Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells. (1/2132)

CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.  (+info)

Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl)retinamide. (2/2132)

Epithelial cells from non-cancerous mammary tissue in response to exposure to chemical carcinogens or transfection with oncogenes exhibit hyperproliferation and hyperplasia prior to the development of cancer. Aberrant proliferation may, therefore, represent a modifiable early occurring preneoplastic event that is susceptible to chemoprevention of carcinogenesis. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR), has exhibited preventive efficacy in several in vitro and in vivo breast cancer models, and represents a promising chemopreventive compound for clinical trials. Clinically relevant biochemical and cellular mechanisms responsible for the chemopreventive effects of HPR, however, are not fully understood. Experiments were performed on preneoplastic human mammary epithelial 184-B5/HER cells derived from reduction mammoplasty and initiated for tumorigenic transformation by overexpression of HER-2/neu oncogene, to examine whether HPR inhibits aberrant proliferation of these cells and to identify the possible mechanism(s) responsible for the inhibitory effects of HPR. Continuous 7-day treatment with HPR produced a dose-dependent, reversible growth inhibition. Long-term (21 day) treatment of 184-B5/HER cells with HPR inhibited anchorage-dependent colony formation by approximately 80% (P < 0.01) relative to that observed in the solvent control. A 24 h treatment with cytostatic 400 nM HPR produced a 25% increase (P = 0.01) in G0/G1 phase, and a 36% decrease (P = 0.01) in S phase of the cell cycle. HPR treatment also induced a 10-fold increase (P = 0.02) in the sub-G0 (apoptotic) peak that was down-regulated in the presence of the antioxidant N-acetyl-L-cysteine. Treatment with HPR resulted in a 30% reduction of cellular immunoreactivity to tyrosine kinase, whereas immunoreactivity to p185HER remained essentially unaltered. HPR exposure resulted in time-dependent increase in cellular metabolism of the retinoid as evidenced by increased formation of the inert metabolite N-(4-methoxyphenyl)-retinamide (MPR) and progressive increase in apoptosis. Thus, HPR-induced inhibition of aberrant proliferation may be caused, in part, by its ability to inhibit HER-2/neu-mediated proliferative signal transduction, retard cell cycle progression and upregulate cellular apoptosis.  (+info)

Busulphan is active against neuroblastoma and medulloblastoma xenografts in athymic mice at clinically achievable plasma drug concentrations. (3/2132)

High-dose busulphan-containing chemotherapy regimens have shown high response rates in children with relapsed or refractory neuroblastoma, Ewing's sarcoma and medulloblastoma. However, the anti-tumour activity of busulfan as a single agent remains to be defined, and this was evaluated in athymic mice bearing advanced stage subcutaneous paediatric solid tumour xenografts. Because busulphan is highly insoluble in water, the use of several vehicles for enteral and parenteral administration was first investigated in terms of pharmacokinetics and toxicity. The highest bioavailability was obtained with busulphan in DMSO administered i.p. When busulphan was suspended in carboxymethylcellulose and given orally or i.p., the bioavailability was poor. Then, in the therapeutic experiments, busulphan in DMSO was administered i.p. on days 0 and 4. At the maximum tolerated total dose (50 mg kg(-1)), busulphan induced a significant tumour growth delay, ranging from 12 to 34 days in the three neuroblastomas evaluated and in one out of three medulloblastomas. At a dose level above the maximum tolerated dose, busulphan induced complete and partial tumour regressions. Busulphan was inactive in a peripheral primitive neuroectodermal tumour (PNET) xenograft. When busulphan pharmacokinetics in mice and humans were considered, the estimated systemic exposure at the therapeutically active dose in mice (113 microg h ml(-1)) was close to the mean total systemic exposure in children receiving high-dose busulphan (102.4 microg h ml(-1)). In conclusion, busulphan displayed a significant anti-tumour activity in neuroblastoma and medulloblastoma xenografts at plasma drug concentrations which can be achieved clinically in children receiving high-dose busulphan-containing regimens.  (+info)

Conjugated linoleic acid inhibits differentiation of pre- and post- confluent 3T3-L1 preadipocytes but inhibits cell proliferation only in preconfluent cells. (4/2132)

