Jeff M. P. Holly

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.To investigate in vivo adipose tissue production of tumor necrosis factor-α (TNF-α), 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/m2, and %body fat of 42.5 (28.5-51.2) %. Although there was not a significant difference in the arteriovenous concentrations of TNF-α ( 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-α 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.

Insulin-like Growth Factor-binding Protein (IGFBP-3) Predisposes Breast Cancer Cells to Programmed Cell Death in a Non-IGF-dependent Manner

Insulin-like growth factor (IGF) -independent growth inhibition of human breast cancer cells, Hs578T, by IGF-binding protein-3 (IGFBP-3) has previously been demonstrated. Cell growth is a balance between proliferation and programmed cell death (apoptosis). We have investigated whether IGFBP-3 can affect apoptosis of Hs578T cells. As no induction of apoptosis was found, we also investigated its effect on the response to ceramide, an intracellular second messenger that mediates the signal for apoptosis. Using the cell permeable ceramide analogue, C2, induction of apoptosis was established by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, trypan blue uptake, morphological criteria, and flow cytometry. Incubation of cells with non-glycosylated IGFBP-3 (ngIGFBP-3; 0.5–100 ng/ml) resulted in no growth inhibition or increase in apoptosis; whereas, C2 (1–30 μm) resulted in a dose-dependent induction of apoptosis. Addition of IGFs to the cells, alone or with C2, elicited no response in terms of proliferation or survival, respectively. When the cells were preincubated with ngIGFBP-3 before addition of C2 (2–5 μm), apoptosis was accentuated in a dose-dependent manner (at 100 ng/ml IGFBP-3, apoptosis increased from 11 to 88%). In conclusion, we found that IGFBP-3 had no direct inhibitory effect on Hs578T cells but could accentuate apoptosis induced by the physiological trigger ceramide in an IGF-independent manner.

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. J. Cell. Biochem. 75:652–664, 1999.

A prospective study of serum insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-3 and breast cancer risk

The associations between serum concentrations of insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins (IGFBP)-3 and risk of breast cancer were investigated in a nested case–control study involving 117 cases (70 premenopausal and 47 postmenopausal at blood collection) and 350 matched controls within a cohort of women from the island of Guernsey, UK. Women using exogenous hormones at the time of blood collection were excluded. Premenopausal women in the top vs bottom third of serum IGF-I concentration had a nonsignificantly increased risk for breast cancer after adjustment for IGFBP-3 (odds ratio (OR) 1.71; 95% confidence interval (CI): 0.74–3.95; test for linear trend, P=0.21). Serum IGFBP-3 was associated with a reduction in risk in premenopausal women after adjustment for IGF-I (top third vs the bottom third: OR 0.49; 95% CI: 0.21–1.12, P for trend=0.07). Neither IGF-I nor IGFBP-3 was associated with risk in postmenopausal women and serum IGF-II concentration was not associated with risk in pre- or postmenopausal women. These data are compatible with the hypothesis that premenopausal women with a relatively high circulating concentration of IGF-I and low IGFBP-3 are at an increased risk of developing breast cancer.

Tumor necrosis factor-?-induced apoptosis is associated with suppression of insulin-like growth factor binding protein-5 secretion in differentiating murine skeletal myoblasts

Wasting of muscle and fat during cachexia exceeds that explained by reduced food intake alone. This wasting may result from an imbalanced cytokine environment, which could lead to increased protein catabolism. Supporting this, tumor necrosis factor‐α (TNF‐α) is raised in several animal models of cachectic muscle wasting. Therefore, we assessed the effects of TNF‐α and its second messenger, ceramide, on the proliferation, differentiation, and survival of murine C2 skeletal myoblasts. Because insulin‐like growth factor binding protein‐5 (IGFBP‐5) and insulin‐like growth factor‐II (IGF‐II) are potent regulators of myoblast proliferation and differentiation, we monitored the ability of exogenous TNF‐α to manipulate this system. Fibroblast growth factor (FGF) ceramide, or TNF‐α suppressed differentiation of C2 cells compared with controls. All treatments suppressed IGF‐II production but only TNF‐α blocked IGFBP‐5 secretion. TNF‐α increased apoptotic cell death, which otherwise remained basal (low serum differentiation medium (LSM), FGF) or low (ceramide). Suppression of both IGFBP‐5 and IGF‐II secretion may explain why of all triggers tested, only TNF‐α not only blocked differentiation, but also promoted cell death. This suggests a fundamental role of IGFBP‐5 for maintaining muscle survival. Supporting this hypothesis, no increase in apoptosis was seen in IGFBP‐5 cDNA tranfected C2 cells after TNF‐α treatment. In summary, the IGF system is essential for maintaining skeletal muscle cell survival and differentiation, and its suppression by TNF‐α is fundamental regarding muscle wasting, and may be associated in vivo with cancer cachexia. J. Cell. Physiol. 183:330–337, 2000.

