Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury

Abstract

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

Figure 1.

Simultaneous LIVE/DEAD and TUNEL assays for discriminating between apoptosis and ACD. (Right and left micrographs are of the same cells.) Photomicrographs are of control NHEKs (A and B) and NHEKs exposed to 58°C (C and D), 60°C (E and F), 62°C (G and H), and 72°C (I and J) for 1 second each, 24 hours posttreatment. Staining was with the LIVE/DEAD Viability/Cytotoxicity Kit (A, C, E, G, and I) to differentiate live (green) from dead (red) cells followed by TUNEL labeling (B, D, F, H, and J) to indicate cells with fragmented DNA (red). Small arrowheads: Viable cells with fragmented DNA. Large arrowheads: Dead TUNEL-positive cells.

Figure 2.

Cell death after thermal injury. NHEKs were exposed to 58°C (A), 60°C (B), 62°C (C), or 72°C (D) for 1 second. Data are expressed as a percentage of TUNEL-positive cells (▪) and ethidium homodimer-1-positive cells (•). Data from at least three experiments were pooled. Values are mean ± SD of five representative areas of a minimum of 200 cells in at least five different microscope fields.

Figure 3.

Detection of histone-associated DNA fragments with in situ cell death ELISA. NHEK cell culture medium was analyzed 36 hours after heating to temperatures of 56 to 62°C for 1 second. Data are shown as optical density at 405 nm for 2,2′-azino-di[3-ethylbenzthiazolin-sulfonate] supplied as substrate for peroxidase conjugated to monoclonal anti-DNA antibody. Values are mean ± SD of nine samples of medium from wells containing coverslips with NHEKs.

Figure 4.

Detection of DNA laddering by Southern blot of DNA isolated from medium. DNA was isolated from NHEK cell culture medium 36 hours after heating to temperatures of 56°C (A), 58°C (B), or 59°C (C) for 1 second and labeled with biotin dCTP followed by streptavidin alkaline phosphatase and chemiluminescence detection with dioxetane. Arrowheads indicate size of DNA fragments in base pairs.

Figure 5.

MitoTracker Orange evaluation of mitochondrial transmembrane potential 24 hours after thermal injury. Photomicrographs are of control NHEKs (A, D, G, and J) and cells heated to 58°C (B, E, H, and K) or 62°C (C, F, I, and L). Staining with calcein AM (A to C) to identify live (green) cells and MitoTracker Orange (D to F) to visualize functional mitochondria (bright orange) was followed by TUNEL labeling (G to I) to mark cells with fragmented DNA (red). J to L: Staining pattern of a single cell with MitoTracker Orange. Arrowheads indicate live, TUNEL-positive cells with functional mitochondria (B, E, and H) or after mitochondrial permeability transition (C, F, and I).

Figure 6.

Transmission electron micrographs of control NHEKs and thermally injured NHEKs. Shown are control NHEKs (A) and NHEKs 24 hours after 1-second exposure to 58°C (B), 59°C (C), or 60°C (D). Bars indicate 1.0 μm.

Figure 7.

Transmission electron micrographs of mitochondria in control or thermally injured NHEKs. Shown are mitochondria in control NHEKs (A), in NHEKs 20 hours after exposure to 58°C for 1 second (B), and 12 hours (C) or 24 hours (D) after 1-second exposure to 60°C. Bars indicate 1.0 μm.

Figure 8.

Morphological changes in thermally injured keratinocytes (58°C for 1 second). A: Fluorescence LIVE/DEAD image taken 24 hours posttreatment. One cell undergoing budding and three rounded-up cells can be identified. B and C: Transmission electron micrographs of cells undergoing budding at 12 hours (B) and 24 hours (C) after thermal injury. Bars indicate 1.0 μm.

Figure 9.

Synthesis of hsp70 and p53 in NHEKs, as a function of temperature, 8 hours posttreatment. A, top: Western blot of hsp70; graph shows the relative amount of hsp70 detected by densitometry. B, top: Western blot of p53; graph shows the relative amount of p53 detected by densitometry.