I Insulin-like Growth Factor Research Articles

Resveratrol inhibits Erk1/2‐mediated adhesion of cancer cells via activating PP2A–PTEN signaling network

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin-like growth factor (IGF)-1-stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant-negative mitogen-activated protein kinase kinase 1 (MKK1) strengthened resveratrol's inhibition of the basal or IGF-1-stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)-Akt were implicated in resveratrol-inactivated Erk1/2-dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant-negative PP2A rendered resistance to resveratrol's suppression of the basal or IGF-1-stimulated phospho-Erk1/2 and cell adhesion, whereas expression of wild-type PP2A enhanced resveratrol's inhibitory effects. Overexpression of wild-type PTEN or dominant-negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol's inhibition of the basal or IGF-1-stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol's inhibitory effects on the basal and IGF-1-induced inhibition of PP2A-PTEN, activation of Akt-Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A-PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.

Interaction between cyclooxygenase-2 and insulin-like growth factor in breast cancer: A new field for prevention and treatment

The objective of this study was to evaluate the correlation between cyclooxygenase-2 (COX-2) and markers of cell proliferation and apoptosis, including, Bcl-2, Bax, Ki-67 and the type I insulin-like growth factor (IGF) receptor (IGF1-R) in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC), present in the same surgical specimen. A total of 110 cases were evaluated using tissue microarrays. Cases were classified in scores from 0 to 3 according to pre-defined methods. The results showed that the positivity rates were COX-2 in 87% of cases in DCIS and IDC; Bcl-2 in 55% of cases in DCIS and IDC; Bax in 23% of cases in IDC and 19% in DCIS, IGF-1 in 24% of cases in DCIS and IDC; and Ki-67 in 81% of cases in DCIS and IDC. We also observed a positive correlation between the expression of COX-2 and IGF1-R (p=0.045). Our results demonstrate a positive correlation between the expression of COX-2 and IGF1-R in DCIS and IDC, demonstrating that they are involved in breast cancer carcinogenesis. Further studies are required to prove the effectiveness of COX-2 and IGF1-R inhibitors for the prevention and treatment of breast cancer, as well as to explain their mechanism of action.

How to Train Your Biomarker

Type I insulin-like growth factor (IGF) receptor (IGF1R) inhibitors are new cancer therapies. Pitts and colleagues used in vitro data to "train" a predictive biomarker for an IGF1R tyrosine kinase inhibitor. Given the complexity of IGF signaling, additional layers of biomarker analysis will likely be needed to develop predictive factors.

A candidate targeting molecule of insulin-like growth factor-I receptor for gastrointestinal cancers

Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage. One new group of targets is tyrosine kinase receptors, which can be treated by several strategies, including small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs). Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal (GI) carcinomas. The concept of targeting specific carcinogenic receptors has been validated by successful clinical application of many new drugs. Type I insulin-like growth factor (IGF) receptor (IGF-IR) signaling potently stimulates tumor progression and cellular differentiation, and is a promising new molecular target in human malignancies. In this review, we focus on this promising therapeutic target, IGF-IR. The IGF/IGF-IR axis is an important modifier of tumor cell proliferation, survival, growth, and treatment sensitivity in many malignant diseases, including human GI cancers. Preclinical studies demonstrated that downregulation of IGF-IR signals reversed the neoplastic phenotype and sensitized cells to anticancer treatments. These results were mainly obtained through our strategy of adenoviruses expressing dominant negative IGF-IR (IGF-IR/dn) against gastrointestinal cancers, including esophagus, stomach, colon, and pancreas. We also summarize a variety of strategies to interrupt the IGFs/IGF-IR axis and their preclinical experiences. Several mAbs and TKIs targeting IGF-IR have entered clinical trials, and early results have suggested that these agents have generally acceptable safety profiles as single agents. We summarize the advantages and disadvantages of each strategy and discuss the merits/demerits of dual targeting of IGF-IR and other growth factor receptors, including Her2 and the insulin receptor, as well as other alternatives and possible drug combinations. Thus, IGF-IR might be a candidate for a molecular therapeutic target in human GI carcinomas.

Role of the insulin receptor in IGF-system signaling and biology.

