Histamine-stimulated expression of insulin-like growth factors in human glioma cells

Glioma tumour growth is associated with the expression of insulin-like growth factors I and II (IGFs) and of both type I and type II IGF receptors. It has also been shown that IGFs can stimulate proliferation of cultured glioma cells. We previously reported that histamine too can stimulate the growth of glioma cells in vitro. In this report, we study whether the histamine-induced growth of G47 glioma cells is mediated by the IGFs. We found that histamine stimulates the expression of both IGF-I and IGF-II mRNAs, as determined by a semiquantitative in situ hybridization analysis. Furthermore, incubation of G47 cells with histamine also induced cellular immunostaining for IGF-II. It could be shown that IGF-I-stimulated proliferation is inhibited by IGFBP-3, which decreases the availability of IGFs for binding to the IGF receptors, and by beta-galactosidase, which may decrease IGF binding to the type II IGF receptor, but is not inhibited by the anti-type I IGF receptor monoclonal antibody alphaIR3. However, neither IGFBP-3 nor beta-galactosidase nor alphaIR3 inhibited the histamine-induced proliferation. These results show that the growth-stimulatory effect of histamine is accompanied by the induction of IGFs. This histamine-induced growth stimulation is not mediated by activation of cell surface IGF receptors, although intracrine activation of type II IGF receptors may be involved.     

A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer

The 5-year survival rate for women with metastatic breast cancer is only 25-30%; thus, the need to improve treatment is apparent. Overexpression of insulin-like growth factor-I receptor (IGF-IR) correlates with poor prognosis and local recurrence. In this study, we addressed whether functional impairment of IGF-IR affects adhesion, invasion, and metastasis of breast cancer. Impairment of IGF-IR function was achieved by transfecting a dominant negative form of the receptor, termed 486stop, into MDA-MB-435 metastatic breast cancer cells. The protein product of 486stop is secreted extracellularly, resulting in a bystander effect. Cellular adhesion to laminin and collagen was inhibited 94 and 88%, respectively. Furthermore, 486stop inhibited insulin-like growth factor-I-stimulated invasion through collagen IV by 75%. The dominant negative receptor was secreted, as evidenced by the observation that MDA-MB-435 and MDA-MB-231 cells were prevented from binding to laminin by 90% when treated with conditioned medium (CM) from 486stop-transfected cells. CM also inhibited the invasion of MDA-MB-231 cells across collagen IV by 80%. Finally, CM made MDA-MB-231 cells 30% more sensitive to Taxol-induced cell death. Growth in soft agar was suppressed by 486stop, but growth in monolayer was unaffected. When injected into the mammary fat pad, 486stop did not significantly suppress growth of the primary tumor, but metastasis to the lungs, livers, lymph nodes, and lymph vessels was significantly decreased compared to the vector control. In conclusion, inhibition of IGF-IR resulted in suppression of adhesion, invasion, and metastasis, providing a mechanistic rationale for targeting IGF-IR in the treatment of metastatic breast cancer.     

Redundancy of autocrine loops in human osteosarcoma cells

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.     

The insulin-like growth factor-I receptor signaling pathways are important for tumorigenesis and inhibition of apoptosis

The biological actions of the insulin-like growth factors IGF-I and IGF-II are mediated by their activation of the IGF-IR, a transmembrane tyrosine kinase linked to the ras-raf-MAPK cascade. Functional IGF-IRs are required for the cell to progress through the cell cycle. Most importantly, cells lacking this receptor cannot be transformed by any of a number of dominant oncogenes, a finding that proves that the presence of the IGF-IR is important for the development of a malignant phenotype. Consistent with this role, the IGF-IR displays a potent antiapoptotic effect, both in vitro and in vivo. Because of its key role in the transformation process, the IGF-IR is actively studied as a potential therapeutic target in different types of neoplastic growth.     

