The control of proliferation and epithelial restitution are processes that are poorly understood. The effects of (n-3), (n-6) and trans fatty acids on proliferation of subconfluent IEC-6 cultures and restitution of wounded IEC-6 monolayers were investigated. Incorporation of supplemented fatty acids into cellular phospholipid was also assessed. Sulforhodamine B protein dye binding assay was utilized to assess the proliferative effects of fatty acids on growth of IEC-6 cultures. Incorporation of supplemental fatty acids into cellular phospholipid was examined by thin-layer chromatography combined with gas chromatography. The modulation of epithelial restitution was examined by razor blade wounding confluent IEC-6 monolayers grown in media supplemented with various fatty acids. Inhibition of eicosanoid synthesis by indomethacin during the wounding assay was also assessed. Both (n-3) and (n-6) fatty acids significantly inhibited growth of this intestinal epithelial cell model at concentrations above 125 micromol/L. The trans fatty acid, linoelaidate 18:2(n-6)trans, inhibited growth of IEC-6 cells at concentrations above 250 micromol/L. Another trans fatty acid, elaidate 18:1(n-9)trans, was well-tolerated at concentrations as high as 500 micromol/L. Eicosapentanoic 20:5(n-3), linoleic 18:2(n-6), alpha-linolenic 18:3(n-3), gamma-linolenic 18:3(n-6) and arachidonic 20:4(n-6) acids all significantly enhanced cellular migration in the IEC-6 model of wound healing. Eicosapentanoate, linoleate, alpha-linolenate, gamma-linolenate and arachidonate are all capable of improving reconstitution of epithelial integrity following mucosal injury. Inhibition of eicosanoid synthesis reduced the enhancement of restitution by n-6 fatty acids back to control levels.

We wanted to examine the effects of an oil rich in docosahexaenoic acid (DHA), without eicosapentaenoic acid, on the composition of membrane phospholipid in a variety of tissues. Our in vitro studies had previously shown that DHA could modify glucose and nucleoside transport in cells in culture and also increase selectivity of the nucleoside drug, arabinosylcytosine (araC) toward tumor cells. Here we wanted to examine what effect DHA supplementation would have in the whole animal in terms of the chemosensitivity of normal bone marrow, the dose-limiting tissue during chemotherapy, to araC. The purpose was to determine whether fatty acid supplementation might be useful as an adjuvant to chemotherapy. We fed diets containing 5% (w/w) low fat-corn oil (LF-CO group), 10% moderate fat-safflower oil (MF-SO group), or 10% DHASCO (MF-DHA group) to weanling Fischer 344 rats for 8-9 wk. Feed intake and growth were not different between the different diets. Similarly, treatment of animals with the chemotherapeutic drug araC did not differentially affect growth, feed intake, or tissue fatty acid composition for the different diet groups. Fatty acid compositions of bone marrow, liver, red blood cells, plasma phospholipid and triglyceride, as well as skeletal and cardiac muscle, were substantially different between the dietary groups. The DHASCO oil contained 46% DHA (22:6n-3) and resulted in profound incorporation of DHA in all tissues examined. The most dramatic response was seen in skeletal muscle of MF-DHA fed animals where DHA represented 46% of membrane phospholipid fatty acids. This is likely to have consequences to muscle function. Although DHASCO contains a similar level of saturated fatty acids (42%), few differences in saturates were noted between the various dietary groups for most of the tissues examined. Both LF-CO and MF-SO diets were hypercholesterolemic, and the LF-CO was also hypertriglyceridemic compared to the chow-fed animals. Animals fed the MF-DHA diet had the lowest triglyceride levels of any of the treatment groups and cholesterol levels comparable to chow-fed animals. MF-DHA had substantially higher numbers of colony-forming units-granulocyte macrophage (CFU-GM) as reflected in a twofold higher bone marrow cellularity than either chow or LF-CO animals, suggesting expansion of the bone marrow compartment with DHA feeding. Although higher than LF-SO, the number of CFU-GM in MF-SO animals was not significantly higher than animals fed chow. Bone marrow from LF-CO animals appeared to be more resistant to araC treatment than either MF group. Thus, DHA, fed as DHASCO, has advantages over low or moderate n-6 diets and chow as it is has both hypolipidemic- and bone marrow-enhancing properties in weanling Fischer 344 rats. This suggests that DHA supplementation may be useful in adjuvant chemotherapy.

We have shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) primes NB4 cells, the only available acute promyelocytic leukemia cell line, for 12-O-tetradecanoyl-phorbol-13-acetate-induced monocytic differentiation. Here, we have used isomers of 1,25(OH)2D3 to investigate the role of 1,25(OH)2D3 and its putative nuclear receptor (VDR) in NB4 cell monocytic differentiation. 1 beta,25-dihydroxyvitamin D3 (HL), a specific antagonist of only the nongenomic signals of 1,25(OH)2D3, attenuated the priming effect of 1,25(OH)2D3. The 6-cis conformer of 1,25(OH)2D3 (HF), which is unable to bind to VDR, was 20 times more potent than 1,25(OH)2D3 as a priming agent for monocytic differentiation. This response was also blocked by the HL antagonist. Unlike myelocytic HL-60 cells, which respond to 1,25(OH)2D3 with increases in VDR expression and monocytic differentiation, neither HF nor 1,25(OH)2D3 regulated VDR expression in NB4 cells. In the monocytic differentiation of acute promyelocytic leukemia cells, 1,25(OH)2D3 appears to signal through a pathway independent of VDR/VDRE action.

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na ؉ , guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na ؉ buffer decreased by 75% with the addition of 1 M NBMPR and the IC 50 for NBMPR inhibition was 0.7 ؎ 0.1 nM. Replacement of Na ؉ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na ؉ -dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na ؉ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mM competing nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of the csg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.Purine and pyrimidine nucleotides, and their related metabolic products, participate in numerous biological processes.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.