Abstract Abstract #4055 The role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in breast cancer has been extensively studied, and has led to the development of several IGF1R inhibitors currently in clinical trials. The closely related insulin receptor (IR) has been shown to contribute to IGF signaling through the formation of IGF1R/IR-hybrid receptors and through a homodimer of an alternatively spliced form of the IR (IR-A). Understanding the role of the IR in cancer biology will help determine which receptor(s) in the IGF system should be targeted to efficiently inhibit IGF signaling. Our previous work has shown that selective downregulation of IGF1R by siRNA resulted in enhanced sensitivity to insulin through increasing the numbers of insulin receptor homodimers. To determine how downregulation of IR affected IGF1R signaling, we used T47D cells, a breast cancer cell line expressing both the IR and IGF1R, as our model system. We transiently downregulated IR levels using siRNA and evaluated signaling. 5 nM of insulin activated downstream signaling molecules, such as insulin receptor substrate 1(IRS-1) and Akt in control cells, but this response was diminished in the IR-downregulated cells. In contrast, 5 nM of IGF-I did not result in differences in signaling between IR-downregulated cells and control cells. Using IR shRNA, we created stable clones with IR levels reduced by more than 72%. IR shRNA clones demonstrated decreased insulin binding, as measured by 125I-insulin binding assay. Additionally, clones showed decreased response to insulin signaling. 5 nM of insulin activated IRS-1 and Akt in parental T47D cells, but this response was attenuated in the IR-downregulated clones. Proliferation was also decreased in response to 5 nM insulin in the clones compared to parental cells. In contrast, IGF-I mediated signaling was not altered in clones when compared to vector control cells. Further, no change in proliferation was observed in response to IGF-I between wild-type and IR shRNA clones. We also generated stable IR shRNA clones using LCC6 cells. Insulin signaling was reduced in LCC6 clones when compared to wild-type LCC6 cells. However, in contrast to the T47D clones, IGF-I signaling was increased in LCC6 IR shRNA clones. Thus, the role of the insulin receptor in cancer biology may be dependent upon the ratio of IR, IGF1R, and the hybrid receptor. If cells contain mostly homodimers of IGF1R and IR, then downregulation of IR may affect only insulin response. However, if there are high levels of hybrid receptor, then downregulation of IR could affect, and even augment, IGF1R signaling. Our data suggest that the ratio and confirmation states of both IR and IGF1R should be understood to predict the effect of receptor downregulation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4055.

Comparative breast tumor imaging to identify the expression of type I IGF receptor (IGF1R) by anti-IGF1R antibody-conjugated small molecule fluorophore or quantum dots in vivo.

Abstract Abstract #6001 The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane receptor tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several antibodies against IGF1R, including some in clinical trials, downregulate the level of IGF1R both in cell line studies and in in vivo animal experiments. Thus, this downregulation is a biodynamic marker of antibody delivery to the tumor. To date, identification of IGF1R expression and its downregulation in vivo in a non-invasive way is still challenging. In this study, we sort to use two different fluorescent technologies, small molecule fluorophores, or quantum dots (QDs), to detect receptor expression and its downregulation by antibodies in vivo. Alexa 680 is a small molecule fluorophore that can be excited with a peak emission at 705 nm. QDs are nanocrystals that emit fluorescence upon excitation, and QDs with a peak emission at 705 nm was used in this study. Both Alexa 680 and QDs were conjugated with AVE-1642, a humanized anti-IGF1R antibody developed by sanofi-aventis. After conjugation, they both were able to detect the expression of IGF1R and its downregulation in in vitro cell line studies. We used mouse xenograft tumor models to examine their efficacies in vivo. AVE-1642 conjugated Alexa 680 or QDs (705 nm) was intravenously delivered to mice bearing mammary xenograft tumors that express IGF1R. After 24 hours of circulation, Alexa 680 fluorescence was shown to solely localize to the xenograft tumor, with little non-specific targeting to other tissue or organs in mice. In addition, Alexa 680 fluorescence was diminished in tumors that had downregulated IGF1R by antibody pretreatment. In contrast, conjugated QDs fluorescence was mainly localized to liver, spleen, bone marrow and lymph nodes, with very small amount of QDs in the xenograft tumors. The QDs fluorescence in xenograft tumors remained unchanged even when IGF1R was downregulated by antibody pretreatment. Furthermore, we identified that QDs were mainly localized to the hepatic sinusoids, and subsequently engulfed by Kupffer cells. Taken together, our data suggest that small molecule fluorophores, such as Alexa 680, are useful agents to detect the expression and downregulation of IGF1R in vivo. Our data provide valuable insight not only into the clinical development of anti-IGF1R antibodies, but also to other targeted therapy as well. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6001.

