Regulation of insulin-like growth factor (IGF) binding protein-2 synthesis and degradation by platelet-derived growth factor and the IGFs is enhanced by serum deprivation in vascular smooth muscle cells

Growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-1) stimulate proliferation and migration of vascular smooth muscle cells (SMC). IGF-l bioactivity is modulated by high-affinity binding proteins (IGFBP) which are important regulators of these processes. Procine vascular SMC synthesize IGFBP-2 and IGFBP-4 in vitro. In the present study, levels of IGFBP-2 in conditioned media (CM) were increased approximately 1.6 to 2.2-fold when cells were exposed to PDGF (20 ng.ml) or insulin (5 μg/ml) for 24 hr following a 24 hr incubation in serum-free media, or following a 72 hr exposure to either growth factor. Similar increases in IGFBP-2 mRNA levels were observed. Exposure of cells to PDGF for 24 hr without prior serum deprivation resulted in smaller (47 ± 11%) increases in IGFBP-2 protein levels but failed to alter mRNA levels. IGF-1, FGF-b? and EGF failed to increase IGFBP-2 using either experimental paradigm. In contrast, IGFBP-2 protein levels were consistently decreased (75 ± 14%) after 72 hr of exposure to IGF-II without corresponding decreases in IGFBP-2 mRNA levels. Immunoprecipitation of [³⁵S] methionine-labeled IGFBP-2 indicated that this decrease was not due to a decrease in synthesis of IGFBP-2. Immunoblot analysis of CM from cells treated with IGF-II indicated that the decrease in intact protein corresponded with an increase in two non-IGF binding IGFBP-2 fragments of 22 and 14 kD. Increased abundance of these fragements was also observed following IGF-I exposure, although corresponding decreases in intact IGFBP-2 were not usually observed. The relative abundance of these fragments did not appear to be affected by treatment with PDGF or insulin. In contrast to IGFBP-2, regulation of the levels of IGFBP-4 in CM did not appear to be altered by serum deprivation. Insulin consistently increased IGFBP-4 mRNA and protein levels under all situations. PDGF tended to increase IGFBP-4 protein levels, although this effect was less consistent and not as great as the increase observe with insulin. Treatment with IGF-I or -ll consistently decreased IGFBP-4 levels in CM but tended to increase their mRNA levels under all situations. These data indicate that insulin, PDGF, and the IGFs regulate both IGFBP-2 and IGFBP-4. While PDGF and insulin stimulate IGFBP-2 and 4 synthesis, the IGFs appear to activate protease(s) which regulate IGFBP-2 and -4 levels post-translationally. The regulation of IGFBP-2 levels by each of these mechanisms appears to be amplified by serum deprivation, but this is not observed with IGFBP-4. © 1995 Wiley-Liss, Inc.     

Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.     

Green microalgae in intermittent light: a meta-analysis assisted by machine learning

Journal of Applied Phycology - Microalgal biotechnology still needs to alleviate the productivity bottleneck before achieving the full extent of its promises. With this goal in mind, many studies...     

Effect of amino acids on acrylamide formation and elimination kinetics

The effect of amino acids other than asparagine on acrylamide (AA) formation/elimination kinetics was studied in an asparagine-glucose model system (0.01 M, pH 6) heated at temperatures between 140 and 200 degrees C. Addition of cysteine or lysine to the model significantly lowered the AA yield, whereas addition of glutamine had a strong promoting effect and of alanine a rather neutral effect on the AA formation. This was also reflected by AA formation/elimination kinetics, which for all model systems studied could be modeled by two consecutive first-order reactions. The ratio of the elimination to the formation rate constant increased from the systems to which glutamine or alanine was added, over the control model system, to the model systems that contained lysine or cysteine.     

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition,cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.     

Ricin A-chain requires c-Jun N-terminal kinase to induce apoptosis in nontransformed epithelial cells

Ricin is a toxin isolated from castor beans that has potential as a weapon of bioterrorism. This glycoprotein consists of an A-chain (RTA) that damages the ribosome and inhibits protein synthesis and a B-chain that plays a role in cellular uptake. Ricin activates the c-Jun N-terminal kinase (JNK) and p38 signaling pathways; however, a role for these pathways in ricin-induced cell death has not been investigated. Our goals were to determine if RTA alone could activate apoptosis and if the JNK and p38 pathways were required for this response. Comparable caspase activation was observed with both ricin and RTA treatment in the immortalized, nontransformed epithelial cell line, MAC-T. Ribosome depurination and inhibition of protein synthesis were induced in 2–4 h with 1 μg/ml RTA and within 4–6 h with 0.1 μg/ml RTA. Apoptosis was not observed until 4 h of treatment with either RTA concentration. RTA activated JNK and p38 in a time- and concentration-dependent manner that preceded increases in apoptosis. Inhibition of the JNK pathway reduced RTA-induced caspase activation and poly(ADP-ribose) polymerase cleavage. In contrast, inhibition of the p38 pathway had little effect on RTA-induced caspase 3/7 activation. These studies are the first to demonstrate a role for the JNK signaling pathway in ricin-induced cell death. In addition, the MAC-T cell line is shown to be a sensitive in vitro model system for future studies using RTA mutants to determine relationships between RTA-induced depurination, ribotoxic stress, and apoptosis in normal epithelial cells.     

