PubMed Health⌕ Search

Biomedical subjects

C P Redfern

Publications and source records attributed to C P Redfern.

At least 19 recordsLinked to original sources
PubMed2001

RXR beta isoforms in neuroblastoma cells and evidence for a novel 3'-end transcript.

RXR beta is predominantly involved in retinoid responses in neuroblastoma cells, in particular the N-type SH SY 5Y cells and the S-type SH S EP cells, both derivatives of a mixed phenotype neuroblastoma cell line. The aim of this study was to identify RXR beta isoforms expressed in neuroblastoma cells and to characterise a putative novel RXR beta transcript. RXR beta 1 and RXR beta 2 were expressed in these neuroblastoma cells. An isoform with an insertion into the ligand binding domain, RXR beta(SLSR) (referred to in previous studies as RXR beta 3), was expressed at a similar level to RXR beta. A novel RXR beta transcript was identified by RNase protection assays and was at least as abundant as the expected RXR beta transcript and expressed in other cell types. Evidence suggests that this novel transcript was transcribed from an internal promoter between exons 5 and 6, contained a retained intron (intron 6) and was alternatively spliced with and without the SLSR insertion. These data show that the pattern of RXR beta expression is complex. The relative abundance of the novel RXR beta transcript suggests that it may be an important aspect of RXR beta function or regulation in a range of cell types.

Base Sequence↗
PubMed2001

Apoptosis in neuroblastomas induced by interferon-gamma involves the CD95/CD95L pathway.

BACKGROUND AND PROCEDURE: The CD95/CD95 ligand (CD95L) system is a key regulator of apoptosis. To evaluate a possible role of the CD95/CD95L system in human neuroblastoma (NB) cells, we investigated the constitutive and interferongamma (INFgamma)-induced expression of CD95 and CD95L, and CD95-mediated cell death in the SK-N-BE(2) cell line. RESULTS: Modulation of CD95/CD95L expression and triggering of an autocrine apoptotic mechanism by IFNgamma suggest a potential role for INFgamma as a therapeutic agent for NB. CONCLUSIONS: The evidence that retinoids induce apoptosis via tissue transglutaminase (tTG) and that N-methyl-D-aspartate (NMDA) and gp120 act through the nitric oxide synthase (NOS) activation pathway, indicates the existence of different molecular mechanisms of action, whose pharmacological exploitation might be used in an additive fashion.

Apoptosis↗
PubMed2001

The vitamin A analogues: 13-cis retinoic acid, 9-cis retinoic acid, and Ro 13-6307 inhibit neuroblastoma tumour growth in vivo.

BACKGROUND: Neuroblastoma, a childhood tumour of the sympathetic nervous system, may undergo spontaneous differentiation or regression due to apoptosis after no or minimal therapy. However, the majority of neuroblastomas are diagnosed as metastatic tumours with a poor prognosis in spite of intensive multimodal therapy. Vitamin A and its analogues (retinoic acid, RA) play an important role in normal cel lular differentiation and programmed cell death. RA regulates neuroblastoma growth and differentiation in vitro, and has shown activity against human neuroblastoma in vivo. PROCEDURE: Recently, 9-cis RA was shown to induce apoptosis in vitro in neuroblastoma using a 5 days short-term treatment and subsequent washout. In the present study, nude rats with human neuroblastoma SH-SY5Y xenografts were treated with 13-cis RA (4 mg po daily), 9-cis RA (5 mg po daily) or the novel analogue Ro 13-6307 (0.3 mg po daily) using either a continuous or short-term schedule. RESULTS: ALL three different retinoids decreased neuroblastoma growth significantly in terms of tumour weight after 8-12 days when compared to untreated controls (P < 0.05). Minor signs of toxicity in 13-cis RA treated rats were observed. However, severe toxicity with significant weight loss was seen in all rats treated with 9-cis RA and Ro 13-6307. Toxicity was more pronounced with the continuous regimen. CONCLUSIONS: We conclude that different retinoids reduce neuroblastoma tumour growth in vivo. Drug scheduling and dosage may affect both therapeutic efficacy and toxic side effects. Further in vivo studies are warranted, including pharmacokinetic and molecular analyses, before clinical trials with promising retinoids like 9-cis RA and Ro 13-6307 can be started in children with neuroblastoma.

Alitretinoin↗
PubMed2001

Differential gene regulation by 9-cis and all-trans retinoic acid in neuroblastoma cells.