Detection and downregulation of type I IGF receptor expression by antibody-conjugated quantum dots in breast cancer cells

The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several monoclonal antibodies directed against the receptor are in clinical trials. In order to develop a methodology to detect and measure IGF1R levels in breast cancer cells, we covalently conjugated an IGF1R antibody, AVE-1642, with quantum dots (Qdots), which are nanocrystals that emit fluorescence upon excitation. AVE-1642 Qdots only bound to cells that express IGF1R, and measured IGF1R levels by fluorescence emission at 655 nm. After binding to the cell surface, AVE-1642 Qdots underwent receptor mediated endocytosis, localized to endosome, and later translocated into the nucleus. Treating MCF-7 cells with AVE-1642 Qdots, but not unconjugated Qdots alone, downregulated IGF1R levels and rendered cells refractory to IGF-I stimulation. Furthermore, cell proliferation was slightly inhibited by AVE-1642 Qdots, but not the unconjugated Qdots. Our data suggest that AVE-1642 Qdots can be used to detect IGF1R expression and measure changes in cell surface receptor levels. In addition, the inhibitory effect of AVE-1642 Qdots to cell proliferation implies that it may serve as a traceable therapeutic agent.

Vitamin C suppresses proliferation of the human melanoma cell SK‐MEL‐2 through the inhibition of cyclooxygenase‐2 (COX‐2) expression and the modulation of insulin‐like growth factor II (IGF‐II) production

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.

Differential regulation of insulin receptor substrate-1 degradation during mannitol and okadaic acid induced apoptosis in human neuroblastoma cells

Insulin receptor substrate (IRS) proteins are major docking molecules for the type I insulin like growth factor (IGF) receptor (IGF-IR) and mediate their effects on downstream signaling molecules. In this report, we investigated IRS-1 regulation during apoptosis in human neuroblastoma SH-EP cells. Treatment of SH-EP cells with mannitol or okadaic acid (OA) induces apoptosis with the typical characteristics of anoikis. Mannitol treatment results in IRS-1 degradation with concomitant appearance of smaller fragments, likely representing caspase cleavage products. In contrast OA-induced IRS-1 degradation is accompanied by a mobility shift in IRS-1, suggesting IRS-1 serine/threonine phosphorylation. Mannitol-induced, but not OA-induced, degradation is blocked by IGF-I. Pretreatment of the cells with caspase or proteasome inhibitors also partially blocks mannitol-induced IRS-1 degradation. These results suggest two independent pathways are involved in IRS-1 degradation; one pathway is dependent on caspase activation and is blocked by IGF-I, while a second pathway is caspase-independent and IGF-I-insensitive.

A Dominant Negative Type I Insulin-like Growth Factor Receptor Inhibits Metastasis of Human Cancer Cells

We have previously shown that LCC6 wild-type (WT) cells, a metastatic variant of MDA-MB-435 cancer cells originally derived from a breast cancer patient, exhibit enhanced motility in response to IGF-I compared with the parent MDA-MB-435 cells. To further understand the role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in cancer metastasis we inhibited signaling via IGF1R using a C-terminal-truncated IGF1R. The truncated receptor retains the ligand binding domain but lacks the autophosphorylated tyrosine residues in the carboxyl terminus. Cells stably transfected with this truncated receptor (LCC6-DN cells) overexpressed the truncated IGF1R messenger RNA nearly 50-fold over endogenous receptor. The truncated receptor in the LCC6-DN cells behaved in a dominant negative manner to inhibit endogenous IGF1R activation by IGF-I. Compared with the LCC6-WT cells, LCC6-DN cells failed to phosphorylate the adaptor proteins insulin receptor substrate-1 and -2 in response to IGF-I and did not activate Akt after exposure to IGF-I. Unlike LCC6-WT cells, LCC6-DN cells did not show enhanced motility in response to IGF-I. To assay for metastasis, LCC6-WT and LCC6-DN cells were injected into the mammary fat pads of mice, and the primary xenograft tumors were removed after 21 days. Mice sacrificed 5 weeks later showed multiple lung metastases derived from LCC-WT xenografts, whereas mice harboring LCC6-DN xenografts showed no lung metastases. Our data show that IGF1R can regulate several aspects of the malignant phenotype. In these cells, metastasis but not proliferation requires IGF1R function.