Endogenous IGFBP‐3 is required for both growth factor‐stimulated cell proliferation and cytokine‐induced apoptosis in mammary epithelial cells

TNF‐α and IGF‐I exert opposing effects on mammary epithelial cell (MEC) growth and survival. However, both increase IGF binding protein‐3 (IGFBP‐3) expression, a multifunctional protein that plays both IGF‐dependent as well as independent roles in these processes. We have reported that IGF‐I utilizes the PI3‐K and MAPK pathways to induce IGFBP‐3 expression in bovine MEC. Here we show that TNF‐α requires the SAPK pathway p38, but not JNK, to induce IGFBP‐3 expression. Contrary to reports in cancer cell lines, TNF‐α retained its ability to decrease DNA synthesis in cells transfected with IGFBP‐3 siRNA. It also retained its ability to inhibit IGF‐I‐stimulated DNA synthesis in these cells. In contrast, the ability of IGF‐I to increase DNA synthesis was attenuated with IGFBP‐3 knockdown. IGFBP‐3 knockdown also decreased basal DNA synthesis, indicating that a certain level of IGFBP‐3 may be required for cell proliferation. While TNF‐α alone failed to induce apoptosis, it increased cell death when added with the JNK agonist anisomycin (ANS). TNF‐α and ANS were unable to induce apoptosis when either IGFBP‐3 or JNK‐2 was knocked‐down, suggesting that both JNK and IGFBP‐3 may interact with a downstream molecule central to apoptosis. There are reports that IGFBP‐3 promotes either cell proliferation or apoptosis in different cell systems. However, this is the first report that endogenous IGFBP‐3 is required for the action of both stimulatory and inhibitory factors within the same cell line. Therefore, the actions of IGFBP‐3 are not pre‐determined, but instead governed by cellular context such as JNK activation. J. Cell. Physiol. 220: 182–188, 2009. © 2009 Wiley‐Liss, Inc.     

IGF binding protein‐3 mediates stress‐induced apoptosis in non‐transformed mammary epithelial cells

Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF‐I increases IGF binding protein‐3 (IGFBP‐3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP‐3 mRNA and protein in a dose‐ and concentration‐dependent manner that mirrors activation of caspase‐3 and ‐7, with significant increases in both IGFBP‐3 protein and caspase activation observed by 3 h. Knock‐down of IGFBP‐3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock‐down of IGFBP‐2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP‐3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP‐3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF‐I and the apoptotic inducer ANS increase IGFBP‐3 production in MEC, we proposed that cellular localization might determine IGFBP‐3 action. While both IGF‐I and ANS stimulated the release of IGFBP‐3 into conditioned media, only ANS induced nuclear localization of IGFBP‐3. A pan‐caspase inhibitor had no effect on ANS‐induced nuclear localization of IGFBP‐3, indicating that nuclear entry of IGFBP‐3 precedes caspase activation. Treatment with IGF‐I had no effect on ANS‐induced nuclear localization, but did block ANS‐induced apoptosis. In summary, our data indicate that IGFBP‐3 plays a role in stress‐induced apoptosis that may require nuclear localization in non‐transformed MEC. J. Cell. Physiol. 228: 734–742, 2013. © 2012 Wiley Periodicals, Inc.     

Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX?

Background

NY-ESO-1 protein formulated in ISCOMATRIX? results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial.

Methods

Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252?C1,155?days (median?=?681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX? adjuvant at three protein doses 10???g, 30???g or 100???g (n?=?14); 100???g NY-ESO-1 protein (n?=?8) or placebo (n?=?6), together with 1???g of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2?days after DTH skin testing with NY-ESO-1 protein (1???g) or peptides (10???g). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17.

Results

Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX? adjuvant. In contrast, immunity only persisted in 3/14 who received 100???g un-adjuvanted NY-ESO-1 protein (3/8) or 2???g DTH protein (0/6) P?=?0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred.

Conclusion

Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX?. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.