Insulin-like growth factor I receptor prevents apoptosis and enhances neuroblastoma tumorigenesis

Autocrine stimulation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-II is one mechanism that allows cancer cells to maintain unregulated growth and to resist programmed cell death (PCD). SH-SY5Y and SHEP cells are cloned human neuroblastoma (NBL) lines originating from a single primary tumor. SH-SY5Y cells, which express abundant cell surface IGF-IR and produce IGF-II, exhibit serum independent growth and resist PCD due to hypoxia and hyperosmolar conditions. In contrast, SHEP cells, which produce no IGF-II and express five-fold fewer IGF-IRs, die in serum-free media or following exposure to metabolic stressors. To better understand the roles of IGF-IR and its ligand, IGF-II, in NBL carcinogenesis, we stably transfected SHEP cells with either IGF-II or IGF-IR. Unregulated expression of IGF-II did not alter the growth characteristics of SHEP/human IGF-II transfectants. In contrast, overexpression of IGF-IR allowed SHEP/IGF-IR transfectants to survive in media supplemented only by IGF-II. IGF-IR abundance correlated in a graded fashion with resistance to PCD in response to three different death-inducing paradigms: mitogen withdrawal, hyperosmolar metabolic stress, and treatment with etoposide. Our results suggest that adjuvant therapy aimed at reducing IGF-IR abundance may enhance chemotherapy-coupled apoptosis in the treatment of NBL.     

Deficient processing and activity of type I insulin-like growth factor receptor in the furin-deficient LoVo-C5 cells

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A small amount of successfully cleaved alpha/beta heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 x 10(3) sites/cell; Kd, 1.9 nM for IGF-I and 7.0 nM for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 x 10(4) sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as beta-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, suggesting that in LoVo-C5 cells only the small amount of mature type I IGF-IR binds IGFs with high affinity. To provide evidence for this idea, we showed that mild trypsin treatment of living LoVo-C5 cells partially restored alpha/beta cleavage of IGF-IR, and greatly enhanced (6-fold) the IGF-I binding capacity of LoVo-C5 cells, but did not restore IGF-IR signaling activity. Moreover, LoVo-C5 cells were totally unresponsive to IGF-I in terms of cell migration, in contrast to fully processed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved in the endoproteolytic processing of the IGF-IR and suggest that this posttranslational event might be crucial for its ligand binding and signaling activities. However, our data do not exclude that other proprotein convertases could participate to IGF-IR maturation.     

Structure-function of the human insulin-like growth factor-I receptor: a discordance of somatotroph internalization and signaling

Insulin-like growth factor-I (IGF-I), a GH-dependent growth factor, suppresses GH secretion by pituitary cells. To clarify the role of ligand-mediated receptor internalization for IGF-I signaling to GH, human IGF-I receptor (IGF-IR) cDNAs mutated in the beta-subunit were stably transfected into GC rat pituitary cells. Overexpression of wild-type IGF-IR markedly enhanced IGF-I suppression of GH 4-fold (P < 0.005) compared to that of untransfected cells. A mutant IGF-IR with a 943Tyr-->Ala substitution in the IGF-IR submembrane domain only partially suppressed GH (73% of IGF-IR wild type), while replacement of 957Tyr-->Ala or 940Gly-->Ala produced IGF-IRs that retained enhanced IGF-I signaling to GH. Substitution of 950Tyr-->Ala or 1003Lys-->Ala in the human IGF-IR beta-subunit failed to enhance IGF-I signaling to GH above that of untransfected cells. Intracellular phosphorylation of insulin-responsive substrate-I by these mutant IGF-IRs paralleled the observed IGF-I suppression of GH, with no phosphorylation of IRS-I by 950Tyr-->Ala. Ligand-mediated receptor internalization, however, was not reduced by substitution of either 943Tyr-->Ala or 950Tyr-->Ala. In contrast, substitution of 957Tyr-->Ala reduced the internalization of labeled IGF-I to 35% that of wild-type IGF-IR. Substitution of 1003Lys-->Ala abolished IGF-IR internalization, as expected. These results demonstrate that both 950Tyr and 943Tyr are important for IGF-I signaling to GH and that IGF-IR internalization is discordant for IGF-I signaling to the GH gene.     

Activation of protein kinase C-zeta and phosphatidylinositol 3'-kinase and promotion of macrophage differentiation by insulin-like growth factor-I

