The Pattern of Protein Synthesis Induced by Heat Shock of HeLa Cells

Eur. J. Biochem. 117, 341-346 (1981) 0 FEBS 1981

The Pattern of Protein Synthesis Induced by Heat Shock of HeLa Cells

Adrian SLATER, Andrew C. B. CATO, Gillian M. SILLAR, Joanna KIOUSSIS, and Roy H. BURDON

Department of Biochemistry, University of Glasgow

(Received January 16, 1981)

While incubation of HeLa cells for 5 - I0 min at 45 "C does not affect subsequent cellular growth and DNA synthesis at 37"C, there is an increased synthesis of specific proteins in the molecular weight region of 100000, 72000-74000 and 37000. The synthesis of these proteins reaches a maximum 2 h after the heat shock and while the proteins themselves are stable, their synthesis declines to normal levels 4 h afterwards. The increased synthesis is blocked by actinomycin D but the addition of cycloheximide or NaF during the 'induction' period is without effect.

When Drosophila larvae, or their excised tissues, are incubated at an elevated temperature (for example, 40 min at 37 "C, the normal culture temperature being 25 "C) puffs are induced at several specific polytene chromosome bands [l, 21. The induction of the puffs occurs within 1 min of the increase in temperature and the puffs continue to increase in size for 30 - 40 min at 37 "C before regressing. The maximum size of the puffs are a function of the severity of the temperature shock [3].

The heat shock also results in the production of a set of RNAs transcribed from the specific chromosome puffs. Some of these RNAs are preferentially translated into a set of polypeptides known as the heat-shock polypeptides [4]. Unlike the puffing, the synthesis of the heat-shock polypeptides is detected 10 min after the start of the heat shock and continues for several hours afterwards [5] .

In general there appears to be no tissue specificity in the number and sizes of the induced proteins even though some charge heterogeneity and size polymorphism has been reported. This may be due to post-translational protein modification and/or aberrant transcription at the elevated temperature [6,7].

A response similar to that in Drosophila has been reported for cultured chick embryo fibroblasts [8]. In this report we show that cultured human cells respond to heat shock by increased synthesis of at least three classes of proteins. Studies using the inhibitors actinomycin D and cycloheximide suggests that control of this response in HeLa cells may be exercised at the transcriptional level.

MATERIALS AND METHODS

Cell Culture

HeLa cells and HT1080 cells (a human fibrosarcoma cell line) [9] were grown in culture as monolayers in the Glasgow modification of Eagle's minimal essential medium (Bio-cult Laboratories Ltd, Paisley) supplemented with 10 "/, (v/v) calf serum (Bio-cult, Glasgow).

Heat Shock and Labelling of Cells with ~ - ( ~ ~ S ] M e t h i o n i n e

0.5 x 10" cells were grown overnight in the bottom of glass scintillation vials and heat shocked by immersion of the vial in a water bath at 45°C. The cells were washed with minimal essential medium minus methionine containing 10 '%; (v/v) dialysed serum, then labelled with 10 pCi ~.-[~~S]methionine (1150 Ci/mmol, New England Nuclear, Boston, MA) in the same medium. The radioactive medium was then removed by washing the monolayers in balanced salt solution and the cells were lysed by addition of 0.25 ml of dodecylsulphate sample buffer ( 2 % sodium dodecylsulphate, 1O'x glycerol, 3 "/, 2-mercaptoethanol, 50 mM Tris/HCl pH 6.8, 0.1 mM phenylmethylsulphonyl fluoride) [ 8 ] . The lysate was sonicated using a Dawes soniprobe at 7 A for 10 s and then heated to 100 "C for 2 min prior to addition of bromophenol blue (0.01 %) as a marker for subsequent dodecylsulphate/polyacrylamide gel electrophoresis.

