Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.     

Differential cellular distribution of retinoic acid during staurosporine potentiation of retinoic acid-induced granulocytic differentiation in human leukemia HL-60 cells

Pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor, was found to potentiate the granulocytic differentiation induced by a brief (2 h) retinoic acid treatment. By cell cycle analysis, staurosporine was found to have little effect on the cell cycle. Retinoic acid was distributed equally in the nuclei (40%) and in the plasma membrane (40%) of staurosporine-pretreated cells while less than 20% of retinoic acid was found in the membrane of control non-staurosporine-pretreated cells during the retinoic acid-induced differentiation. These results indicate that the enhancing effect of staurosporine may be somehow associated with the localization of retinoic acid in the plasma membrane of the cell. -1This work is dedicated to Prof. Harris Busch (Baylor College of Medicine, Tex., USA) for his 33 wonderful years at Baylor and 50 years in medicine.     

Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.     

Beta-carotene interaction with NNK in the AJ-mouse model: effects on cell proliferation, tumor formation and retinoic acid responsive genes

We studied the influence of beta-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low beta-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans. beta-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by beta-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARbeta isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, beta-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.     

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

Effects of tunicamycin on the differentiation of F9 cells induced by either retinoic acid or retinoic acid and dibutyryl cyclic AMP

Tunicamycin (0.5 micrograms/ml) inhibited differentiation of F9 cells treated either with retinoic acid or with retinoic acid and dibutyryl cyclic AMP, as monitored by the activity of alkaline phosphatase and expression of cytokeratins. On the other hand, the pattern of the polysaccharide chain synthesis changed drastically with the treatment irrespective of the presence of tunicamycin. Therefore, phenotypes induced with retinoic acid are dissociated into two categories, one that is directly induced by the drug and the other that is induced indirectly by a mechanism in which glycoproteins play a role.     

Anti-tumor effects of all-trans retinoic acid are enhanced by genistein

The effects of all-trans retinoic acid (ATRA) on cancer are complex. ATRA has anti-cancer effects as it promotes cancer cell differentiation. However, ATRA also up-regulates expression of vascular endothelial growth factor (VEGF) in cancer cells, which leads to angiogenesis and can, thus, facilitate cancer growth. Genistein, a crucial non-nutrient component in soybean, exhibits anti-cancer effects by inhibiting protein tyrosine kinase that is involved in up-regulation of VEGF. We hypothesized that genistein, applied simultaneously with ATRA, would counter its undesired angiogenic effects and, thus, enhance the anti-cancer effects of ATRA. The purpose of this study was to document potential synergistic effects of genistein and ATRA in A549 lung adenocarcinoma cells. We further explored the role of genistein on countering the ATRA-induced VEGF expression. We demonstrate that genistein enhances the ATRA-induced growth inhibition of A549 cells by promoting apoptosis. Further, the combined use of ATRA and genistein leads to cancer cell arrest in G0/G1 and G2/M cell cycle phases. Finally, expression of VEGF (both mRNA and protein) was diminished in A549 cells exposed to both ATRA and genistein. In conclusion, our results demonstrate that genistein effectively enhances anti-cancer effects of ATRA, particularly, by countering the ATRA-induced up-regulation of VEGF. Our study provides an experimental basis for combined use of ATRA and genistein in the treatment of lung cancer.     

Expression of granulocytic functions by leukemic promyelocytic HL-60 cells: differential induction by dimethylsulfoxide and retinoic acid

Recently, a novel approach has been used in the treatment of leukemia: induction of the leukemic cells to undergo terminal differentiation. Based on its in vitro ability to induce differentiation in several myeloid leukemic cell lines, retinoic acid (RA) has been applied clinically in cases of myelodysplastic syndromes and acute myeloid and promyelocytic leukemia. In the present study we have determined in detail the ability of RA to induce expression of granulocytic functions in a human promyelocytic leukemia cell line (HL-60) and compared it with that of dimethylsulfoxide (DMSO). Several granulocytic characteristics (phagocytosis, surface adherence and generation of free radicals in response to phorbol-ester) were induced to the same degree by both agents. Other normal neutrophil functions, including lysozyme accumulation, spontaneous migration, chemotactic activity toward zymosan-activated serum (containing C5a), the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and spontaneous motility in semi-solid medium were induced by DMSO, but they were absent or incompletely expressed in RA-induced cells. In contrast, only RA induced migration toward leukotriene B4 (LTB4). Simultaneous treatment with RA and DMSO proved synergistic with respect to morphological maturation and several functions (e.g. NBT reduction), but complementary stimulation of other activities (e.g. chemotaxis, lysozyme content) could not be demonstrated. Furthermore, characteristics induced by DMSO (i.e., expression of C5a and FMLP receptors and accumulation of lysozyme) were inhibited by the addition of RA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor

Recently, many lines of evidence have been accumulated indicating that thyroid hormone receptor (TR) and retinoic acid receptor (RAR) undergo a ligand-dependent conformation change. Since most of these results were obtained by either gel-shift assay or circular dichroism spectroscopic studies, it was not clear which part of the receptor bore the major conformational change. Moreover, it is not clear whether the formation of heterodimer between TR or RAR and retinoic X receptor (RXR) has any effects on this structural change. Utilizing partial proteolytic analysis, we demonstrated that thyroid hormone and retinoic acid induce a specific protease-resistant conformation to their cognate receptors. Studies of various deletion mutants reveal that the entire ligand binding domain of these receptors is involved in this change, and suggest that ligand may induce a more compact structure in its binding domain. Evidence from native gel electrophoresis supports this notion. This conformational change occurs in the absence of DNA and occurs indenpendently of other domains in the receptor. Heterodimerization between TR or RAR and the RXR has little effect on receptor conformation in the absence of hormone but does enhance the ligand-dependent structural change. Interestingly, dual hormone treatment, i.e. thyroid hormone and 9-cis RA, intensifies this enhancement. We suggest that the observed protease-resistant conformation may introduce a different configuration to the receptor and therefore may effect the receptor in various ways, but most likely is involved in converting the receptor from a negative regulator to a positive activator.