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Identification of Protein Markers of Lithium Radio- and Neuroprotective Effect

2007; Elsevier BV; Volume: 69; Issue: 3 Linguagem: Inglês

10.1016/j.ijrobp.2007.07.234

1879-355X

Eugenia M. Yazlovitskaya, Dinesh Thotala, Amy K.Y. Fu, Dennis E. Hallahan,

Protein Hydrolysis and Bioactive Peptides

Purpose/Objective(s)Neurocognitive deficits are devastating effects of cranial irradiation in cancer patients. Lithium has been effectively used as a protector in various neurological injuries. Identification of protein markers of lithium protective effects helps to discover novel pharmaceutical targets for enhanced neuroprotection from radiation. Novel radiation protecting drugs that preserve neurocognitive function in the brain require validation in pre-clinical cellular models. We have established a series of biomarkers expressed in the hippocampal neurons due to radio- and neuroprotective effect of lithium.Materials/MethodsMouse hippocampal neuronal cells HT-22 were treated with 3 mM LiCl for 7 days prior to irradiation with 3 Gy. For Microarray analysis, total RNA was isolated from LiCl-treated and control HT-22 cells. Microarray services were performed by the Microarray Shared Resource (http://www.vmsr.net) using MEEBO mouse genome set (http://mmc.ucsf.edu/Meebo.html). Data were organized in Excel file according to the specific molecular functions of proteins represented by identified genes (e.g., transcriptional factors) or to the involvement in different cellular processes (e.g., apoptosis). Microarray experimental details are available for public review at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/query/entry) with accession number E-MEXP-521. Real Time PCR and Western blot analyses were performed to confirm expression of two identified lithium-regulated genes: birc1f (also, neuronal apoptosis inhibitory protein, NAIP) and decorin.ResultsThe effect of lithium on the expression of over 30,000 mouse genes in HT-22 cells was analyzed. LiCl induced a greater than two-fold increase in several dozen genes involved in the anti-apoptotic signaling, DNA repair mechanism and neurogenesis; or greater than two-fold reduction in expression of genes with pro-apoptotic activity. The results of Real Time PCR confirmed an increase in expression of anti-apoptotic genes decorin and birc1f (NAIP) of 2.5- and 1.4-fold correspondingly. In Western blot analysis, lithium treatment caused a significant increase of 1.6-fold in decorin protein expression. In both untreated and LiCl-treated cells, we detected full-length and several proteolytic fragments of NAIP showing elevation from 1.4 to 2.2-fold in all protein forms. Interestingly, the 60 kDa NAIP fragment demonstrated the highest increase of 2.2-fold in response to lithium. In direct contrast, the 60 kDa fragment was substantially reduced following irradiation (33% decrease; 1.7 vs. 2.2-fold), suggesting a potentially unique involvement of NAIP in lithium radioprotective effect for neurons.ConclusionsThe importance of the pre-clinical models is that the potential radioprotectors must enter neurons within the brain and induce the expression of proteins that prevent radiation-induced apoptosis. The identified protein markers, NAIP and decorin, could be useful for validation of lead radioprotecting agents that are planned for clinical trials to prevent radiation-induced neurocognitive deficit in patients receiving brain irradiation. Purpose/Objective(s)Neurocognitive deficits are devastating effects of cranial irradiation in cancer patients. Lithium has been effectively used as a protector in various neurological injuries. Identification of protein markers of lithium protective effects helps to discover novel pharmaceutical targets for enhanced neuroprotection from radiation. Novel radiation protecting drugs that preserve neurocognitive function in the brain require validation in pre-clinical cellular models. We have established a series of biomarkers expressed in the hippocampal neurons due to radio- and neuroprotective effect of lithium. Neurocognitive deficits are devastating effects of cranial irradiation in cancer patients. Lithium has been effectively used as a protector in various neurological injuries. Identification of protein markers of lithium protective effects helps to discover novel pharmaceutical targets for enhanced neuroprotection from radiation. Novel radiation protecting drugs that preserve neurocognitive function in the brain require validation in pre-clinical cellular models. We have established a series of biomarkers expressed in the hippocampal neurons due to radio- and neuroprotective effect of lithium. Materials/MethodsMouse hippocampal neuronal cells HT-22 were treated with 3 mM LiCl for 7 days prior to irradiation with 3 Gy. For Microarray