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Biomedical subjects

J M Holly

Publications and source records attributed to J M Holly.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on protein metabolism.

In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R(3)IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.

Adult↗
PubMed1999

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.

Antineoplastic Agents↗
PubMed1999

Increased tyrosine kinase activity but not calcium mobilization is required for ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.

Animals↗
PubMed1999

Changes in the levels of integrin and focal adhesion kinase (FAK) in human melanoma cells following 532 nm laser treatment.

Despite the increase in laser therapy, concern remains that sublethal treatment of pre-malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the beta1 integrin in laser vs. non-laser treated cells. We treated cells with laser generated from a frequency doubled Q-switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with beta1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4- and 11.2-fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both alpha3 and 4 integrins were up-regulated, by approximately 4-fold and 7- to 9-fold, respectively. In addition, the beta1 integrin was proteolysed in both cell lines and the levels of FAK associated with beta1 was increased (2.1- and 2.7-fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology.

Cell Adhesion Molecules↗
PubMed1999

Estrogen replacement therapy modulation of the insulin-like growth factor system in monkey knee joints.

OBJECTIVE: Epidemiologic studies have suggested that estrogen replacement therapy may lower the risk of osteoarthritis in women, but the mechanism of this effect is unknown. Since estrogen acts in other tissues in part through regulation of the insulin-like growth factor (IGF) system as well as cytokines including interleukin-6 (IL-6), we determined whether estrogen replacement regulates the levels of these factors in synovial fluid (SF). METHODS: Levels of IGF-1, IGF-2, IGF binding proteins (IGFBP) 1-3, and IL-6 were measured in SF samples obtained from 67 female adult cynomolgus monkeys that had been ovariectomized and treated for 30 months in 1 of 3 groups. Group 1 (n = 24) had no estrogen replacement (control), group 2 (n = 22) received estrogen (Premarin) at the human equivalent of 0.625 mg/day, and group 3 (n = 21) received estrogen at the same dose as group 2, plus progesterone (Provera) at the equivalent of 2.5 mg/day. RESULTS: Compared with controls, estrogen-treated monkeys had 2-fold higher SF levels of IGF-1 (P < 0.001), 1.7-fold higher IGF-2 (P < 0.006), 5.9-fold higher IGFBP-1 (P < 0.02), and 2.5-fold higher IGFBP-3 (P < 0.001). Estrogen plus progesterone-treated monkeys had SF levels of IGF-1, IGF-2, IGFBP-1, and IGFBP-3 that were intermediate between the levels in the control and estrogen groups, except that the level of IGFBP-3 was significantly greater than that in the control group (P < 0.001). SF levels of IGFBP-2 and IL-6 did not differ by treatment group. Treatment group did not affect the serum levels of IGF-1 and IL-6, but IGF-2 and IGFBP-3 were increased by 1.6- and 1.8-fold, respectively, in the estrogen group (P < 0.001). There was no correlation between changes in serum and SF levels of IGF components, except for a weak correlation for IGFBP-3 levels from control (r = 0.464, P = 0.04) and estrogen-treated (r = 0.577, P = 0.008) animals. CONCLUSION: This study demonstrates a significant effect of estrogen replacement on IGF system components in synovial fluid, of which at least some are distinct from any systemic changes observed. The results indicate a potential stimulatory effect of estrogen on joint tissues in vivo.

Animals↗
PubMed1999

Effects of treatment with megestrol acetate on the insulin-like growth factor system: time and dose dependency.

