Metabolic plasticity in CLL: adaptation to the hypoxic niche.

Metabolic transformation in cancer is increasingly well understood. However, little is known about the metabolic responses of cancer cells that permit their survival in different microenvironments. We have used a nuclear magnetic resonance based approach to monitor metabolism in living primary chronic lymphoid leukemia (CLL) cells and to interrogate their real-time metabolic responses to hypoxia. Our studies demonstrate considerable metabolic plasticity in CLL cells. Despite being in oxygenated blood, circulating CLL cells are primed for hypoxia as measured by constitutively low level hypoxia-inducible factor (HIF-1α) activity and modest lactate production from glycolysis. Upon entry to hypoxia we observed rapid upregulation of metabolic rates. CLL cells that had adapted to hypoxia returned to the 'primed' state when re-oxygenated and again showed the same adaptive response upon secondary exposure to hypoxia. We also observed HIF-1α independent differential utilization of pyruvate in oxygenated and hypoxic conditions. When oxygenated, CLL cells released pyruvate, but in hypoxia imported pyruvate to protect against hypoxia-associated oxidative stress. Finally, we identified a marked association of slower resting glucose and glutamine consumption, and lower alanine and lactate production with Binet A0 stage samples indicating that CLL may be divided into tumors with higher and lower metabolic states that reflect disease stage.

Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production.

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity.

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

Treatment of primary CLL cells with bezafibrate and medroxyprogesterone acetate induces apoptosis and represses the pro-proliferative signal of CD40-ligand, in part through increased 15dDelta12,14,PGJ2.

B-cell chronic lymphocytic leukemia (CLL), the most common leukemia in older adults, remains largely incurable and novel treatments are urgently required. We previously reported powerful pro-apoptotic actions of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against Burkitts lymphoma cells. Here, we demonstrate that BEZ and MPA individually, and more potently when combined (BEZ+MPA), induce apoptosis of unsorted and CD19(+ve)-selected CLL cells and abrogate the pro-proliferative activity of CD40(L). This action was tumor cell specific, as the drugs had little impact on normal donor cells. The antiproliferative actions of BEZ+MPA were associated with the generation of reactive oxygen species (ROS), and the proapoptotic actions were associated with the generation of both ROS and mitochondrial superoxide (MSO). BEZ increased prostaglandin D(2) (PGD(2)) synthesis by CLL cells, and treatment with PGD(2) and its antineoplastic derivative 15dDelta(12,14,)PGJ(2) recapitulated BEZ-induced antiproliferative and proapoptotic actions. The PGD(2) receptor antagonist, BW868C, did not block BEZ or PGD(2) activity against CLL cells. The potency of BEZ+MPA against CLL cells mirrored that of chlorambucil, and BEZ+MPA combined with chlorambucil was more potent than either treatment alone. Given the known safety profiles of BEZ and MPA, our data warrant further investigation of their potential as novel therapy for CLL.

Induction of sodium iodide symporter gene and molecular characterisation of HNF3 beta/FoxA2, TTF-1 and C/EBP beta in thyroid carcinoma cells.

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 beta (HNF3 beta)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein (C/EBP beta). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed decreased expression of HNF3 beta/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3 beta/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBP beta was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBP beta in the cytoplasm, suggesting that a large proportion of C/EBP beta protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells.

Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors.

Histone deacetylase inhibitors (HDIs) are a new class of drugs with significant antileukemic activity. To explore mechanisms of disease-specific HDI activity in acute myeloid leukaemia (AML), we have characterised expression of all 18 members of the histone deacetylase family in primary AML blasts and in four control cell types, namely CD34+ progenitors from umbilical cord, either quiescent or cycling (post-culture), cycling CD34+ progenitors from GCSF-stimulated adult donors and peripheral blood mononuclear cells. Only SIRT1 was consistently overexpressed (>2 fold) in AML samples compared with all controls, while HDAC6 was overexpressed relative to adult, but not neo-natal cells. HDAC5 and SIRT4 were consistently underexpressed. AML blasts and cell lines, exposed to HDIs in culture, showed both histone hyperacetylation and, unexpectedly, specific hypermethylation of H3 lysine 4. Such treatment also modulated the pattern of HDAC expression, with strong induction of HDAC11 in all myeloid cells tested and with all inhibitors (valproate, butyrate, TSA, SAHA), and lesser, more selective, induction of HDAC9 and SIRT4. The distinct pattern of HDAC expression in AML and its response to HDIs is of relevance to the development of HDI-based therapeutic strategies and may contribute to observed patterns of clinical response and development of drug resistance.

