EXPERIMENTAL

CELL

RESEARCH

202,

The Relationshi

507-518

(1992)

between hsp 70 Localization

Section of Cancer Biology, Radiation Oncology Center, Mallinckrodt Znstitute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63108

structure is highly conserved during evolution [4 Studies examining the intracellular localization of 70 in Drosophila and mammalian cells h.ave shown that hsp 70 is diffusely localized thro~gb~~t the cytoplasm and nuclei at normal growth t~rn~er~t~~@~ and that it is translocated into the nuclei and nucleoli by heat shock etails of this process [7-g]. Although the biochemical have not been studied in detail, genetic studies have identified specific portions of the C termmal end of the hsp 70 molecule responsible for the heat-induced nuclear/nucleolar association of the human bsp 70 [ 101. An important aspect concerning the localization of hsp 70 which has not been explored in detail is whether or not such localization is altered in cells that display transient or permanent thermal res~sta~~~~ Prokaryotic and eukaryotic cells, when exposed to a nonlethal heat shock, acquire the transient resistance to a subsequent heat challenge, a phenomenon that has been called thermotolerance [3, 14 ]* Hn mammalian cells, the enhanced synthesis of the Ps, especially the members of the hsp 70 family [X3, 141 is closely related to the development of thermotolerance. However, the role and/or function of hsp 70 in the phenomenon of thermotolerance is not clear at this time. Nevertheless, the isolation of beat-resistant variants of Chinese hamster fibroblasts in which the cognate form of hsp 70, hse 72, was found to be synthesized at greater levels under normal growth conditions and the ability of transfeeted human hsp 70 to confer thermal resistance on rat fibroblasts lends strong support to the notion that this partieular HSP plays a role in thermal resistance ]I%, 161. In this report we have examined the time/temperature dependence of the localization of h 70 in normal cells, transiently heat-resistant cells (re ered thermotolerant by a mild heat treatment or a brief exposure to sodium arsenite), or the perrna~e~~~~ heat-resistant cells which express elevated levels of hse 72 1153. The localization of hsp 70 was monitore rescent techniques with hsp c antibodies. Using these techniques, we exa kinetics of aecumulation and removal of hsp 70 in nuclei of normal, permanently heat-resistant, and transiently thermotolerant HA-I cells after heat shocks of various duration at various temperatures.

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-B Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in tbe nuclei were found to be timeltemperature dependent at all temperatures tested (42-45°C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37°C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heatresistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular 0 1992 Academic Press, Inc. alterations.

INTRODUCTION All organisms examined thus far respond to a variety of environmental stresses by synthesizing a family of proteins, the so-called heat shock proteins (HSP), or stress proteins [P-3]. Among these proteins, the 70,000Da heat shock protein (hsp 70) is most abundant and its

’ Present address: Laboratory of Experimental Radiology, Aichi Cancer Center Research Hnstitute, Chikusa-Ku, Nagoya 464, Japan. ‘To whom correspondence and reprint requests should be addressed at Section of Cancer Biology, 4511 Forest Park, Suite 419, St. Louis, MO 63108. 507

OQi4-4827/92$5.00

Copyright Q 1992 by Academic Press, Inc. AI1 rights of reproduction. in any form reserved.

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MATERIALS

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METHODS

Cells and culture conditions. Chinese hamster fibroblasts (HA-l) and their 3012 heat-resistant variants [15] were grown on either 35mm plastic dishes (Falcon) or on glass coverslips in the same dishes in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum (Hazelton), 50 pg gentamycin/ml, and nonessential amino acids. The 9 x 9 mm coverslips (Bellco) were pretreated with a few drops of acetic acid in glass distilled water, rinsed, and stored in 70% ethanol. They were flamed briefly before use. Cells in the exponential phase of growth were used throughout these studies. Heating. Before heating tissue culture dishes containing monolayers or coverslips, the medium was changed, the plates were gassed briefly with sterile 5% CO,, 95% air, sealed with parafilm, and then were immersed into a 37°C waterbath for 10 min. After this incubation, the plates were immersed into a precision controlled waterbath at 45, 44, 43, 42.5, 42, or 41°C (20.05”C) for appropriate lengths of time. Under these conditions, the temperature elevation was achieved in 2.5 min (t,,2 = 45 s). In the experiments in which cells were allowed to recover at 37°C after the heat challenges, the sealed plates were incubated in the 37°C waterbath for 10 min, followed by a medium change before return to the 37°C incubator. The state of transient thermotolerInduction of thermotolerance. ante was induced in two ways. Cells were exposed to 45°C for 15 min and then allowed to recover for 12 h at 37°C. Alternatively, cells were exposed to 100 @f sodium arsenite in complete medium for 55 min, washed three times with warm (37’C) PBS, and were allowed to recover for 12 h at 37°C. Clonogenic survival curves for the induction and decay of thermotolerance were generated as described [15]. Briefly, posthyperthermia cell survival was determined by plating appropriate numbers of cells to generate 100-200 clones per 60-mm culture dish (Falcon) after various treatments. The dishes were incubated for 9-10 days at 37”C, fixed in Carnoys, and were stained with crystal violet. Colony counts were corrected for plating efficiency, which was routinely 70 to 80%. Gel electrophoresis and immunoblotting. After various treatments, cells or isolated nuclei were lysed in SDS-sample buffer (17) and boiled for 5 min. The cell lysates were then analyzed by SDS-polyacrylamide gel electrophoresis in gels containing 12.5% acrylamide formulated according to [17]. After electrophoresis, the proteins in the gels were transferred to a nitrocellulose membrane [18]. The membrane was then processed for immunoperoxidase staining using a monospecific rabbit antiserum against murine hsp 70 which reacts with murine hsp 68 and hsc 69, as described [19]. Rabbit antiserum at a l/200 dilution was used as the first antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel-Organon) at a l/1000 dilution as the second antibody. Indirect immunofluorescence. For the analysis of the intracellular localization of hsp 70, the cells were grown on glass coverslips (9 X 9 mm, Be&o). After various treatments, cells were washed with PBS three times, fixed for 10 min in 3.7% formaldehyde in PBS containing 0.2% Triton X-100, washed three times with PBS, all at room temperature, and then were extracted with absolute acetone at -20°C for 10 min, followed by three washes with PBS at room temperature. Cells were then incubated with the anti-murine hsp 70 antiserum [19] at a 1:60 dilution. Affinity-purified, FITC-conjugated goat anti-rabbit antibody (Sigma), at a 1:lQO dilution was used to detect the primary antibody. The coverslips were mounted in Gelvatol and observed with a Laborlux II fluorescence microscope (Leitz, Germany). Photography was performed with Kodak T-Max 400 film rated at an exposure index of 400 and developed in T-Max developer. The number of cells with brightly stained nuclei were counted as “nuclear stained” cells (this is further discussed under Results). Each datum point represents the examination of 200 to 400 cells. These experiments were