This study was designed to evaluate time and dose dependency of alterations in insulin-like growth factor (IGF)-I, free IGF-I and the functional status of IGF-binding protein (IGFBP)-3 in breast cancer patients during treatment with megestrol acetate (MA). In 16 patients receiving MA 160 mg daily, total IGF-I levels increased gradually (significant after 3 days on treatment) by a maximum of 2.66-fold after 5-6 months on treatment. However, free (readily dissociable) IGF-I levels increased to a smaller extent (1.23-2.15-fold). This discrepancy may be due to an increase in intact IGFBP-3 determined by Western ligand blotting (WLB). Similar findings were observed in 12 patients treated with MA in escalating doses from 40-800 mg daily. A dose-dependent increase in IGF-I was observed up to a dose level of 120 mg daily. We conclude that treatment with MA caused a profound increase in plasma levels of total IGF-I accompanied by a moderate increase in free IGF-I. This may explain the anabolic effects of MA in patients suffering from cachexia, but refute the hypothesis that alterations in the IGF-system may contribute to the antitumour effects of MA in breast cancer patients.

Aged↗
PubMed1999

Activation of integrin and ceramide signalling pathways can inhibit the mitogenic effect of insulin-like growth factor I (IGF-I) in human breast cancer cell lines.

Cell counting, cell cycle analysis and Western immunoblotting were used to examine the effects of non-apoptotic doses of a ceramide analogue, C2, and a synthetic arginine-glycine-aspartic acid (RGD)-containing peptide, RGD, in MCF-7 and T47D cells to determine whether activation of these signalling pathways could alter the mitogenic potential of insulin-like growth factor I (IGF-I). IGF-I alone increased total cell number in both cell lines, associated with a rise in the percentage of cells in the S-phase of the cell cycle and a co-incident increase in cyclin A production. Treatments alone had no effects on cell number or cyclin A production relative to controls. C2 inhibited IGF-I-induced mitogenesis in both lines, whereas RGD was only effective in the T47D line. Despite inhibition of cell proliferation, IGF-I stimulation of cells in S-phase and of cyclin A levels were unaffected; however, an IGF-I-induced increase in cyclin B1 levels was inhibited by 30%. Low-dose induction of integrin and ceramide signalling pathways causes cells to be blocked in S-phase, thereby inhibiting the normal cycle of events associated with the IGF-I-induced mitotic signal. Activating these pathways may not only restrict tumour growth by induction of apoptosis but they may also directly inhibit IGF-I-induced cell proliferation.

Breast Neoplasms↗
PubMed1999

Insulin-like growth factor binding protein-1 in NIDDM: relationship with the insulin resistance syndrome.

OBJECTIVE: In order to examine the role of insulin-like growth factors in the pathogenesis of accelerated macrovascular disease in noninsulin-dependent diabetes mellitus (NIDDM), we investigated the relationship between the insulin resistance syndrome and the IGF axis. DESIGN: Cross-sectional analysis of the relationship between insulin resistance syndrome variables and concentrations of IGF-1, IGF-2, IGFBP-1 and IGFBP-3 in 80 subjects with NIDDM. RESULTS: After correcting for age, sex and body mass index, concentrations of IGFBP-1, correlated with those of HDL-cholesterol (r = 0.40; P < 0.001), triglycerides (r = -0.24; P = 0.04), insulin (r = -0.39; P < 0.001), intact proinsulin (r = -0.32; P = 0.006), des 31,32 proinsulin (r = -0.40; P = 0.001), and with insulin sensitivity (r = 0.38; P = 0.001) and PAI-1 activity (r = -0.24; P = 0.05); IGF-1 levels only correlated with those of HDL-cholesterol (r = -0.33; P = 0.005), and this was not explained by IGFBP-1 or insulin sensitivity. With additional correction for insulin, concentrations of IGFBP-1 still correlated with HDL-cholesterol (r = 0.40; P < 0.001), but not those of triglycerides or PAI-1 activity. There were no significant relationships between levels of IGF-2 and any of the variables investigated, and IGFBP-3 levels only correlated with those of total cholesterol (r = 0.24, P = 0.04). CONCLUSIONS: In NIDDM, concentrations of IGFBP-1 are related to those of insulin, insulin sensitivity, serum lipoproteins and PAI-1 activity. The relationship between concentrations of IGFBP-1 and HDL-cholesterol is not explained by insulin. Concentrations of IGF-1 are linked to HDL-cholesterol, and this is not explained by levels of IGFBP-1. IGFBP-1 concentrations were related to PAI-1 activity, and this may be explained by insulin, which regulates the production of IGFBP-1 and PAI-1.

