., Feldman E., Ahmed T., Chiao J. W. The differentiation and maturation mediator for human myeloid leukemia cells shares homology with neuroleukin or phosphoglucose isomerase. Blood, 87: 4502-4506, 1996.[Abstract/Free Full Text]
  5. Jeffery C. J. Moonlighting proteins. Trends Biochem. Sci., 24: 8-11, 1999.[Medline]
  6. Shimizu K., Tani M., Watanabe H., Nagamachi Y., Niinaka Y., Shiroishi T., Ohwada S., Raz A., Yokota J. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett., 456: 295-300, 1999.[Medline]
  7. Nakamori S., Watanabe H., Kameyama M., Imaoka S., Furukawa H., Ishikawa O., Sasaki Y., Kabuto T., Raz A. Expression of autocrine motility factor receptor in colorectal cancer as a predictor for disease recurrence. Cancer (Phila.), 74: 1855-1862, 1994.[Medline]
  8. Maruyama K., Watanabe H., Shiozaki H., Takayama T., Gofuku J., Yano H., Inoue M., Tamura S., Raz A., Monden M. Expression of autocrine motility factor receptor in human esophageal squamous cell carcinoma. Int. J. Cancer, 64: 316-321, 1995.[Medline]
  9. Otto T., Bex A., Schmidt U., Raz A., Rubben H. Improved prognosis assessment for patients with bladder carcinoma. Am. J. Pathol., 150: 1919-1923, 1997.[Abstract]
  10. Takanami I., Takeuchi K., Naruke M., Kodaira S., Tanaka F., Watanabe H., Raz A. Autocrine motility factor in pulmonary adenocarcinomas: results of an immunohistochemical study. Tumour Biol., 19: 384-389, 1998.[Medline]
  11. Taniguchi K., Yonemura Y., Nojima N., Hirono Y., Fushida S., Fujimura T., Miwa K., Endo Y., Yamamoto H., Watanabe H. The relation between the growth patterns of gastric carcinoma and the expression of hepatocyte growth factor receptor (c-met), autocrine motility factor receptor, and urokinase-type plasminogen activator receptor. Cancer (Phila.), 82: 2112-2122, 1998.[Medline]
  12. Liotta L. A., Mandler R., Murano G., Katz D. A., Gordon R. K., Chiang P. K., Schiffmann E. Tumor cell autocrine motility factor. Proc. Natl. Acad. Sci. USA, 83: 3302-3306, 1986.[Abstract/Free Full Text]
  13. Baumann M., Kappl A., Lang T., Brand K., Siegfried W., Paterok E. The diagnostic validity of the serum tumor marker phosphohexose isomerase (PHI) in patients with gastrointestinal, kidney, and breast cancer. Cancer Investig., 8: 351-356, 1990.[Medline]
  14. Guirguis R., Javadpour N., Sharareh S., Biswas C., el-Amin W., Mansur I., Kim J. S. A new method for evaluation of urinary autocrine motility factor and tumor cell collagenase stimulating factor as markers for urinary tract cancers. J. Occup. Med., 32: 846-853, 1990.[Medline]
  15. Filella X., Molina R., Jo J., Mas E., Ballesta A. M. Serum phosphohexose isomerase activities in patients with colorectal cancer. Tumour Biol., 12: 360-367, 1991.[Medline]
  16. Patel P. S., Raval G. N., Rawal R. M., Patel G. H., Balar D. B., Shah P. M., Patel D. D. Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer. Neoplasia, 42: 271-274, 1995.