Dodecylsulphate/Polyacrylamide Gel Electrophoresis

8.75 yi (w/v) polyacrylamide slab gels overlaid with a 3 '%, (w/v) polyacrylamide stacking gel were prepared by the method of Le Stourgeon and Beyer [lo]. After application of the sample a current of 20 mA was applied until the bromophenol blue dye front passed into the resolving gel, then the current was increased to 40 mA for 2-3 h. Molec- ular weight (20000- 200000) calibration proteins (Combi- thek, Boehringer, Mannheim) comprising Escherichia coli RNA polymerase ( E 39000, f l 155000, f i ' 165000), bovine serum albumin (68000, and soya bean trypsin inhibitor (21 500) were also run. In certain cases, gels were stained with 0.25 "/, (w/v) Coomassie brilliant blue in 9 acetic acid, 45 ';;; methanol before fluorography.

Fluorography

For fluorography, gels were equilibrated with dimethyl- sulphoxide and then impregnated with 2,5-diphenyloxazole (PPO) by immersion in 4 vol. 20% (w/v) PPO in dimethyl-

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sulphoxide for 3 h. The gels were then soaked in water and dried under vacuum [Il l . The gel was exposed to Fuji RX X-ray film (Fuji photo film Co. Ltd, Tokyo) at - 70°C. The density of the film image was determined using a Joyce- Loebl microdensitometer. The relative fraction of an individual protein band was calculated as a measure of the area under an individual peak divided by the total area under the scan that included all the protein bands in a particular track [8].

Two- Dimensional Gel Electrophoresis

Two-dimensional electrophoretic analyses followed the procedure of O'Farrell [12] with the modification that the ampholytes used for the first-dimensional isoelectric focusing consisted of 1 ampholytes (pH 5 - 7) and 1 "/, ampholytes (pH 3.5 - 10) respectively.

Labelled proteins were precipitated from dodecylsulphate- sample buffer with 9 vol. acetone and dissolved in 10 pl of lysis buffer [9.5 M urea, 2% (w/v) NP-50, 2 % ampholytes (pH 3.5- lo), 5 % 2-mercaptoethanol]. Following electro- focusing at 400 V for 18 h at room temperature, the isoelectric focusing gel was stored in 5 ml dodecylsulphate-sample buffer at - 70 "C for the second-dimension polyacrylamide electrophoresis. The isoelectric focusing gel was thawed and fused with 1% agarose to the top of a polyacrylamide slab gel. Electrophoresis in the second dimension and fluorography were performed as described above.

Heat Shock and Labelling o j Cellular DNA with (3H]Thymidine

Monolayer cultures of HeLa cells were set up in tissue- culture dishes (60 x 15 mm) in 5 ml minimal essential medium, 10% calf serum ( 5 x lo4 cells/dish). After culture for 18 h at 37"C, they were then exposed to a temperature of 45°C for 10 min and returned to 37 "C. At specific times thereafter duplicate cultures were labelled for I-h periods with 10 pCi t3H]thymidine (20 Ci/mmol). The labelled cells were then washed with balanced salt solution and, after adding 3 ml 5 ice-cold trichloroacetic acid, the precipitated cells were collected onto Whatman GFjC glass filters. The precipitated cells were solubilized by the addition of 0.5 ml hyamine hydroxide (1 M solution in methanol) and incubated for 20 min at 60 "C. The radioactivity was estimated in toluene scintillator (0.5 "/: diphenyloxazole in toluene).

Growth of HeLa Cells after Heat Shock

Monolayer cultures of HeLa cells were set up in tissue- culture dishes (60 x 15 mm) at 5 x lo4 cells per dish in 5 ml minimal essential medium, 10% calf serum. After 18 h at 37°C duplicate cultures were exposed to a temperature of 45°C for varying periods. They were allowed to grow for 3 days at 37 "C and then the cell number per dish was determined after trypsinisation using a haemacytometer.