Analysis of Variance↗
PubMed1999

The relationship between insulin, IGF-I and weight gain in cystic fibrosis.

OBJECTIVE: In cystic fibrosis, reduced body mass is related to low levels of IGF-I and changes in the IGF binding proteins. Our aim was to determine whether these abnormalities are linked to pancreatic endocrine dysfunction. PATIENTS AND DESIGN: We measured serum levels of insulin, IGF-I, IGFBP-I, IGFBP-3 and IGF bioactivity in 77 fasting subjects (43 male) mean age 9.6 years (range 2.99-17.98 years). Data were analysed with respect of body mass, puberty and stature and compared with control data established in the same laboratory. RESULTS: The mean height standard deviation score (SDS (SD)) was -0.54 (0.97) and the body mass index SDS -0.24 (1.09). Both body mass index SDS (r = -0.40, P = 0.0003) and IGF-I SDS (r = - 0.32, P = 0.009) declined with age. Insulin levels were also low and correlated with IGF-I and IGFBP-3 (r = 0.42, P = 0.0004, and r = 0.45, P = 0.0002, respectively) whereas levels of IGFBP-I were inversely related to those of IGF-I and insulin (r = - 0.43, P = 0. 0004, r = - 0.52, P < 0.0001). IGF bioactivity was reduced and inversely related to IGFBP-I (r = - 0.31, P = 0.009). In multiple regression analysis, body mass index SDS was negatively related to age (P < 0.0001) and positively related to insulin and IGF-I (P = 0. 04, P = 0.03, respectively). Height SDS was correlated with IGF bioactivity (P = 0.003) and negatively with IGFBP-I (P = 0.01). CONCLUSIONS: We conclude that progressive insulin deficiency may result in reduced IGF-I levels and IGF-bioactivity and may determine weight gain and statural growth in cystic fibrosis.

Adolescent↗
PubMed1999

An immunohistochemical study of type I insulin-like growth factor receptors in the placentae of pregnancies with appropriately grown or growth restricted fetuses.

Insulin-like growth factors (IGFs) and their receptors in the fetus are essential for growth and postnatal survival but the role of maternal IGFs is less well understood. Animal and in vitro evidence suggests that maternal IGF-I may have important effects on placental function. Recent work in humans suggests that although there is no relationship between maternal serum IGF-I and normal fetal growth, levels are low in pregnancies complicated by fetal growth restriction due to placental dysfunction. A prospective and observational study was undertaken of the distribution and concentration of placental type I IGF receptors (IGF-IR) in women with small for gestational age (n=26) or appropriately grown (n=14) fetuses. Women were scanned biweekly from 24 weeks to delivery and cases (birthweight <5th centile) were assigned to two groups: 'fetal growth restriction' (FGR; umbilical artery pulsatility index [UAPI] > +2 s.d.; n=16) and 'normal small for gestational age' (SGA; normal UAPI, growth velocity and amniotic fluid; n=10). Immunohistochemistry of the IGF-IR was performed on formol saline-fixed placental biopsies obtained at delivery. In control pregnancies IGF-IR were present in villous endothelium and stroma, trophoblast and decidua and their distribution and density were unchanged in both SGA and FGR pregnancies. We hypothesize that a therapeutic elevation of maternal IGF-I in FGR pregnancy might lead to enhanced placental function and so fetal growth. Our findings of normal localization and density of placental IGF-IR in FGR encourage us to extend our work to look at the effects of maternal IGF-I on the transport of glucose and amino acids.

Chorionic Villi↗
PubMed1999

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.

Animals↗
PubMed1999

Maternal serum insulin-like growth factor binding protein-2 and -3 and fetal growth.