Insulin-like growth factor binding proteins in follicular fluid from morphologically distinct healthy and atretic bovine antral follicles.

In bovine follicles 2-5 mm in diameter, two morphologically distinct types of healthy follicles and two types of atretic follicles have been described recently. Healthy follicles either have columnar basal granulosa cells with follicular basal lamina composed of many layers or 'loops' or they have rounded basal cells with a conventional single-layered, aligned follicular basal lamina. In atretic follicles, cell death either commences at the basal layer and progresses to the antrum (basal atresia) with macrophage penetration of the membrana granulosa or death progresses from the antrum in a basal direction (antral atresia). Little is known about how these different phenotypes develop. To determine whether insulin-like growth factor binding protein (IGFBP) levels in follicular fluid differ between these different types of follicles, we measured IGFBP levels in fluids from these follicles. A total of 61 follicles were assessed by light microscopy and characterized by morphological analysis as either healthy, with columnar or rounded basal granulosa cells, or as undergoing antral or basal atresia. The IGFBP concentration in the follicular fluid of individual follicles from the four groups (n = 12-20 per group) was identified by Western ligand blots using (125)I-insulin-like growth factor (IGF)-II as a probe. Insulin-like growth factor binding proteins 2, 3 (44 and 40 kDa), 4 (glycosylated and non-glycosylated) and 5 were observed. The levels (per volume of fluid) of IGFBPs 2, 4 and 5 were greater in atretic follicles than in healthy follicles. However, there were no statistical differences in levels of each IGFBP between either the two types of healthy follicle or between the two types of atretic follicles. Thus, IGFBP levels are not related to the different types of healthy or atretic follicles.

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.

Insulin-like growth factors control the regulation of oestrogen and progesterone receptor expression by oestrogens

Ligands for the type I insulin-like growth factor (IGF) receptor interact with oestrogens to control the proliferation of oestrogen responsive breast cancer cells. The aim of this study was to determine the involvement of ligands for the type I IGF receptor in the regulation of oestrogen receptor (OR) expression by oestrogens and antioestrogens in these cells. Oestrogen decreased OR mRNA levels in MCF-7 cells whereas it increased them in T47D, EFM-19 and ZR-75 cells. In MCF-7 cells, IGF-I and insulin lowered further OR expression in the presence of oestrogen. In the presence of IGF-I or insulin, the induction of progesterone receptor mRNA by oestradiol was considerably attenuated in MCF-7 cells, showing that the enhanced down-regulation of OR mRNA levels influenced the expression of oestrogen-regulated genes. The oestrogen agonist activity of the antioestrogens tamoxifen and ICI 182 780 for the down-regulation of OR expression in MCF-7 cells was modulated by type I IGF receptor ligands. Overall these experiments show that OR expression is differentially regulated by oestrogen in individual oestrogen-responsive breast cancer cell lines. Ligands for the type I IGF receptor can modulate regulation of OR expression by oestrogens and antioestrogens principally in cells in which oestrogens down-regulate OR expression.

Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol.

Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5-fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor.

Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol

Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5-fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor.

Type I insulin-like growth factor receptors in human colorectal cancer

Type I insulin-like growth factor (IGF) receptors have been recently characterised in human colorectal cancers. The aim of this study was to determine whether type I IGF receptor concentration may be related to prognostic variables in colorectal cancers. Saturation experiments with [125I]IGF-I were performed on membrane preparations of 46 frozen specimens (20 tumours, 26 controls) and analysed according to the Scatchard method. In all the studied cases, we found a single class of high affinity binding sites in both normal and malignant colorectal tissues (median 0.17 and 0.15 nmol/l, respectively). Using paired analysis, we found no significant difference in terms of type I IGF receptor concentration between malignant and normal colorectal tissues. There was also no relationship between type I IGF receptors and any of the tumour characteristics studied. This study does not support a critical role of the type I IGF receptors in the clinical management of colorectal cancers.

Insulin-like growth factor-I (IGF-I) and its binding protein IGFBP-4 in human prostatic hyperplastic tissue: gene expression and its cellular localization.

