Assessment of Cellular Response to Thermal Laser Injury Through Bioluminescence Imaging of Heat Shock Protein 70¶†

Abstract

Assessment of laser-induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser-induced injury. A stable cell line (NIH-3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium-yttrium aluminum garnet laser (lambda = 2.1 microm, taup = 250 micros, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum bioluminescence over a range of 500-700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8-12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant-temperature water bath and laser experiments were carried out.

Invited Review Article: Current State of Research on Biological Effects of Terahertz Radiation

“…For example, cells exposed to 45°C for 15 min were observed to undergo transient G 2 block, while when exposed to 45°C for 30 min, they exhibited a long lasting G 2 phase and a late S-phase block [73]. The CSR mechanism is well documented in the literature [66][67][68][69][74][75][76][77], and the general consensus is that cells activate these mechanisms to combat and survive hyperthermic stress. CSR mechanisms involve many intracellular pathways, including redox, DNA sensing and repair, molecular chaperones, proteolysis, energy metabolism, and apoptosis [78].…”

“…These proteins, collectively referred to as minimal stress proteins, are significantly upregulated by cells immediately after exposure to stress. The most widely studied family of minimal stress proteins are the heat shock proteins (Hsps), which includes Hsp70, Hsp40, Hsp60, and Hsp105 [66][67][68][69][74][75][76][77] (Fig. 4a, c).…”

Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle

“…Among members of the Hsp family, Hsp70 (namely, HSP70.1 and Hsp70.2) is the most abundant as well as temperature sensitive (Beckham et al, 2004). It is an important "molecular chaperon" that can protect cells from heat damage as well prevent protein denaturation and apoptosis (Sonna et al, 2002).…”

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures

“…Future efforts to confirm localized heating in cells during UVA photoactivation could be analyzed through heat shock protein expression, similar to studies with longer wavelengths of light. 47 Cell density has previously been shown to be significant in studying the mode of cell death for confluent cells and microcolonies. 31 By extrapolating this information, we confirmed that cell confluence also affected HeLa cell sensitivity to UVA light.…”