2

1

C

H

C

H

FIG. 1. Immunoblotting of extracts of wild-type HA-1 and heatresistant 3012 cells with the hsp 70-specific antibody used in this study. (1) Wild-type HA-l cells, (2) heat-resistant cells. (C) Control, nonheated cells, (H) cells heated at 45°C for 15 min and allowed to recover at 37°C for 12 h. The upper band corresponds to the cognate form of hsp 70, hsc 72, and the lower band to the heat-inducible form of hsp 70. Cell extracts were prepared and immunoblotting with a rabbit polyclonal antibody against the murine hsp 70 family [19] was performed as described under Materials and Methods.

performed in a blind fashion with consistent results.

and were repeated at least three times

RESULTS

Localization of hsp 70 in Wild-Type Heat-Resistant Cells

HA-l

and 3012

The term hsp 70 is used in many different ways in the literature and this usage is often confusing. Therefore, we first define how this term is used in this report. In rodent cells, at least two forms of hsp 70 are detected, i.e., an inducible form (hsp 70) and a constitutive or cognate form (hsc 72) [6]. In the cells used in this study, Chinese hamster HA-l fibroblasts, hsp 70 is not present under normal growth conditions; it is strictly heat inducible and does not respond to other inducers [ 151. On the other hand, hsc 72 is abundant in normal, nonstressed cells and it is also inducible by heat, sodium arsenite, and amino acid analogs [E, 201. These two major members of the hsp 70 gene family are highly homologous structurally [5, 6, 211. With this background in mind, in this report we use the general term hsp 70 to refer of both forms of the protein, and hsc 72 refers to the cognate form of the protein. As reported earlier [15], one of the members of the hsp 70 family, hsc 72 (which has been referred to in the literature variably either as cognate hsp 70 or constitutively synthesized hsp 70), was synthesized at greater levels in the 3012 heat-resistant variants than in the HA-l cells at normal growth conditions. The immunoblotting analysis of total cellular extracts from the wildtype and heat-resistant cells illustrated in Fig. 1 confirmed our earlier conclusions based on two-dimensional gel analysis and in vitro translation [ 151. The data in this figure demonstrate that higher levels of hsc 72 are expressed both under normal growing conditions and following a mild heat shock in the permanently heat-resistant variant of HA-l cells, 3012. More signifi-

hsp 70 LOCALIZATION

AND

HEAT

RESISTANCE

509

FIG. 2. Localization of hsp 70 in HA-1 cells (a and b) and in 3012 heat-resistant cells (c and d). Cells growing at 37’C (a and c), or heated at 45°C for 15 min and fixed immediately after heating (b and d), were processed for immunoflnorescence as described under Materials and Methods. The photographs represent equal exposures. Bar, 10 Frn.

cantly for this report, these results also demonstrate that the polyclonal antibody used in this study, which was obtained against the mouse mastocytoma hsp 70 family [19], cross-reacts well with both the constitutively expressed hsc 72 and the heat-inducible hsp 70 found in the HA-1 cells. We next examined the localization of hsp 70 in normal and heated wild-type HA-1 and heat-resistant 3012 cells using this polyclonal antibody. When exponentially growing cells, control or exposed to 45°C for 15 min, were fixed and stained with this antibody, the results illustrated in Fig. 2 were obtained. In unheated wild-type HA-1 (Fig. 2a) and heat-resistant 3012 cells (Fig. 26) the staining pattern is similar: dif-

fuse staining throughout the cytoplas clear staining. There is a slight phology of the two cell lines: t cells are slightly rounder, as reported earlier 1X]. This pattern of hsp 70 localization is similar to that which has been reported by others in rat chicken embryonic fibroblasts [23] ness of the nuclei in relation to t nonheated cells may be due to plasm compared to the thickn wild-type HA-I (Fig. 2c) and halt-resi~ta.~t 3012 cells (Fig. 2d) are exposed ts 45’C for 15 min, the nuclear staining becomes predominant with very little cytoplas-

