The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during proliferation of human K562 leukemia cells

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.     

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.     

c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.     

The insulin-like growth factor mRNA binding-protein IMP-1 and the Ras-regulatory protein G3BP associate with tau mRNA and HuD protein in differentiated P19 neuronal cells

Tau mRNA is axonally localized mRNA that is found in developing neurons and targeted by an axonal localization signal (ALS) that is located in the 3'UTR of the message. The tau mRNA is trafficked in an RNA-protein complex (RNP) from the neuronal cell body to the distal parts of the axon, reaching as far as the growth cone. This movement is microtubule-dependent and is observed as granules that contain tau mRNA and additional proteins. A major protein contained in the granule is HuD, an Elav protein family member, which has an identified mRNA binding site on the tau 3'UTR and stabilizes the tau message and several axonally targeted mRNAs. Using GST-HuD fusion protein as bait, we have identified four proteins contained within the tau RNP, in differentiated P19 neuronal cells. In this work, we studied two of the identified proteins, i.e. IGF-II mRNA binding protein 1 (IMP-1), the orthologue of chick beta-actin binding protein-ZBP1, and RAS-GAP SH3 domain binding protein (G3BP). We show that IMP-1 associates with HuD and G3BP-1 proteins in an RNA-dependent manner and binds directly to tau mRNA. We also show an RNA-dependent association between G3BP-1 and HuD proteins. These associations are investigated in relation to the neuronal differentiation of P19 cells.     

Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression

Several experimental and epidemiological studies have suggested a role for the use of cyclooxygenase (COX)-2 inhibitors in the prevention of breast cancer. The relative lack of toxicity associated with these compounds favors their use as chemopreventive agents, but the underlying mechanism of their chemopreventive effect remains unclear. We have observed that the COX-2 inhibitor celecoxib inhibits growth and induces apoptosis in the immortalized breast epithelial cell line 184htert. Microarray gene expression analysis of 184htert cells treated with 50 microM celecoxib for 6h revealed the modulation of several genes of interest, including a significant induction of expression of the mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 is a potent pro-apoptotic protein and growth inhibitor of breast cancer cells, which acts mainly by inhibiting the access of the mitogens IGF-I and IGF-II to their cell surface receptor, but also via IGF-independent effects. Quantitative real-time RT PCR demonstrated that 50 microM celecoxib induced a approximately 3-fold increase in expression of IGFBP-3 mRNA after 6h. Furthermore, ligand blot analysis revealed that celecoxib treatment was associated with the upregulation of IGFBP-3 at the protein level. IGFBP-3 (500 ng/ml) treatment of 184htert cells inhibited IGF-I and serum-induced proliferation, but had no effect on cell growth under serum-free conditions, indicating that IGF-independent effects of IGFBP-3 are not observed in this system. Our results suggest that celecoxib may decrease IGF-I-associated breast cancer risk by a mechanism involving induction of expression of IGFBP-3 and subsequent reduced proliferation of at-risk breast epithelial cells.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.     

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Background

Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.

Methodology/Principal Findings

In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.

Conclusions/Significance

This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.     

Ionizing radiation-induced apoptosis in ataxia-telangiectasia fibroblasts. Roles of caspase-9 and cellular inhibitor of apoptosis protein-1

Ionizing radiation (IR) has been shown to induce apoptosis to a greater extent in a fibroblast cell line AT5BIVA derived from an individual with ataxia-telangiectasia (AT) than in control fibroblasts. However, the signaling pathway that underlies IR-induced apoptosis in AT cells has remained unknown. The mechanism of apoptosis in response to gamma-irradiation has now been examined in three AT fibroblast lines (AT3BIVA, AT4BIVA, and AT5BIVA) derived from different individuals with AT. The apoptotic indexes of these cell lines at 72 h after irradiation were 12, 31, and 35%, respectively, compared with a value of 2.3% for control fibroblasts. Immunoblot analysis and fluorometric assays revealed that the extents of IR-induced activation of caspase-3 and caspase-9 were markedly greater in AT4BIVA and AT5BIVA cells than in AT3BIVA and control cells. Furthermore, the basal abundance of the apoptotic inhibitor, a cellular inhibitor of apoptosis proteins (c-IAP-1), was markedly reduced in AT4BIVA and AT5BIVA cells compared with that in AT3BIVA and control cells. The overexpression of either caspase-9 mutant forms or recombinant c-IAP-1 in AT5BIVA cells inhibited the IR-induced activation of caspases-3 and 9 and reduced the apoptotic index of the irradiated cells. These results indicate that the extent of IR-induced apoptosis in different AT cell lines is inversely related to the abundance of c-IAP-1 and directly related to the extent of activation of caspases-3 and 9.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Regulation of the Akt/Glycogen synthase kinase-3 axis by insulin-like growth factor-II via activation of the human insulin receptor isoform-A

Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Developmental and steroid hormonal regulation of insulin-like growth factor II expression

We have investigated the influence of steroid hormones on insulin-like growth factor II (IGF-II) expression. Hepatic IGF-II mRNA decreased gradually during postnatal development, reaching adult levels at 3 weeks of age. Treatment of 1-day-old rats for 4 days with 10 micrograms/day of the glucocorticoid dexamethasone (DEX) reduced IGF-II mRNA levels 10-fold in liver and inhibited body weight gain. Estradiol and testosterone did not affect IGF-II expression. A dose-response relationship between IGF-II mRNA levels and the different amounts of DEX injected was seen. IGF-II levels remained low after withdrawal of DEX, indicating an irreversible effect. Albumin expression was increased in newborn rat livers after DEX treatment. Our results suggest that glucocorticoids play an important role in the regulation of IGF-II expression. The mechanism for glucocorticoid-induced reduction of IGF-II mRNA is still unclear; however, our findings indicate that DEX inhibits IGF-II by causing premature differentiation of the liver.     

Roles of insulin-like growth factor II in cardiomyoblast apoptosis and in hypertensive rat heart with abdominal aorta ligation

Although IGF-II activating the IGF-II receptor signaling pathway has been found to stimulate cardiomyocyte hypertrophy, the role of IGF-II in cardiac cell apoptosis remains unclear. This study aimed to identify the roles of IGF-II and/or IGF-II receptors (IGF-II/IIR) in cardiomyoblast apoptosis and in hypertensive rat hearts with abdominal aorta ligation. Cultured rat heart-derived H9c2 cardiomyoblasts and excised hearts from Sprague-Dawley rats with 0- to 20-day complete abdominal aorta ligation, a model of ANG II elevation and hypertension, were used. IGF-II/IIR expression, caspase activity, DNA fragmentation, and apoptotic cells were measured by RT-PCR, Western blot, agarose gel electrophoresis, and TUNEL assay following various combinations of ANG II, IGF-II/IIR antibody, CsA (calcineurin inhibitor), SP-600125 (JNK inhibitor), SB-203580 (p38 inhibitor), U-0126 (MEK inhibitor), or Staurosporine (PKC inhibitor) in H9c2 cells. ANG II-induced DNA fragmentation and TUNEL-positive cells were blocked by IGF-II/IIR antibodies and antisense IGF-II, but not by IGF-II sense. IGF-II-induced apoptosis was blocked by IGF-IIR antibody and CsA. The increased gene expressions of IGF-II and -IIR induced by ANG II were reversed by U-0126 and Sp600125, respectively. Caspase 8 activities induced by ANG II were attenuated by U-0126, SP-600125, and CsA. DNA fragmentation induced by ANG II was totally blocked by SP-600125, and CsA and was attenuated by U-0126. In rats with 0- to 20-day complete abdominal aorta ligation, the increases in IGF-II/IIR levels in the left ventricle were accompanied by hypertension as well as increases in caspase 9 activities and TUNEL-positive cardiac myocytes. ANG II-induced apoptosis was reversed by IGF-II/IIR blockade and coexisted with increased transactivation of IGF-II and -IIR, which are mediated by ERK and JNK pathways, respectively, both of which further contributed to cardiomyoblast apoptosis via calcineurin signaling. The increased cardiac IGF-II, IGF-IIR, caspase 9, and cellular apoptosis were also found in hypertensive rats with abdominal aorta ligation.     

