Abstract
Molt-4 leukemia cells undergo p53-dependent apoptosis accompanied by accumulation of de novo ceramide after 14 hours of γ-irradiation. In order to identify the potential mediators involved in ceramide accumulation and the cell death response, differentially expressed genes were identified by Affymetrix Microarray Analysis. Molt-4-LXSN cells, expressing wild type p53, and p53-deficient Molt-4-E6 cells were irradiated and harvested at 3 and 8 hours post-irradiation. Human genome U133 plus 2.0 array containing >47,000 transcripts was used for gene expression profiling. From over 10,000 probes, 281 and 12 probes were differentially expressed in Molt-4-LXSN and Molt-4-E6 cells, respectively. Data analysis revealed 63 (upregulated) and 20 (downregulated) genes (>2 fold) in Molt-4-LXSN at 3 hours and 140 (upregulated) and 21 (downregulated) at 8 hours post-irradiation. In Molt-4-E6 cells, 5 (upregulated) genes each were found at 3 hours and 8 hours, respectively. In Molt-4-LXSN cells, a significant fraction of the genes with altered expression at 3 hours were found to be involved in apoptosis signaling pathway (BCL2L11), p53 pathway (PMAIP1, CDKN1A and FAS) and oxidative stress response (FDXR, CROT and JUN). Similarly, at 8 hours the genes with altered expression were involved in the apoptosis signaling pathway (BAX, BIK and JUN), p53 pathway (BAX, CDKN1A and FAS), oxidative stress response (FDXR and CROT) and p53 pathway feedback loops 2 (MDM2 and CDKN1A). A global molecular and biological interaction map analysis showed an association of these altered genes with apoptosis, senescence, DNA damage, oxidative stress, cell cycle arrest and caspase activation. In a targeted study, activation of apoptosis correlated with changes in gene expression of some of the above genes and revealed sequential activation of both intrinsic and extrinsic apoptotic pathways that precede ceramide accumulation and subsequent execution of apoptosis. One or more of these altered genes may be involved in p53-dependent ceramide accumulation.
Highlights
Chemotherapeutics [1] and irradiation [2,3] are genotoxic stressors widely used in the treatment of various cancers and known to stimulate cell cycle arrest or apoptosis depending on the cell type
Over 10,000 probes, 12 probes were differentially expressed in Molt-4-E6 cells and 281 probes in Molt-4-LXSN cells
Upon γ-irradiation (5Gy) of Molt-4 cells, 12 probes were differentially expressed in Molt-4-E6 cells and 281 probes in Molt-4-LXSN cells
Summary
Chemotherapeutics [1] and irradiation [2,3] are genotoxic stressors widely used in the treatment of various cancers and known to stimulate cell cycle arrest or apoptosis depending on the cell type. The tumor suppressor p53 plays a central role in the response to genotoxic stress and is critical in mediating apoptosis during cancer therapy. Capable of inducing apoptosis and cell cycle arrest in a wide variety of cancer and normal cell types, may accumulate following exposure to γ-irradiation or chemotherapeutic agents, in both p53-dependent and p53-independent manners [6,7,8]. Ceramide generated downstream of p53 presents an interesting therapeutic target in p53-deficient cancer cells as it may play an important role in p53-independent apoptosis
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