The Adenosine Receptor and Serum Deprivation-Induced Neuronal Differentiation
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Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation. On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
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serum deprivation-induced neuronal differentiation