Re-induction of hsp70 synthesis: An assay for thermotolerance

Abstract

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 C, 4 h) and then incubated at 37 C for 0-72 h. At various times after the first heat treatment, cells were either challenged with a 45 C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [ 35 S]methionine before or after an additional test heat dose (e.g. 43.5 C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.

Role of Pluronic block copolymers in modulation of heat shock protein 70 expression

“…Up-regulation of Hsp70 is clinically relevant because it enhances thermotolerance of tumour tissues [16] and results in decreased apoptosis and necrosis, which facilitates cancer survival. Hsp70s are omnipresent, chaperone molecules that assist in protein folding in the active confirmation and are expressed rapidly as a result of cellular stresses such as hyperthermia, hypoxia, ionising radiation, and the like.…”

Can nanomedicines kill cancer stem cells?

“…Furthermore, in another study it was shown that Pluronic in combination with low-grade hyperthermia depleted the intracellular ATP and decreased the expression of heat shock protein 70 (HSP70) [258]. It is known that heat shock proteins, in particular HSP70, increases the thermoresistance of the tumors [259]. Notably, the reduction of HSP70 expression by Pluronic L61 was observed both in vitro and in vivo [258].…”

Hyperthermia classic article commentary: ‘Re-induction of hsp70 synthesis: An assay for thermotolerance’ by Gloria C. Li and Johnson Y. Mak,International Journal of Hyperthermia1989;5:389-403

“…Therefore, it was highly important to develop an assay which could assess the presence and severity of thermotolerance so that fractionated hyperthermia treatment could be optimized. In other words, the motivation for performing the studies that led to the above referenced article [31] derived from the need to develop a rapid assay to monitor hsp70 and the level of retained thermotolerance during a course of fractionated hyperthermia. The feasibility studies were first performed in vitro [31], then validated in animal model systems [32,33], and finally translated into clinical trials.…”

“…In other words, the motivation for performing the studies that led to the above referenced article [31] derived from the need to develop a rapid assay to monitor hsp70 and the level of retained thermotolerance during a course of fractionated hyperthermia. The feasibility studies were first performed in vitro [31], then validated in animal model systems [32,33], and finally translated into clinical trials. The series of events associated with this translational and clinical research are briefly described in the following paragraphs.…”

“…Specifically, using a murine tumor (SQ-1) and a human tumor cell line (HCT-8), Li and Mak studied the relationship between the retained thermotolerance after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro [31]. The working hypotheses behind this study are: (1) when tolerance is at its maximum, the cells' ability to re-initiate synthesis of hsp70 in response to a second heat shock is less than that of control cells; and (2) when tolerance gradually decays, the cells' ability to re-initiate synthesis of hsp70 in response to a second test heat shock will return to that of control cells.…”