Fibrates and medroxyprogesterone acetate induce apoptosis of primary Burkitt's lymphoma cells and cell lines: potential for applying old drugs to a new disease.

Current therapies for Burkitt's lymphoma (BL) utilise combined cytotoxic chemotherapy, but these treatments are not always available in areas where the disease is endemic and are also markedly less successful in AIDS-related BL. Therefore, additional therapies are urgently required. We demonstrate here that combined fibrates and MPA exert powerful, antiproliferative actions against well-characterised Daudi, Raji and L3055 BL cell lines and primary BL cells. Detailed studies in L3055 demonstrated that this activity was mediated by induced apoptosis and confirmed by observations that overexpression of the antiapoptotic genes bcl-2 or bcl-x(L) conferred significant protection against the drugs. Importantly, since fibrates and MPA are inexpensive and stable with minimal-associated toxicities, we suggest that these drugs should be considered as adjuncts to currently available treatments for BL in endemic and AIDS-related disease.

Collagen or collagen-related peptide cause (Ca2+)i elevation and increased tyrosine phosphorylation in human megakaryocytes.

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

Bistratene A induces a microtubule-dependent block in cytokinesis and altered stathmin expression in HL60 cells.

Bistratene A is a cyclic polyether which affects cell cycle progression and can induce phosphorylation of cellular proteins. Treatment of HL60 cells with 100 ng/ml bistratene A was found to inhibit cytokinesis but had no effect on DNA synthesis and nuclear division. Consequently, bistratene A-treated cells became polyploid and multinucleate. In association with the development of this phenotype, the cytoplasmic protein stathmin was biphasically phosphorylated and levels of expression were doubled. Immunostaining of binucleate cells (bistratene A for 24 h) revealed increased alpha-tubulin localization where the cleavage furrow might be expected to form, i.e., along the equatorial plane. Treatment of these binucleate cells with the microtubule depolymerizing agent nocadazole promoted cleavage furrow formation and partially ameliorated the bistratene A-induced block in cell division. These findings implicate the polymerization status of microtubules and stathmin function in the regulation of cytokinesis.

Clofibric acid: a potential therapeutic agent in AML and MDS.

Differentiation therapy using retinoic acids (RAs) or 1alpha25-dihydroxyvitamin D3 (D3) is an attractive alternative to chemotherapy in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). However, with the exception of RA therapy for acute promyelocytic leukaemia (APL), RAs and D3 are not potent enough at doses that can be tolerated by patients. We demonstrate that clofibric acid (CA) enhances the response of HL60 cells to all-trans RA and D3. Our findings and those of others in the field lead us to suggest that combination therapy using all-trans RA and CA should be considered as potential therapy for AML and MDS.

Estrone potentiates myeloid cell differentiation: a role for 17 beta-hydroxysteroid dehydrogenase in modulating hemopoiesis.

Hormones such as 1 alpha, 25-dihydroxy vitamin D3 (D3), all-trans retinoic acid, and 9-cis retinoic acid stimulate differentiation of myeloid progenitor cells via their interaction with specific hormone receptors. However, the sensitivity of cells to these agents is not merely governed by the expression of their receptors and the availability of ligand to bind them. Recent studies from our group suggested that the actions of D3 and retinoids on myelopoiesis also are influenced by endogenous mechanisms involving other steroid hormones. In this study we examined the influence of local estrogen metabolism on the differentiation of HL60 cells and normal primitive myeloid progenitor cells. Quantitative thin-layer chromatography (TLC) analyses showed that HL60 and normal cells are able to generate estrone (E1) from estradiol (E2). Neither cell population generated significant amounts of E2 from E1. Reverse transcriptase polymerase chain reaction and Northern analyses confirmed that normal and leukemic myeloid progenitor cells expressed mRNA for the type I and IV isoforms of 17 beta-hydroxysteroid dehydrogenase. Conversion of E2 to E1 was upregulated within 24 hours when HL60 cells were treated with either all-trans retinoic acid or D3 at doses that induce their differentiation toward neutrophils or monocytes, respectively. Similarly, D3-induced monocyte differentiation of normal myeloid progenitor cells was associated with increased capacity to generate E1 from E2. When HL60 cells or normal myeloid progenitor cells were exposed to exogenous E1 they became more sensitive to the differentiation-inducing effects of D3. Data presented provide further evidence for the local modulation of myelopoiesis by intracrine mechanisms. In particular, our findings suggest that local metabolism of steroids by normal as well as leukemic myeloid cells influences their responsiveness to D3 and retinoids.