RESULTS

When examined under the light microscope, HeLa cells do not show any gross morphological changes after incubation at 45 "C in normal growth medium for periods up to 1 h. However when these cells are left for more than 10 min at 45"C, subsequent growth at 37°C over the next 72 h is im-

0 10 20 30 40 50 60 Time at 4 5 O C (rnin)

Fig. 1. The growth of HeLa cells after heat shock at 45 C. Monolayer cultures of HeLa cells were set up in tissue-cultures dishes (60 x 15 mm) at 5 x lo4 cells per dish. After 18 h at 37 "C, duplicate cultures were exposed to a temperature of 45 "C for varying periods. They were then allowed to grow for 3 days at 37°C and the number of cells per dish determined after trypsination using a haemacytometer

---- - 0 12 24 36 42

Time ( h )

Fig. 2. The incorporation of (3H]thymidine into DNA of normal and heat- shocked HeLa cells. Monolayer cultures of HeLa cells were set up in dishes (60 x 25 mm) at 5 x 104cells per dish. After culture for 18 h at 37 "C, duplicate cultures were exposed to a temperature of 45 "C for 10 min and then returned to 37 "C. At specific times thereafter they were labelled for 1 h with [3H]thymidine. The labelled cells were then washed with balanced salt solution and precipitated onto Whatman GF/C glass filters with 5 trichloroacetic acid and radioactivity estimated by liquid scintillation counting

paired. As shown in Fig. 1, after 72 h at 37 "C control cells had multiplied tenfold while the growth of cells held at 45 "C for 40 min was reduced by 50 %. Even treatment at 45 "C for 20 min still caused a 9 % loss in viability of the cells. In subsequent experiments therefore heat treatment at 45 "C was only carried out for 10 min or less.

To determine whether any alterations in DNA synthesis resulted from heat shock, HeLa cells were labelled for 1 h at various temperatures after treatment at 45 "C for 10 min and the incorporation of [3H]thymidine into DNA estimated (Fig. 2). There was no immediate change in the rate of DNA synthesis following this heat shock, nor indeed any change in the DNA synthetic rate up to 48 h later. Similarly, there was no major change in the gross protein synthetic rate in heat-shocked cells over the control cells as measured by the hourly level of incorporation of ["'Slmethionine into total polypeptide at various times after heat treatment (results not shown).

The protein synthesised after heat shock can, however, be resolved partially by polyacrylamide gel electrophoresis in sodium dodecylsulphate.

When the cells are incubated at 45°C for 5 min, then returned to 37 "C for a period of 2 h and then labelled for 1 h

343

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+ 1 0 3 a, .-

6 8 - a 3 u a, 0

- - z

'? 39- 0

-

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Fig. 3. Fluorogram of a dodt~cylsulphatelpolycrylumide slab gel of ["'Sl- n7ethionin~-labelled proteins /rom normal and heat-shocked HeLa cel1.r. (A) Proteins from cell cultured at 37 "C and labelled for 1 h with [35S]-

methionine. (B) Proteins from cells heat-shocked at 45 "C for 5 min and transferred to 37°C for 2 h prior to labelling for 1 h with ["S]- methionine. The arrows (a), (b) and (c) refer to the heat-shock protein bands in the M , regions of 100000, 72000-74000 and 37000

72-74 1

t ul Y)

\ bottom Migration top

Fig. 4. Densilometric .scan of J'uoroxraphic pattern of a dodecylsulphatel polyacrylamide slab gel of' HeLa cell /'5S/methionine-lahelled proteins. (- -) Protein paltern from normal cells labelled with [35S]methionine for 1 11 at 37 'C; (- -) protein pattern from cells heat-shocked at 45 'C for 5 min, transferred to 37°C for 2 h and then labelled for 1 h with [3sS]methionine. The numbers indicate molecular weight x

with [35S]methionine, there is a noticeable increase in the synthesis of three particular protein bands. These protein bands have molecular weights in the region of 100000,72000 - 74000 and 37000 (Fig.3). Of these the 72000-74000-M, band is always reasonably well resolved from other cellular proteins. However a scan of the fluorographic pattern de- monstrates more clearly the presence of the 100000-M, and the 37000-M, bands (Fig. 4).