BACKGROUND: 9-cis retinoic acid (RA) is more effective than all-trans RA at inducing neuroblastoma differentiation in vitro, and has distinct biological properties with respect to its ability to promote apoptosis in N-type neuroblastoma cells. The cellular effects of 9-cis RA may, in part, result from activation of retinoid X receptor (RXR) homodimers. If this hypothesis is correct, 9-cis RA may control the expression of a different subset of retinoid-regulated genes compared to all-trans RA. PROCEDURE: We have therefore used differential mRNA display to identify genes differentially expressed in neuroblastoma cells in response to all-trans and 9-cis RA. RESULTS: The majority of cDNAs differentially expressed in response to all-trans or 9-cis RA matched to nonredundant Genbank sequences or EST database sequences. Differential-display profiles were similar in SH SY 5Y and SH S EP cells, clonal derivatives of the mixed neuroblastoma cell line SK N SH, although there were apparent differences between these cell lines with respect to the retinoid-regulation of specific RT-PCR cDNA fragments. CONCLUSIONS: These data support the view that 9-cis and all-trans RA act via different receptor mechanisms.

Alitretinoin↗
PubMed2001

The role of AP-1 in glucocorticoid resistance in leukaemia.

Glucocorticoids are used in the treatment of acute lymphoblastic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL) but many patients develop glucocorticoid resistance on relapse. The ligand-activated glucocorticoid receptor inhibits activity of the AP-1 transcription factor and the purpose of this study was to test the hypothesis that up-regulation or overexpression of AP1-binding activity may be an important mechanism of glucocorticoid resistance in ALL and CLL. In vitro sensitivity of patient blasts to prednisolone was measured using th

Adult↗
PubMed2001

The N363S polymorphism of the glucocorticoid receptor: potential contribution to central obesity in men and lack of association with other risk factors for coronary heart disease and diabetes mellitus.

Considerable evidence suggests that diabetes mellitus and hypertension are influenced by genetic factors. Studies in humans have associated glucocorticoid receptor (GR) polymorphisms with high blood pressure, insulin sensitivity, body mass index, increased visceral fat, and variations in tissue-specific steroid sensitivity. The N363S polymorphism of the GR results in an asparagine to serine amino acid substitution in a modulatory region of the receptor. Phosphorylation of serine residues in this region has been shown to enhance transactivation of GR responsive genes. The aim of this study was to investigate the association between the 363S allele and risk factors for coronary heart disease and diabetes mellitus in a population of European origin living in the northeast of the United KINGDOM: Blood samples from 135 males and 240 females were characterized for 363 allele status. The overall frequency of the 363S allele was 3.0%, 23 heterozygotes (7 males and 16 females) but no 363S homozygotes were identified. The data show a significant association of the 363S allele with increased waist to hip ratio in males but not females. This allele was not associated with blood pressure, body mass index, serum cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein cholesterol levels, and glucose tolerance status. The results of this study suggest that this GR polymorphism may contribute to central obesity in men. Further studies are required to elucidate the properties of GR(363S) at a molecular level.

Adult↗
PubMed2000

Synergistic induction of apoptosis of neuroblastoma by fenretinide or CD437 in combination with chemotherapeutic drugs.

Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Effector mechanisms of fenretinide-induced apoptosis in neuroblastoma.

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Receptor mechanisms mediating differentiation and proliferation effects of retinoids on neuroblastoma cells.

The aim of this study was to clarify retinoid receptor mechanisms mediating the effects of 9-cis retinoic acid (RA) and investigate the ability of RAR- and RXR-specific analogues to induce differentiation and inhibit proliferation in neuroblastoma cells. Differentiation and the inhibition of proliferation by 9-cis RA, but not all-trans RA, were inhibited by the RXR-homodimer antagonist LG745. The RXR-specific agonist LGD1069 was ineffective at inducing differentiation or inhibiting proliferation, but showed marked synergism with RAR-specific agonists with respect to inhibiting proliferation. These data suggest that the effects of 9-cis RA are mediated via both RXR-homodimers and heterodimers. However, combinations of RAR- and RXR-selective analogues were not as effective at promoting differentiation. This study indicates that different receptor mechanisms are involved in retinoid-induced differentiation and inhibition of proliferation in neuroblastoma cells.

Alitretinoin↗
PubMed2000

Distinct properties of fenretinide and CD437 lead to synergistic responses with chemotherapeutic reagents.

