No abstract

Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.

Expansion of extracellular matrix with fibrosis occurs in many tissues as part of the end-organ complications in diabetes, and advanced glycosylation end products (AGE) are implicated as one causative factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as insulin-like growth factor-binding protein-related protein-2 (IGFBP-rP2), is a potent inducer of extracellular matrix synthesis and angiogenesis and is increased in tissues from rodent models of diabetes. The aim of this study was to determine whether CTGF is up-regulated by AGE in vitro and to explore the cellular mechanisms involved. AGE treatment of primary cultures of nonfetal human dermal fibroblasts in confluent monolayer increased CTGF steady state messenger RNA (mRNA) levels in a time- and dose-dependent manner. In contrast, mRNAs for other IGFBP superfamily members, IGFBP-rP1 (mac 25) and IGFBP-3, were not up-regulated by AGE. The effect of the AGE BSA reagent on CTGF mRNA was due to nonenzymatic glycosylation of BSA and, using neutralizing antisera to AGE and to the receptor for AGE, termed RAGE, was seen to be due to late products of nonenzymatic glycosylation and was partly mediated by RAGE. Reactive oxygen species as well as endogenous transforming growth factor-beta1 could not explain the AGE effect on CTGF mRNA. AGE also increased CTGF protein in the conditioned medium and cell-associated CTGF. Thus, AGE up-regulates the profibrotic and proangiogenic protein CTGF (IGFBP-rP2), a finding that may have significance in the development of diabetic complications.

Expansion of extracellular matrix with fibrosis occurs in many tissues, including skin, as part of the end-organ complications in diabetes. Advanced glycosylation end-products (AGEs) have been implicated as a pathogenic factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as IGF-binding protein-related protein-2, induces extracellular matrix. We have recently shown that CTGF mRNA and protein are up-regulated by AGE treatment of cultured human dermal fibroblasts. The aim of this study was to determine whether CTGF is an autocrine mediator in the induction of fibronectin (FN) by AGE. Primary cultures of nonfetal human dermal fibroblasts in confluent monolayer were treated with synthesized soluble AGE BSA, 0-200 microg/ml. Analysis of mRNA, by quantitative real-time RT-PCR and conditioned media from treated cultures, showed that FN mRNA was increased by approximately 4-fold at 48 h, and FN protein levels by Western immunoblot and FN ELISA were doubled, compared with control. In the same system, added recombinant human CTGF (0-500 ng/ml) induced FN mRNA and protein levels dose dependently and in a rapid time course. To test whether AGE BSA acts through cell-derived CTGF to induce FN, a CTGF neutralizing antibody was shown to significantly attenuate, but not fully inhibit, the AGE induction of FN mRNA. A pan-specific PKC inhibitor, GF109203X, at 0.2 microM, inhibited the induction of FN mRNA by AGE BSA. Although the same inhibitor did not significantly affect the induction of CTGF mRNA by AGE, it blocked the induction of FN mRNA by recombinant human CTGF. In summary, the induction of FN by AGE is partly mediated by the AGE-induced up-regulation of cell-derived CTGF and is dependent on PKC activity. These results have potential implications for the expansion of extracellular matrix in diabetes mellitus by advanced glycosylation end products.

Optic pathway/hypothalamic pilocytic astrocytomas in children are usually treated with chemotherapy following a surgical biopsy. In this report, we retrospectively considered the role of surgical intervention. In a series of 25 patients without neurofibromatosis type 1, the median age at initial treatment was 3.1 years (range, 0-15 years). Twenty cases were verified by histology, and five cases were diagnosed by MRI findings. Twenty-three patients received chemotherapy. All patients were alive at median follow-up of 66 months. Aims of surgery at the initiation of treatment were biopsy in 12 cases (1 stereotactic and 11 craniotomies) and debulking in 7 cases. The 11 open biopsies revealed pilocytic astrocytoma; however, noticeable complications occurred in five children after the biopsies. Review of preoperative MRIs showed that all had typical findings indicating pilocytic astrocytoma. The open biopsy offered no noteworthy benefit for the patients despite surgical risk and delay of chemotherapy. The extent of the seven resection surgeries was 70% or less removal, and postoperative adjuvant therapy was needed for six of the seven patients. The remaining six children who did not undergo surgery obtained remission with chemotherapy alone. After relapse in nine patients, 15 bulk-reduction surgeries were performed. Surgical resection was not curative in any patient. In five patients, mostly older children, cystic expansion of tumor was partially resected, resulting in additional remission. In conclusion, considering the risk of open surgery and the effectiveness of chemotherapy, the role of surgical intervention is restricted to bulk-reduction surgery only when it is inevitable, especially at relapse after chemotherapy.