Since each of these particular protein bands could well result from a mixture of different polypeptides of the same or similar molecular weights, two-dimensional gel analysis was carried out according to the method of O'Farrell [12]. This method combines isoelectric focusing in 8 M urea in the

first dimension and dodecylsulphate/polyacrylamide electrophoresis in the second. Firstly, the results confirmed the observations already made in the one-dimensional gel analysis. Secondly, the 72000- 74000-M, band was resolved into at least seven polypeptide spots and the 100000-M, and 37000-M, bands were resolved into at least two polypeptide spots each (Fig.5). All the polypeptide spots found in the two-dimensional gel analysis of proteins from the heat- shocked cells also appear in the analysis of proteins from control cells although at considerably lowcr intensities. In addition some minor polypeptides (not seen on one-dimensional gels) appear more strongly labelled after heat-shock. Unfortunately their appearance is not always as clear cut as is the case with the major species already discussed.

A question arises whether these proteins of HeLa cells, whose synthesis is increased in response to heat treatment, are unique to HeLa cells or common to human cells in general. To investigate this point another human cell line, HT 1080, was heat-shocked and labelled under identical conditions to those used for HeLa cells. The labelled proteins were fractionated on one-dimensional dodecylsulphate/polyacrylamide gels and it was clear that the proteins whose synthesis was increased by heat shock have similar molecular weights to those found in HeLa cells (results not shown).

When actinomycin D is added prior to heat shock the absence of increased synthesis of proteins in the 72000- 74000-M, region was particularly noticeable, suggesting an element of regulation at the transcriptional level. The eflect on the l00000-M, and 37000-M, regions was similar but less easy to see. On the other hand, actinomycin D did not appear to have any other very major effect on the pattern of protein synthesised in cells maintained at 37 'C (Fig. 6, lane 1). During the actual heat shock if levels of cycloheximide and NaF sufficient to block protein synthesis were incubated in the medium the appearance of the heat-shock proteins in the prominent 72000 - 74000-M, region was not afyected, provided the inhibitors were removed prior to return to 37 'C and addition of label. Cycloheximide and NaF block the elongation of protein chains [I31 and the initiation of protein chains [I41 respectively. Thus the increases in synthesis of proteins after heat shock do not appear to require specific protein synthesis, at least during the actual heat-shock periods.

Another question is how soon and for how long after heat shock, do the HeLa cells increase the level of heat-shock protein synthesis. Cells were incubated in normal medium at 45 "C for 5 min and at various times thereafter the cells were labelled for 1 h and the pattern of protein synthesised examined by one-dimensional gel analysis. In order to quantify changes in protein synthesis following the heat treatment, densitometric analysis was applied to the fluorographic pattern of the "S-labelled proteins separated in these gels. The relative fraction of the heat shock protein bands was calculated as a measure of the area under the individual peak divided by the total area under the scan that included all the protein bands in a particular gel track [8]. In the case of the proteins with M , of I00000 and 72000-74000, synthesis reaches a maximum by 2 h after return lo normal growth temperature but thereafter the level of synthesis declines (Fig. 7). However to decide whether the heat-shock proteins themselves are metabolically labile, HeLa cells heat shocked for 5 min at 45 'C, were returned to 37°C and after 2 h were labelled for 1 h with [35S]methionine and then incubated at 37 "C in medium containing unlabelled methionine for 6 h. Since the intensities of the heat-shock protein