Multifaceted roles of TNF-α in myoblast destruction: A multitude of signal transduction pathways

In catabolic conditions, such as cancer cachexia, a balance favouring a cytokine environment culminates in muscle destruction. Utilising an in vitro model to mimic muscle wasting, we elucidate here the multifaceted roles that one such cytokine, TNF‐α, invokes in the degeneration process. Treatment of C2 skeletal myoblasts with TNF‐α not only suppresses morphological and biochemical differentiation, but following an initial wave of proliferation, and of survival (24 h), induces apoptosis. Investigating the mechanisms underlying these diverse actions of TNF‐α, we demonstrate that cell replication is dependent on rapid and sustained activation of MAP kinase. Map kinase is not, however, central to the death process, which is associated with a progressive rise in caspase‐8 activity, and is accompanied by sustained activation of JNK1 and transient activation of JNK2. Caspase inhibition caused a dose responsive reduction in cell death, while inhibition of the JNKs caused a significant increase in apoptosis. We further report that PI3 kinase is not involved in conferring early protection against TNF‐α‐induced death. By contrast, inhibition of NF‐κB in the presence of TNF‐α culminates in increased cell cycle progression, decreased gadd45β expression and significant and precociously increased cell death, when compared with TNF‐α alone. Our results begin to characterise the mechanisms underlying the acute mitogenic and anti‐apoptotic roles of TNF‐α, which appear to be defined by a balance between MAP kinase, Jun kinase (JNK), NF‐κB and gadd45β. They establish that inhibition of any one of these molecules, as may occur following caspase activation, could eliminate vital stem cells required for skeletal muscle regeneration during chronic catabolic conditions. J. Cell. Physiol. 198: 237–247, 2004© 2003 Wiley‐Liss, Inc.

Effects of oral and transdermal oestrogen replacement therapy on plasma levels of insulin-like growth factors and IGF binding proteins 1 and 3: a cross-over study

OBJECTIVE Conflicting results have been reported on the effects of oral and transdermal oestrogen replacement therapy on IGF‐I, while little information exists regarding the effects on IGFBP ‐1 and ‐3. In this study we evaluated the effects of oral and transdermal oestrogens on these parameters in post‐menopausal women in a randomized cross‐over study.

Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin

The insulin-like growth factor binding proteins (IGFBPs) have IGF-independent differential effects on cell function. We investigated whether they can affect integrin-receptor-mediated cell attachment to different extracellular matrix (ECM) components in Hs578T cells. Cell attachment to a general ECM gel was unaffected by IGFBP-1 and -6 but was significantly increased by IGFBP-4 and -5 and decreased by IGFBP-2 and -3. Similar results were obtained for attachment to laminin or collagen type IV. Attachment to fibronectin, however, was increased by IGFBP-3 and decreased by IGFBP-5. The actions of IGFBP-3 and -5 on cell attachment to ECM were lost in the presence of a soluble Arg-Gly-Asp (RGD)-containing fibronectin fragment. Thrombospondin reversed the actions of IGFBP-3 on cell attachment, but IGFBP-5 still increased cell attachment. On plastic, neither IGFBP-3 nor -5 alone affected cell viability; although ceramide-induced apoptosis was enhanced by IGFBP-3 but reduced by IGFBP-5. The presence of RGD reversed the action of IGFBP-5 on cell death but attenuated that of IGFBP-3. With cells grown on fibronectin, the action of IGFBP-3 was reversed, and it conferred cell survival, whereas the survival effect of IGFBP-5 was lost. In summary we have demonstrated that IGFBP-3 and -5 both have intrinsic effects on cell survival. In each case the presence of fibronectin or fibronectin fragments determines whether susceptibility to apoptosis is increased or decreased. These effects on cell survival are paralleled by acute effects on integrin receptor function; IGFBP-3 and -5 were able to either enhance or inhibit cell attachment in the presence of fibronectin. Cell survival is tightly controlled by cues from the ECM and from growth factors, particularly the IGFs. Our findings indicate that, in addition to being crucial modulators of IGF actions, the IGFBPs have direct actions on cell attachment and survival that are specific and dependent upon the matrix components present.

The differential regulation of the circulating levels of the insulin-like growth factors and their binding proteins (IGFBP) 1, 2 and 3 after elective abdominal surgery

OBJECTIVES Patients undergoing abdominal surgery often suffer from morbidity associated with increased protein catabolism. Therapeutic recombinant human insulin‐like growth factor (rhIGF)‐I has been proposed as a means of reversing this process. As IGFBPs modulate the bioavailability of the IGFs, we have studied the changes in the circulating levels of these peptides during surgery.

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) POTENTIATES PACLITAXEL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

Variability in response to chemotherapy is poorly understood. Paclitaxel‐induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay , cell counting, morphological features and flow cytometry. Pre‐dosing cells with non‐glycosylated insulin‐like growth factor binding protein‐3 (ngIGFBP‐3) had no effect on the cells per se but accentuated paclitaxel‐induced apoptosis. The apoptotic pathway was further examined by measuring caspase‐3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase‐3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP‐3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP‐3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP‐3, which was further increased if the cells had been pre‐dosed with ngIGFBP‐3. These findings suggest that IGFBP‐3 may be an important modulator of paclitaxel‐induced apoptosis. Int. J. Cancer 88:448–453, 2000.