  17. Niinaka Y., Paku S., Haga A., Watanabe H., Raz A. Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells. Cancer Res., 58: 2667-2674, 1998.[Abstract/Free Full Text]
  18. Silletti S., Raz A. Autocrine motility factor is a growth factor. Biochem. Biophys. Res. Commun., 194: 446-457, 1993.[Medline]
  19. Leclerc N., Vallee A., Nabi I. R. Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum. J. Neurosci. Res., 60: 602-612, 2000.[Medline]
  20. Luo Y., Long J. M., Lu C., Chan S. L., Spangler E. L., Mascarucci P., Raz A., Longo D. L., Mattson M. P., Ingram D. K., Weng N. P. A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78. J. Neurochem., 80: 354-361, 2002.[Medline]
  21. Gurney M. E., Apatoff B. R., Spear G. T., Baumel M. J., Antel J. P., Bania M. B., Reder A. T. Neuroleukin: a lymphokine product of lectin-stimulated T cells. Science (Wash. DC), 234: 574-581, 1986.[Abstract/Free Full Text]
  22. Parrizas M., Saltiel A. R., LeRoth D. Insulin-like growth factor-1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J. Biol. Chem., 272: 154-161, 1997.[Abstract/Free Full Text]
  23. Bardelli A., Longati P., Albero D., Goruppi S., Schneider C., Ponzetto C., Comoglio P. M. HGF receptor associates with the anti-apoptotic protein BAG-1 and prevents cell death. EMBO J., 15: 6205-6212, 1996.[Medline]
  24. Harrington E. A., Bennett M. R., Fanidi A., Evan G. I. c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J., 13: 3286-3295, 1994.[Medline]
  25. Bikfalvi A., Klein S., Pintucci G., Rifkin D. B. Biological roles of fibroblast growth factor-2. Endocr. Rev., 18: 26-45, 1997.[Abstract/Free Full Text]
  26. Descamps S., Toillon R. A., Adriaenssens E., Pawlowski V., Cool S. M., Nurcombe V., Le Bourhis X., Boilly B., Peyrat J. P., Hondermarck H. Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways. J. Biol. Chem., 276: 17864-17870, 2001.[Abstract/Free Full Text]
  27. Okamoto T., Schlegel A., Scherer P. E., Lisanti M. P. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J. Biol. Chem., 273: 5419-5422, 1998.[Free Full Text]
  28. Anderson R. G. The caveolae membrane system. Annu. Rev. Biochem., 67: 199-225, 1998.[Medline]
  29. Koleske A. J., Baltimore D., Lisanti M. P. Reduction of caveolin and caveolae in oncogenically transformed cells. Proc. Natl. Acad. Sci. USA, 92: 1381-1385, 1995.[Abstract/Free Full Text]
  30. Racine C., Belanger M., Hirabayashi H., Boucher M., Chakir J., Couet J. Reduction of caveolin 1 gene expression in lung carcinoma cell lines. Biochem. Biophys. Res. Commun., 255: 580-586, 1999.[Medline]
  31. Nabi I. R., Watanabe H., Raz A. Identification of B16-F1 melanoma autocrine motility-like factor receptor. Cancer Res., 50: 409-414, 1990.[Abstract/Free Full Text]
  32. Le P. U., Guay G., Altschuler Y., Nabi I. R. Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J. Biol. Chem., 277: 3371-3379, 2002.[Abstract/Free Full Text]
  33. Franke T. F., Yang S. I., Chan T. O., Datta K., Kazlauskas A., Morrison D. K., Kaplan D. R., Tsichlis P. N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell, 81: 727-736, 1995.[Medline]
  34. Alessi D. R., Andjelkovic M., Caudwell B., Cron P., Morrice N., Cohen P., Hemmings B. A. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J., 15: 6541-6551, 1996.[Medline]
  35. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA, 90: 5889-5892, 1993.[Abstract/Free Full Text]
  36. Mathupala S. P., Rempel A., Pedersen P. L. Glucose catabolism in cancer cells: identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J. Biol. Chem., 276: 43407-43412, 2001.[Abstract/Free Full Text]
  37. Hallbook F., Persson H., Barbany G., Ebendal T. Development and regional expression of chicken neuroleukin (glucose-6-phosphate isomerase) messenger RNA. J. Neurosci. Res., 23: 142-151, 1989.[Medline]
  38. Zhi J., Sommerfeldt D. W., Rubin C. T., Hadjiargyrou M. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation. J. Bone. Miner. Res., 16: 1994-2004, 2001.[Medline]
  39. Yoon D. Y., Buchler P., Saarikoski S. T., Hines O. J., Reber H. A., Hankinson O. Identification of genes differentially induced by hypoxia in pancreatic cancer cells. Biochem. Biophys. Res. Commun., 288: 882-886, 2001.[Medline]
  40. Niizeki H., Kobayashi M., Horiuchi I., Akakura N., Chen J., Wang J., Hamada J. I., Seth P., H, Katoh H., Watanabe H., Raz A., Hosokawa M. Hypoxia enhances the expression of autocrine motility factor and the motility of human pancreatic cancer cells. Br. J. Cancer, 86: 1914-1919, 2002.[Medline]