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C

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FIG. 3. Immunoblotting of nuclei isolated from control and heated cells. Nuclei isolated from control (C) and heated (45”C, 15 min, H) wild-type HA-l (1) and heat-resistant 3012 (2) cells were processed as described in the legend for Fig. 1. The band corresponding to hsc 72 is observed only since the nuclei were isolated immediately after heating of exponentially growing cells.

mic staining; note that all the spread cells display this pattern of localization. At this level of resolution, we could not detect any significant differences in the pattern of localization of hsp 70 in either control or heated wild-type or heat-resistant cells. Thus, the increased heat resistance conferred by elevated expression of hsp 70 in the HR 3012 cells is not accompanied by any obvious alteration in the localization of hsp 70. An increase of hsp 70 was also observed when we compared nuclei isolated from heated and control wild-type or heat-resistant cells using immunoblotting techniques with the same antibody (Fig. 3). This result indicates that the increased nuclear fluorescence associated with the nuclei in heated cells is a consequence of increased amounts of hsp 70, and not only the extinction of the ability of the cytoplasmic hsp 70 protein and/or an increase in the availability of the nuclear hsp 70 to crossreact with our antibody. Although our experiments do not completely eliminate the later possibility or the possibility that the cytoplasmic material is lost from the heated cells, before or during fixation, they do convincingly demonstrate an increase in the mass of hsp 70 associated with the nuclei in heated cells. Our data and the data of others are consistent with the notion of some process of translocation of hsp 70 from the cytoplasm to the nucleus in heated cells. For the purposes of this report we use the term “enhanced nuclear localization” (ENL) of hsp 70 to describe this observable phenomenon of heat-induced translocation of hsp 70 to the nucleus without implying any putative biochemical and/or biophysical mechanisms that may be involved.

AND

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lengths of time. At the various time points, the relative fraction of cells displaying ENL of hsp 70 as illustrated in Fig. 2c and Fig. 2d was determined. The results of a representative experiment are illustrated in Fig. 4. It is clear that the fraction of cells displaying heat-induced ENL of hsp 70 is both time and temperature dependent. In other experiments, we could not observe significant increase in the fraction of cells displaying ENL of hsp 70 in cells incubated at 41°C for up to 6 h of heating (data not shown). At 42°C the process appears to be very slow; after 60 min, the time frame of the experiment illustrated in Fig. 4, only about 20% of the cells displayed ENL of hsp 70. In other experiments we found that a 3-h exposure at 42’C was required for 90% of the cells to display ENL (data not shown). However, at temperatures of 42.5”C and above, the kinetics of the heatinduced increase in the fraction of cells displaying ENL of hsp 70 appears to speed up. The response is saturated (approximately 90% of the cells displaying ENL) within the limitations of our assay by 60 min at 43°C 30 min at 44”C, and 15 min at 45°C. The different responses at temperatures below and above 42.5”C is very reminiscent of the observations concerning the kinetics of heatinduced cell killing in Chinese hamster cell lines [24, 251, which also display this differential behavior above and below 42.5”C. Indeed, the formalism of rate processes used by Westra and Dewey to examine heat-induced cell killing [24] can be used to obtain an Arrhenius plot for the process of the heat-induced ENL of hsp 70. Using the data from Fig. 4, the logarithm of the recip-

10

The Effect of Various Heat Treatments on the Localization of hsp 70 in Normal, Transiently Thermotolerant, and Permanently Heat-Resistant Cells To further understand the heat-induced ENL of hsp 70, the localization of hsp 70 was determined immediately after the exposure of exponentially growing HA-l cells to temperatures from 42 to 45°C for various

20

30 Minutes

40

50

60

FIG. 4. The kinetics of heat-induced ENL of hsp 70 in wild-type HA-l cells at various temperatures. Exponentially growing wild-type HA-l cells were exnosed to 42°C (0).,, 42.5”C (0). 43°C (0).,_44°C (A).,. and 45’C (0), for various lengths of time. Cells were fixed and processed for immunofluorescence immediately after heating as described under Materials and Methods. The fraction of cells with brightly stained nuclei resulting from the heat-inducedENL of hsp 70 were counted as “% Staining.” At least 400 cells were observed for each point.

hsp 70 LOCALIZATION

AND

rocal of the rate of increase in the fraction of cells displaying ENL of bsp 70 was plotted against the reciprocal of the absolute temperature (Fig. 5). (The rate of increase in the fraction of the cells displaying ENL of hsp 70 was obtained from the slope of the linear parts of the curves presented in Fig. 4.) The Arrhenius plot obtamed in this manner has an inflection point around 43°C; using the slope of the Arrhenius plot we calculate that the activation energy for the heat-induced ENL of hsp 70 above 43°C was approximately 120 kcal/mol. These thermodynamic parameters are very similar to those associated with the cell killing effect of heat [24, 251. The latter have been interpreted to indicate that protein denaturation may be a rate limiting step in the process of beat-induced cell killing. Arguing by analogy, it may be said that the heat-induced ENL of hsp 70 is rate limited by a step(s) involving protein denaturation. The similarity in activation energies of these two processes does not necessarily imply the heat-induced ENL of hsp 70 is related to cell death or that the mechanism of translocation of hsp 70 in nuclei is related to cell death. Indeed, the relationship between these two processes is not clear. Under heating conditions in which the heat-induced ENL of hsp 70 is saturated as measured by our immunofluorescence assay, cell survival continues to decrease in an exponential fashion in a time/temperature-dependent manner in the HA-1 ceil Eine [15]* However, the similarity of the activation energies associated with cell killing and ENL of hsp 70 does

1/Tx105 FIG. 5. An Arrhenius plot for the heat-induced increase in ENL of hsp 70. The plot was generated from the data illustrated in Fig. 4, as described in the text.

