Functional expression cloning reveals proapoptotic role for protein phosphatase 4.

Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.

Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre.

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.

Changes in antigen expression on differentiating HL60 cells treated with dimethylsulphoxide, all-trans retinoic acid, alpha1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl phorbol-13-acetate.

We describe changes in antigen expression on HL60 cells with differentiation into granulocytes induced by all-trans retinoic acid (ATRA) or dimethylsulphoxide (DMSO), into monocytes by alpha1,25-dihydroxyvitamin D3 (D3), or into macrophages by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Undifferentiated cells expressed CD13, CD14 (at a low level), CD15, CDw17, CD32, CD33, CD49e, CD63, CD64, CDw65, CD71 and CD87 antigens and bound the unclustered mAb D171 and Mo5. Differentiated and undifferentiated cells were negative for CD16, CD34, CD61, CD66abcde, CD68, CD88, CDw90 and CD93. Four panels of markers were identified whose expression changes significantly following differentiation. CD15, CD49e, CD63, CDw65, CD71 and mAb D171 and IGR-2,1A6 for DMSO; CD13, CD15, CDw17, CD49e, CD63, CDw65, CD71, CD87, CDw92 and mAb D171 and IGR-2,1A6 for ATRA; CD14, CD31, CD35, CD71, CD87, CDw92 and mAb D171 and BRIC18 for D3; CDw12, CD13, CD15, CD31, CD35, CD49e, CD71, CD87, CDw92 and mAb D171 for TPA. These will be useful for analyzing the pathways that regulate differentiation, whether the observed changes are consequences of differentiation or more direct effects of the inducers. HL60 cells provide a model for investigating the regulation of these antigens.

recessive spotting: a linked locus that interacts with W/Kit but is not allelic.

BACKGROUND: The murine coat-colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto-oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan-rs. RESULTS: The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk-1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit. CONCLUSIONS: rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk-1 or Txk, but Tec remains a candidate for rs.

Transcriptional repression by the promyelocytic leukemia protein, PML.

Acute promyelocytic leukemia is characterized by the presence of a t(15; 17) chromosomal translocation which results in the expression of a chimeric gene product, PMLRAR alpha, consisting of an N-terminal-truncated retinoic acid receptor-alpha fused to a C-terminal-truncated PML. Several structural features, and regions of homology to known transcription factors, suggest that PML may be involved in the regulation of gene expression. In this study we have analyzed the transcriptional regulatory activity of PML using chimeric GAL4/PML constructs and GAL4-responsive reporter plasmids. The data presented demonstrate that PML, when fused to the DNA-binding domain of GAL4 (GAL4/PML), inhibits transcription from GAL4-responsive promoters. The magnitude of this repression is cell type and promoter dependent, and deletion studies show that the putative coiled-coil and part of the serine-rich regions of PML are required for this activity. These regions are also shown to be responsible for the repression of transcription activity from the EGFR promoter. The data presented also demonstrate that GAL4/PML can recruit PMLRAR alpha resulting in the retinoid-inducible transcriptional activation of a GAL4-responsive promoter, a function dependent on the presence of the coiled-coil region of PMLRAR alpha.

Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123).

It is well established that the fluorescent probes dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydrorhodamine 123 (H2R123) can be used to detect the respiratory burst response of mature myeloid cells. We describe a simple, fast and quantitative assay for myeloid differentiation based on the oxidation of these probes, which can be performed from start to finish in 96-well dishes. A bis(acetoxymethyl) ester of H2DCF-DA, 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CODCF-DA) is also capable of detecting the respiratory burst, but is less suitable than H2DCF-DA or H2R123 in our system. The amount of fluorescence produced can be quantified using a calibration curve, and values can be normalised to cell numbers using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylte-trazolium bromide (MTT) cell proliferation assay. Results are expressed as 'equivalents of soluble fluorescein' (ESF) produced per cell under the defined reaction conditions. The extent to which HL60 cells reduce MTT is unaffected by differentiation induced by retinoic acid or 1 alpha,25-dihydroxyvitamin D3, and normalisation of fluorescence values using the MTT assay appears to be valid for a wide range of myeloid cell lines and differentiation inducers or cytokines.

Retinoid receptors and acute promyelocytic leukaemia.

