Biomedical subjects

Philip J Hughes

Publications and source records attributed to Philip J Hughes.

6 recordsLinked to original sources

PubMed2006

1Alpha,25-dihydroxyvitamin D3-mediated stimulation of steroid sulphatase activity in myeloid leukaemic cell lines requires VDRnuc-mediated activation of the RAS/RAF/ERK-MAP kinase signalling pathway.

1Alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of steroid sulphatase (STS) in myeloid cells [Hughes et al., 2001, 2005]. This was attenuated by inhibitors of phospholipase D (PLD) (n-butanol, 2,3-diphosphoglyceric acid, C(2)-ceramide) and phosphatidate phosphohydrolase (PAP) (propranolol and chlorpromazine), but was unaffected by inhibitors of phospholipase C. The 1alpha,25(OH)(2)D(3)-induced STS activity was also attenuated by inhibitors of protein kinase Calpha and protein kinase Cdelta (Go 6976, HBDDE and rottlerin), but not by an inhibitor of protein kinase Cbeta (LY379196). Additionally, 1alpha,25(OH)(2)D(3)-induced STS activity was attenuated by inhibitors of RAS (manumycin A), RAF (GW5074), MEK (PD098059 and U1026) and JNK (SP600125), but not p38 (PD169316). 1alpha,25(OH)(2)D(3) produced a rapid and long lasting stimulation of the ERK-MAP kinase signalling cascade in HL60 myeloid leukaemic cells. This 'non-genomic' effect of 1alpha,25(OH)(2)D(3) blocked by pharmacological antagonists of nuclear vitamin D receptors (VDR(nuc)) and does not appear to require hetero-dimerisation with the retinoid-X receptor (RXR). Inhibitors of the Src tyrosine kinase (PP1), RAS (manumycin A), RAS-RAF interactions (sulindac sulphide and RAS inhibitory peptide), RAF (GW5074 or chloroquine), and protein kinase Calpha (HBDDE) abrogated the 1alpha,25(OH)(2)D(3)-stimulated increase in ERK-MAP kinase activity. Taken together, these results show that 1alpha,25(OH)(2)D(3)/VDR(nuc) activation of the RAS/RAF/ERK-MAP kinase signalling pathway plays an important role in augmenting STS activity in human myeloid leukaemic cell lines.

Calcium Signaling↗

PubMed2006

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARalpha and RXR and involves the phosphoinositide 3-kinase and ERK-MAP kinase pathways.

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.

Cells, Cultured↗

PubMed2005

1alpha,25-dihydroxyvitamin D3 stimulates steroid sulphatase activity in HL60 and NB4 acute myeloid leukaemia cell lines by different receptor-mediated mechanisms.

Steroid sulphatase is a key enzyme in the biosynthesis of bioactive estrogens and androgens from highly abundant inactive circulating sulphated steroid precursors. Little is known about how the expression/activity of this enzyme is regulated. In this article, we show that of 1alpha,25(OH)2D3 stimulates an increase steroid sulphatase activity in the HL60 myeloid leukaemic cell line that is inhibited by a specific nuclear VDR (VDRnuc) antagonist and unaffected by plasma membrane-associated vitamin D receptor (VDRmem) agonists and antagonists. 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells was augmented by RXR agonists, blocked by RXR-specific antagonists, and RAR specific agonists and antagonists had no effect. In contrast, the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in the NB4 myeloid leukaemic cell line was unaffected by the specific VDRnuc and RXR antagonists, but was blocked by a VDRmem-specific antagonist and was increased by VDRmem-specific agonists. The findings reveal that VDRnuc-RXR-heterodimers play a key role in the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells. However, in NB4 cells, VDRnuc-derived signals do not play an obligatory role, and non-genomic VDRmem-derived signals are important.

Calcitriol↗

PubMed2004

Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle.

Inositol polyphosphates other than Ins(1,4,5)P3 are involved in several aspects of cell regulation. For example, recent evidence has implicated InsP6, Ins(1,3,4,5,6)P5 and their close metabolic relatives, which are amongst the more abundant intracellular inositol polyphosphates, in chromatin organization, DNA maintenance, gene transcription, nuclear mRNA transport, membrane trafficking and control of cell proliferation. However, little is known of how the intracellular concentrations of inositol polyphosphates change through the cell cycle. Here we show that the concentrations of several inositol polyphosphates fluctuate in synchrony with the cell cycle in proliferating WRK-1 cells. InsP6, Ins(1,3,4,5,6)P5 and their metabolic relatives behave similarly: concentrations are high during G1-phase, fall to much lower levels during S-phase and rise again late in the cycle. The Ins(1,2,3)P3 concentration shows especially large fluctuations, and PP-InsP5 fluctuations are also very marked. Remarkably, Ins(1,2,3)P3 turns over fastest during S-phase, when its concentration is lowest. These results establish that several fairly abundant intracellular inositol polyphosphates, for which important biological roles are emerging, display dynamic behaviour that is synchronized with cell-cycle progression.

Animals↗

PubMed2003

Cell differentiation and proliferation--simultaneous but independent?

Despite studies over many years, it is still not clear to what extent cellular controls on proliferation and on differentiation are interrelated. For example, the idea that exit from cell cycle into G1/G0 is a necessary-or at least frequent-prelude to differentiation developed partly from studies of haemopoietic cell maturation, often using cell lines as models. The responses of cells to treatment with differentiating agents suggested that exit from cell cycle into G1/G0 occurs quite quickly, with functional differentiated characteristics acquired later, and so promoted the notion that cyclin-dependent kinase inhibitors (CDKIs) might be important initiators of normal differentiation. However, recent work has made it clear that differentiation and cell proliferation are regulated simultaneously but independently, that cells often start differentiating long before they stop dividing, and that the launching of differentiation is not restricted to any particular segment of the cell cycle. This combination of attributes allows expansion of cell numbers and acquisition of differentiated function to occur in parallel, generating abundant effector cells. Given this dichotomy, future studies to develop a more exact picture of the events that initiate and drive differentiation might be simplified by studying differentiation under experimental conditions that divorce it from concerns about cell cycle control.

Animals↗

PubMed2002

HL60 cells halted in G1 or S phase differentiate normally.

Differentiating agents regulate the proliferation and myeloid maturation of HL60 cells by mechanisms that are at least partly independent (Drayson et al., (2001), Exp. Cell Res. 266, 126-134). We have investigated whether halting HL60 cells in G1 or S phase influences their commitment to or maturation along the neutrophil and monocyte pathways. Early G1 and S phase cells were isolated separately by elutriation. Quinidine was used to block the cell cycle progression of G1 cells and aphidicolin to greatly retard the progression of S phase cells. Neutrophilic (in response to all-trans-retinoic acid) or monocytic (to 1 alpha,25-dihydroxyvitamin D(3)) differentiation were assessed by induction of CD11b, M-CSF receptor and CD14 expression, acquisition of granulocyte-colony stimulating factor responsiveness, capacities to phagocytose yeast and reduce nitroblue tetrazolium, and down-regulation of CD30 and transferrin receptor expression. The cell-cycle-blocked cells differentiated at normal rates, mostly without incorporating bromodeoxyuridine. These observations establish: (a) that neither transit through the cell cycle nor a cell's position in the cell cycle substantially influences execution of the neutrophilic and monocytic differentiation programs by HL60 cells; and (b) that individual HL60 cells are genuinely bipotent.

Antineoplastic Agents↗