Phosphoinositides that are phosphorylated at the D3 position have been reported to activate an atypical, Ca2-independent protein kinase C (PKC) isoform designated PKC-zeta, and overexpression of this enzyme leads to monocytic differentiation. In this study, we cultured human HL-60 promyeloid cells with vitamin D3 and insulin-like growth factor-I (IGF-I), a 70-amino-acid peptide that activates phosphatidylinositol 3'-kinase (PI 3-kinase) in murine promyeloid cells. Two days later, the proportion of cells differentiating into macrophages in serum-free medium, as assessed by expression of the alpha-subunit of the beta2 integrin CD11b, increased from 5 +/- 1% to 25 +/- 3%. Addition of IGF-I increased the proportion of cells differentiating into CD11b-positive macrophages to 78 +/- 5%. In the absence of vitamin D3, IGF-I did not induce expression of CD11b (6 +/- 1%). The IGF-I-promoted macrophage differentiation was blocked specifically by preincubation of HL-60 cells with a mAb (alphaIR3) directed against the IGF type I receptor. Similarly, pretreatment of cells with either alphaIR3 or an IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b expression when cells were cultured with vitamin D3 in serum-containing medium. IGF-I, but not vitamin D3, caused a sevenfold increase in the enzymatic activity of both PI 3-kinase and atypical PKC-zeta. Inhibition of IGF-I-inducible PI 3-kinase with either wortmannin or LY294002 abrogated the IGF-I-induced activation of PKC-zeta and totally blocked the enhancement in macrophage differentiation caused by IGF-I. These data establish that PKC-zeta is a putative downstream target of PI 3-kinase that is activated during IGF-I-promoted macrophage differentiation.     

The IGF-I receptor in mitogenesis and transformation of mouse embryo cells: role of receptor number

The type 1 receptor for insulin-like growth factors (IGF-IR) plays an important role in the growth and transformation of several types of cells. We have investigated the role of IGF-IR number in IGF-I-mediated mitogenesis and transformation of mouse embryo fibroblasts. We have used R- cells (3T3-like cells originating from mouse embryos with a targeted disruption of the IGF-IR genes) transfected with a plasmid expressing the human IGF-IR cDNA to generate clones with receptor numbers ranging from zero to 10(6) receptors per cell. In this model, between 15,000 and 22,000 receptors per cell are sufficient to render mouse embryo cells competent to grow in serum-free medium supplemented solely with IGF-I. For growth in soft agar, 30,000 receptors per cell seem to be the minimum requirement. These experiments indicate that a small increment in the number of receptors per cell, well within the physiological range, can modulate the mitogenic and transforming activities of the IGF-IR in 3T3-like cells.     

The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions--comparison to other cytokines and growth factors

The role of insulin (INS), and insulin-like growth factor-I (IGF-I) in the regulation of human erythropoiesis is not completely understood. To address this issue we employed several complementary strategies including: serum free cloning of CD34+ cells, RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). In a serum-free culture model, both INS and IGF-I enhanced survival of CD34+ cells, but neither of these growth factors stimulated their proliferation. The influence of INS and IGF-I on erythroid colony development was dependent on a combination of growth factors used for stimulating BFU-E growth. When BFU-E growth was optimally stimulated with erythropoietin (EpO) + kit ligand (KL) the large erythroid colonies developed normally even in the absence of INS or IGF-I. However, the addition of both of these growth factors slightly enhanced colony size. On the other hand, if erythroid colonies were stimulated suboptimally with EpO + IL-3 only, INS or IGF-I increased the number of small erythroid bursts by approximately 30%. Both INS and IGF-I activated signal transduction in maturing human erythropoietic cells as determined by phosphorylation of the insulin receptor substrate-2 (IRS-2) protein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34+, c-kit-R+ marrow cells, and in cells isolated from BFU-E and CFU-GM colonies. Expression of INS-R protein on these cells was subsequently confirmed by cytofluorometry. In contrast, the receptor for insulin-like growth factor-I (IGF-IR) was not detected on CD34+ cells, and was first easily detectable on more differentiated cells derived from day 6 BFU-E and CFU-GM colonies. We conclude that INS and IGF-I may be survival factors for human CD34+ cells, but are not required during early erythropoiesis. In contrast, both growth factors may play some role at the final stages of erythroid maturation.     

Demonstration of interleukin-1 (IL-1) as an autocrine growth inhibitor in renal cancer cell line, TC-1

In this report, we demonstrated that Interleukin-6 (IL-6) production could be induced by stimulating renal cell carcinoma cell lines, namely ACHN, Caki-1 and TC-1 cells with Interleukin-1 beta (IL-1 beta). IL-1 beta had no effects on cell proliferation in ACHN cells. However, IL-1 beta could suppress cell proliferation in Caki-1 and TC-1 cells. Flow cytometric cell cycle analysis by double staining method with propidium iodide and Proliferating cell nuclear antigen (PCNA) disclosed IL-1 beta caused cell accumulation at G1 phase. Fine granules were visualized in perinuclear area of TC-1 cells treated with IL-1 beta under microscopy. High electron density granules and spherically dilated rough endoplasmic reticula were observed by electron microscopic examinations. In TC-1 cell culture, IL-1 beta excretion into the supernatant was demonstrated by bioassay and ELISA. These results suggest that IL-1 beta functions as an "autocrine growth inhibitor" against TC-1 cells. Half-maximal inhibition of IL-1 beta and IFN-alpha was 6.5 pg/ml, and 720 U/ml, respectively for TC-1 proliferation and combination of these cytokines showed enhanced activity in cell growth inhibition.     