IIEAT

511

RESISTANCE

iflutes FIG. 6. The kinetics of heat-induced ENL of hsp 70 in heat-resistant 3012 (20) cells. Exponentially growing 3012 cells were exposed to 42°C (V), 42.5”C (0), 43°C (Cl), 44°C (A), and45”C (O), for various lengths of time. Following heating, cells were processed for immunofluorescence and the data are presented as described in the legend for Fig. 4.

indicate that these two processes do have a ;similar temperature dependence and that perhaps bate-limiting steps with similar temperature se~s~~~v~~i~~may be involved in both. We examined next the effect of beat resistance, both the permanent kind associated with t e stable heat-resistant variant and the transient kin associated witb induced thermotolerance, on the time/t@m~eratnre dependence of the heat-induced ENL of hsp 70. First, we examined the heat-induced increase in the fraction of cells displaying ENL of hsp 70 in t e 30’12 heat-resistant variant (Fig. 6). If these re ts are compared with those obtained for the wild-ty nt hne (Fig. 4), then it is clear that no sign% ik’ferences are observed with respect ts this response ween the two cell lines at the temperatures we tested e then examined onse in cells in which t clonogenic tbermotolerance was induced by exposure to a mild heat treatment (45’@ for 15 min, fo~~owea by 12 h recovery at S7'C) or exposure to 100 pA.4sodium arsenite (55 min at 37”C, fOllowed by recovery for 12 h at 37”‘C), at which times thermotolerance was maximally d~~elQ~ed [26]” We used these two different protocols because of the reported difference between the ~eat-~~a~~ed and arsenite-induced states of thermot~~e~~~ce [26, 33, 343. The time-dependent mcrease an t fraction of eehs that display ENL of hsp 70 in the ree different cell types is illustrated in Fig. 7. These data indicate that at both 45 and 43°C there is no time/temperature dependence of fraction of cells displaying transiently tbermQt~~era~t, sistant cells. The results also show that the response of

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0 n 6 A

Normal TThas, TT.. 2diHR)

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FIG. 7. A comparison of the kinetics of accumulation of hsp 70 in the nuclei of control and thermotolerant HA-1 cells and control heatresistant 3012 cells. Control HA-l wild-type cells (0, l ), thermotolerant wild-type HA-l cells, in which thermotolerance was induced by a 45”C, 15-min treatment followed by a 12-h recovery at 37°C (0, n ), thermotolerant wild-type HA-l cells in which thermotolerance was induced by a 55-min exposure to 100 &4 sodium arsenite followed by a 12-h recovery at 37°C (0, +) and control heat-resistant 3012 cells (A, A) were exposed to 45°C (0, 0, 0, A) or 43°C (0, n , +, A) for various lengths of time. Cells were then processed for immunofluorescence and the data are presented as described in the legend for Fig. 4.

the heat-resistant variants is similar if not identical to the response of transiently thermotolerant cells. The data in Fig. 7 lend additional support to the notion stated above that the heat-induced ENL of hsp 70 is probably not related directly to heat-induced cell killing, since cells which display as much as three orders of magnitude differences in clonogenic survival do not significantly vary in the time/temperature dependence of the heat-induced increase in the number of cells displaying ENL of hsp 70. Moreover, the heat-induced increase in the fraction of cells displaying ENL of hsp 70 is not altered by the presence of elevated levels of hsp 70, which are found in both the transiently thermotolerant and permanently heat-resistant cells [ 13, 151.

AND

LASZLO

(For brevity, we call this process “recovery from ENL”). When cells were returned to 37°C after heating at 45°C for 10, 15, 20, or 30 min, the fraction of cells that displayed ENL of hsp 70 returned to that observed in control unheated cells, within 6,12,24, or more than 24 h of recovery at 37”C, respectively. The reappearance of the cytoplasmic localization of hsp 70 is not only due to the synthesis of new hsp 70 molecules as a consequence of the initial heat shock, because this process of “recovery” has been also observed in cells in which protein synthesis had been inhibited by cycloheximide in both HeLa and HA-l cells ([9]; Ohtsuka and Laszlo, manuscript in preparation). The time required for recovery from the heat-induced ENL of hsp 70 was then examined in cells with various thermal responses (Fig. 9). In both the permanently heat-resistant variant and in transiently thermotolerant cells (induced either by prior heating or prior treatment with sodium arsenite), the time required for this process of recovery was significantly shorter than that which was observed in normal HA-1 cells. In the case of an initial heat challenge of 45°C for 15 min, the halftime of recovery was between 6 and 8 h in HA-l cells with complete recovery by 12 h, while in the permanently heat-resistant variants and in the transiently thermotolerant cells, the halftime for recovery was only 3-4 h with complete recovery by 8 h. Again, the response of the permanently heat-resistant cells was very similar to the response of transiently thermotolerant cells. The time required for this recovery process was also examined in transiently thermotolerant cells exposed to different initial heat shocks at 45°C: the time required for

