The Repressive Effect Of Nf-κb On P53 By Mot-2 Is Involved In Neoplastic Transformation Induced By Low Levels Of Arsenite In Human Cells

Arsenic is a widespread environmental toxicant that has been associated with numerous human health problems. Sodium arsenite (NaAsO ) is a main existing form of arsenic.Epidemiologic evidences have implicated exposure to arsenic in the causation of human cancers of the skin, lung, and bladder. Skin and lung are thought to be the most important sites for arsenic toxicity. Although arsenic has been recognized as a human carcinogen, underlying the mechanisms of its carcinogenesis remain unclear because the models of arsenic-induced tumorigenesis in experimental animals have not been successfully established.In mammalian cells, p⁵³ is involved in the maintenance of genome stability. In response to diverse cellular stresses, p⁵³ transactivates downstream target genes required for DNA repair, cell cycle arrest, and apoptosis. Thus, functional inactivation of p⁵³ is a common characteristic of tumors. Data relating to the effects of arsenic on p⁵³ are conflicting, although high levels of arsenic always induce p⁵³, the effects of low levels of arsenic, however, are dependent on the compound, concentration, duration of treatment, and cell type. Inactivation of p⁵³ function plays a role in carcinogenesis induced by low levels of arsenic, the molecular mechanisms, however, remain largely investigated.Because the tumor suppressor function of p⁵³ must be compromised before a cell undergoes arsenic-mediated transformation and because defining the molecular mechanism underlying the inactivation of p⁵³ in low levels of arsenic-induced neoplastic transformation of human cells is necessary for a complete understanding of the oncogenesis caused by arsenic. In the present study, we exposed human embryo lung fibroblast (HELF) cells and human keratinocytes (HaCaT) to low levels of sodium arsenite to evaluate oncogenic transformation and further using two-dimensional electrophoresis (2-DE) to investigate underlying mechanisms of arsenite-mediated effects on p⁵³ function and neoplastic transformation of these cells.Methods1. Neoplastic transformation of human cells induced by low levels of arseniteAfter cells were cultured in fresh medium for 24 h, HELF cells were exposed to DMEM medium containing 0.0, 0.5, 1.0, or 2.0μM NaAsO₂, and HaCaT cells were exposed to 1640 medium containing 0.0 or 1.0μM NaAsO₂ for 30 passages (about 15 weeks).2. Anchorage-independent growthSoft agar dishes were prepared with under-layers of 0.70% agarose in fresh medium. To test for soft-agar colony growth capacity, cells were plated in triplicate at a density of 1×104 in 2 mL of 0.35% agarose over the agar base. Cultures were fed every three days, after 4 weeks, colonies were examined microscopically. 3. Tumorigenicity in nude miceTo confirm neoplastic transformation, 1×10⁷/0.2 mL of treated cells were injected subcutaneously into the right armpit of nude/BalbC mice. Four weeks later, the tumor tissues were removed, fixed with 4% formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and analyzed by light microscopy.4. Determine the mutations of the coding sequences of p⁵³ in transformed HELF cellsDNA fragments, including all the coding sequences of p⁵³, were amplified by PCR, and then were sequencing using primers of PCR amplification and determined by blast analysis.5. Identification of proteins differentially expressed in transformed HELF cellsDifferences in levels of cellular proteins between passage control and transformed HELF cells were detected by 2-DE. We chosed the proteins, which involved in inactivation of p⁵³ for further investigation.6. Western blotCell lysates were subjected to SDS-PAGE and transferred to NC membranes. Immune complexes were detected by enhanced chemiluminescence.7. Southwestern blotCell extracts were separated by SDS-PAGE and transferred to NC membranes. After transferring, the filters were hybridized with the biotin-labeled probe (promoter of mot-2). The positions of the biotin-labeled oligonucleotides were detected by a chemiluminescent reaction. 