analysis, total RNA was isolated from LiCl-treated and control HT-22 cells. Microarray services were performed by the Microarray Shared Resource (http://www.vmsr.net) using MEEBO mouse genome set (http://mmc.ucsf.edu/Meebo.html). Data were organized in Excel file according to the specific molecular functions of proteins represented by identified genes (e.g., transcriptional factors) or to the involvement in different cellular processes (e.g., apoptosis). Microarray experimental details are available for public review at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/query/entry) with accession number E-MEXP-521. Real Time PCR and Western blot analyses were performed to confirm expression of two identified lithium-regulated genes: birc1f (also, neuronal apoptosis inhibitory protein, NAIP) and decorin. Mouse hippocampal neuronal cells HT-22 were treated with 3 mM LiCl for 7 days prior to irradiation with 3 Gy. For Microarray analysis, total RNA was isolated from LiCl-treated and control HT-22 cells. Microarray services were performed by the Microarray Shared Resource (http://www.vmsr.net) using MEEBO mouse genome set (http://mmc.ucsf.edu/Meebo.html). Data were organized in Excel file according to the specific molecular functions of proteins represented by identified genes (e.g., transcriptional factors) or to the involvement in different cellular processes (e.g., apoptosis). Microarray experimental details are available for public review at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/query/entry) with accession number E-MEXP-521. Real Time PCR and Western blot analyses were performed to confirm expression of two identified lithium-regulated genes: birc1f (also, neuronal apoptosis inhibitory protein, NAIP) and decorin. ResultsThe effect of lithium on the expression of over 30,000 mouse genes in HT-22 cells was analyzed. LiCl induced a greater than two-fold increase in several dozen genes involved in the anti-apoptotic signaling, DNA repair mechanism and neurogenesis; or greater than two-fold reduction in expression of genes with pro-apoptotic activity. The results of Real Time PCR confirmed an increase in expression of anti-apoptotic genes decorin and birc1f (NAIP) of 2.5- and 1.4-fold correspondingly. In Western blot analysis, lithium treatment caused a significant increase of 1.6-fold in decorin protein expression. In both untreated and LiCl-treated cells, we detected full-length and several proteolytic fragments of NAIP showing elevation from 1.4 to 2.2-fold in all protein forms. Interestingly, the 60 kDa NAIP fragment demonstrated the highest increase of 2.2-fold in response to lithium. In direct contrast, the 60 kDa fragment was substantially reduced following irradiation (33% decrease; 1.7 vs. 2.2-fold), suggesting a potentially unique involvement of NAIP in lithium radioprotective effect for neurons. The effect of lithium on the expression of over 30,000 mouse genes in HT-22 cells was analyzed. LiCl induced a greater than two-fold increase in several dozen genes involved in the anti-apoptotic signaling, DNA repair mechanism and neurogenesis; or greater than two-fold reduction in expression of genes with pro-apoptotic activity. The results of Real Time PCR confirmed an increase in expression of anti-apoptotic genes decorin and birc1f (NAIP) of 2.5- and 1.4-fold correspondingly. In Western blot analysis, lithium treatment caused a significant increase of 1.6-fold in decorin protein expression. In both untreated and LiCl-treated cells, we detected full-length and several proteolytic fragments of NAIP showing elevation from 1.4 to 2.2-fold in all protein forms. Interestingly, the 60 kDa NAIP fragment demonstrated the highest increase of 2.2-fold in response to lithium. In direct contrast, the 60 kDa fragment was substantially reduced following irradiation (33% decrease; 1.7 vs. 2.2-fold), suggesting a potentially unique involvement of NAIP in lithium radioprotective effect for neurons. ConclusionsThe importance of the pre-clinical models is that the potential radioprotectors must enter neurons within the brain and induce the expression of proteins that prevent radiation-induced apoptosis. The identified protein markers, NAIP and decorin, could be useful for validation of lead radioprotecting agents that are planned for clinical trials to prevent radiation-induced neurocognitive deficit in patients receiving brain irradiation. The importance of the pre-clinical models is that the potential radioprotectors must enter neurons within the brain and induce the expression of proteins that prevent radiation-induced apoptosis. The identified protein markers, NAIP and decorin, could be useful for validation of lead radioprotecting agents that are planned for clinical trials to prevent radiation-induced neurocognitive deficit in patients receiving brain irradiation.