While a great deal has been learned about APL over the last few years, many important questions remain unanswered. It has become clear that the PML/RAR-alpha fusion protein is expressed in most cases of APL, and this protein presumably contributes to leukaemia initiation and/or progression. PML/RAR-alpha appears to specifically block the further differentiation of myeloid progenitor cells, although the mechanism of its action is not known. It may inhibit the transcription of RAR- or PML-regulated genes, in which case expression must be restored in the presence of therapeutic RA concentrations. However, the possibility remains that PML/RAR-alpha may have a novel function. In order to elucidate the molecular pathogenesis of APL, several important questions remain to be answered. These include whether PML is a transcription factor; the identification of its target genes and response elements, and the role of PML/RAR-alpha and RA in their regulation. Also whether the expression of PML/RAR-alpha in bone marrow cells (either by itself or in combination with other oncogenes) alters their tumourigenicity or differentiation potential. It is also important to determine the function(s) of PLZF and PLZF/RAR-alpha, and determine whether other APL patients with mutations involving PML or RAR-alpha (but not both) respond to therapy with all-trans-RA. Finally, it is important both for the understanding of the molecular biology of APL and its therapy, to determine the effects of other regulatory factors involved in the control of myeloid cell differentiation such as granulocyte colony stimulation factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) on APL cells in vitro and in vivo, both at presentation and in the RA-resistant patients in relapse.

Long-term use of depot-norethisterone enanthate: effect on blood lipid fractions.

This is the third report of a metabolic study on 56 long-term users (24 for 2-5 yr; 32 for over 5 yr) of the injectable contraceptive norethisterone enanthate (Net-En) and deals with the effects on the blood levels of lipoprotein fractions. There was no significant difference between this group and a group of 30 non-users in serum concentrations of triglycerides, total cholesterol, low density and very low density lipoproteins. There was a significant reduction in mean high density lipoprotein levels between the controls and the user groups (16% for the intermediate duration and 12% for the longer duration). Age, ethnic group, body-mass index and a close family history of cardiovascular disease were taken into account, as were various lifestyle factors: diet, exercise, alcohol consumption and smoking. In a smaller group, levels of apoproteins A and B were also assayed. There was a significant reduction in apoprotein A between controls and all users, but the significance was lost on adjusting for confounding variables. Consideration was given to the ratio of total cholesterol to high density lipoprotein cholesterol as an index of coronary risk. There were no significant differences in the total cholesterol:HDL-C ratio between controls and user groups. There was a significant interaction with ethnic group (Caucasian or Afro-Caribbean) in the response to duration of use.

Stimulation of proliferation of HL60 cells by low concentrations of 12-O-tetradecanoylphorbol-13-acetate and its relationship to the mitogenic effects of insulin.

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA (greater than 1 nM) cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived of insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. A TPA concentration between 0.03 and 0.3 nM will approximately double the long-term rate of thymidine incorporation into DNA and the rate of increase in cell density. Low-TPA becomes progressively less able to stimulate further proliferation as the insulin concentration is increased and is virtually without effect on cells stimulated by an optimal insulin concentration (5 micrograms ml-1). Insulin itself stimulates proliferation to a greater extent than low-TPA, increasing the long-term rate of thymidine incorporation and the rate of increase in cell density by three- to fourfold. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine (during a 1-h pulse after 1 or 2 days incubation) by three- to fourfold, as compared to a sevenfold stimulation by insulin. The proliferation response to low TPA concentrations provides a useful model for dissecting the signalling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.

Phorbol ester-induced macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells occurs independently of transferrin availability.

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate into macrophage-like cells by the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Addition of this agent to HL-60 cells causes a rapid internalization of surface transferrin receptor, followed by long-term receptor down-regulation at the level of gene expression. These effects precede the inhibition of proliferation and the acquisition of differentiation markers, and it has been suggested that transferrin receptor down-regulation may play a mediating role in these later events. Here we show that HL-60 cells will grow indefinitely in serum-free medium supplemented with either 5 micrograms ml-1 transferrin or 300 microM ferric citrate and that TPA inhibits cell proliferation (assayed by cell density and rate of thymidine incorporation) and induces macrophage-like differentiation (assayed by induction of cell adhesion and increased nonspecific esterase activity) with identical dose curves in both media. Furthermore, a neutralizing anti-transferrin antibody completely inhibits transferrin-dependent cell proliferation but has no effect on differentiation in the presence or absence of transferrin. We conclude that TPA-induced down-regulation of transferrin binding and internalization does not mediate the subsequent growth arrest and differentiation of HL-60 cells.

