The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia.

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC(50)=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC(50) 1 µM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

Retinoids are endogenous to the porcine blastocyst and secreted by trophectoderm cells at functionally-active levels.

Retinoic acid and its isomers are the major morphogens in vertebrate development. For mammals, it was previously considered that circulating retinoic acid was recruited from the uterine environment, to influence embryonic differentiation, morphogenesis and development. Here we report that retinoic acid is endogenous to the blastocyst of the domestic pig, Sus scrofa, as detected by high pressure liquid chromatography. Furthermore, using a continuous, normal line of porcine trophectoderm cells, TE1, we have identified the trophoblast as a major source of retinoids. Endogenous retinoic acid was found at a concentration of approximately 35 nM in extracts of entire blastocysts, and at a similar concentration in extracts of TE1 cells. Retinoids in explants of blastocysts and in conditioned medium from the TE1 cell line were found to be functionally-active, inducing gene expression from a retinoic-acid-responsive enhancer element in an in vitro assay system. In conclusion, we propose that there is a morphogenetic role for endogenous, and trophoblast-derived, retinoids in the early development of the pre-gastrulation porcine embryo; and that the TE1 cell line therefore provides a useful in vitro system for the study of retinoid metabolism. Furthermore, an implication of this study is that endogenous retinoids may play an active role in the pre-implantation embryology of other species, such as the human.

Protein kinase C-alpha and -epsilon are enriched in growth cones of differentiating SH-SY5Y human neuroblastoma cells.

SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like growth-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C-alpha (PKC-alpha), PKC-epsilon, and PKC-zeta as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC-alpha and PKC-epsilon were enriched. In contrast, PKC-zeta was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 microM) differentiate poorly and continue to proliferate. In those cells, PKC-alpha and PKC-epsilon were found to be down-regulated while PKC-zeta remained present. Thus, down-regulation of PKC-alpha and PKC-epsilon appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin-like growth factor I. Growth cones isolated from such cells are also enriched in PKC-alpha and PKC-epsilon, but not in PKC-zeta. Based on the subcellular distribution of PKC-alpha and epsilon, and that PKC substrates like GAP-43 and pp60c-src are enriched in SH-SY5Y growth cones, a role during neurite growth is suggested.

Protein synthesis and mRNA in isolated growth cones from differentiating SH-SY5Y neuroblastoma cells.

The human neuroblastoma cell line, SH-SY5Y, differentiates into a neuronal, sympathetic phenotype in the presence of phorbol ester and serum. Growth cones prepared from differentiating SH-SY5Y cells have characteristics similar to those of growth cones from embryonic rat brain. In addition, SH-SY5Y growth cones contain ribosomes. In this study we show, by metabolic labeling of isolated growth cones, that local protein synthesis occurred in these structures. The pattern of labeled proteins was very similar to that of the corresponding cell body fraction. RNA was shown to be transported to the growth cone compartment, and by in situ hybridization. beta-actin mRNA could be visualized in intact neuritic growth cones. Comparison by Northern blot hybridizations of RNA prepared from growth cones and cell bodies, respectively, showed that mRNAs coding for growth-associated protein 43, microtubule-associated protein 2, actin, neuropeptide tyrosine, and glyceraldehyde-3-phosphate dehydrogenase were present in both fractions. In contrast, mRNAs coding for the nuclear proteins c-jun and N-myc were virtually absent in the growth cone, but readily detectable in the cell body preparation. The selective distribution of mRNAs to the growth cones was not restricted to stable, abundant mRNA species, since mRNA coding for the insulin-like growth factor I receptor was stable, but not present in growth cones. Thus, differentiating SH-SY5Y cells can sort and transport RNA to the growth cone compartment, suggesting that this system of clonal cells could be useful to unravel mechanisms involved in the compartmentalization of mRNA.

Basic FGF and IGF-I promote differentiation of human SH-SY5Y neuroblastoma cells in culture.

Phorbolester-triggered differentiation of SH-SY5Y neuroblastoma cells requires serum and a prolonged activation of protein kinase C (PKC). Under serum-free conditions development of a mature phenotype requires phorbolester in combination with a member of either the insulin-like growth factor (IGF) or the platelet-derived growth factor family. Here we report that basic and acidic fibroblast growth factor (FGF) and epidermal growth factor, but not nerve growth factor, synergistically potentiate phorbolester-induced differentiation. Alone these factors induced a mitogenic response which varied in magnitude, with basic FGF and IGF-I being the two most potent mitogens. However, a combination of basic FGF and IGF-I induced differentiation as judged by morphology and the increase in growth associated protein (GAP-43) and neuropeptide tyrosine mRNA levels. In contrast to the phenotype obtained in the presence of phorbolester, bFGF and IGF-I-treated SH-SY5Y cells retained their capacity to proliferate. Finally, in these cells, the phosphorylation of the endogenous PKC substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), was slightly increased during several days, suggesting an involvement of PKC in the bFGF and IGF-I-induced differentiation.

Activation of the human NPY gene during neuroblastoma cell differentiation: induced transcriptional activities of AP-1 and AP-2.

