[CANCER RESEARCH 52, 2854-2861, May 15, 1992| Thermotolerance in the Absence of Induced Heat Shock Proteins in a Murine Lymphoma1 Barbara Fisher,2 Peggy Kraft, George M. Hahn, and Robin L. Anderson3 Department of Radiation Oncology, Stanford University, Stanford, California 94305 [B. F., P. K., G. M. ff.J, and Peter MacCallum Cancer Institute, 4SI Lt. Lonsdale St., Melbourne, yictoria 3000, Australia [R. L. A.] ABSTRACT Three murine lymphoma cell lines, CHI, a B-cell lymphoma, and VL3 and RDM4, both T-cell lymphomas, were tested for their ability to induce heat shock protein synthesis and thermotolerance after heat shock. All three lines could develop thermotolerance, but the persistence of tolerance was less than can be measured in nonlymphoid cell lines. Analysis of protein synthesis patterns by one-dimensional gel electrophoresis sug gested that only the VL3 cells were capable of the induction of heat shock proteins. After two-dimensional gel analysis, however, the induc tion of one heat shock protein was evident in RDM4 cells. No induced heat shock proteins could be detected in the CHI cells. These data provide strong evidence that, while the induction of heat shock proteins may be sufficient for development of thermotolerance, they are not necessary and that another mechanism is available to cells. INTRODUCTION The development of thermotolerance, a transient resistance to heat induced by prior heating or by other inducers of the heat shock response (1), is nearly always accompanied by the induction of a class of proteins known as the HSP4 or stress proteins (2). The good temporal correlation between the ap pearance and turnover of the HSP and the development and decay of thermotolerance (3-5) is strong circumstantial evi dence that the HSP protect cells from subsequent damage from heat or other stress-inducing agents such as ethanol, arsenite, transition metals, release from hypoxia, and various inhibitors of oxidative phosphorylation. Further evidence for the involvement of HSP in heat protec tion comes from the characterization of heat-resistant lines selected from rodent cell lines. Heat-resistant cells derived from Chinese hamster ovary cells contain elevated levels of HSP73 (6), those from a transformed subclone of Chinese hamster lung fibroblasts contain elevated levels of HSP27 (7), and those from the murine RIF-1 tumor contain elevated levels of several major HSP (8). Landry et al. (9) have gone on to show that transfec- tion of the human gene for HSP27 into the Chinese hamster lung cells confers resistance to heat shock. Other experiments have shown that microinjection of antibodies against HSP73/ 72 (10) or introduction of a large number of copies of the heat shock element (11) results in increased heat sensitivity, provid ing further evidence for the involvement of this family of proteins in heat protection. (There are two major HSP in murine cells with subunit molecular weights of approximately 70,000: HSP73, which is constitutively expressed; and HSP72, which is induced after stress.) On the other hand, several studies have shown that HSP are Received 3/26/90; accepted 3/10/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grant CA4466S from the National Cancer Institute and by the Anti-Cancer Council of Victoria, Australia. 2 Present address: London Regional Cancer Centre, 790 Commissioners Rd. E., London, Ontario N6A 4L6, Canada. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: HSP, heat shock protein(s); CHO, Chinese hamster ovary. not essential for the expression of thermotolerance. For exam ple, under certain circumstances, cells treated with cyclohex- imide can still express thermotolerance despite the lack of de novo protein synthesis (12-15). However, these experiments are confounded by the fact that cycloheximide itself can confer heat protection (16, 17). Other research suggests that cyclohex imide does inhibit thermotolerance (18). There are other examples of thermotolerance in the absence of HSP induction that do not involve the use of protein synthe sis inhibitors. CHO cells heated slowly to 42°Cover a 3-h period show tolerance to 42°Cheating without an increase in the amount of HSP, but tolerance to 45°Cis minimal (19). Exposure of cells to hydrocortisone for 12 to 24 h also induces thermotolerance without the induction of HSP (20). Other examples include thermal adaptation of CHO cells at 40°C (21), exposure of pollen tubes from Tradescantia to gradually increasing temperatures (22), and mild heat exposure in snail larvae (23). Taken together, these studies suggest that, while HSP are sufficient for expression of thermotolerance, they are not necessary and that another mechanism(s) can also confer heat protection. Data from our laboratory (19), from Hallberg (14), and from Lee and Dewey (24) suggest that the tolerance that develops in the absence of increased HSP synthesis does not confer the same degree of heat protection, especially at higher temperatures, as can be achieved in the presence of elevated HSP. One approach to investigating the importance of induced HSP in the development of thermotolerance is to study cells with a defective heat shock response. Aujame (25) has shown that some murine plasmacytomas of both B- and T-cell origin and some murine erythroleukemia cell lines (26) fail to show induction of HSP 72, but induction of the other HSP is normal. Aujame and Furko (27) went on to show that the plasmacyto mas do express thermotolerance. Early embryonic cells also fail to show induction of HSP until the embryo reaches the blasto- cyst stage (28) and, in parallel with these observations, Müller e/ al. (29) have demonstrated that these cells fail to express thermotolerance. Some embryonal carcinoma cell lines (28, 30) and a murine erythroleukemic line, MEL (31), are also deficient in expression of HSP, but the response of these cells to heat has not been tested. Some species of Hydra, but not all, also fail to exhibit thermotolerance and induction of HSP (32). A cell line with an inability to synthesize any heat-induced HSP was discovered in our laboratory during a study of the effect of hyperthermia on the lateral diffusion of H-2Kk (33). The CHI cells are from an early murine B-cell lymphoma isolated by Lanier et al. (34) by inoculation of BlO-H-FH-Sp/ Wts mice with spleen cells or sera from syngeneic donors hyperimmunized with sheep erythrocytes. The cells lack the T- cell-specific antigen (Thy-1) but express cell surface immunog- lobin and the H-2K, H-2D, and la specificities determined by the H-2°haplotype. We have now analyzed the response of CH1 cells to heat shock in more detail and compared them to two other murine lymphoma lines, VL3 and RDM4. In all three lines, thermotolerance can be expressed, but it decays more rapidly than in fibroblast cell lines. Analysis of protein 2854 Research. on October 24, 2021. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from