Conjugated linoleic acid (CLA; 18:2) is a group of isomers (mainly 9-cis, 11-trans and 10-trans, 12-cis) of linoleic acid. CLA is the product of rumen fermentation and can be found in the milk and muscle of ruminants. Animals fed CLA have a lower body fat content. The objective of this study was to establish the possible mechanisms by which CLA affects adipogenesis. 3T3-L1 is a well-established cell line that is used extensively in studying adipocyte biology. These cells typically grow in a culture medium until they reach confluence, at which time they are induced to differentiate by hormonal treatment (d 0). Treatment of 3T3-L1 cells with 25 to 100 micromol/L CLA inhibited differentiation in a dose-dependent manner, while linoleic acid treatment did not differ from DMSO-treated controls. Continuous treatment from d -2, -1, 0 or 2 to d 8 and treatment from d -2 to d 0 and from d 0 to d 2 inhibited differentiation. Differentiation was monitored morphologically (oil Red-O staining), enzymatically (reduction of activity of glycerol-3-phosphate dehydrogenase), and by northern analysis of peroxisome proliferator-activated receptor gamma2, CCAAT/enhancer binding protein alpha and adipocyte specific protein 2 mRNA. CLA inhibited cell proliferation of nonconfluent cells but did not affect cell division of confluent cells, as indicated by 5-bromo-2'-deoxyuridine incorporation and mitochondria metabolism. Therefore, CLA inhibited differentiation before confluence and during induction. However, cellular proliferation was only inhibited prior to induction. These results imply that fat reduction caused by CLA treatment may be attributed to its inhibition of both proliferation and differentiation of preadipocytes in animals.  (+info)

Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. (5/2132)

Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.  (+info)

In vitro cell cycle arrest, in vivo action on solid metastasizing tumors, and host toxicity of the antimetastatic drug NAMI-A and cisplatin. (6/2132)

The effects of NAMI-A (imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate) are compared with cisplatin on tumor cells cultured in vitro at doses of 1 to 100 microM and on tumor metastases in vivo at maximum tolerated doses. Using mouse tumors that metastasize to the lungs, NAMI-A given i.p. for 6 consecutive days at 35 mg/kg/day, was effective independently of the tumor line being treated and of the stage of metastasis growth. Conversely, cisplatin (2 mg/kg/day for 6 days) was as effective as NAMI-A on MCa mammary carcinoma and TS/A adenocarcinoma and less effective than NAMI-A on Lewis lung carcinoma. Cisplatin reduced body weight gain and spleen weight during treatment and was much more toxic than NAMI-A on liver sinusoids, kidney tubules, and lung epithelium. In vitro NAMI-A caused a transient cell cycle arrest of tumor cells in the premitotic G2/M phase, whereas cisplatin caused a progressive dose-dependent disruption of cell cycle phases. Correspondingly, NAMI-A did not modify cell growth, whereas cisplatin caused a dose-dependent reduction of cell proliferation, as determined by sulforhodamine B test. Thus, NAMI-A, unlike cisplatin, is a potent agent for the treatment of solid tumor metastases as well as when these tumor lesions are in an advanced stage of growth. NAMI-A is endowed with a mechanism of action unrelated to direct tumor cell cytotoxicity, and such mechanism of action is responsible for a reduced host toxicity.  (+info)

Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation. (7/2132)

With dimethyl sulphoxide instead of butanol in the thiobarbituric acid assay for sialic acid, a non-fading chromophore with lambdamax. = 549 nm was produced in a homogeneous solution, allowing dilution of the test mixture in case of high colour yield. This test adapted well to studies on alkaline de-O-acetylation. Bovine and rat submaxillary mucins, and rabbit Tamm-Horsfall urinary sialoproteins contain O-acetyl isomers of neuramine acid that are resistant to the thiobarbituric acid assay. Alkaline de-O-acetylation converted resistant O-acetylneuraminic acid into thiobarbituric acid-reactive sialic acid, and such conversion paralleled de-O-acetylation as measured by the ferric hydroxamate method. The colour increment was similar when the alkaline treatment of bovine submaxillary mucin either preceded or followed the acid hydrolysis. Only alkaline preptreatment was effective with rat submaxillary mucin. By selecting optimal conditions for alkaline de-O-acetylation, O-acetyl isomers can be accurately assessed by the thiobarbituric acid assay.  (+info)

Regulation of IL-8RA (CXCR1) expression in polymorphonuclear leukocytes by hypoxia/reoxygenation. (8/2132)

Interleukin-8 (IL-8) is an important mediator of neutrophil (PMN) function and the type A IL-8 receptor (IL-8RA) mediates these pro-inflammatory signals. Hypoxia or hypoxia/reoxygenation (H/R) affects the production of IL-8, but no data is available regarding its effect on IL-8RA expression. The purpose of this study was to determine the effects of hypoxia and/or H/R on the expression of IL-8RA in PMN. We demonstrated that IL-8RA mRNA levels were similar under normoxic and hypoxic conditions but H/R resulted in a significant reduction in mRNA expression between 30 and 60 min. IL-8RA protein also decreased with reoxygenation of whole blood, which was altered by the addition of specific antioxidants. Therefore, H/R appears to attenuate the effect of IL-8 by down-regulating IL-8RA in PMN. These data show that changes in oxygen tension within the wound site not only affect the expression of inflammatory cytokines, but also control their actions by regulating their receptors.  (+info)