  41. Amraei M., Nabi I. R. Evolutionary acquisition of the cytokine activity of phosphoglucose isomerae. FEBS Lett., 525: 151-155, 2002.[Medline]
  42. Beckner M. E., Stracke M. L., Liotta L. A., Schiffmann E. Glycolysis as primary energy source in tumor cell chemotaxis. J. Natl. Cancer Inst. (Bethesda), 82: 1836-1840, 1990.[Abstract/Free Full Text]
  43. Nguyen T. N., Wang H. J., Zalzal S., Nanci A., Nabi I. R. Purification and characterization of ß-actin-rich tumor cell pseudopodia: role of glycolysis. Exp. Cell Res., 258: 171-183, 2000.[Medline]
  44. Beutler E., West C., Britton H. A., Harris J., Forman J. Glucosephosphate isomerase (GPI) deficiency mutations associated with hereditary nonspherocytic hemolytic anemia (HNSHA). Blood Cells Mol. Dis., 23: 402-409, 1997.[Medline]
  45. Kugler W., Breme K., Laspe P., Muirhead H., Davies C., Winkler H., Schroter W., Lakomek M. Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency. Hum. Genet., 103: 450-454, 1998.[Medline]
  46. Funasaka T., Haga A., Raz A., Nagase H. Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility. Biochem. Biophys. Res. Commun., 285: 118-128, 2001.[Medline]
  47. Ishikawa F., Miyazono K., Hellman U., Drexler H., Wernstedt C., Hagiwara K., Usuki K., Takaku F., Risau W., Heldin C. H. Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. Nature (Lond.), 338: 557-562, 1989.[Medline]
  48. Lisanti M. P., Scherer P., Tang Z. L., Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol., 4: 231-235, 1994.[Medline]
  49. Engelman J. A., Zhang X. L., Lisanti M. P. Genes encoding human caveolin-1 and -2 are colocalized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers. FEBS Lett., 436: 403-410, 1998.[Medline]
  50. Hurlstone A. F., Reid G., Reeves J. R., Fraser J., Strathdee G., Rahilly M., Parkinson E. K., Black D. M. Analysis of the caveolin-1 gene at human chromosome 7q31. 1 in primary tumours and tumour-derived cell lines. Oncogene, 18: 1881-1890, 1999.[Medline]
  51. Galbiati F., Volonté D., Engelman J. A., Watanabe G., Burk R., Richard G., Pestell R. G., Lisanti M. P. Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. EMBO J., 17: 6633-6648, 1998.[Medline]
  52. Benlimame N., Le P. U., Nabi I. R. Localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum. Mol. Biol. Cell, 9: 1773-1786, 1998.[Abstract/Free Full Text]
  53. Nguyen M., Watanabe H., Budson A. E., Richie J. P., Hayes D. F., Folkman J. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J. Natl. Cancer Inst. (Bethesda), 86: 356-361, 1994.[Abstract/Free Full Text]
  54. Karsan A., Yee E., Poirier G. G., Zhou P., Craig R., Harlan J. M. Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms. Am. J. Pathol., 151: 1775-1784, 1997.[Abstract]
  55. Delrieu I., Arnaud E., Ferjoux G., Bayard F., Faye J. C. Overexpression of the FGF-2 24-kDa isoform up-regulates IL-6 transcription in NIH-3T3 cells. FEBS Lett., 436: 17-22, 1998.[Medline]
  56. Bikfalvi A., Klein S., Pintucci G., Quarto N., Mignatti P., Rifkin D. B. Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms. J. Cell Biol., 129: 233-243, 1995.[Abstract/Free Full Text]
  57. Chan T. O., Rittenhouse S. E., Tsichlis P. N. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu. Rev. Biochem., 68: 965-1014, 1999.[Medline]
  58. Dudek H., Datta S. R., Franke T. F., Birnbaum M. J., Yao R., Cooper G. M., Segal R. A., Kaplan D. R., Greenberg M. E. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science (Wash. DC), 275: 661-665, 1997.[Abstract/Free Full Text]
  59. Khwaja A., Rodriguez-Viciana P., Wennstrom S., Warne P. H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J., 16: 2783-2793, 1997.[Medline]
  60. Erhardt P., Schremser E. J., Cooper G. M. B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol. Cell. Biol., 19: 5308-5315, 1999.[Abstract/Free Full Text]
  61. Delehedde M., Seve M., Sergeant N., Wartelle I., Lyon M., Rudland P. S., Fernig D. G. Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and I{kappa}B degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts. J. Biol. Chem., 275: 33905-33910, 2000.[Abstract/Free Full Text]
  62. Kohn E. C., Liotta L. A., Schiffmann E. Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells. Biochem. Biophys. Res. Commun., 166: 757-764, 1990.[Medline]
  63. Datta S. R., Brunet A., Greenberg M. E. Cellular survival: a play in three Akts. Genes Dev., 13: 2905-2927, 1999.[Free Full Text]



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