The RARbeta/gamma-selective retinoids fenretinide and CD437 induce caspase-dependent apoptosis but generate free radicals independently of caspases. Apoptosis, but not free radical generation, induced by these retinoids is inhibited by RARbeta/gamma-specific antagonists. Both fenretinide and CD437 induce apoptosis synergistically with cisplatin, carboplatin, or etoposide. However, antioxidants inhibit this synergy to the level obtained with chemotherapeutic drugs alone, and this implies that free radical generation is important in the synergistic response. Since apoptosis induced by fenretinide or CD437 is mediated by apoptotic pathways involving RARs and/or mitochondria and differs from mechanisms of chemotherapy-induced apoptosis this may explain the strong synergistic response seen between these synthetic retinoids and chemotherapeutic drugs. These results suggest that fenretinide or CD437 may be useful adjuncts to neuroblastoma therapy.

Antineoplastic Agents↗
PubMed1999

Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma.

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Glucocorticoid resistance and the AP-1 transcription factor in leukaemia.

Glucocorticoids have been used in the treatment of acute lymphoblastic leukaemia (ALL) for many years, initially as the only agent and then as part of multiagent chemotherapy. In ALL 20% of patients are resistant to glucocorticoids at presentation but this rises to greater than 70% on relapse. It has recently been reported that the glucocorticoid receptor inhibits activity of the AP-1 transcription factor by the ligand-dependant binding of glucocorticoid receptor (GR) to the fos and jun components of AP-1. Since AP-1 is necessary for cell proliferation, the upregulation or over-expression of AP-1 may be a mechanism of resistance to glucocorticoids. The aim of the study was to investigate whether AP-1 levels correlate with in vitro glucocorticoid resistance. In vitro sensitivity to glucocorticoids was measured using the MTT assay. AP-1 levels were quantified using gel shift analysis: a consensus sequence for the AP-1 binding site was synthesised, labelled with 32P and incubated with nuclear extracts of leukaemic blasts from 14 ALL and 26 CLL patients. Leukaemic blasts were treated with prednisolone or with vehicle alone before preparation of nuclear extracts. The gels were dried and bands quantified using a phosphorimager, using an appropriate internal standard and correcting for protein loading and cytoplasmic contamination of nuclear extracts. The patient samples fell into two distinct groups with respect to their sensitivity to glucocorticoids: AP-1 levels were significantly higher (p < 0.02) in sensitive blasts than resistant ones. There was no significant change in AP-1 levels after treating blasts for 4 hours with 0.2 mM prednisolone. No change was seen in CLL samples. These data show that glucocorticoid resistance is not associated with increased AP-1. Conversely, glucocorticoid resistance in these samples was apparently associated with decreased AP-1 levels in ALL samples. Whether this has any causal relationship to glucocorticoid resistance is unknown. Clearly, further studies on the role of AP-1 and related transcription factors is essential for understanding the control of proliferation and apoptosis in ALL.

Adult↗
PubMed1998

Absorptive apical amiloride-sensitive Na+ conductance in human endometrial epithelium.

1. Human endometrial epithelial cells cultured on porous tissue culture supports formed tight, polarized epithelial monolayers with features characteristic of tight epithelia. Endometrial epithelial layers generated significant transepithelial electrical resistance (750 Omega cm2) and potential difference (15.3 mV), with an inward short-circuit current (Isc; 20.5 microA cm-2). 2. The Isc was linearly proportional to the external Na+ concentration and was abolished in the absence of Na+. The Isc was sensitive to apical amiloride. Net 22Na+ flux was in the absorptive apical to basolateral direction and fully accounted for the inward Isc. In addition, apical to basolateral and net 22Na+ transport were reduced in the presence of amiloride. 3. The Isc was also sensitive to addition of ouabain and Ba2+ to the basal solution, consistent with a role for basolateral Na+-K+-ATPase and K+ channels in generation of the current. 4. These data demonstrate that human endometrial epithelial cells in primary culture produce tight, functional monolayers on permeable supports. We provide the first evidence that human endometrial epithelial cells have an inward Isc accounted for by an amiloride-sensitive Na+ conductance. The Na+-absorptive function of the endometrium may provide an appropriate environment for sperm function and embryo growth.

Absorption↗
PubMed1998

Retinoid-induced differentiation of neuroblastoma: comparison between LG69, an RXR-selective analogue and 9-cis retinoic acid.