344

A

1st dimension

1st dimension 0

C 0 In c a,

._

E ._ U

U C N

B

Fig. 5. Fluorogram of iwo-dimensional polyacrylarnide gels of (35S]methionine-labeiledproteins,frorn normal and heat-shocked HeLa cells. (A) Proteins from cell culture at 37°C and labelled with [35S]methionine for 1 h at 37 "C; (B) proteins from cells heat-shocked at 45°C for 5 min, transferred to 37°C for 2 h and then labelled for 1 h with [35S]methionine. The arrows (a), (b) and (c) refer to the low level of synthesis of the 100000-M,, 72000-74000 M , and 37000-M, proteins at 37°C whilst a', b' and c' refer to the increased synthesis of these proteins after the heat shock

bands seen on dodecylsulphate/polyacrylamide gels before and after this chase procedure were identical, it seems that although the cells have lost their ability for elevated synthesis of heat-shock proteins by 4 h, the proteins synthesised in response to heat shock are metabolically stable.

DISCUSSION

The increased synthesis of polypeptides after heat shock has now been studied not only in insects but in a wide variety of other animals [ & I 5 - 191. However differences exist in the

345

temperature required to induce the synthesis of these proteins and the viability of the cells following the heat treatment.

Heat-shock proteins are produced by yeasts when the temperature of growth cultures is shifted from 23 "C to 36 'C [15]. Yeast cells can grow at 36°C although with a shorter generation time than if grown at 23°C. Drosophilu cells, on the one hand, can be heat-shocked for 40 min at 37°C [2] while chick embryo fibroblasts can be held at 45°C for 1 h or even for periods up to 6 h in some cases [8]. However, about 90% of HeLa cells incubated at 45°C for 1 h failed to grow after transfer of the cells to their normal temperature of growth (37 "C). On the whole the mammalian cells we have examined can be maintained at 45 "C for 10 min without greatly aflecting the viability of the cells thereafter. But slight differences exist even in the viability of mammalian cells following heat shock at 45°C. For example, baby hamster kidney (BHK) and mouse fibroblasts (L929) tend to be more sensitive to the heat treatment than do HeLa cells (un- published observation).

Following heat shock in Drosophilu cells [2], chick embryo fibroblasts [8] and yeasts [15], there is a decrease in protein synthesis evident in the repression or disappearance of a number of protein bands which were present in the protein synthetic pattern from control cells. In HeLa cells, there is no such noticeable change in the synthesis of other cellular proteins. Also unlike the other organisms, HeLa cells require about 2 h before the maximum synthesis of the heat-shock proteins is observed. The reason for these differences is un- known at present.

Two-dimensional gel analysis of proteins from heat- shocked HeLa cells revealed two or more polypeptide spots for each heat-shock protein band observed in one dimension. These spots may either be unique polypeptide species or they may occur due to differences brought about by various modifications of these proteins. Some of the Drosophilu heat- shock proteins are known to be highly modified [7,20].

We find that, even though the HeLa heat-shock proteins are stable for over 6 h at 37 "C, the ability of the cells to make these proteins declines 4 h after a 5-min incubation at 45 "C. It could be that the messenger RNAs for these proteins have short half lives. The existence of short-lived populations of RNA in HeLa cells has been demonstrated [21] and it is possible that the mRNAs for the heat-shock proteins may belong to such populations. Alternatively these mRNAs could have the normal half-live of other cellular messengers but the limited time of translation could be due to a control mechanism brought about by the heat shock and exercised at the level of translation.

Though there is as yet no biological activity associated with the heat-shock proteins, the fact that the heat-shock phenomenon occurs ubiquitously in many organisms suggests that it may be important in the regulation of gene expression.

Fig. 6. Fluorogram of a dodecylsulphate/polyacrylamide slab gel of ("Sl- methionine-labelled proteins from normal and heat-shocked HeLa cells treated with actinomycin, cycloheximide or NaF. ( -) Cells were cultured at 37 "C and labelled with [35S]methionine for 1 h; (+) cells were heat- shocked at 45 "C for 5 min and returned to 37 "C for 2 h and then labelled for 1 h with [35S]methionine. Cells were either treated with actinomycin D (1 pg/ml) throughout the heat-shock period as well as the subsequent 2-h period at 37 'C and the labelling period (tracks 1, 2), or cells were treated with 25 pg/ml of cycloheximide (tracks 3, 4) and 20 mM NaF (tracks 5 , 6) only during the actual heat treatment at 45 "C. Thereafter they were washed with balanced salt solution and labelled in the absence of the drug. Control and heat-shocked cells labelled as above but without any drug treatment, were also run (tracks 7.8). The arrows a. b, c show the positions of the 100000-M,, 72000-74000-M, and 37 000-M, proteins