Removal of hsp 70 from Nuclei after Heat Shock in Normal, Permanently Heat-Resistant, and Transiently Thermotolerant Cells As reported previously, by ourselves and others, the hsp 70 accumulated in nuclei and nucleoli immediately after heat shock returned gradually to the cytoplasm within several hours after heat shock in HeLa cells [9] and rat fibroblasts [27]. We examined the localization of hsp 70 in HA-l cells which were exposed to 45°C for various lengths of time and then allowed to recover at 37°C (Fig. 8). The time required to reduce the fraction of cells displaying ENL of hsp 70 to the levels observed in normal cells was dependent on the original heat dose.

” 0

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6 Hours

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‘24

FIG. 8. Recovery from heat-induced ENL of hsp ‘70 in HA-1 cells exposed to 45°C. Wild-type HA-l cells exposed to 45°C for 10 min (O), 15 min (II), 20 min (A), and 30 min (0) were allowed to recover at 37°C in fresh medium for various lengths of time. After 0,2,4,&S, 10, 12, and 24 h of recovery, the cells were processed for immunofluorescence as described in the legend for Fig. 4. The data are presented as described in the legend for Fig. 4.

bsp 70 LOCALIZATION

al return to the initial levels of nuclear association of hsp was shorter in the transiently thermotolerant cells than in the control cells at every heat dose tested (Fig. 10; compare with Fig. 8). Similar results were also obtained with initial heat shocks of various length at 43°C (data not shown). A mild heat shock has been shown to induce transient thermotolerance in the permanently heat-resistant 3012 variant (15); under these conditions, the time required for return to the control fraction of cells displaying ENL of hsp 70 in the thermotolerant heat-resistant cells was slightly faster than that observed in the control heat-resistant variant cells (data not shown). Thus, although the time/temperature dependence of the initial increase in the fraction of cells displaying heat-induced ENL of hsp 70 was found to be similar in normal, permanently heat-resistant, and transiently thermotolerant cells, the time required for a return to control levels of nuclear association of hsp 70 was dramatically shortened in transiently thermotolerant and permanently heat-resistant cells. Therefore, it appears that the length of time required for return to control numbers of cells displaying ENL of hsp 70 after a heat challenge correlated well with the thermoresistant state as defined by clonogenic survival. The Relationship between Transient Thermotolerance and the i%f~re Rapid Recovery from Heat-Induced ENL

of hsp 70

The data presented above indicate that the thermoresistant state associated with transient thermotolerance

0 Normal

HA-l HA-l 0 TT,, HA-l A 20(HR) q TTheat

L

0

2

4

6 8 10 12 24 Hours FIG. 9. Recovery from heat-induced ENL of hsp 70 in control, tbermotolerant, and heat-resistant cells after heating at 45°C for 15 min. Control wild-type HA-1 cells (O), heat-induced thermotolerant wild-type HA-1 cells (U), sodium arsenite-induced thermotolerant wild-type HA-1 cells (O), and heat-resistant 3012 cells (A) were all exposed to 45°C for 15 min and were allowed to recover in fresh medium at 37°C for various lengths of time. After 0,2,4,6,8,10,12, and 24 h of recovery, cells were processed for immunofluorescence as described in the legend for Fig. 4. The data are presented as described in the legend for Fig. 4.

AND

HEAT

513

RESISTANCE

HoiJrs FIG. 10. Recovery from heat-induced ENL of bsp 70 in beat-induced tbermotolerant cells exposed to 45°C. Exponentially growing HA-1 cells were exposed to 45’C for 15 min and allowed to recover at 37°C 12 b. The thermotolerant cells were then exposed to 45’C for 10 min (o), 15 min (Cl), 20 min (A), and 30 mm (0) and were allowed to recover at 37°C for various lengths of time. After 0, 2, 4, 6,8, 10,12, and 24 h of recovery the cells were processed for immunofluorescence as described in the legend for Fig. 4. The data are presented as described in the legend for Fig. 4.

is characterized by a more r recovery from similar initial perturbations in the ization of hsp 70. We ensured that these observations were under conditions of maximum thermotolerance b periments after 12 h recovery fol ments that induce transient ther exposure to 45°C for 15 min or I for 55 min (261. We examined next the relationship of the maintenance and decay of the heat-induced transiently thermotolerant state to the maintenance and decay of the capacity for faster recovery from heat-induced perturbations in the localization following experiment. Exponentially cells were exposed to 45°C for 15 mm allowed to recover at 37°C for up to 9 h. j&er 12,24,4$, 72, and 96 h of recovery, the cells were exposed to 45°C the beat-i~duced infor 15 min, and the recovery fro crease in the fraction of cells dis~~ayi~~ was determined for up to 12 h after such 8 second heat challenge (Fig. 11). The shortest time for the recovery from the heat-induced ENL of hsp 70 was observed at 12 h of recovery at 37°C. The time from the ENL of hsp 70 was sli h after the initial heat shock. time required for the recovery intermediate between that observed in maximally thermotolerant cells and in control cells, and by 96 h it was essentially identical to that observed in the control cells. (Since the recovery from the ENL of hsp 70 by the original heat shock used to induce t~er~~~~~~ra~ce takes 12

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FIG. 11. The kinetics of recovery from heat-induced ENL of hsp 70 during the period of the decay of heat-induced clonogenic thermotolerance. Exponentially growing wild-type HA-l cells were heated at 45°C for 15 min, then were allowed to recover at 37°C for 12 h (0), 24 h (A), 48 h (Cl), 72 h (O), and 96 h (0). At these times, the cells were heated again at 45°C for 15 min and then allowed to recover at 37°C for various lengths of time. After 0,2,4,6,8, 10,12, and 24 h recovery, the cells were processed for immunofluorescence as described in the legend for Fig. 4. The data are presented as described in the legend for Fig. 4. The dashed line labeled normal represents the data obtained with control wild-type HA-1 cells illustrated in Fig. 9.

