Cancer cell Thermogenesis and Uncoupling Proteins
The nonshivering process of thermogenesis implicates brown adipose tissue (BAT) where oxidative metabolism is uncoupled from ATP production in a process that energy is expended. Brown adipose tissue (BAT) is distinct from white adipose tissue (WAT) for its ability to burn, rather than store, energy. BAT uniquely expresses uncoupling protein-1 (abbreviated as UCP1), which diverts the energy produced by cellular respiration to generate heat. UCP1 is widely expressed in BAT and its main role is in thermogenesis process. UCP2 is expressed ubiquitously in human body whereas UCP3 is expressed in skeletal muscle and BAT. They have similar roles such us thermogenic, regulation of REDOX state, regulation of fatty acid and glucose metabolism and control of ROS. Finally, UCP4 and UCP5 are expressed in neural tissues and their role implicates thermogenesis and ROS control. UCPs levels and activity is regulated by modulators of cellular metabolism at multiple points (transcription, translation, modulation of catalytic activity and protein degradation). However, UCPs also play important role in the reprogramming of cancer cell metabolism. Due to increased metabolic activity of cancer cells high temperature (1–2°C) is observed within solid tumors usually translated with increased, dicreased or in general abnormal activity of UCPs with different expression patterns depending on the nature of the tumor. In particular, increased expression of UCPs have been found in breast tumors cells, leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors.
For immunofluorescence staining, cells were grown on No. 1.5 glass coverslips, fixed in 3.7% paraformaldehyde/PBS pH 7.4 for 20 min at 37°C and then permeabilized in PBS/0.1% v/v Triton X-100 pH 7.4 for 5 min at room temperature. In addition, cells were blocked in PBS/5% w/v BSA pH 7.4 for 20 min and stained with various primary antibodies: anti-UCP1 rabbit polyclonal (1:200; Abcam) and anti-UPC3 rabbit polyclonal (1:200; Abcam) for 1 h at RT. Cells were washed in PBS pH 7.4, incubated with appropriate CF 564 secondary antibody at RT and DNA was counterstained with Hoechst 33342 (1 µg/ml; Sigma-Aldrich). After final washes coverslips were mounted in homemade Mowiol mounting medium. Imaging was performed on a customized Andor Revolution Spinning Disk Confocal System built around a stand (IX81; Olympus) with a 60x lens and a digital camera (Andor Ixon+885) (CIBIT Facility, MBG-DUTH). Image acquisition was performed in Andor IQ 2 software. Optical sections were recorded every 0.3 µm. All confocal microscopy images presented in this work are 2D maximum intensity projections of z-stack images , and image analysis for the obtained data sets has been performed using ImageJ 1.47v (National Institute of Health, USA).
Whole-cell lysates were prepared in lysis buffer with sucrose based lysis buffer (0.25 M sucrose, 25mM Tris-HCl, pH 7,4) containing protease inhibitors and phosphatase inhibitors. Proteins of each lysate were resolved by discontinuous sodium dodecyl sulfate (SDS) gels and transferred to PVDF membranes. Following, blocking in 5% non-fat dried milk for 2 hours at room temperature, membranes were hybridized at 4 οC overnight with the appropriate primary antibody. The membranes were then hybridized for 2 hours at room temperature with the secondary antibody. The images of the blots were captured utilizing ChemiDoc™ MP imaging system (Bio-Rad, Hercules, CA), quantification was performed using Image J software (ImageJ 1.49v National Institute of Health, USA).
Mitotic Catastrophe of HeLa cells following exposure to an experimental drug
Formation of atypical mitotic spindles has been described as an indication of mitotic catastrophe. The effect of Drug -1 on spindle organization and chromosome alignment has been examined in HeLa cells using immunofluorescence and confocal microscopy. Following a 24h treatment, the effect on spindle formation and chromosome alignment was quite severe, demonstrating mitotic defects with chromosome misalignment and multipolar spindle formations. This cell–based method is used in our lab for screening several drugs implicated in mitotic catastrophe.
Partial body irradiation of mice
Handling and positioning of mice in the syringes and the PMMA apparatus. (A) A 3D reconstruction of whole body CT-scans illustrating the front of the device and the heads of the five animals. (B) An up-front view of the PMMA device. (C) The syringe and the appropriately shaped plunger permitting the tails of the animals to be placed outside the chamber. (D) A mouse is gently picked up. (E) The mouse is gently pushed head first into the syringe. (F) The mouse typically moves to place its nose to the tip of the syringe to breath. (G) The plunger is placed at the caudal end of the mouse and gently pushed to immobilize the mouse, while the tail stays outside the chamber. (H) The syringe is placed in the drilled hole of the PMMA apparatus. (I) The PMMA apparatus with five mice is placed on the table of the LINAC.
A 15 min video of a BALB/C mouse in a syringe in order to observe the movement behavior of the mouse during irradiation. Noted that the irradiation time is 3-5 min, so at least a 3 fold longer time of observation is allowed. The two drawn lines on te syringe show the limits between head/thorax ad chest/abdomen, defining the limits of the three different fields of radiotherapy for partial body irradiation (head, chest, abdomen) . Partial rotational movement can be noted but this does not affect the body area located within and outside the drawn lines. The forward/backward movements that would displace the target body area outside the radiation portal, or that would allow irradiation of non-target body area, was calculated to be less than ±2mm in the head/chest and less than 1mm in the chest/abdomen areas. Such a displacement is certainly a slight disadvantage of the device when compared to immobilization by anesthesia. Nevertheless, uncertainties of radiation dose that would have been produced within such a small tissue areas, are considered of limited value when whole organ post-irradiation toxicity or implanted tumor shrinkage is the goal of the experiments.