Protective effect of the insulin-like growth factor I receptor on apoptosis induced by okadaic acid

Okadaic acid (OKA), a potent inhibitor of serine phosphatases at concentrations as low as 20-25 nM, induces apoptosis of R- mouse embryo fibroblasts, which are 3T3-like cells devoid of type 1 insulin-like growth factor receptors (IGF-IRs). From R- cells, we have generated (by stable transfection) cell lines with IGF-IR numbers ranging from 0 (R- cells) to >10(6) receptors per cell. The wild-type IGF-IR protects R- cells from OKA-induced apoptosis, its protective effect being exquisitely dependent on the number of receptors. A small increment in wild-type receptor number (from 15 x 10(3) to 22 x 10(3) receptors/cell) is sufficient to change R(-)-derived cells from sensitive to resistant to apoptosis. We have also studied the effect of various mutations of the IGF-IR on its ability to protect R(-)-derived cells from OKA-induced apoptosis. Our data indicate a correlation between protection from apoptosis and the ability of the receptor to respond to insulin-like growth factor I with mitogenesis.     

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.     

Luteinizing hormone-releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgen-independent prostate cancer cells

We have previously shown that LHRH agonists exert a direct and specific inhibitory action on the proliferation of the androgen-independent DU 145 prostate cancer cell line; however, the cellular mechanisms mediating this antiproliferative action are not well defined. It is well known that the insulin-like growth factor (IGF) system plays a crucial role in the local regulation of the growth of androgen-independent prostate cancer. The present experiments were performed to evaluate whether LHRH agonists might exert their antimitogenic effect by interfering with the activity of the locally expressed IGF system. To this purpose, the effects of the LHRH agonist Zoladex (LHRH-A) on 1) the mitogenic action of IGF-I, 2) the tyrosine phosphorylation of type 1 IGF-I receptor (IGF-IR), 3) the concentration of IGF-IR, and 4) the secretion of IGF-binding protein-3 were studied. The results obtained show that in DU 145 cells, LHRH-A 1) counteracts the mitogenic action of IGF-I in a dose-dependent manner, 2) prevents the IGF-I-induced tyrosine phosphorylation of the IGF-IR, 3) reduces the concentration of IGF-IR without affecting its Kd value, and 4) does not affect the secretion of IGF-binding protein-3 in the conditioned medium from these cells. These data suggest that LHRH agonists may inhibit the proliferation of human androgen-independent prostate tumor cells by interfering with some of the cellular mechanisms mediating the stimulatory action of the IGF system.     

p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced tyrosine phosphorylation in an osteosarcoma cell line: interaction between p53 and Sp1

The insulin-like growth factor-I receptor (IGF-IR) is involved in tumorigenesis. The aim of the present study was to investigate whether the IGF-IR is a physiological target for p53 in osteosarcoma cells. The p53-induced regulation of IGF-IR levels was studied in a tetracycline-regulated expression system. When expressed in Saos-2, osteosarcoma cells that lack p53, wild-type p53 decreased, whereas mutated p53 increased IGF-IR expression, and IGF-I-induced tyrosine phosphorylation of the IGF-IR. Similarly, wild-type p53 decreased IGF-I-induced tyrosine phosphorylation of IRS-1. A functional and physical interaction between p53 and Sp1, in the regulation of the IGF-R, was studied in osteosarcoma cells. Expression of p53 decreased IGF-IR promoter activity, whereas no effect on promoter activity was seen by Sp1 expressed alone. However, Sp1 counteracted the inhibitory effect of p53 on IGF-IR promoter activity in a dose-dependent manner. Furthermore, wild-type and mutated p53 were coimmunoprecipitated with Sp1, indicating a physical interaction between p53 and Sp1. In conclusion, p53 regulates IGF-IR expression, as reflected by a reduction in IGF-IR protein and a parallel reduction in IGF-I-induced tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma cell line. These data indicate that the IGF-I receptor is a physiological target for p53 in osteosarcoma cells. Furthermore, data supporting an interaction between p53 and Sp1 in the regulation of the promoter activity of IGF-IR are presented.     