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tained in avian cells [23]. However, such fixation conditions also led to severe loss of cells from the coverslips, making quantitation of the heat-induced increase in nucleolar association of hsp 70 not feasible. In any case, our notion is that while there is a nucleolar association of hsp 70 after heat treatment of the nonpreheated HA1 cells after heat shock, it is obscured by the extensive nucleoplasmic localization of hsp 70 under the fixation conditions used in this report. More recently, we have obtained monoclonal antibodies which react specifically with the cognate form of hsp 70, hsc 72. Using these antibodies, we have detected nucleolar association of hsc 72 even in nonheated cells [41]. These and other features of the nucieolar association of members of the hsp 70 family in a variety of rodent and human cells will be described in detail elsewhere (Laszlo, Ohtsuka et al., manuscript in preparation). In all mammalian cells studied thus far, hsp 70 is a member of a gene family, members of which share high sequence homology [4-61. The expression of the various members of the hsp 70 family under normal growth con-

loo

I I

h, we could not perform this experiment at any earlier time points.) The time course of development and decay of the more rapid recovery from the heat-induced ENL of hsp 70 closely paralleled the time course for the development and decay of clonogenic thermotolerance in exponentially growing HA-1 cells under our experimental conditions (Fig. 12). DISCUSSION

An inherent association of hsp 70 with nuclei in nonheated cells, which is increased upon exposure to elevated temperatures has been reported in many cell types, including Drosophila [7], avian [23], and various mammalian cells [8, 9, 271. In some cell lines, mainly of primate origin, the increased nuclear localization of hsp 70 in heated cells is accompanied by association with distinct structures such as the nucleoli [8,9]. In the experiments described in this report using a rabbit polyclonal antibody against mouse mastocytoma hsp 68 and hsc 69 [9], we observed mainly nuclear localization of hsp 70 after exposure to elevated temperatures. Under more severe experimental conditions, such as the pretreatment of cells with high concentrations of nonionic detergents (0.5% Triton X-100 or NP-40) before fixation, we have observed nucleolar association of hsp 70 in the heated cells (Laszlo and Hu, unpublished observations). This observation is reminiscent of results ob-

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96

lhrl

FIG. 12. The kinetics of development and decay of clonogenic thermotolerance. Expontentially growing wild-type HA-l cells were exposed to 45°C for 15 min. After various times of recovery at 37”C, the cells were challenged with a 45min exposure to 45°C and clonogenie survival was determined as described under Materials and Methods. The triangle represents the clonogenic survival obtained after exposure of control, nonpreheated wild-type HA-1 cells to 45°C for 45 min.

hsp 70 LOCALIZATION

ditions is cell line specific. On the one hand, in some cell lines the constitutive expression of several of the members of the hsp 70 family can be increased further after exposures that elicit the beat shock response, while on the other hand, in some other cell lines, there are members of the hsp 70 family that are only expressed after heat shock or other stresses. In the cells used in this study, HA-1 Chinese hamster fibroblasts, we have found a species of hsp 70 that is expressed under normal growing conditions and is induced further by beat shock, sodium arsenite, and amino acid analogs 115, 201. In previous reports we have called this species of hsp 70 ‘“constitutive hsp 70,” or hsp 70, 1151. The constitutively expressed member of the hsp 70 family has often been called “cognate hsp 70” or hsc 72-73 in other mammalian cells [4,5,21]; it has been found to be only slightly heat inducible in human [4] and rat cells [21]. IIowever, recent molecular cloning data from our laboratory indicates that the hamster constitutive hsp 7Q has excellent sequence homology with the cloned rat and human hsc 72 and is in fact the hamster hsc 72 (M-S. @hen and A. Laszlo, manuscript in preparation). The novel feature of the hamster hsc 72 is that it is heat inducible, in contrast to the hsc 72 gene product found in buman and rat cells. In this report, as a consequence of this new information, we have referred to this member of the bsp 70 family in MA-1 cells as “cognate hsp 70” or hsc 72. Given the foregoing complex overall picture, we need to define precisely the members of the hsp 70 gene family which were involved in the beat-induced ENL of hsp 70 that we studied in the experiments reported here. Since we examined the localization of hsp 70 in cells under normal growth conditions immediately after exposure to elevated temperatures, we were examining the localization of hsc 72, which is present at significant levels under these conditions 1151. An elevated level of hsc 72 and the presence of the heat-inducible form of lnsp 70 were found in the heat-induced thermotolerant cells (Fig. I), but only elevated levels of hsc 72 were found in the arsenite-induced thermotolerant cells [ 151. However, as demonstrated by the immunoblots presented in Fig. 1, in beat-induced thermotolerant cells, the total fraction of the hsp 70 family represented by the heat-inducible form of hsp 78 is less than lo%, in agreement with estimates obtained from labeling experiments 1151. Thus it is probable that in the studies reported here we have mainly observed the localization of hsc 72 in both control, beat-resistant, and transiently thermotolerant cells. Nevertheless, for the sake of simplicity, the term ““ENL of hsp 70” encompasses all the members of the hsp 70 family. The studies presented here have focused on the observation that upon exposure to elevated temperatures, there is an increase in the association of preexisting hsc

