Multifunctional Role of Bcl-2 in Malignant Transformation and Tumorigenesis of Cr(VI)-Transformed Lung Cells

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI)

From Data to Software to Science with the Rubin Observatory LSST

editorial reviewedThe Vera C. Rubin Observatory Legacy Survey of Space and Time (LSST) dataset will dramatically alter our understanding of the Universe, from the origins of the Solar System to the nature of dark matter and dark energy. Much of this research will depend on the existence of robust, tested, and scalable algorithms, software, and services. Identifying and developing such tools ahead of time has the potential to significantly accelerate the delivery of early science from LSST. Developing these collaboratively, and making them broadly available, can enable more inclusive and equitable collaboration on LSST science. To facilitate such opportunities, a community workshop entitled "From Data to Software to Science with the Rubin Observatory LSST" was organized by the LSST Interdisciplinary Network for Collaboration and Computing (LINCC) and partners, and held at the Flatiron Institute in New York, March 28-30th 2022. The workshop included over 50 in-person attendees invited from over 300 applications. It identified seven key software areas of need: (i) scalable cross-matching and distributed joining of catalogs, (ii) robust photometric redshift determination, (iii) software for determination of selection functions, (iv) frameworks for scalable time-series analyses, (v) services for image access and reprocessing at scale, (vi) object image access (cutouts) and analysis at scale, and (vii) scalable job execution systems. This white paper summarizes the discussions of this workshop. It considers the motivating science use cases, identified cross-cutting algorithms, software, and services, their high-level technical specifications, and the principles of inclusive collaborations needed to develop them. We provide it as a useful roadmap of needs, as well as to spur action and collaboration between groups and individuals looking to develop reusable software for early LSST science

Molecular Mechanisms of Chromium (VI) - Induced Toxicity and Carcinogenesis

Cr(VI) compounds are redox cycling carcinogens that induce apoptosis as a primary mode of cell death. Since defects in apoptosis regulatory mechanisms are considered to be indispensable foundation of carcinogenesis it is critical to understand the molecular effectors involved. Cellular energy powerhouses, the mitochondria, house the effectors involved in signaling cascades comprising commitment to apoptosis and are reported to be dysregulated in variety of tumors. The objective of this study was to characterize the key signaling effectors in Cr(VI)-induced apoptosis and determine the relationship between those effectors and mode of cellular energy production in cancerous vs. non-cancerous cells. Our data show that Cr(VI) induced mitochondrial ROS dependent apoptosis in human lung cancer (H460) and non-cancerous (BEAS-2B) cells. The intensity of mitochondrial ROS production and consequently apoptosis was highly dependent on the energy substrate available to the cells. Forcing of oxidative phosphorylation-only energy metabolism abrogated apoptosis resistance in cancerous H460 cells vs. non-cancerous BEAS-2B cells. Collectively, these findings present a mechanistic advancement in the understanding of molecular effectors underlying the Warburg\u27s mitochondrial dysregulation hypothesis and put mitochondrial ROS as key mediators causal agents of the commitment phase of programmed cell death in a model of Cr(VI)-induced toxicity. In addition, long-term exposure to Cr(VI) leads to malignant transformation of human lung epithelial BEAS-2B cells, as indicated by their increased cell migration, invasion, proliferation, and colony formation activities. Cr(VI)-transformed cells induced tumorigenesis in nude mice comparable to that of well established human lung cancer H460 cells, whereas passage-matched control BEAS-2B cells showed no tumor development. To provide a mechanistic insight to the tumorigenic process, stable knockdown mutants of Cr(VI)-transformed cells and H460 cells exhibiting downregulated Bcl-2 phenotype were generated and evaluated for tumor associated properties in vitro and in vivo. The Bcl-2 knockdown mutants showed a substantial decrease in the rates of colony formation, invasion, migration and proliferation, as well as tumor formation in mice, in comparison to vector controls. These results indicate a novel role of Bcl-2, beyond apoptosis regulation, to be a key regulator of malignant transformation and tumorigenesis in vivo. The model described here may provide utility to other xeno-carcinogenesis studies, including those of heavy metals which currently lack effective in vivo tumor development experimental means

Effect of Bcl-2 knockdown on cell invasion and migration of BEAS-Cr and H460 cells.

<p>(<i>A</i>) BEAS-2B, BEAS-Cr and H460 cells (1×10⁵ cells) were added to Transwell® inserts coated with Matrigel® and incubated for 24 h. Invading cells were stained and counted under a light microscope. Plots show relative invasion of BEAS-2B, BEAS-Cr and H460 cells. (<i>B</i>) Effect of Bcl-2 on cell invasion of the respective cell lines and their mutants. Experiments were repeated with the indicated cell lines and analyzed for cell invasion. (<i>C</i>) Confluent monolayers of BEAS-2B, BEAS-Cr and H460 cells were wounded, and the cells were allowed to migrate for 24 h. Wound space was visualized by light microscopy and analyzed by comparing the relative change in wound space as compared to control cell monolayers. (<i>D</i>) Effect of Bcl-2 on cell migration of the respective cell lines and their mutants. Cells were wounded and analyzed for cell migration over a 24 h period. (<i>E</i>) Representative micrographs of cells stained for invasion are shown. Values are means (± SD) (<i>n</i> = 4). *<i>P</i><0.05 versus passage-control BEAS-2B cells. ^#<i>P</i><0.05 versus the respective BEAS-Cr and H460 cells.</p

Apoptosis response to Cr(VI) treatment in BEAS-2B, BEAS-Cr, and H460 cells.

<p>(<i>A</i>) BEAS-2B, BEAS-Cr and H460 cells were treated with or without Cr(VI) (20 µM) for 12 h and apoptosis was determined by Hoechst 33342 assay. (<i>B</i>) and (<i>C</i>) Effect of Bcl-2 knockdown on Cr(VI)-induced apoptosis of the respective cell lines and their mutants. Values are means (± SD) (<i>n</i> = 4). *<i>P</i><0.05 versus non-treated control. ^#<i>P</i><0.05 versus Cr(VI)-treated passage-control BEAS-2B cells. **<i>P</i><0.05 versus Cr(VI)-treated respective BEAS-Cr and H460 cells.</p