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.     

Effect of the insulin-like growth factor I receptor on ionizing radiation-induced cell death in mouse embryo fibroblasts

We have investigated the effect of the insulin-like growth factor I receptor (IGF-IR) on ionizing radiation (IR)-induced cell death using the following two mouse embryo fibroblast cell lines: (i) R- cells with a null mutation of the IGF-IR gene, therefore expressing no endogenous IGF-IR; (ii) R+ cells derived from R- cells, a stable transfectant overexpressing the human IGF-IR. Numbers of R- cells began to detach from dishes and float into the medium about 48 h after 10 Gy of X-irradiation. Internucleosomal DNA fragmentation detected by agarose gel electrophoresis, which is characteristic of apoptosis, was observed in the floating R- cells, but not in the attached cells. Unexpectedly, morphological analysis of the floating cells 72 h after irradiation revealed that only about half of them showed apoptotic death and the rest showed a nonapoptotic, presumably necrotic, one. On the other hand, R+ cells retained more than 90% viability even 4 days after irradiation, and very few floating cells were observed. The G2 arrest was induced in both cell lines following irradiation and G2/M fractions similarly returned to normal levels by around 20 h after irradiation, indicating that the cell death which appeared thereafter in R- cells is mediated through mitosis. Significant induction of p53 following irradiation was not detected by Western blot analysis in either R- or R+ cells. Collectively, these results demonstrate that signal transduction pathways originating from the IGF-IR may be involved in preventing IR-induced apoptosis and necrosis without affecting cell cycle arrest or p53 pathways.     

Affinity for the insulin-like growth factor-II (IGF-II) receptor inhibits autocrine IGF-II activity in MCF-7 breast cancer cells

We have investigated the autocrine regulation of insulin-like growth factor-II (IGF-II) signaling by the insulin-like growth factor-I receptor (IGF-IR) and the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) in MCF-7 breast cancer cells, employing retroviruses encoding both IGF-I, IGF-II, and IGF-I and II mutants with reductions in affinity for either the IGF-IR or the IGF-IIR. These studies revealed reciprocal roles for IGF-IR and IGF-IIR affinity in the regulation of autocrine IGF-II activity. IGF-IR affinity was required for serum-free proliferation but also for efficient IGF-II secretion. In contrast, cellular proliferation, receptor tyrosine kinase-dependent signaling, and extracellular IGF-II protein accumulation were all reduced in the presence of IGF-IIR affinity. Inhibition of IGF-II signaling appeared to be the sole consequence of IGF-IIR affinity, as no cellular responses attributable to selective IGF-IIR binding by a reduced IGF-IR affinity IGF-II mutant could be detected. By operating as an IGF-II antagonist, the IGF-IIR has tumor suppressor-like properties, a suggestion consistent with reports of loss of heterozygosity at the IGF-IIR locus in a variety of human malignancies.     

The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions

The goals of this study were to determine the cellular sites of insulin-like growth factor (IGF) and IGF type-I receptor (IGF-IR) expression and to begin to elucidate functional roles for the IGFs during postnatal development of the murine mammary gland. Using in situ hybridization analyses, we determined that IGF-I, IGF-II, and IGF-IR messenger RNAs were expressed in the highly proliferative terminal end buds during pubertal ductal growth. Consistent with these data, IGF-I (in combination with mammogenic hormones) promoted ductal growth in pubertal stage mammary glands cultured in vitro. During postpubertal and pregnancy stages, IGF-II and IGF-IR continued to be expressed in ductal epithelium. Expression of IGF-II in ductal and alveolar epithelium correlated with the pattern of rapidly proliferating cells, as determined by incorporation of 5-Bromo-2'-deoxyuridine, suggesting a potential autocrine or paracrine role for IGF-II as a mitogen for ductal epithelial cells. IGF-I expression was reinitiated in mammary epithelium in the differentiated alveoli at the end of pregnancy, suggesting an additional role for this factor in maintenance of the alveoli during lactation. Taken together, these data support an in vivo role for locally-produced IGFs in promoting ductal growth during puberty and suggest that IGF-I and IGF-II may have distinct functions during pregnancy-induced alveolar development.