Differential insulin‐like growth factor (IGF)‐independent interactions of IGF binding protein‐3 and IGF binding protein‐5 on apoptosis in human breast cancer cells. Involvement of the mitochondria.

We have demonstrated previously in Hs578T cells that insulin‐like growth factor binding protein (IGFBP)‐3 can significantly accentuate ceramide (C2)‐induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine‐glycine‐aspartic acid (RGD)‐containing peptide]. In contrast we found that IGFBP‐5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF‐independent actions of IGFBP‐3 and IGFBP‐5 on C2 and RGD‐induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 μmol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 μmol/L) significantly inhibited C2‐induced apoptosis, whereas it significantly accentuated RGD‐induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP‐3 but is not affected by IGFBP‐5. These data indicate that IGFBP‐3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP‐5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria. J. Cell. Biochem. 80:248–258, 2000. © 2000 Wiley‐Liss, Inc.     

Insulin-like growth factor (IGF)-independent effects of IGF binding protein-4 on human granulosa cell steroidogenesis

The ovarian insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system operates to permit maximal stimulation of steroidogenesis in the dominant follicle. In atretic follicles, the predominant IGFBPs are IGFBP-2 and IGFBP-4, which appear to be selectively cleaved in healthy follicles. We have recently demonstrated potent inhibition by IGFBP-4 of both theca and granulosa cell steroid production. The degree to which the inhibition occurred suggested that it was greater than might be expected by sequestration of IGF alone. Our study was designed to test this idea. Granulosa cells were harvested from follicles dissected intact from patients undergoing total abdominal hysterectomy and bilateral salpingoophorectomy. Granulosa cells were incubated with or without gonadotropins and IGFBP-4 in the presence or absence of either the IGF type I receptor blocker alphaIR3 or excess IGFBP-3 to remove the effects of endogenous IGF action. Steroid accumulation in the medium was assessed. IGFBP-4 continued to exert potent inhibitory effects when the action of endogenous IGF was removed from the system, demonstrating that its actions are independent of IGF binding. There was no effect on cell metabolism, and the effects on steroidogenesis were reversible after IGFBP-4 removal from the culture medium. No similar effects were seen with IGFBP-2. These reasults are the first evidence of IGF-independent IGFBP-4 actions and the first evidence of IGF-independent actions of any IGFBPs in the ovary.     

Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.     

Oncogenic ras causes resistance to the growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3) in breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-ras oncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic ras expression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

Promotion of cancer cell migration: an insulin-like growth factor (IGF)-independent action of IGF-binding protein-6

A family of six high affinity IGF-binding proteins (IGFBPs 1-6) plays an important role in modulating IGF activities. Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. However, very little is known about possible IGF-independent actions of IGFBP-6. We have generated a non-IGF-binding IGFBP-6 mutant by substituting Ala for four amino acid residues (Pro(93)/Leu(94)/Leu(97)/Leu(98)) in its N-domain IGF-binding site. A >10,000-fold loss of binding affinity for IGF-I and IGF-II was observed using charcoal solution binding assay, BIAcore biosensor, and ligand blotting. Wild-type and mutant IGFBP-6, as well as IGF-II, induced cell migration in RD rhabdomyosarcoma and LIM 1215 colon cancer cells. Cell migration was mediated by the C-domain of IGFBP-6. Transient p38 phosphorylation was observed in RD cells after treatment with IGFBP-6, whereas no change was seen in phospho-ERK1/2 levels. Phospho-JNK was not detected. IGFBP-6-induced cell migration was inhibited by SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of ERK1/2 MAPK activation. In contrast, SP600125, a JNK MAPK inhibitor, had no effect on migration. Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells.

Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.     

Regulation of insulin-like growth factor (IGF) bioactivity by sequential proteolytic cleavage of IGF binding protein-4 and -5

The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.     

The effects of insulin-like growth factor binding protein-3 (IGFBP-3) on T47D breast cancer cells enriched for IGFBP-3 binding sites

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca2+) and magnesium (Mg2+) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg(-1), I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg(-1) urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca2+ and Mg2+ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 +/- 2.42 mg dl(-1) (n = 44) and >500 mg dl(-1) (n = 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 +/- 0.05 ul min(-1) (n = 10) and 1.28 +/- 0.16 ul min(-1) (n = 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca2+]i in control and diabetic fura-2-loaded acinar cells was 109.40 +/- 15.41 nM (n = 15) and 130.62 +/- 17.66 nM (n = 8), respectively. CCK-8 (10(-8)M) induced a peak response of 436.55 +/- 36.54 nM (n = 15) and 409.31 +/- 34.64 nM (n = 8) in control and diabetic cells, respectively. Basal [Mg2+]i in control and diabetic magfura-2-loaded acinar cells was 0.96 +/- 0.06 nM (n = 18) and 0.86 +/- 0.04 nM (n = 10). In the presence of CCK-8 (10(-8)) [Mg2+]i in control and diabetic cells was 0.80 +/- 0.05 nM (n = 18) and 0.60 +/- 0.02 nM (n = 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca2+ and Mg2+ homeostasis.     

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.     