8. Co-immunoprecipitationCell extracts were incubated with IP antibody and subsequently with A+G sepharose beads at 4°C overnight. To determine protein-protein interaction or to determine DNA-protein interaction, the immunoprecipitates were analyzed by Western blot or by Southwestern blot.9. Immunostaining analyses of the nuclear translocation of p⁵³Treated cells were stained with rabbit p-p⁵³ antibody for 24 h. They were then incubated with Cy3-conjugated goat-anti-rabbit secondary antibody for 1 h. To stain the nuclei, DAPI was added for 15 min, and the cells were observed under a fluorescence microscope. The images were analyzed by use of an Image-Pro Plus 6.0. The fluorescence intensities were analyzed with a TECAN multimode microplate reader.Results1. low levels of arsenite induced changes of cell biological characteristicsHELF cells were exposed to 0.0, 0.5, 1.0, or 2.0μM NaAsO₂, and HaCaT cells were exposed to 0.0 or 1.0μM NaAsO₂. After 30 passages, transformed cells showed an unorganized morphology; the doubling time of the transformed cells were significantly shorter than that of passage control cells. These changes were the most significant in 1.0μM arsenite-transformed HELF cells.2. Neoplastic transformation and tumorigenesis induced by low levels of arseniteHELF cells were exposed to 0.0, 0.5, 1.0, or 2.0μM NaAsO₂, and HaCaT cells were exposed to 0.0 or 1.0μM NaAsO₂. After 30 passages, in agar, colonies were formed in trandsformed cells and A549 cells. In contrast, passage control cells showed no anchorage-independent growth. Tumor incidences in the groups injected with transformed cells and in the positive control group injected with A549 carcinoma cells were all 100%. These changes were the most marked in 1.0μM arsenite-transformed HELF cells.3. Determine the mutations of the coding sequences of p⁵³ in arsenite-induced neoplastic transformed HELF cellsIn transformed HELF cells, all the coding sequences of p⁵³ were a 100% match to p⁵³ cDNA after DNA fragments including all the coding sequences of p⁵³ were sequencing using primers of PCR amplification, which indicated that no mutations of p⁵³ were found in arsenite-induced transformed HELF cells.4. Identification of proteins differentially expressed in transformed HELF cellsOf 5671 protein spots, 126 were different between the passage control and transformed HELF cells. Of these, nuclear factor-κB repressing factor (NKRF, an NF-κB inhibitor) and mot-2 (a p⁵³ inhibitor) were selected for further analysis. The NKRF in transformed HELF cells was lower than in passage control cells, while mot-2 in transformed HELF cells was higher than in passage control cells.5. Levels of NKRF, mot-2, and RelA in transformed HELF cellsVerifying the results of 2-DE, Western blots confirmed the down-expression of NKRF and up-expression of mot-2 in transformed cells. Further, phosphorylation of the RelA (p-RelA), indicating an activation of NF-κB, were higher in transformed HELF cells than in passage control cells. With increased time of exposure to arsenite, there were more malignant HELF cells and greater down-expression of NKRF and up-expression of p-RelA and mot-2. There were no such changes, however, in passage control HELF cells. 