This was a prospective observational study of maternal insulin-like growth factor binding protein-2 and -3 and fetal growth in 141 pregnant women after 24 weeks gestation who were scanned and venesected fortnightly. Cases (birthweight <5th centile) were sub-divided into those with growth restriction due to placental dysfunction (n = 25) and normal small (n = 27) and there were 89 normally grown controls. Maternal binding protein-3 was measured by radioimmunoassay and the overall pattern of the binding proteins and their proteolytic modifications were assessed by Western ligand blotting and immunoblotting followed by densitometric analysis. In controls, there was no correlation between binding protein-3 and birthweight, and binding protein-3 was elevated in the normal small but not the placental dysfunction group. Complete proteolysis of the 40 kDa doublet of binding protein-3 was observed in all pregnancies. Maternal serum binding protein-2 concentrations were unchanged in normal pregnancy compared to non-pregnant controls but elevated in the growth-restricted group and in all pregnancies binding protein-2 was predominantly present as a 14 kDa proteolysed fragment. These results suggest that compensatory changes in binding protein-2 and -3 or their proteolysis do not increase bioavailability and so do not confound the low maternal insulin-like growth factor-I in growth restricted pregnancies.

Case-Control Studies↗
PubMed1999

Production of soluble tumor necrosis factor receptors by human subcutaneous adipose tissue in vivo.

To investigate in vivo adipose tissue production of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and their soluble receptors: TNF receptor type I (sTNFR-I), TNF receptor type II (sTNFR-II), and IL-6 receptor (sIL-6R), we determined arteriovenous differences in their levels across abdominal subcutaneous adipose tissue in obese subjects. Subjects had a median (interquartile range) age of 44.5 (27-51.3) yr, body mass index (BMI) of 32.9 (26. 0-46.6) kg/m(2), and %body fat of 42.5 (28.5-51.2) %. Although there was not a significant difference in the arteriovenous concentrations of TNF-alpha (P = 0.073) or sTNFR-II (P = 0.18), the levels of sTNFR-I (P = 0.002) were higher in the vein compared with artery, suggesting adipose tissue production of this soluble receptor. There was a significant arteriovenous difference in IL-6 (P < 0.001) but not in its soluble receptor (P = 0.18). There was no relationship between TNF-alpha levels and adiposity indexes (r(s) = 0.12-0.22, P = not significant); however, levels of both its soluble receptor isomers correlated significantly with BMI and %body fat (sTNFR-I r(s) = 0.42-0.72, P < 0.001; sTNFR-II r(s) = 0.36-0.65, P < 0.05- <0. 001). IL-6 levels correlated significantly with both BMI and %body fat (r(s) = 0.51, P = 0.004, and r(s) = 0.63, P < 0.001), but sIL-6R did not. In conclusion, 1) soluble TNFR-I is produced by adipose tissue, and concentrations of both soluble isoforms correlate with the degree of adiposity, and 2) IL-6, but not its soluble receptor, is produced by adipose tissue and relates to adiposity.

Abdomen↗
PubMed1999

Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity.