Overexpression of insulin-like growth factor II (IGFII) in ZR-75-1 human breast cancer cells: higher threshold levels of receptor (IGFIR) are required for a proliferative response than for effects on specific gene expression

Previous transfection experiments using a zinc-inducible expression vector have shown that overexpression of insulin-like growth factor II (IGFII) in MCF7 human breast cancer cells can reduce dependence on oestrogen for cell growth in vitro (DALY RJ, HARRIS WH, WANG DY, DARBRE PD. (1991) Cell Growth Differentiation 2, 457-464.). Parallel transfections now performed into another oestrogen-dependent human breast cancer cell line (ZR-75-1) yielded three clones of transfected ZR-75-1 cells that produced levels of zinc-inducible IGFII mRNA and secreted mature IGFII protein similar to those found in the transfected MCF7 cells. However, unlike in MCF7 cells, no resulting effects were found on cell growth in the ZR-75-1 clones, even though the ZR-75-1 clones possessed receptors capable of binding 125I-IGFI and showed a growth response to exogenously added IGFII. Medium conditioned by the ZR-75-1 clones could stimulate growth of untransfected MCF7 cells, indicating that the secreted IGFII protein was bioactive. Furthermore, zinc-induced IGFII was capable of increasing both pS2 mRNA levels and CAT activity from a transiently transfected AP1-CAT gene in the ZR-75-1 clones. Constitutive co-overexpression of the protein processing enzyme PC2 resulted in reduced levels of large forms of zinc-inducible IGFII, but zinc treatment still produced no effect on cell growth rate. Finally, however, constitutive co-overexpression of the type I IGF receptor (IGFIR) did result in zinc-inducible increased basal cell growth and reduced dependence on oestrogen for cell growth. These results demonstrate that while overexpression of IGFII per se was sufficient to deregulate MCF7 cell growth, the ZR-75-1 cells are limited in their proliferative response by their intrinsic receptor levels. However, although the proliferative response was limited, molecular responses (expression of pS2 and AP1-CAT) were not limited, indicating that different cellular responses can have different threshold receptor level requirements.     

Expression and function of insulin-like growth factor receptors on anti-CD3-activated human T lymphocytes.

The pattern of expression of receptors for insulin-like growth factors (IGF-I and IGF-II) and insulin was studied on monocyte-depleted human peripheral blood T cells activated via anti-CD3. Binding assays demonstrated the sequential appearance of receptors for IGF-I, IGF-II, and insulin on activated T cells. IGF-IR appeared early, their expression reaching maximum levels at or before the peak of cellular proliferation. IGF-IIR expression generally followed that of the IGF-IR and was more transient, with increases and decreases in expression paralleling the rise and decline of cellular proliferation. Insulin receptor expression remained low throughout the activation time course. The identity of the IGFR on anti-CD3-activated T cells was confirmed in affinity cross-linking experiments. These data demonstrated a 135,000 Mr peptide that specifically binds radiolabeled IGF-I and corresponds to the alpha subunit of the type I IGF-IR, and a 260,000 Mr peptide that specifically binds radiolabeled IGF-II and corresponds to the type II IGFR. We have additionally found that IGF-I and IGF-II (in nanomolar concentrations) produce as much as a threefold enhancement of T cell proliferation early in the activation process, correlating with the early appearance of IGF-IR. The effect of both IGF appeared to be mediated through the type I receptor, since an antibody (alpha IR3), which blocks binding to the alpha subunit of this receptor, inhibited enhancement by up to 83%. Furthermore, we have found expression of IGF-IR on T cells after activation to be associated with both CD4+ and CD8+ T cell subpopulations. These observations provide a foundation for investigating the contribution of IGF in regulating T cell proliferation, differentiation, and effector function.     

Reversal of cathepsin D routing modulation in MCF-7 breast cancer cells expressing antisense insulin-like growth factor II (IGF-II).

We previously demonstrated that expression of IGF-II modulates the routing of cathepsin D in MCF-7 cells. In our present study, we transfected antisense IGF-II into IGF-II secreting MCF-7 cells to test the hypothesis that blocking IGF-II may reduce the secretion of cathepsin D in breast cancer cells. The concentration of IGF-II in media conditioned by the antisense clone was reduced to almost undetectable levels. Likewise, Northern blotting analysis revealed that IGF-II mRNA was nearly undetectable in the antisense transfected cells. Metabolic labeling experiments performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes demonstrated that cathepsin D secretion was dramatically reduced. Similarly, a significant reduction in cathepsin D was observed when conditioned media and cell extracts were examined by Western blotting after a 48 h incubation. No changes in cathepsin D mRNA in antisense cells were detected by Northern blot analysis. We conclude that endogenous IGF-II may modulate the routing of cathepsin D by interfering with receptor trafficking in MCF-7 cells, and that this modulation is reversible. Abnormally high levels of IGF-II may alter this homeostasis, conferring on breast cancer cells an advantageous mechanism that promotes rapid growth, and may facilitate metastasis.