6. NF-κB activation induced by low levels of arseniteThe levels of reactive oxygen species (ROS), p-IKKβ, p-IκBα, p-RelA and mot-2 were increased by 1.0μM NaAsO and 1.0μM hydrogen peroxide (H O ), which could be blocked by catalase. Data indicated that arsenite activated NF-κB by ROS, and mot-2 expression consistented with the NF-κB activation.7. NF-κB activation as a regulator of mot-2 in arsenite-exposed cells1) The interaction of mot-2 with NF-κB in arsenite-exposed HaCaT cellsBased on the fact that the sequence of the mot-2 promoter is similar to kappaB DNA elements, the possibility that the mot-2 increases were caused by NF-κB activation was investigated. Interaction of the promoter of mot-2 with NF-κB was established by Southwestern and Western blot.2) NF-κB activation as a regulator of mot-2 in arsenite-exposed HELF cellsThe levels of p-IκBα, p-RelA, and mot-2 were elevated by 1.0μM NaAsO₂. Inhibition of NF-κB by Bay11-7082 or RelA siRNA, however, blocked the arsenite-induced increases of the mot-2 protein and mRNA levels, which indicated that arsenite-induced mot-2 expression was regulated by NF-κB in HELF cells.3) NF-κB activation as a regulator of mot-2 in arsenite-exposed HaCaT cellsThe levels of mot-2 were elevated by 1.0μM NaAsO₂. Inhibition of NF-κB by Bay11-7082, however, blocked the arsenite-induced increases of the mot-2 levels, which indicated that NF-κB up-regulated mot-2 expression in arsenite-treated HaCaT cells. 8. Involvement of NF-κB and mot-2 in the phosphorylation and nuclear translocation of p⁵³ in arsenite-treated cells1) Involvement of NF-κB and mot-2 in the phosphorylation and nuclear translocation of p⁵³ in HELF cellsBlockage of NF-κB or mot-2 further increased the arsenite-induced elevation of p-p⁵³. In parallel with these results, inhibition of either NF-κB or mot-2 increased the nuclear translocation of p⁵³ induced by arsenite in HELF cells, which indicated that NF-κB blocked p⁵³ activation and nuclear translocation by mot-2 in arsenite-treated HELF cells.2) Involvement of NF-κB and mot-2 in the phosphorylation and nuclear translocation of p⁵³ in HaCaT cellsInhibition of NF-κB or blockage of mot-2 further increased the arsenite-induced elevation of p-p⁵³,. In parallel with these results, inhibition of either NF-κB or mot-2 increased the nuclear translocation of p⁵³ induced by arsenite in HaCaT cells, which indicated that NF-κB blocked p⁵³ activation and nuclear translocation by mot-2 in arsenite-treated HaCaT cells.9. The repressive effects of mot-2 on p⁵³ changes the binding preference of CBP to p⁵³ in HELF cells1) NF-κB regulated the repressive effects of mot-2 on p⁵³Arsenite increased the binding of mot-2 to p⁵³ in HELF cells, which was attenuated by blocking of NF-κB activity, which indicated that NF-κB up-regulated the interaction between mot-2 and p⁵³ in arsenite-treated HELF cells.2) Mot-2 mediated interaction of NF-κB or p⁵³ with CBP In mot-2 normal HELF cells, with a blocked NF-κB signal pathway, 1.0μM arsenite promoted binding of the CBP co-activator to p⁵³ instead of NF-κB; the change, however, was not evident in mot-2-knockdown HELF cells, which indicated that the repressive effects of mot-2 on p⁵³ change the binding preference of CBP.10. NF-κB inhibition blocked the arsenite-induced neoplastic transformation of human cellsDuring transformation, Bay11-7082 effectively blocked arsenite-induced NF-κB activation. After 15 weeks, in agar, colonies were formed from cells exposed to 1.0μM NaAsO₂ and from A549 carcinoma cells, respectively. Tumor incidences of the group injected with cells exposed to 1.0μM NaAsO₂ and the group injected with A549 carcinoma cells were 100%; In contrast, unexposed cells and cells exposed to Bay11-7082 with or without 1.0μM NaAsO₂ showed no anchorage independent growth and tumorigenesis in nude mice, which indicated that inhibition of NF-κB blocked the arsenite-induced neoplastic transformation of human cells.Conclusions1. HELF and HaCaT cells are transformed by chronic exposure to low levels of sodium arsenite, and transformed cells possess oncogenicity.2. In transformed HELF cells induced by low levels of sodium arsenite, there is no mutation in the coding sequences of p⁵³.3. By mot-2, NF-κB blocks the phosphorylation and nuclear translocation of p⁵³ in low levels of sodium arsenite-treated HELF and HaCaT cells.4. Inhibition of NF-κB prevents the neoplastic transformation of HELF and HaCaT cells induced by low levels of sodium arsenite.