Plasmapheresis in familial hypercholesterolemia.

Twelve years' experience confirms that plasma exchange beneficially influences the course of homozygous familial hypercholesterolemia. Pilot studies with low density lipoprotein (LDL) apheresis using a dextran sulphate affinity column suggest that this procedure has advantages over plasma exchange, which it may eventually replace. Combination therapy with lovastatin and LDL apheresis is currently being assessed as a means of inducing regression of coronary lesions. Preliminary results of a pilot study in four patients with heterozygous familial hypercholesterolemia are encouraging.

High prevalence of hypertriglyceridaemia and apolipoprotein abnormalities in coronary artery disease.

Serum lipids and apolipoproteins A-I and B were measured in 174 men aged less than 60 with angiographically confirmed coronary artery disease and in 572 healthy control men. Two thirds of the patients had raised age-corrected values of fasting serum cholesterol and/or triglyceride and/or a low high density lipoprotein (HDL) cholesterol compared with the controls. Eighteen (30%) of the 61 normolipidaemic patients had a concentration of serum apolipoprotein A-I below the 5th percentile of 233 controls. In normolipidaemic patients on beta blockers the relative prevalence of serum low density lipoprotein (LDL)-apolipoprotein B values above the 95th percentile of 339 controls was significantly increased. Discriminant function analysis showed that a raised concentration of serum triglyceride was the best discriminant between patients and controls, with raised LDL-apolipoprotein B and reduced apolipoprotein A-I coming second only to triglyceride in analyses where each was separately compared with all the lipid variables. These associations were highly significant and were independent of other influences, including beta blockade. These findings re-emphasise the importance of hypertriglyceridaemia as a risk factor and confirm that apolipoprotein abnormalities occur frequently in coronary disease, even in normolipidaemic patients.

Metabolic investigations with Femodene--an oral contraceptive containing gestodene and ethinyloestradiol.

The metabolic effects of a new oral contraceptive Femodene (SHD 356C) containing 75 micrograms gestodene (delta-15-levonorgestrel) and 30 micrograms ethinyloestradiol were studied in two groups of women. Group 1 consisted of women not currently using oral contraceptives; Group 2 consisted of women switching to Femodene from their current oral contraceptive. Changes in lipid metabolism were assessed by measuring serum levels of cholesterol, triglycerides, LDL-C, VLDL-C, HDL-C, HDL2-C and HDL3-C. Minimal changes occurred in lipid metabolism apart from increases in triglyceride concentrations. Women in Group 1 showed a 105% increase in SHBG levels and a 51% increase in caeruloplasmin levels compared to increases of 33% and 2% in women in Group 2. A comparison of the two groups of women suggested that the gestagen in Femodene exerted a less anti-oestrogenic effect than most of the gestagens currently used in oral contraceptives. No significant changes occurred in liver function (assessed by estimation of gamma-glutamyl transferase) or in the coagulation factors, Factor X and antithrombin III. Minor effects on glucose tolerance as assessed by blood glucose and plasma insulin levels were noted. These minimal effects on metabolism, combined with its high efficacy and acceptability shown in clinical trials, makes Femodene an ideal alternative to currently used oral contraceptives.

Paradoxical elevation of LDL apoprotein B levels in hypertriglyceridaemic patients and normal subjects ingesting fish oil.

Twenty-three hypertriglyceridaemic patients treated with 15 g/day of a fish oil concentrate (Maxepa) showed the expected reduction in serum triglyceride concentration but levels of LDL apoprotein B (apoB), measured by radial immunodiffusion, increased significantly. Increases in LDL apoB did not correlate with lipoprotein phenotype or changes in serum triglyceride. Studies in eight normal volunteers demonstrated that the effect of fish oil on LDL apoB was not restricted to hypertriglyceridaemic subjects. In view of the evidence that LDL apoB may be a risk factor for coronary heart disease these findings raise questions regarding the use of fish oil in the treatment of hypertriglyceridaemia.

Efficacy of mevinolin as adjuvant therapy for refractory familial hypercholesterolaemia.

