Monitoring Bip promoter activation during cancer cell growth by bioluminescence imaging at the single-cell level

Abstract

Cancer cells require the regulation of organelle-specific unfolded protein responses, such as endoplasmic reticulum (ER) stress, because of their increased metabolic activity during rapid proliferation and cell growth, which are executed through the activation of diverse signaling pathways. In this study, we focused on the dynamic regulation of ER stress in accordance with cancer cellular demand, and we performed real-time monitoring of the activation of the binding immunoglobulin protein (Bip) promoter, which is one of the most responsive genes to ER stress during cancer cell growth, in two and three dimensional (2D and 3D) cell culture using bioluminescence imaging at the single-cell level. Bioluminescence images were obtained from living single cancer cells after transient transfection of the reporter gene, and we observed Bip promoter activation during cell growth. Bip promoter activation was also observed in 2D and 3D culture using stably transfected glioblastoma cancer cells with the reporter gene. The Bip promoter was activated especially in dividing cells during cell growth. We then performed real-time monitoring of Bip promoter activation by bioluminescence imaging in tissue slices obtained from U251/pBipPro-Luc tumors. Luminescence intensity was not constant and was different in individual regions of the tumor slices, and the Bip promoter was activated in several regions during monitoring in vitro. These results show that real-time monitoring by bioluminescence imaging at the single-cell level is a suitable tool for not only gene analysis of signal transduction and regulation of the dynamics of the unfolded protein response in cancer cells but also for the evaluation of the efficacy of anti-cancer agents, and could provide additional information that has been difficult to obtain using conventional assays.

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

“…We first evaluated the effects of several chemical chaperones on Bip promoter activity in glioblastoma cells by bioluminescence imaging at the single cell level. As shown in Figure , the Bip promoter was activated during cancer cell growth, with activation peaks observed several times during long‐term observation of cancer cell growth, as reported previously . When we performed real‐time monitoring of Bip promoter activity by bioluminescence imaging at the single cell level in the presence of FA, 4PBA, γ‐oryzanol, silybin, kojic acid, or rutin, Bip promoter activity was affected and several patterns of promoter activity were observed with the chemical chaperones (Figure b).…”

“…Transient transfection with the constructed vectors was performed using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. U251 cells stably transfected with Mock, pNLF1C alone, or CD63NLuc (Mock/U251 or CD63NLuc/U251) were established through the selection in cell culture medium containing hygromycin B (Nacalai Tesque) as described previously …”

“…Bioluminescence and morphology was detected and observed at the single cell level using this system, and was important in selecting live cells and excluding false positives. This technique is a suitable and available tool not only for gene analysis related to the regulation of unfolded protein responses in cancer cells but also for evaluation of anti‐cancer agent efficacy . The main advantages in using bioluminescence imaging from luciferin–luciferase reaction emissions are its lower background, and higher quantification .…”

“…Previously, we have monitored Bip/GRP78 promoter activity during cancer cell growth by real-time imaging at the level of the single cell using a bioluminescence microscopy system, in which a cooling charge coupled device (CCD) camera was successfully combined with a microscope as a new optical system. [18] Bioluminescence and morphology was detected and observed at the single cell level using this system, and was important in selecting live cells and excluding false positives. This technique is a suitable and available tool not only for gene analysis related to the regulation of unfolded protein responses in cancer cells but also for evaluation of anti-cancer agent efficacy.…”

“…This technique is a suitable and available tool not only for gene analysis related to the regulation of unfolded protein responses in cancer cells but also for evaluation of anti-cancer agent efficacy. [18] The main advantages in using bioluminescence imaging from luciferin-luciferase reaction emissions are its lower background, and higher quantification. [19,20] In addition there is low damage to living cells compared with the use of fluorescent proteins or fluorescein isothiocyanate, as an excitation light source is not needed for detection.…”