Abstract. LIM homeodomain transcription factors regulate many aspects of development in multicellular organisms. LHX4/Lhx4 is a protein that is essential for pituitary development and motor neuron specification in mammals. In human, a heterozygous splicing mutation of the LHX4 gene was reported in a family with combined pituitary hormone deficiencies (CPHD). In addition to CPHD, these patients were characterized by small sella turcica and chiari malformation. Here we report a Japanese patient with CPHD (GH, PRL, TSH, LH, FSH, and ACTH deficiency) due to a novel missense mutation (P366T) of the LHX 4 gene. She showed severe respiratory disease and hypoglycemia soon after birth. Brain MRI demonstrated hypoplastic anterior pituitary, ectopic posterior lobe, a poorly developed sella turcica, and chiari malformation. Sequence analysis of the LHX 4 gene identified a heterozygous missense mutation (P366T) in exon 6, which was present in LIM4 specific domain. Neither of the patient's parents harbored this mutation, indicating de novo mutation.

Dietary factors play an important role in both the development and prevention of human cancers, including breast carcinoma. One dietary micronutrient, sodium butyrate (NaB), is a major end product of dietary starch and fiber, produced naturally during digestion by anaerobic bacteria in the cecum and colon. NaB is a potent growth inhibitor and initiates cell differentiation for many cell types in vitro. In this study, we investigated the effects of NaB on three human mammary epithelial cells and regulation of the IGF axis, specifically, IGF-binding protein-3 (IGFBP-3), a known growth regulator in human mammary cells, and IGFBP-related protein 2 (IGFBP-rP2)/connective tissue growth factor. NaB inhibited DNA synthesis, as measured by [ 3 H]thymidine incorporation, in estrogen-responsive (MCF-7) and estrogen-non-responsive (Hs578T) breast cancer cells, and normal human mammary epithelial cells (HMEC) to a similar degree (up to 90% inhibition at 1-10 mM concentrations). Treatment of cells with NaB induced histone hyperacetylation, suggesting that NaB exerts its biological effects, at least in part, as a histone deacetylase inhibitor in mammary epithelial cells. Treatment of Hs578T cells with NaB caused an induction of apoptotic cell death. NaB treatment resulted in increased levels of p21Waf1/Cip1 mRNA and protein in Hs578T cells and distinct upregulation of p27 Kip1 in HMEC, suggesting that NaB activates different genes involved in cell cycle arrest, depending upon the cell type. In the same context, among the IGFBP superfamily members tested, NaB specifically upregulated the expression of IGFBP-3 and IGFBP-rP2. These two proteins are known to be involved in inhibition of mammary epithelial cell replication. Northern blot analysis showed that NaB treatment at 1-10 mM concentrations caused a dose-dependent stimulation of IGFBP-3 mRNA expression in cancerous cells and IGFBP-rP2 mRNA expression in both cancerous and non-cancerous cells. Protein data from Western ligand blot and immunoblot analyses demonstrated parallel results.In summary, we have demonstrated that NaB (i) uniformly suppresses DNA synthesis in both cancerous and non-cancerous mammary cells, and (ii) upregulates IGFBP-3 and IGFBP-rP2 mRNA and protein levels in cancerous and non-cancerous mammary cells. These results provide the first demonstration that butyrate regulates the IGFBP system in the human mammary system.

In Sapporo, Japan, a neonatal screening program for congenital hypothyroidism (CH) has employed measurement of free thyroxine (T4) and TSH in the same filter-paper blood spot. This system has enabled us to identify primary CH and central CH during the neonatal period. The aim of this study was to clarify the prevalence and clinical characteristics of central CH. For this purpose, the screening program requested serum from infants with free T4 concentrations below the cut off value regardless of the TSH levels. Between January 2000 and December 2004, 83,232 newborns were screened and six central CH patients were detected as a result of follow-up of low free T4 and non-elevated TSH screening (1:13,872). This frequency is higher than in other studies. Four patients showed multiple pituitary hormone deficiency with pituitary malformations on magnetic resonance imaging. One patient was diagnosed as having Prader-Willie syndrome. The remaining patient was considered to have isolated central CH. Our study demonstrated that the frequency of central CH is 1:13,872. Free T4 measurement would also be advantageous in early recognition of multiple pituitary hormone deficiency.