Philip J. Hughes

Sulfation of “Estrogenic” Alkylphenols and 17β-Estradiol by Human Platelet Phenol Sulfotransferases

We have investigated the ability of alkylphenols to act as substrates and/or inhibitors of phenol sulfotransferase enzymes in human platelet cytosolic fractions. Our results indicate: (i) straight chain alkylphenols do not interact with the monoamine-sulfating phenol sulfotransferase (SULT1A3); (ii) short chain 4-n-alkylphenols (C < 8) are substrates for the phenol-sulfating enzymes (SULT1A1/2), which exhibit two activity maxima against substrates with alkyl chain lengths of C1–2 and C4–5; (iii) long chain 4-n-substituted alkylphenols (C ≥ 8) are poor substrates and act as inhibitors of SULT1A1/2; (iv) human platelets contain two activities, of low and high affinity, capable of sulfating 17β-estradiol, and 4-n-nonylphenol is a partial mixed inhibitor of the low affinity form of this activity. We conclude that by acting either as substrates or inhibitors of SULT1A1/2, alkylphenols may influence the sulfation, and hence the excretion, of estrogens and other phenol sulfotransferase substrates in humans.

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.

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.

1α,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

1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) 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, C2‐ceramide) and phosphatidate phosphohydrolase (PAP) (propranolol and chlorpromazine), but was unaffected by inhibitors of phospholipase C. The 1α,25(OH)2D3‐induced STS activity was also attenuated by inhibitors of protein kinase Cα and protein kinase Cδ (Go 6976, HBDDE and rottlerin), but not by an inhibitor of protein kinase Cβ (LY379196). Additionally, 1α,25(OH)2D3‐induced STS activity was attenuated by inhibitors of RAS (manumycin A), RAF (GW5074), MEK (PD098059 and U1026) and JNK (SP600125), but not p38 (PD169316). 1α,25(OH)2D3 produced a rapid and long lasting stimulation of the ERK‐MAP kinase signalling cascade in HL60 myeloid leukaemic cells. This ‘non‐genomic’ effect of 1α,25(OH)2D3 blocked by pharmacological antagonists of nuclear vitamin D receptors (VDRnuc) 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 Cα (HBDDE) abrogated the 1α,25(OH)2D3‐stimulated increase in ERK‐MAP kinase activity. Taken together, these results show that 1α,25(OH)2D3/VDRnuc activation of the RAS/RAF/ERK‐MAP kinase signalling pathway plays an important role in augmenting STS activity in human myeloid leukaemic cell lines. J. Cell. Biochem. 98: 590–617, 2006. � 2006 Wiley‐Liss, Inc.

The vitamin D receptor-mediated activation of phosphatidylinositol 3-kinase (PI3Kα) plays a role in the 1α,25-dihydroxyvitamin D3-stimulated increase in steroid sulphatase activity in myeloid leukaemic cell lines

In this article we show that 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) stimulates the activity of the class IA phosphatidylinositol 3‐kinase PI3Kα and its downstream target Akt in HL60, U937 and THP‐1 myeloid leukaemic cell lines. Furthermore, we show that the classical nuclear vitamin D receptor (VDRnuc) is involved in this activation of the PI3K/Akt signalling in these cell lines. We have previously shown that the activity of steroid sulphatase is stimulated in HL60, U937 and THP‐1 myeloid leukaemic cell lines by 1α,25(OH)2D3 (Hughes et al., [ 2001 ] Biochem J 355:361–371; Hughes et al., [ 2005 ] J Cell Biochem 94:1175–1189; Hughes and Brown [ 2006 ] J Cell Biochem 98:590–617). In this article we show that the 1α,25(OH)2D3‐stimulated increase in signalling via the PI3K/Akt pathway plays a role in the increase in steroid sulphatase activity in the HL60 U937 and THP‐1 cell lines. We used a variety of pharmacological and biochemical approaches to show that activation of PI3Kα mediates the 1α,25(OH)2D3‐stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. We also show that the PI3K/Akt dependent activation of NF‐κB plays a role in the 1α,25(OH)2D3‐stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. J. Cell. Biochem. 103: 1551–1572, 2008.

Novel inositol containing phospholipids and phosphates: their synthesis and possible new roles in cellular signalling.

Details of the widely employed PtdIns(4,5)P2 hydrolysis receptor-stimulated signalling pathway continue to be elucidated rapidly. However, it has recently become apparent that numerous other inositol lipids and phosphates are widespread and are likely to have important cellular functions. In this review, we focus particularly on three rapidly progressing areas: the synthesis and possible functions of 3-phosphorylated inositol lipids, particularly phosphatidylinositol 3,4,5-trisphosphate; the roles of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in coordinating intracellular Ca2+ mobilization and Ca2+ influx in stimulated cells; and the metabolism and possible functions of other inositol polyphosphates and of inositol polyphosphate pyrophosphates.

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARα 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 α (RARα). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARα/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 PKCα and PKCδ 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.

Retinoid Differentiation Therapy for Common Types of Acute Myeloid Leukemia

Many cancers arise in a tissue stem cell, and cell differentiation is impaired resulting in an accumulation of immature cells. The introduction of all-trans retinoic acid (ATRA) in 1987 to treat acute promyelocytic leukemia (APL), a rare subtype of acute myeloid leukemia (AML), pioneered a new approach to obtain remission in malignancies by restoring the terminal maturation of leukemia cells resulting in these cells having a limited lifespan. Differentiation therapy also offers the prospect of a less aggressive treatment by virtue of attenuated growth of leukemia cells coupled to limited damage to normal cells. The success of ATRA in differentiation therapy of APL is well known. However, ATRA does not work in non-APL AML. Here we examine some of the molecular pathways towards new retinoid-based differentiation therapy of non-APL AML. Prospects include modulation of the epigenetic status of ATRA-insensitive AML cells, agents that influence intracellular signalling events that are provoked by ATRA, and the use of novel synthetic retinoids.

1α,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 1α,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. 1α,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 1α,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 1α,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.

The versatility of haematopoietic stem cells: implications for leukaemia.

To understand the origins, and disease progression, of leukaemia we first need a clear idea of how the progeny of haematopoietic stem/precursor cells normally choose their fates. For about 30 years, ‘classical’ models of blood cell development have envisaged a branching tree with two trunks representing the two major families of cells: myeloid/erythroid and lymphoid. Recent debate about this apparent dichotomy has given rise to new models of haematopoiesis and new ways of viewing stem-cell behaviour. These suggest that stem and progenitor cells are more versatile than was first appreciated, so there can be multiple routes to one type of end cell. An important aspect of this versatility during haematopoiesis is that progenitor cells retain an unexpected portfolio of clandestine lineage potentials even when they seem to have progressed quite far along a particular developmental pathway. Here we examine this decision-making process and ask whether, developmentally, leukaemia stem cells are equally or less versatile than their normal counterparts.