Although expression of the IGF-II has been demonstrated within the central nervous system (CNS), past studies have failed to reveal its precise roles or responses subsequent to a traumatic injury. To demonstrate that IGF-II, IGFBP, and IGF receptor (-R) expression alters in response to a penetrating CNS injury, we used the techniques of ribonuclease protection assay, in situ hybridization, immunohistochemistry, Western blotting, and RIA. Under normal physiology, IGF-II expression is restricted to the mesenchymal support structures of the brain, including the choroid plexus, where its expression is coincident with that of IGFBP-2. Between 1-7 days post lesion (dpl), in the acute phase following a penetrant wound to the CNS, IGF-II and IGF-IIR protein, but not messenger RNA, were colocalized, with IGF-I, IGF-IR, and IGFBP-1, -2, -3, and -6, to neurons, macrophages, astrocytes, and microglia within the damaged tissue. Within the cerebrospinal fluid (CSF), levels of IGF-II peptide increased to peak at 7 dpl. IGFBP-2, -3, and -6 were also observed within the CSF, with IGFBP-2 predominating and exhibiting an increase in binding efficiency from 7-10 dpl. In the chronic phase of injury (7-14 dpl), an increase in both IGF-II, IGF-IIR and IGFBP-5 messenger RNA and protein was observed specifically and focally in the marginal astrocytes forming the limiting glial membrane of the wound. Thus, our evidence suggests that there are two mechanisms of action for IGF-II within the injured rat brain. During the acute phase, the secretion of IGF-II from the choroid plexus into the CSF is up-regulated, resulting in increased transport of the peptide to the wound. In the CSF, transported IGF-II is complexed to IGFBP-2 and essentially demonstrates an endocrine mode of action with a balance of locally produced IGFBPs modulating its bioactivity in the wound. Later in the wounding response, levels of IGF-II decline in the CSF and the wound neuropil, possibly with the aid of increased IGFBP-5 levels that may help to locally sequester and down-regulate IGF-II activity. Hence, in the chronic phase of the injury response, IGF-II reasserts itself to a predominantly autocrine/paracrine role restricted to the mesenchymal support structures, including the glia limitans, which may help reestablish and maintain tissue homeostasis.

Animals↗
PubMed1999

The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosis in breast epithelial cells.

We have recently reported that insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was observed in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also previously reported that almost all of the IGFBP-3 outside the circulation in extravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to investigate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apoptosis. In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.

Animals↗
PubMed1999

Effect of insulin-like growth factor binding protein-1 on integrin signalling and the induction of apoptosis in human breast cancer cells.

Interaction of epithelial cells with the extracellular matrix is mediated through integrin receptors, which transmit signals regulating cell growth, differentiation and death. Occupation of these receptors, via Arg-Gly-Asp (RGD) recognition sequences, leads to activation of focal adhesion kinase (FAK). We treated human breast cancer cell lines with RGD-containing peptides, which can disrupt integrin attachment, and investigated alterations in FAK phosphorylation, cell detachment and death. Cells grown in vitro were treated with insulin-like growth factor-binding protein-1 (IGFBP-1) and a small, synthetic RGD-containing peptide (Gly-Arg-Gly-Asp-Thr-Pro) and its negative control peptide RGE (Arg-Gly-Glu-Ser) for either 30 min followed by immunoprecipitation of cell lysates with anti-phosphotyrosine and Western immunoblotting with anti-FAK or for 24 h followed by cell counting, immunocytochemistry and flow cytometry. Both IGFBP-1 (0-800 ng/ml) and the synthetic RGD-containing peptide (1-100 microg/ml) caused significant dephosphorylation of FAK. Furthermore, after 24 h both peptides caused detachment from the matrix and the induction of apoptosis. We conclude from these data that IGFBP-1 can interact with integrin receptors to induce FAK dephosphorylation and subsequently influence attachment and cell death.

Antigens, CD↗
PubMed1999

Growth hormone, IGF-I and cancer. Less intervention to avoid cancer? More intervention to prevent cancer?

The GH/IGF-I axis has a clearly established role in somatic growth regulation and there is much evidence suggesting that it can play a contributing role in neoplastic tissue growth; a number of recent epidemiological reports indicate that it may also be an important determinant of cancer incidence. Whilst there have been previous reports of changes to the axis in patients with established cancers, these new studies are distinct in being prospective and the inferences that can be made from this are outlined in this review. The recent studies are considered within the context of other indirect epidemiological evidence, and together indicate that the GH/IGF-I axis may establish the level of predisposition to a number of common cancers and indeed that such risk may be programmed from early life. There is considerable evidence for a number of possible mechanisms, both direct and indirect, which could account for the associations between GH/IGF-I levels and cancer incidence; these mechanisms are briefly summarised. The implications of the new findings are then discussed in relation to the increasing clinical usage of chronic GH administration and the need for further studies to establish any consequent increase in cancer risk. Finally the opportunities for further work to optimise cancer risk assessment and risk reduction strategies are highlighted.

Adult↗