During functional neuronal differentiation of human SH-SY5Y neuroblastoma cells, induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the mRNA expression of c-fos and c-jun displayed a synchronous and biphasic type of induction for both mRNAs, with an early transient (30 to 120 min) and a later (> 8 h) more persistent increase. This was coupled to increased in vitro DNA binding activity of cFos/cJun AP-1 heterodimers in SH-SY5Y nuclear extracts using the electrophoretic mobility shift assay. Functional AP-1 activity was demonstrated in differentiating SH-SY5Y cells by transient transfection assays using a TPA-responsive reporter plasmid. The second expression phase of these protooncogenes was paralleled by a sustained induction of neuronal differentiation markers, as exemplified by growth-associated protein 43 and neuropeptide tyrosine (NPY) mRNAs. DNA-protein interaction between an evolutionarily conserved region (-73 to -45) of the human NPY promoter, containing potential binding sites for AP-1, AP-2, and Sp1, and nuclear extracts prepared from untreated and TPA-treated SH-SY5Y cells revealed one complex (CI) that was unaffected and three complexes (CII to CIV) that were induced by TPA treatment. Competition for DNA binding using AP-1, AP-2, and Sp1 consensus sequences and an anti-cJun antibody, respectively, revealed cooperative interactions between AP-1, AP-2, and Sp1 transcription factors and the NPY promoter. In addition, TPA-mediated induction of AP-2 DNA binding activity to the NPY promoter was not dependent on increased AP-2 mRNA expression. This high degree of complexity presumably involved in NPY gene expression during neuronal differentiation of SH-SY5Y cells suggests productive cooperative interactions between multiple transcription factors.

Human GAP-43 Gene Expression: Multiple Start Sites for Initiation of Transcription in Differentiating Human Neuroblastoma Cells.

Phorbol ester treatment of human SH-SY5Y neuroblastoma cells, which leads to mature neuron-like cells with a sympathetic phenotype, induces outgrowth of neurites which are terminated by growth cones. The neurite extension is parallelled by an increased expression of the growth-associated protein, GAP-43. At the mRNA level, two GAP-43 mRNA species of 1.4 and 1.6 kb, respectively, were detected in SH-SY5Y cells. Both the low- and high-molecular-weight GAP-43 transcripts cosedimented with a polysomal fraction, indicating translation of both types of transcripts. To structurally characterize these GAP-43 mRNAs, several cDNA clones were isolated. The only difference identified corresponded to various size extensions in the 5'-untranslated region. A human genomic DNA fragment extending 1145 bp 5' of the GAP-43 translation start site, including a putative promoter region of the GAP-43 gene, was also characterized. Comparison of human and rat GAP-43 genomic sequences revealed an 85% identity between the first 900 bp 5' of translation start site. By RNase protection analysis, two clusters of putative transcription start sites, located approximately 200 bp apart, were identified. DNaseI footprinting analyses, using nuclear extracts prepared from untreated and TPA-treated SH-SY5Y cells, revealed specific footprints primarily detected in extracts prepared from differentiating cells. These clustered at positions immediately 5' of the two putative transcription start site regions. GAP-43 mRNA expression was finally studied using a probe which specifically recognizes the high-molecular-weight GAP-43 transcripts. Five tested human neuroblastoma cell lines and human fetal brain tissue expressed these transcripts. Furthermore, during differentiation of SH-SY5Y and LA-N-5 cells, both sizes of GAP-43 transcripts were transiently induced with the larger slightly preceeding the smaller mRNA species.

Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.

Signal transduction alterations in GH(1)2C1 rat pituitary tumour cells following treatment with 5-azacytidine.

In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels.

5-azacytidine-induced alterations in the GH12C1 cells: effects on cellular morphology, chromosome structure, DNA and protein synthesis.

Growth hormone-producing rat pituitary tumor cells (GH12C1) were cultivated in the presence of 5-azacytidine (5-AC). After cessation of treatment the cells were allowed to recover in normal cell culture medium prior to subcultivation. Within hours after the subcultivation the cells underwent several morphological alterations, later followed by changes in growth pattern: (1) cell shape was irreversibly changed from round or spindle shape to different clones of fibroblast-like cells; (2) some of these clones later formed foci; and (3) an extensive generation of multinucleated cells was seen. The demethylation was maximal approximately one week after the first subcultivation in the absence of 5-AC. Severe alterations of the chromosome structure were seen after the first subcultivation. During the following weeks the 5-AC-treated cells showed impaired chromosome condensation, and homogeneous staining with quinacrine mustard. During this period the DNA synthesis is doubled when compared to control cell DNA synthesis. The core histone synthesis increases in parallel with the DNA synthesis, but the majority of nuclear proteins, including the linker histones, remain at the control level. This results in a ratio of linker histone to core histone synthesis at approximately half the control cell level. The altered ratio of synthesis slowly decreased to control levels during a period of five weeks of continuous cultivation in the absence of the drug, and the under-condensed chromosomes could no longer be seen. The induced novel phenotypes with their pleiomorphic appearances were conserved. The growth hormone synthesis remained constant during all phases of the experiment and prolactin synthesis was not induced.