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.

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.