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RESISTANCE

72 molecules with the nut cells, this process is essential immunofluorescent assay serve any states that could (The inability to observe be due to the fact that we could not ~stin~~isb levels of fluorescence intensity associate cleus by the techniques used in this st

515

various

demonstrated to be time and te Biochemical experiments indicated that an increased amount of hsp 7Q was associated with nuclei isolate from heated cells. These results co med our interpretation of the results of the immun rescence studies. They argue against the inability of our amibody to detect cytoplasmic hsp 70 in kea 11s and/or an increased ability of our antibody nucleus due to conform~tiona strate binding-in tained similar results us eluding several 72 (Laszlo, Oht observations) ~ The Arrhenius analysis of the t~~~e~~t~re dependence of the beat-induced increase in the fraction of cells displaying ENL of hsp 70 indicated that the activation energy for this process is very similar to that reported for protein denatnration 1241.The mechanism of this beat-induce ENL of hsp 70, which has been often called “nuclear” translocation by other workers [ 7-91, is not clear at this time. Nevertheless, we speculate that partial denatu~at~on or confo~~~~iona~ cha.nges of hsp 70 induced by beat may allow it to migrate to the nuclei. Alternatively, some unmasking or alteration of hsp 7 by heat shock may result in an increased atlinity for nuclei or the mechanism which rn~~n~a~n~ the equilibrium between cy be modified by idence for SW a mechanism maintaining eqm clear pools of hsc 72 has been presented in studies of Xenopus cmytes under nor and&ions 128, 291. However, the effect of this equilibrium has not yet been exami heless, it is conceivable a rap ux into the ~nucleus with a %343d (due to in could be involved an e haat-induce and the recovery from it. Altbough evidence exists that putative nuclear localization sequences in the hsp 78 molecule play a role i the pattern of hsp 70 localization, the exact nature an location of such sequences within the molecule is not

516

OHTSUKA

clear at this time. In Xenopus oocytes, deletion of the C-terminal end of purified rat hsc 73 led to a decreased influx of cytoplasmically microinjected material into the nucleus and a decreased efflux of microinjected material from the nuclei [29]. Studies of deletions of the Drosophila hsp 70 gene transfected and transiently expressed in COS cells demonstrated that while both the N- and C-terminal domains are capable of accumulating in the nuclei of unstressed cells, only the more conserved N-terminal end was capable of binding to nucleoli in heated cells [30]. On the other hand, specific regions in the C-terminal end of the human heat-inducible form of hsp 70 that are responsible for the heat-induced nuclear and nucleolar localization have been identified [lo]. Moreover, when a synthetic peptide corresponding to a region from the middle of the human heat-inducible form of hsp 70 which resembles the HIVTat I nucleolar localization sequence is attached to pyruvate kinase, this hybrid molecule is then found in the nucleolus in nonheated cells [31]. The heat dose dependence of the increase in the fraction of cells displaying ENL of hsp 70 in HA-l cells indicates that this process does not appear to be directly related to the heat-induced cell killing as assayed by clonogenic survival. The increase in the fraction of cells displaying ENL of hsp 70, as detected by our techniques, saturates very quickly, especially at 45°C. At the saturation point after a 15min exposure to 45°C at least 10 to 20% of the cells will still end up surviving [ 151. Such saturation of the heat-induced increase in the ENL of hsp 70 without the simultaneous saturation of clonogenic cell killing could be explained by invoking the radiobiological concept of potentially lethal damage (PLD) [32]. The rapid accumulation of hsp 70 in the nucleus after heating could represent a cellular response to the accumulation of potentially lethal lesions in the nucleus. Such potentially lethal lesions have two possible fates: they are either repaired or become fixed lethal events. From a conceptual point of view, it can be reasonably hypothesized that the process of repair would be inhibited at elevated temperatures. Since the fixation of PLD events to lethal events could continue during the exposure to elevated temperatures, while the PLD represented by the ENL of hsp 70 is saturated within 15 min at 45°C exponential cell killing would continue upon further incubation at 45°C. It must be noted, however, that the notion of PLD, although useful for conceptual discussion, is still only a model without a firm biochemical basis at this time. The model invoking PLD can also be used to explain the puzzling results that the dose response of the heatinduced increase in the fraction of cells displaying ENL of hsp 70 was found to be similar in cells that have an altered clonogenic survival response to heat, by as much as three orders of magnitude (Fig. 7). The interpretation

