Biochemical analysis of heat-resistant mouse tumor cell strains: a new member of the HSP70 family.

Abstract

A series of heat-resistant mutants selected from a murine tumor cell line, RIF-1, display a markedly increased and stable resistance to heat shock. The mutant cell lines were analyzed for differences that may explain their increased resistance. Membrane lipid analysis showed no change in cholesterol content but an increase in the proportion of saturated fatty acids in the phospholipid fraction. Two-dimensional gel analysis revealed a generally increased constitutive synthesis of several major heat shock proteins (HSP), including HSP90, 68, 60, and 28. In addition, a new protein in the 70-kilodalton region is present in the resistant lines.The new protein has a lower isoelectric point than the constitutive HSP70 does, is only weakly induced by heat shock, and is immunologically cross-reactive with other members of the HSP70 family. After heat shock, the mutants display increases in HSP similar to those seen in the wild-type cells and they develop further transient tolerance to heat. Analysis of these mutants may help in understanding the function of HSP, both in normal growth and after heat shock.The heat shock proteins (HSP), or stress proteins, are synthesized in response to a variety of stresses apart from heat, including ethanol, arsenite, transition metals, release from anoxia, and mutagens (31). The molecular basis for this transient alteration in gene expression is the subject of intensive investigation. In tissue culture and in mouse tumor systems, the appearance of HSP is accompanied by the development of thermotolerance or transient resistance to subsequent heating. The close temporal matching of the appearance and decay of thermotolerance and HSP both in tissue culture (25,30,48) and in vivo (29) has led to the hypothesis that HSP function to protect cells from heat damage. However, an increasing number of examples of thermotolerance in the apparent absence of elevated levels of HSP (11,20,41,50) has raised some doubts about this proposed function. At the very least, these results suggest that cells have alternative methods for developing thermotolerance that do not involve the HSP.One possible alternative mechanism that has received considerable attention is an alteration of the cellular membranes to a more thermostable form (7). Heat increases the fluidity of membranes (28), leading to loss of their selective permeability and possibly to cell death. The importance of the plasma membrane in heat damage is indicated in the synergistic interaction of the membrane-active drug, amphotericin B, with hyperthermia (17) and the enhanced uptake of drugs such as adriamycin in cells held at elevated temperatures (43 to 45°C) (43). An aggregation of membrane proteins in the plasma membrane occurs immediately after heat shock in cells destined to die (42). Several integral membrane proteins, such as the ouabain-sensitive Na+, K' -ATPase (1, 8), the Na+-dependent amino acid transport protein (34), the epidermal growth factor receptor (35), and the insulin receptor (9) all show diminished activity after hyperthermic...

Porphyrin photosensitivity in cell lines expressing a heat-resistant phenotype

“…localization of porphyrin with resulting structural and enzymatic mitochondrial damage after PDT. Recent data suggest that thermal resistance observed in the RIF-TR strains is associated with membrane fluidity alterations (24). Interest ing, we did not observe any significant changes in photosensitivity (comparing the parent RIF-1 and the TR strains) using Photofrin II incubation conditions which would lead to either membrane or mitochondrial damage.…”

“…In addition, we have recently observed that porphyrin-mediated PDT can induce a transient increase in mRNA levels of hemeoxygenase and metallothionein.4 Multiple transcription factors can regulate the activation of these genes, and studies focused on PDT-mediated gene regulation may provide information on both target sites and subcellular mechanisms for photosensitizers. Interestingly, heat-resistant cell lines, which have constitutively elevated levels of members of HSP-70 ( 16,24), did not demonstrate cross-resistance to porphyrin-mediated PDT. Therefore, while HSP-70 may modulate thermal sensitivity, it is unlikely that HSP-70 is associated with modification of cellular sensitivity to PDT.…”

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.

“…Very recently, it has been shown that P-internexin, which is a ubiquitous microtubule-and cytoskeleton-associated protein, also corresponds to hsc70 (10). Recently, Anderson et al (1) described a heat-resistant variant of a murine tumor cell line which also contained an electrophoretically altered form of hsc70 (different alteration from that seen in our mutants). However, in contrast to these mutants, our mutants do not show any altered thermotolerance (R. S. Gupta, unpublished results).…”

Mammalian stress proteins HSP70 and HSP28 coinduced by nicotine and either ethanol or heat.

“…We also exposed cells to the same heat shock but in medium that contained 30 Western blot analysis was then performed to confirm that the 28-kDa protein was HSP28; these results are shown in 2-D gel electrophoresis, but not immunoblotting, shows that HSP70 is coinduced by nicotine and ethanol (or heat). Quantification of proteins by 1-D gels is imprecise; that by 2-D analysis is superior (2). We have therefore repeated all the protein pattern experiments described above with 2-D analysis and quantification of radioactivity of individual spots with an appropriate gel-scanning device (Fig.…”

“…Cellular proteins were labeled with L-[3H]leucine as described above. The proteins were then subjected to isoelectric focusing in a 3-mm-diameter 3.5% polyacrylamide tube gel followed by electrophoretic separation on a SDS-10% polyacrylamide slab gel (2). Following electrophoresis, the gels were treated as described above for the 1-D gels.…”