Differential IGF-independent effects of insulin-like growth factor binding proteins (1-6) on apoptosis of breast epithelial cells

We have demonstrated previously that insulin-like growth factor binding protein (IGFBP)-3 alone has little growth inhibitory effect on Hs578T human breast cancer cells, but that it can dramatically accentuate the apoptotic response to the physiological trigger, ceramide, in an IGF-independent manner. We have now studied the potential of other IGFBPs (1-6) to interact with apoptotic signalling pathways. Hs578T cells were preincubated with a binding protein (100 ng/ml) for 24 h, followed by co-incubation of the binding protein with an apoptotic dose of ceramide or RGD-containing peptide for a further 24 h. Apoptosis was assessed using flow cytometry, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) assay and morphological assessment. Binding protein profiles were determined using ligand and immunoblotting techniques. Each of the IGFBPs (1-6) alone had no significant (P > 0. 05) growth inhibitory effects relative to control cells. In contrast to IGFBP-3, which significantly (P < 0.05) accentuated C2-induced apoptosis, IGFBP-1, -2, and -6 had no effect, whereas IGFBP-4 and -5 each caused marked (P < 0.01) inhibition of ceramide-induced programmed cell death. Apoptosis induced by RGD was also significantly (P < 0.05) reduced by IGFBP-5, whereas IGFBP-3 had no effect. These data provide evidence to suggest that individual IGFBPs have specific IGF-independent effects and act differentially on apoptotic signalling pathways.     

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating the IGF-I receptor to stimulate cell survival and proliferation. To date, IGF-independent actions only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. To assess the general importance of IGF-independent mechanisms, we have generated human IGFBP-3 mutants that cannot bind IGF-I or IGF-II by substituting alanine for six residues in the proposed IGF binding site, Ile(56)/Tyr(57)/Arg(75)/Leu(77)/Leu(80)/Leu(81), and expressing the 6m-hIGFBP-3 mutant construct in Chinese hamster ovary cells. Binding of both IGF-I and IGF-II to 6m-hIGFBP-3 was reduced >80-fold. The nonbinding 6m-hIGFBP-3 mutant still was able to inhibit DNA synthesis in a mink lung epithelial cell line in which inhibition by wild-type hIGFBP-3 previously had been shown to be exclusively IGF-independent. 6m-hIGFBP-3 only can act by IGF-independent mechanisms since it is unable to form complexes with the IGFs that inhibit their action. We next compared the ability of wild-type and 6m-hIGFBP-3 to stimulate apoptosis in serum-deprived PC-3 human prostate cancer cells. PC-3 cells are known to synthesize and respond to IGF-II, so that IGFBP-3 could potentially act by either IGF-dependent or IGF-independent mechanisms. In fact, 6m-hIGFBP-3 stimulated PC-3 cell death and stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence as wild-type hIGFBP-3. These results indicate that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells and may play a wider role in the antiproliferative and antitumorigenic actions of IGFBP-3.     

Growth hormone-dependent insulin-like growth factor (IGF) binding protein from human plasma differs from other human IGF binding proteins

A growth hormone-dependent binding protein for insulin-like growth factors (IGF-I and IGF-II) has been isolated from human plasma. Analyzed on SDS gels, the preparation contained a major protein band of 53 kDa, and a minor band of 47 kDa. After transfer to nitrocellulose, both species bound iodinated IGF-I, and could be detected using an antibody raised against the purified preparation. In contrast, an IGF binding protein purified from human amniotic fluid bound IGF-I but was not detectable immunologically. The amino acid comparison of the plasma binding protein preparation was different from that reported for amniotic fluid and HEP G2 hepatoma proteins, and the unique amino-terminal sequence, Gly-Ala-Ser-Ser-Ala-Gly-Leu-Gly-Pro-Val-, was different from that of the amniotic fluid and hepatoma proteins. This study indicates that the growth hormone-dependent IGF binding protein of human plasma is structurally and immunologically distinct from other IGF binding proteins.     

The aromatase inhibitor letrozole in advanced breast cancer: Effects on serum insulin-like growth factor (IGF)-I and IGF-binding protein-3 levels

Serum insulin-like growth factor (IGF)-I and IGF-binding protein-3 levels were measured in two groups of postmenopausal women with advanced breast cancer, who received the aromatase inhibitor letrozole 0.5 or 2.5 mg p.o. once daily. Blood samples were obtained from 15 patients in each dose group at baseline, and one and three months after starting therapy. Circulating IGF-I and IGFBP-3 concentrations were determined by means of radioimmunoassay. In both dosage groups a statistically significant increase in the IGF-I levels was observed during three months of letrozole treatment (P = 0.003). In addition, the multiple testing procedure yielded in the whole patient population a significant result in the comparison between mean IGF-I values after three months of therapy and those observed at baseline (P = 0.004), the estimated average increase being of 24%. No significant result was obtained in the analysis for the dose effect (P = 0.077) and for the time × dose interaction (P = 0.208). Circulating IGFBP-3 levels did not appear to be affected by letrozole treatment in either of the dose groups. This is the first report concerning the short-term effects of letrozole on components of the IGF system in breast cancer patients; further investigations are warranted in order to confirm these preliminary data.     

NMR 15N relaxation of the insulin-like growth factor (IGF)-binding domain of IGF binding protein-5 (IGFBP-5) determined free in solution and in complex with IGF-II.

15N NMR relaxation rates of mini-IGFBP-5, an N-terminal insulin-like growth factor binding domain of the insulin-like growth factor binding protein 5 (IGFBP-5), were analysed at three field strengths using the Lipari-Szabo procedure (see below) and reduced spectral density methods. Isotropic and anisotropic Lipari-Szabo models were analysed and an analytical formula for the overall correlation time for anisotropic molecules is presented. Mini-IGFBP-5 was found to be mainly rigid on fast ps time scales except for 11 unstructured flexible residues at the C-terminus. The insulin-like growth factor binding loop in the apo-protein exhibits small amounts of flexibility on fast time scales (ps to ns) but several loop residues show significant exchange broadening. These loop residues display no exchange broadening in the complex of IGF-II/mini-IGFBP-5. The isotropic overall tumbling time in solution at 31 degrees C of mini-IGFBP-5 complexed to IGF-II is tauc = 18.4 +/- 0.2 ns indicating a strong tendency for aggregation.