AND

LASZLO

of these results along the lines of the PLD model would be that the rate of accumulation of the PLD represented by an increase in the fraction of cells displaying ENL of hsp 70 is independent of the eventual clonogenic survival. Thus, the various heat sensitivities can be explained by altered abilities to repair PLD lesions and/or various degrees of inhibition of the fixation of PLD lesions to lethal ones. The length of time after hyperthermia required for the return of the fraction of cells displaying ENL of hsp 70 to levels found in control cells was also found to be time/temperature dependent. We have called this process as “recovery from ENL,” without implying the involvement of any specific mechanisms. However, in terms of the PLD model, such recovery from ENL would be considered as a “repair” process. We found that the process of recovery from ENL of hsp 70 was different in cells that exhibited different thermal sensitivities. In both permanently heat-resistant and transiently thermotolerant cells, the recovery process was found to be faster than that in nonheated wild-type cells. The more rapid recovery in the transiently thermotolerant cells was independent of the mode of induction of thermotolerance, either a mild heat shock or exposure to sodium arsenite, even though it has been reported that the states of thermotolerance resulting from these two types of treatment are different [26,33,34]. In terms of the PLD model described above, the results concerning recovery from heat-induced ENL suggest that the process of repair of PLD is ameliorated in the heat resistant cells. It could be argued that the recovery from ENL is a function of a differential increase in the total amount of hsp 70 in the cytoplasm after the heat challenge in the thermotolerant or permanently heat-resistant cells. The observation that the thermotolerant state is associated with a significant reduction of the induction of hsp 70 in after a heat challenge [35] is a strong argument against this possibility. Moreover, our scoring system focused on the nuclear association of hsp 70, not on the relative amounts associated with the nucleus or cytoplasm. The observation mentioned earlier, namely that the recovery from heat-induced ENL is independent of protein synthesis, also supports our interpretation. Faster recovery from heat-induced ENL of hsp 70 in thermotolerant and heat-resistant cells was also confirmed in biochemical studies; these results will be presented elsewhere (Laszlo, Hu, and Landry, manuscript submitted for publication). The relationship of the transiently heat-resistant thermotolerant state as determined by clonogenic survival to the more rapid recovery from the heat-induced ENL of hsp 70 was also examined. The decay of the more rapid return to the control levels of nuclear association of hsp 70 paralleled the decay of increased clono-

hsp 70 LOCALIZATION

genie survival associated with transient thermotolerance. Furthermore, heat-resistant cells which have lost their heat-resistant phenotype did not display the more rapid recovery from the heat-induced ENL of hsp 70 (Wu and Laszlo, unpublished observations). Overall, these results indicate a good correlation between the beat-resistant state, whether transient or permanent, and the more rapid decay of the nuclear association of hsp 70. More rapid decay of the heat-induced association of hsp 70 with nucleoli was reported in cells which had been exposed to a mild heat shock previously [27]. However, in this latter report, the recovery process was not quantitated and the relationship of its kinetics to clonogenic thermotolerance was not determined. The molecular basis of the mechanisms responsible for the permanent and transient heat-resistant state is not clear at this time. There is a wealth of evidence that the heat shock proteins, especially the hsp 70 family, play a key role in these processes [13-161. The demonstration of the association of a permanently heat-resistant phenotype with increased expression of an endogenous hsp 70 [15], the association of a heat-resistant phenotype with the transfection of a heterologous hsp 70 gene [16] and the observation that the microinjection of anti-hsp 70 antibodies makes cells incapable to surviving exposures of elevated temperatures [36], all provide conclusive evidence that members of the hsp 70 family do play a pivotal role in the cell’s strategy for survival after exposure to hyperthermia. Recently, an ATPase activity has been shown to be associated with hsc 72 [37, 381, and members of the hsp 70 family have been shown to play a role in protein transport and folding (reviewed in 1391). However, given the limitation that the exposure of mammalian cells to elevated temperatures has pleiotropic effects and thus the mechanisms of heat-induced cell killing are obscure [40,41], it is difficult to delineate the specific mechanisms by which the members of the hsp 70 family play tbeir heat-protective role. However, we can consider at least two global features of the putative mechanisms by which elevated levels of the HSP, especially Asp 70, can mediate protection from thermal killing. This process could involve either protection from beat-induced perturbations or damage, or more efficient repair of similar levels of initial perturbations or damage. A combination of both of these mechanisms is also a distinct possibility. The PLD model outlined earlier is useful in considering these issues. As almentioned, the initial heat-induced ENL of hsp 70 represents some form of heat-induced damage, or a reflection of such damage. Such initial damage is similar in normal, permanently heat-resistant and transiently thermotolerant cells, which display vastly different heat sensitivities in terms of cell killing. In contrast, the recovery from such similar initial perturbations in the localization of hsp 70 was more rapid in transiently ther-

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517

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motolerant and permanently bead-~es~sta~~t cells. parallel decay of the capacity for faster recovery and clonogenic thermotolerance lends strong su.pport to the model involving faster recovery from similar initial damage. On the other hand, alternative ~Oss~~iI~ties also exist. It could be argued that the beat-induced damage in this case involves inhibition of the repair of PLD. Such inhibition would manifest as a delay in the return of the excess nuclear hsp 70 to the qt. m heat-induced repair mechanism were protec eatat,then a more damage in cells which are resistan rapid recovery from ENL of hsp 70 woul this were the cas

physiological understanding duced ENL of hsp 70.

iochemical an of the process, the heat-in-

We thank Dr. J. L. Roti Roti for help with the generation of the Arrhenius plot and many helpful discussions. We aI;so thank Ms. T. Davidson and Ms. K. Bles for proofreaang the manuscript. Tbis research was supported by NIH CA-42591 and CA-49018.

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