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells.

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

Vitamin D and hematopoiesis.

Analysis of the nonclassic actions of vitamin D(3) has highlighted a wide range of target tissues for the hormone 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Systemic or locally produced 1,25(OH)(2)D(3) may play a role in modulating cell development processes such as hematopoiesis. The mechanisms by which 1,25(OH)(2)D(3) achieves this are discussed in this review. In particular, data from our laboratories suggest that 1,25(OH)(2)D(3) does not provide a deterministic signal for monocyte differentiation. Rather, the hormone acts as a permissive agent for myeloid precursor cells to enter a genetically determined terminal maturation pathway. The effiacy of 1,25(OH)(2)D(3) in leukemia therapy has been improved by the development of novel vitamin D analogues that have potent antiproliferative activity and low hypercalcemic side effects. Another solution to the problem of side effects is to enhance specifically the antiproliferative effects of 1,25(OH)(2)D(3). A novel mechanism within hematopoiesic cells that governs their responsiveness to the antiproliferative/differentiative actions of 1,25(OH)(2)D(3) outlined.

Triiodothyronine blocks potentiation of HL60 monocyte differentiation by anti-inflammatory agents and by steroids and induces apoptosis of all-trans retinoic acid "primed" cells.

Sensitivity of the human promyeloid cell line HL60 to physiological differentiating agents [e.g. all-trans retinoic acid (all-trans RA) and 1 alpha, 25-dihydroxyvitamin (D3)] is increased by exposure of cells to "anti-inflammatory agents" (e.g. indomethacin) and to steroids [e.g. medroxyprogesterone acetate (MPA)] and post "priming" with a low dose (10(-8) M) of all-trans RA. Co-treatment of serum free-grown HL60 cells (HL60-ITS) with indomethacin and D3 reduces the dose of D3 required for monocyte differentiation from 10(-7) to 6.25 x 10(-9) M. This potentiating effect was observed to be almost absent when experiments where undertaken using serum-grown HL60 cells (HL60-FCS). The agent present in serum that interferes with indomethacin- and MPA-potentiation of the sensitivity of HL60 cells to D3 has been identified as the thyroid hormone 3,5,3'-L-triiodothyronine (T3). "Priming" of HL60-ITS cells with a low dose of all-trans RA reduces the amount of D3 required for the induction of monocyte differentiation to the same degree as co-treatment with either indomethacin or MPA (to 5 x 10(-9) M). However, the combined effect of all-trans RA "priming" and T3 treatment of HL60-ITS cells was induction of apoptosis. Treatment with either agent alone did not result in increased levels of apoptotic cells. These data reveal that T3 has an important influence on the capacity of HL60 cells to undergo differentiation and can promote apoptosis of these cells. Drug combinations, such as a differentiation potentiating agent, for example, indomethacin or MPA, and a differentiation inducer, for example, all-trans RA or D3, may have important therapeutic significance. Serum levels of T3 would be anticipated to influence the outcome.

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents.

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.

Potentiation of myeloid differentiation by anti-inflammatory agents, by steroids and by retinoic acid involves a single intracellular target, probably an enzyme of the aldoketoreductase family.

HL60 cells are human promyeloid cells that can be induced to differentiate by physiological stimuli (e.g. all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (D3), granulocyte colony-stimulating factor (G-CSF)) and by non-physiological agents such as dimethysulphoxide (DMSO) and protein kinase C-activating phorbol esters. The sensitivity of HL60 cells to physiological differentiating agents, but not to DMSO, is enhanced when cells are exposed to 'anti-inflammatory agents' (e.g. indomethacin) or are 'primed' (pretreated) with a small amount of ATRA: alone, neither treatment induces differentiation. We earlier suggested that indomethacin might act by inhibiting the endogenous formation of a differentiation-suppressing prostanoid (Bunce, C.M., et al. (1994) Leukemia 8, 595-604). Studies of the formation of prostanoids by HL60 cells and of the effects of prostanoids on these cells failed to identify any prostanoid that could be implicated in sensitization by indomethacin. 3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) is another target of such 'anti-inflammatory agents'. Steroid inhibitors of 3 alpha-HSD sensitized HL60 cells to inducers of differentiation in a manner similar to indomethacin. 3 alpha-HSD is a member of the aldoketoreductase enzyme family, which comprises many enzymes of similar size and primary sequence. A protein that was recognised by an antiserum to 3 alpha-HSD was found in HL60 cells, but the cells showed no detectable 3 alpha-HSD activity. The 3 alpha-HSD-like protein was strikingly down-regulated by 'priming' doses of ATRA. When treatment with a differentiation-sensitizing 'anti-inflammatory agent' or steroid was combined with ATRA "priming', the effects of the different treatments were not additive: the resulting increase in sensitivity equalled that achievable by either treatment alone. We conclude that interference with a single intracellular regulatory mechanism underlies the increases in sensitivity of cells to differentiating agents that are caused by anti-inflammatory agents, by certain steroids and by 'priming' with ATRA. Decreased activity of a yet-to-be-identified member of the aldoketoreductase family of dehydrogenases is likely to be a central feature of a previously unrecognised mechanism that controls the responsiveness of cells to environmental stimuli such as retinoids and D3.

