Hyperthermia has promise as a means of cancer therapy, at least for tumors refractory to other means of treatment but situated such that they can be effectively heated to 43-45oC. Most likely it will be used in combination with radiation or certain chemotherapeutic agents. the molecular effects of hyperthermia are not well understood, in particular the target (if there is a single one) of hyperthermia and the molecular basis of thermotolerance are not known. These two problems are intimately related since thermotolerance is probably due to protection or stabilization of the target.
The specific aims of this proposal are to determine if the nucleus and subnuclear components (i.e. nuclear matrix and nucleolus), plasma membrane, and cytoskeleton contain proteins denaturing at the predicted transition temperature of the critical target (46oC), and if the critical target is stabilized in thermotolerant cells, that is if its denaturation temperature is shifted to higher values (47.3oC). Thermostability will be assayed by measuring the temperature at which protein denaturation occurs in these organelles isolated from control and thermotolerant cells and in whole CHL V79 cells using high sensitivity differential scanning calorimetry (DSC) and fluorescence spectroscopy. These experiments will give information about which cellular organelles can contain the critical target for hyperthermia and whether this target, and thus the organelle containing it, are more thermostable in thermotolerant cells. Whether or not there is an increase in the level of heat shock proteins (HSP) in any organelle demonstrating increased thermostability will be determined. Thus, the hypothesis that HSP's are responsible for thermotolerance by directly stabilizing cellular proteins and structures will be tested. The technique of thermal gel analysis will be used to determine the molecular weights of all proteins denaturing during hyperthermia in the isolated organelles. In addition, the cognate form of HSP70 (HSC73) will be added to isolated organelles to determine if it can stabilize these structures. The long term goal is to apply the technique of thermal analysis, that is the measurement of cellular protein denaturation by DSC and other physical techniques, to the problem of understanding thermal damage in mammalian cells and tissue. Thermal analysis has potential as a means of analyzing and predicting the thermostability, hence sensitivity to hyperthermia, of normal and tumor tissue.
|Booth, V K; Roberts, J C; Warters, R L et al. (2000) Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins. Radiat Res 153:813-22|
|Senisterra, G A; Lepock, J R (2000) Thermal destabilization of transmembrane proteins by local anaesthetics. Int J Hyperthermia 16:17-Jan|
|Freeman, M L; Borrelli, M J; Meredith, M J et al. (1999) On the path to the heat shock response: destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification. Free Radic Biol Med 26:737-45|
|Borrelli, M J; Stafford, D M; Rausch, C M et al. (1998) Diamide-induced cytotoxicity and thermotolerance in CHO cells. J Cell Physiol 177:483-92|
|Freeman, M L; Huntley, S A; Meredith, M J et al. (1997) Destabilization and denaturation of cellular protein by glutathione depletion. Cell Stress Chaperones 2:191-8|
|McDuffee, A T; Senisterra, G; Huntley, S et al. (1997) Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response. J Cell Physiol 171:143-51|
|Ali, A; Fernando, P; Smith, W L et al. (1997) Preferential activation of HSF-binding activity and hsp70 gene expression in Xenopus heart after mild hyperthermia. Cell Stress Chaperones 2:229-37|
|Senisterra, G A; Huntley, S A; Escaravage, M et al. (1997) Destabilization of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-specific, heat shock inducers results in thermal denaturation at 37 degrees C. Biochemistry 36:11002-11|
|Borrelli, M J; Lepock, J R; Frey, H E et al. (1996) Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins. J Cell Physiol 167:369-79|
|Borrelli, M J; Stafford, D M; Karczewski, L A et al. (1996) Thermotolerance expression in mitotic CHO cells without increased translation of heat shock proteins. J Cell Physiol 169:420-8|
Showing the most recent 10 out of 29 publications