The aim of this study was to investigate in vitro the effects of all-trans retinoic acid (RA), 9-cis RA and the RXR-selective analogue, LG69, on the morphological differentiation, proliferation and gene expression of neuroblastoma cells. Three different cell lines were cultured with the retinoid for either 9 continuous days or for 5 days followed by 4 days without the retinoid and morphological differentiation was assessed both qualitatively and quantitatively. SH SY 5Y cell proliferation was examined by measuring cell numbers after exposure to the retinoids and RAR-beta gene expression was examined by Northern blot analysis. Morphological differentiation was more effectively induced by all-trans and 9-cis RA than by LG69. SH SY 5Y cells, when treated with 9-cis RA for only 5 of the 9 days of culture, underwent apoptosis, but this was not seen with 9 days continuous exposure nor with LG69. Inhibition of SH SY 5Y cell proliferation by all-trans or 9-cis RA was dose-dependent, but LG69 had little effect. Conversely, LG69 induced higher expression of RAR-beta than all-trans RA, but less than that produced by 9-cis RA. These data suggest that 9-cis RA as a single agent is the most effective modulator of neuroblastoma behaviour and may be the most appropriate therapeutic agent.

Antineoplastic Agents↗
PubMed1997

Apoptosis of N-type neuroblastoma cells after differentiation with 9-cis-retinoic acid and subsequent washout.

BACKGROUND: The overall survival rate for patients with neuroblastoma has improved over the past two decades, but long-term survival for the subgroup of patients with high-risk disease remains low. In recent years, there has been interest in the potential clinical use of drugs able to induce differentiation of neuroblastoma cells. Since 9-cis-retinoic acid induces better and more sustained differentiation of neuroblastoma in vitro than other retinoic acid isomers, this may be a more appropriate retinoid for use in neuroblastoma therapy. PURPOSE: The purpose of this work was to compare the long-term effects of all-trans- and 9-cis-retinoic acid on neuroblastoma differentiation using an N-type (neuroblastic) cell line, SH SY 5Y, as an in vitro model. In addition, we wanted to find out whether 9-cis-retinoic acid would induce programmed cell death (apoptosis) in these N-type neuroblastoma cells and to determine whether the effects of either 9-cis- or all-trans-retinoic acid are dependent on their continued presence in the culture medium. METHODS: SH SY 5Y cells were incubated in either the continued presence of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic acid followed by culture in the absence of retinoid for up to 13 days. Morphologic changes were observed using phase-contrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodide-stained cells and by using terminal deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells. RESULTS: Culture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. These effects were maintained in the continuous presence of each retinoic acid isomer but were more profound in cells treated with 9-cis-retinoic acid. The differentiation of cells treated with all-trans-retinoic acid was reversible once retinoic acid was removed from the medium. Conversely, apoptosis was induced in cells treated with 9-cis-retinoic acid for 5 days and cultured for 9 days (4 days after washout) but not in cells cultured in the continuous presence of 9-cis-retinoic acid. This effect was specific to 9-cis-retinoic acid. CONCLUSIONS: Previous studies have demonstrated differential responses to all-trans-retinoic acid in N- and S-type (substrate-adherent or Schwann-like) neuroblastoma cells: Apoptosis is induced in S-type cells, whereas differentiation occurs in N-type cells. The present results show that, unlike all-trans-retinoic acid, 9-cis-retinoic acid induces both differentiation and apoptosis in N-type SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9-cis-retinoic acid from the culture medium. IMPLICATIONS: Since both differentiation and apoptosis are involved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apoptosis on treatment and subsequent removal of 9-cis-retinoic acid implies that drug scheduling may be an important parameter affecting therapeutic efficacy.

Alitretinoin↗
PubMed1997

Retinoids in neuroblastoma therapy: distinct biological properties of 9-cis- and all-trans-retinoic acid.

We investigated the potential for 9-cis-retinoic acid in the differentiation therapy of neuroblastoma using an N-type neuroblastoma cell line, SH SY 5Y, as an experimental model. In these cells, 9-cis-retinoic acid is more effective than other isomers at inducing the expression of RAR-beta. An RAR-alpha-specific antagonist inhibited the induction of RAR-beta in response to all-trans-but not to 9-cis-retinoic acid. This indicates that the mechanism of gene induction by 9-cis-retinoic acid differs markedly from all-trans-retinoic acid. 9-cis-retinoic acid is also better than all-trans at producing sustained morphological differentiation and inhibition of proliferation of SH SY 5Y cells. Although N-type neuroblastoma cells are not thought to undergo apoptosis in response to all-trans-retinoic acid, we observed a significant degree of apoptosis in SH SY 5Y cells treated with 9-cis-retinoic acid for 5 days and then cultured in the absence of retinoid, an effect not observed in cells treated with the all-trans isomer. These results suggest that 9-cis- and all-trans-retinoic acid have distinct biological properties and that 9-cis retinoic acid may be clinically effective in neuroblastoma by inducing both differentiation and apoptosis under an appropriate treatment regimen.

Alitretinoin↗