Mevinolin, a potent inhibitor of cholesterol synthesis, was used as a therapeutic adjuvant in patients with refractory familial hypercholesterolaemia for an average period of 13 months. Sustained decreases in serum cholesterol of 23 and 31 per cent were achieved by doses of 20 mg and 40 mg/day respectively in 13 heterozygotes already on cholestyramine or after partial ileal bypass. Administration of 80 mg/day to three patients undergoing plasma exchange reduced peak serum cholesterol levels by 11.5 per cent in two homozygotes and by 17 per cent in a double heterozygote for familial hypercholesterolaemia and type III hyperlipoproteinaemia. The decrease in cholesterol was largely confined to low-density lipoprotein and no significant changes occurred in serum triglyceride or high-density lipoprotein cholesterol. Mevinolin was well-tolerated except in one patient who developed myositic symptoms; asymptomatic, transient elevations of serum enzymes were observed in five others. Short and long Synacthen tests showed no evidence that the drug impaired adrenocortical response to ACTH. These results indicate that mevinolin provides a safe and highly effective means of reducing LDL levels in patients with heterozygous familial hypercholesterolaemia refractory to conventional treatment but is less useful in homozygotes.

Long-term effects of intestinal alpha-glucosidase inhibition on postprandial glucose, pancreatic and gut hormone responses and fasting serum lipids in diabetics on sulphonylureas.

Seventeen non-insulin-dependent diabetics poorly controlled by diet and sulphonylurea drugs took part in a long-term (20-52 weeks) trial of the effect of an alpha-glucosidase inhibitor (acarbose 100 mg thrice daily) on postprandial glycaemic and gastro-entero-pancreatic hormone responses. Patients were assessed before, during, and after the trial period with identical 2.2 MJ mixed test meals plus placebo or acarbose 100 mg, and sulphonylurea therapy was continued throughout. Acarbose administration reduced the integrated postprandial plasma responses of glucose to 58 +/- 10% (mean +/- SEM, p less than 0.001), insulin to 61 +/- 10% (p less than 0.01) and gastric inhibitory polypeptide to 45 +/- 8% (p less than 0.001) of control values, increased the enteroglucagon response to 152 +/- 26% (p less than 0.001) of control and slightly prolonged the postprandial release of motilin. Recorded glycosuria was significantly (p less than 0.01) reduced throughout the treatment period. The effects of acarbose on postprandial glycaemic and endocrine responses remained approximately constant throughout the trial period, and responses returned to pre-treatment values within 2 days of stopping treatment.

Coronary artery disease and haemostatic variables in heterozygous familial hypercholesterolaemia.

Haemostatic variables were measured in 61 patients with heterozygous familial hypercholesterolaemia, 32 of whom had evidence of coronary heart disease. Age adjusted mean concentrations of plasma fibrinogen and factor VIII were significantly higher in these patients than in the 29 patients without coronary heart disease, but there were no significant differences in serum lipid concentrations between the two groups. Comparisons in 30 patients taking and not taking lipid lowering drugs showed lower values for low density lipoprotein cholesterol, high density lipoprotein cholesterol and antithrombin III, and a higher high density lipoprotein ratio while receiving treatment. The results suggest that hypercoagulability may play a role in the pathogenesis of coronary heart disease in patients with familial hypercholesterolaemia.

Circulating human C-reactive protein binds very low density lipoproteins.

Native human CRP in solution formed complexes with the abnormal lipoprotein beta-VLDL in serum of patients with type III hyperlipoproteinaemia. CRP also formed complexes in sera from individuals with type IV and type V hyperlipoproteinaemia. The binding was calcium-dependent and inhibitable by free phosphoryl choline. No complexes were demonstrable in sera containing high LDL levels from cases of type IIa hyperlipoproteinaemia. Addition of isolated beta-VLDL, but not of isolated LDL, to acute phase normolipoproteinaemic serum caused the appearance of soluble CRP-lipoprotein complexes. In contrast, addition of an excess of isolated normal VLDL to acute phase serum or to isolated CRP was followed by agglutination (creaming) of the lipoprotein particles. Rabbit CRP, on the other hand, formed soluble complexes both with normal human apoB containing lipoproteins and with the abnormal beta-VLDL. Human CRP complexed with rabbit beta-VLDL but not with normal rabbit serum lipoproteins. These interactions may be important for the role of CRP in health and disease.