RBM3 Research Articles

Assessment of the carcinogenic potential of particulate matter generated from 3D printing devices in Balb/c 3T3-1-1 cells

Recently, there have been reports of sarcoma occurring in a Korean science teachers who used a 3D printer with acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) filaments for educational purposes. However, limited toxicological research data on 3D printing make it challenging to confirm a causal relationship between 3D printing and cancer. Therefore, occupational accidents involving teachers who have developed sarcoma have not been officially recognized. To address this gap, we aimed to evaluate the carcinogenic potential of particulate matter produced from ABS and PLA filaments commonly used in 3D printing. We created a generator mimicking 3D printing to generate particulate matter, which was used as an experimental material. The collected particulate matter was exposed to an in vitro system to investigate genetic damage, effects on cell transformation, and changes in carcinogenesis-related genes. Various assays, such as the comet assay, cell transformation assays, microarray analysis, and glucose consumption measurement, were employed. Cytotoxicity tests performed to determine the exposure concentration for the comet assay showed that cell viability was 83.6, 62.6, 42.0, and 10.2% for ABS at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. PLA showed 91.7, 80.3, 65.1, and 60.8% viability at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. Therefore, 50 µg/mL was set as the highest concentration for both ABS and PLA, and 25 and 12.5 µg/mL were set as the medium and low concentrations, respectively. The comet assay showed no changes in genetic damage caused by the particulate matter. Cytotoxicity results performed to establish exposure concentrations in the transformation assay showed that ABS showed cell viability of 88.0, 77.4, 84.7, and 85.5% at concentrations of 1.25, 2.5, 5, and 10 µg/mL, respectively, but few cells survived at concentrations above 20 µg/mL. PLA showed minimal cytotoxicity up to a concentration of 20 µg/ml. Therefore, in the cell transformation assay, a concentration of 10 µg/mL for ABS and 20 µg/mL for PLA was set as the highest exposure concentration, followed by medium and low exposure concentrations with a common ratio of 2. In cell transformation assays, only one transformed focus each was observed for both ABS and PLA particulate matter-exposed cells. The microarray assay revealed changes in gene expression, with a 41.7% change at 10 µg/mL for ABS and an 18.6% change at 20 µg/mL for PLA compared to the positive control group. Analysis of carcinogenesis-related gene expression changes on days 1, 7, and 25 of the promotion phase revealed that in cells exposed to 5 µg/mL of ABS, RBM3 gene expression increased by 3.66, 3.26, and 3.74 times, respectively, while MPP6 gene expression decreased by 0.33, 0.28, and 0.38 times, respectively, compared to the negative control group. Additionally, the measurement of glucose consumption showed that it increased in cells exposed to ABS and PLA particulate matter. Our findings suggest that the carcinogenic potential of ABS- and PLA-derived particulate matter in 3D printing cannot be completely ruled out. Therefore, further research in other test systems and analysis of additional parameters related to carcinogenesis, are deemed necessary to evaluate the carcinogenic risk of 3D printers using these materials.

Influence of RRM, RGG and Potential Phosphorylated Sites in Cold-Inducible Protein RBM3 on its Subcellular Localization and Neuroprotective Effects.

As a potent mediator of hypothermic neuroprotection, the cold-inducible protein RBM3 is characterized with one RNA-recognition motifs (RRM) and one arginine-glycine-rich (RGG) domain. It is known that these conserved domains are required for nuclear localization in some RNA-binding proteins. However, little is known about the actual role of RRM and RGG domains in subcellular localization of RBM3. To clarify it, various mutants of human Rbm3 gene were constructed. Plasmids were transfected into cells and the localization of RBM3 protein and its varias mutants in cells and role in neuroprotection. In human neuroblastoma SH-SY5Y cells, either a truncation of RRM domain (aa 1-86) or RGG domain (aa 87-157) led to an obvious cytoplasmic distribution, compared to a predominant nuclear localization of whole RBM3 protein (aa 1-157). In contrast, mutants in several potential phosphorylated sites of RBM3, including Ser102, Tyr129, Ser147, and Tyr155, did not alter the nuclear localization of RBM3. Similarly, mutants in two Di-RGG motif sites also did not affect the subcellular distribution of RBM3. Lastly, the role of Di-RGG motif in RGG domains was further investigated. The mutant of double arginines in either Di-RGG motif-1 (Arg87/90) or -2 (Arg99/105) exhibited a higher cytoplasmic localization, indicating that both Di-RGG motifs are required for nucleic localization of RBM3. Our data suggest that RRM and RGG domains are both required for the nuclear localization of RBM3, with two Di-RGG domain being crucial for nucleocytoplasmic shuttling of RBM3.

Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

RBM3 expression is upregulated by NF-κB p65 activity, protecting cells from apoptosis, during mild hypothermia.

The RNA-binding protein RBM3, a cold shock protein whose expression is elevated under hypothermic conditions, plays an important role in cell survival; however, little is known about the mechanism underlying the mild hypothermia-mediated regulation of RBM3 expression and apoptosis. Here we show that the transcription factor NF-κB p65 is phosphorylated at Ser276 and activates RBM3 gene transcription via binding to a particular element within the promoter region in response to induced hypothermia, elevating the protein expression, and suppressing apoptosis. Treatment with caffeic acid phenethyl ester (CAPE), a potent and specific inhibitor that suppresses the translocation of NF-κB p65 from the cytoplasm to the nucleus, resulted in decreased levels of RBM3 mRNA and protein and increased incidence of apoptosis despite cells were cultured under hypothermic conditions. Overexpression of RBM3 abolished the induction of apoptosis in cells treated with CAPE, indicating that NF-κB p65-upregulated RBM3 expression is necessary for the suppression of apoptosis. In addition, experiments with cells overexpressing RBM3 supported the finding demonstrating that the mild hypothermia-mediated higher expression of RBM3 suppressed the induction of apoptosis. Conversely, experiments with cells deficient in RBM3 supported the finding demonstrating that the CAPE-mediated loss of RBM3 induced apoptosis. These results suggest that NF-κB p65 is a critical mediator of mild hypothermia, to which cells are exposed as an extracellular environment, and a central inducer of RBM3 expression, which is responsible for preventing cells from apoptosis. Moreover, CAPE may have a potential for the application to a therapeutic agent for the treatment of cancers.

Effects of moderate and deep hypothermia on RNA-binding proteins RBM3 and CIRP expressions in murine hippocampal brain slices

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5°C) and deep (17°C) hypothermia with normothermia (37°C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2).Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5°C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17°C for 24h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5°C but only in the co-culture at 17°C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature.We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.

Abstract 1746: Expression of the translation regulatory protein RBM3 is associated with a favourable prognosis in epithelial ovarian cancer

Abstract Background: RNA binding proteins are important regulators of gene expression involved in various post-transcriptional processes like mRNA stability and translation. We have recently demonstrated that high expression of RBM3 is associated with a favourable prognosis in breast cancer. Aims and Methods: The aim of the present investigation was to examine the prognostic value of RBM3 in epithelial ovarian cancer (EOC). For this purpose, protein expression of RBM3 was examined by immunohistochemistry in tumour samples from 154 prospectively collected EOC cases (Cohort I). In addition, RBM3 gene expression was examined in an independent cohort with 282 EOC cases (Cohort II). Results: No significant correlation was seen between RBM3 protein expression and any established prognostic clinicopathological parameters. However, survival analysis revealed a significant association between RBM3 nuclear expression and prolonged survival (HR=0.633, 95% CI=0.421-0.952, p=0.028) in cohort I. This association did not remain significant in multivariate analysis. In line with the IHC data, high RBM3 mRNA levels were associated with prolonged survival in Cohort II in both univariate (HR=0.671, 95% CI=0.504-0.894, p=0.006) and multivariate (HR=0.751, 95% CI=0.562-1.004, p=0.053) analysis. Conclusion: In accordance with our previously studies of breast and colon cancer, high nuclear expression of RBM3 was associated with improved survival in patients with ovarian cancer. Further investigations are needed in order to understand the role of RBM3 in tumour progression and to confirm the utility of RBM3 as a prognostic biomarker in various cancer forms. We are currently investigating the role of RBM3 in cisplatin response in ovarian cancer cell lines. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1746.

1212 Computer-aided multiple-point measurement of fetal movements in in vitro mouse fetuses

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5 °C) and deep (17 °C) hypothermia with normothermia (37 °C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2).Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5 °C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17 °C for 24 h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5 °C but only in the co-culture at 17 °C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature.We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.