Particular combinations of signals, by retinoic acid and 1 alpha, 25 dihydroxyvitamin D3, promote apoptosis of HL60 cells.

The promyeloid cell line HL60, when grown in serum-free medium, is induced to differentiate towards either neutrophils or monocytes by treatment with particular concentrations of 9-cis retinoic acid (9-cis RA) and 1 alpha, 25 dihydroxyvitamin D3 (D3). We have investigated whether treatment of HL60 cells with 9-cis RA and D3 can lead to growth arrest and a failure to undergo cell differentiation. This occurred in two circumstances and HL60 cells died rapidly by apoptosis. First, treatment with 5 x 10(-7) M 9-cis RA and 1.25 x 10(-9)-3.1 x 10(-10) M D3 promoted growth arrest and apoptosis of HL60 cells. The amount of 9-cis RA alone promoted significant neutrophil differentiation of HL60 cells. The amounts of D3 alone promoted a very low level of monocyte differentiation. Treatment with each agent alone did not result in increased levels of apoptosis. Second, HL60 cells were treated with concentrations of 9-cis RA (5 x 10(-7) M) and D3 (3.9 x 10(-14) M) that were appropriate for induction of neutrophil differentiation. At the time when they were undergoing commitment to the neutrophil pathway of differentiation (days 1-2), an amount of D3 (1 x 10(-7) M) that promotes monocyte differentiation was added to the cultures. HL60 cells failed to differentiate and died by apoptosis. Hence, certain combinations of signals, elicited by 9-cis RA and D3, promote apoptosis of HL60 cells. This finding has important implications for the use of retinoids and D3 in differentiation therapy.

Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells.

1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.

Treatment of HL60 cells with various combinations of retinoids and 1 alpha,25 dihydroxyvitamin D3 results in differentiation towards neutrophils or monocytes or a failure to differentiate and apoptosis.

It is well documented that treatment of serum-grown HL60 cells with 10(-7) M all-trans retinoic acid (all-trans RA) induces neutrophil differentiation, whereas treatment with 10(-7) M 1 alpha,25 dihydroxyvitamin D3(D3) induces differentiation towards monocytes. In recent investigations, using serum-free grown HL60 cells, we observed that all-trans RA, at 10(-7) M, did not induce neutrophil differentiation and that all-trans RA, at 10(-8) M, reduced the D3 concentration required for monocyte differentiation to 5 x 10(-9) M. In this study, co-operative interactions between all-trans and 9-cis RA and D3 which promote neutrophil and monocyte differentiation of HL60 cells have been analysed in detail. Treatment of serum-free grown HL60 cells with 5 x 10(-7) M all-trans RA or 9-cis RA resulted in sub-optimal neutrophil differentiation (up to 25% mature cells). As shown for all-trans RA, 9-cis RA cooperated with D3 to promote monocyte differentiation. Culture of HL60 cells in 5 x 10(-7) M 9-cis RA together with a wide range of concentrations of D3 resulted in promotion of neutrophil differentiation at 10(-15)-10(-12) D3, a failure to differentiate and apoptosis at 10(-11)-10(-10) M D3, followed by co-operativity between 9-cis RA and 5 x 10(-9) M D3 in inducing monocyte differentiation in the absence of neutrophil differentiation. Similar results were obtained when HL60 cells were treated with 5 x 10(-7) all-trans RA together with a wide range of concentrations of D3. Cross titration analyses of the effects of 9-cis RA and D3 on HL60 cell differentiation were undertaken to determine the boundaries of the concentrations of each agent, alone and in combination, that give rise to optimal neutrophil and monocyte differentiation of HL60 cells. The observed cooperativities between either 9-cis RA or all-trans RA and D3 have important implications for the use of combinations of these agents in differentiation therapy.