It has been previously reported that 1) type I insulin-like growth factor (IGF) receptors are present in the human prostatic tissue; 2) IGF-I receptors are mainly localized in the epithelial cells; 3) IGF-I is a mitogen for prostatic epithelial cells in culture; and 4) IGF-binding proteins (IGFBPs) are released by these cells in the conditioned medium. To add information on the mechanism of IGF-I action in the human prostate, we studied the expression and cellular localization of mRNA encoding IGF-I and IGFBP-4 in human prostatic hyperplastic (BPH) tissue. Northern analysis of total RNA extracted from BPH tissues with cDNA probes containing the entire coding regions for IGF-I and IGFBP-4 documented the presence of multiple IGF-I mRNA transcripts with lengths of 7.5, 1.7, 1.3, and 1.1 kilobases and a single 2.1-kilobase transcript of IGFBP-4 mRNA. In situ hybridization with the cDNA probes used for Northern analysis and with cRNA probes synthesized from the respective cDNA demonstrated that IGF-I mRNA was only localized in the stromal cells, whereas IGFBP-4 mRNA was predominantly expressed by epithelial cells. In addition, immunoreactive IGF-I was measured in BPH tissue extracts after acidification and reverse phase chromatography. The mean (+/- SD) IGF-I content of six BPH tissues was 28.1 +/- 4.0 ng/g tissue. Our results suggest that in the human prostate, the locally secreted IGF-I exerts its principal biological effects with a paracrine mode of action and demonstrate that IGFBP-4 is mainly expressed by IGF-I target cells.

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.

Expression of a type I insulin-like growth factor receptor with low affinity for insulin-like growth factor II.

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.

In vivo regulation of granulosa cell type I insulin-like growth factor receptors: evidence for an inhibitory role for the putative endogenous ligand(s) of the ovarian gonadotropin-releasing hormone receptor.

The putative endogenous occupant(s) of the ovarian GnRH receptor may play a role as an intraovarian regulator. In this communication we explore the possibility that in vivo activation of the ovarian GnRH receptor may prove heteroregulatory to the murine granulosa type I insulin-like growth factor (IGF) receptor complement. To eliminate potentially confounding pituitary involvement, exclusive use was made of hypophysectomized rats, thereby allowing study of the direct ovarian effect(s) of GnRH receptor ligands. Treatment of immature hypophysectomized rats, thereby allowing study of the direct ovarian effect(s) of GnRH receptor ligands. Treatment of immature hypophysectomized diethylstilbestrol-primed rats with a GnRH agonist (25 micrograms/rat, twice daily) for 2.5 days resulted in a 2.1-fold decrease in FSH (10 micrograms/rat, twice daily)-inducible (but not basal) specific [125I]IGF-I binding to isolated granulosa cells. Scatchard analysis of the binding data revealed this effect to be due in large measure to decreased binding capacity (46% inhibition) rather than affinity (2.5 x 10(-9) M). Although in vivo treatment with a GnRH antagonist (25 micrograms/rat, twice daily) by itself proved without significant effect on the specific binding of IGF-I to isolated granulosa cells, the concurrent provision of a minimally effective dose of FSH (1 microgram/rat, twice daily) resulted in a 2.8-fold amplification of the FSH effect consequent to enhanced binding capacity (110%), but not affinity (2.2 x 10(-9) M). Significantly, this level of binding proved comparable to that induced by a maximally effective dose of FSH when used by itself. Combined pretreatment with identical doses of both peptide analogs had little or no effect on granulosa cell IGF-I binding, suggesting stereospecificity of action and mutual neutralization by the opposing actions of the GnRH receptor ligands employed. The ability of ligands of the GnRH receptor to alter the granulosa cell type I IGF receptor complement proved functionally significant, as assessed by corresponding alterations in IGF-I hormonal action. Indeed, pretreatment with a GnRH antagonist resulted in a 2.1-fold enhancement of IGF-I (50 ng/ml)-supported proteoglycan biosynthesis, with the agonistic analog producing a diametrically opposite effect. Moreover, the ovarian effects of GnRH receptor ligands were not limited to type I IGF receptor binding; comparable effects were noted on basal and hCG-stimulated accumulation of progesterone by cultured granulosa cells, ovarian weight, ovarian protein content, as well as size and number of antral follicles.(ABSTRACT TRUNCATED AT 400 WORDS)