-0

.$ 6 5 6 5

W

c W ._ c

2 4 a r

- 3 3 0

$ 2 i

0 1 2 3 4 Time ( h )

7

5

Fig. 7. Relative levels of the heat-shock proteins syntheslred in I h at 37°C at various times after 5-min heat shock at 45 "C. Each point shows the relative amount of [35S]methionine incorporated into the proteins in 1 h at 37 "C after heat shock. The values were computed after scanning with the Joyce-Loebl densitometer and the relative percentage calculated as a measure of the area under an individual peak divided by the total area under the scan that included all the protein bands in a particular track [8]

REFERENCES

1. Ritossa, F. (1962) Experientiu (Basel) 18, 571 -573. 2. Ashburner, M. & Bonner, J. J . (1979) Cell, 17, 241 -254. 3. Ashburner, M. (1970) Chromosoma, 3/, 356-376. 4. Tissieres, A., Mitchell, H. K. & Tracy, V. M. (1974) J . Mol. Biol.

5 . Lewis, M., Helmsing, P. J. & Ashburner, M. (1975) Proc. Nut1

6. Sondermeijei, P. J. A. & Luhsen, N. (1978) Eur. J . Biochem. YX,

84, 389 - 398.

Acad. Sci. USA, 72, 3604-3608.

331 - 339.

346

7. Mirault, M. E., Goldschmidt-Clermont, M., Moran, L., Arrigo, A. P. & Tissieres, A. (1978) Cold Spring Hurbour Symp. Quant. Bid. 42, 819-827.

8. Kelley, P. M. & Schlesinger, M. J. (1978) Cell, IS, 1277-1286. 9. Rasheed, S., Nelson-Kees, W. A., Toth, M., Arnstein, P. & Gardner,

10. Le Stourgeon, W. & Beyer, A. L. (1977) Methods Cell Biol. 16,

11. Bonner, W. M. & Laskey, R. A. (1974) Eur. J . Biochem. 46, 83-88. 12. O’Farrell, P. H. (1975) J. B i d . Chem. 250, 4007-4021. 13. Baliga, B. S., Cohen, S. A. & Munro, H. N. (1970) FEBS Lett. 8,

M. D. (1974) Cancer, 33, 1027-1033.

387-406.

249 -252.

14. Vesco, C. & Colombo, B. (1970) J . Mol. B id . 47, 335-352. 15. McAlister, L., Strausberg, S., Kulaga, A. & Finkelstein, D. B. (1979)

16. Bouche, G., Amalric, F., Caizergues-Ferrer, M. & Zalta, J. P. (1979)

17. Guttman, S. D., Glover, C. V. C., Allis, C. D. & Gorovsky, M. A.

18. Ron, A. & Zeuthen, E. (1980) Exp. Cell. Res. 128, 303-309. 19. Francis, D. & Lin, L. (1980) Dev. Biol. 7Y, 238-242. 20. Arrigo, A. P. (1980) Mol. Gen. Genet. 178, 517-524. 21. Puckett, L., Chambers, S. & Darnell, J. (1975) Proc. Nut1 Acad.

Current Genetics, 1 , 63 - 74.

Nucleic Acids Res. 7, 1739-1747.

(1 980) Cell, 22, 299 - 307.

Sci. USA, 72, 389-393.

A. Slater, A. C. B. Cato, G. M. W a r , J. Kioussis, and R. H. Burdon, Department of Biochemistry, University of Glasgow, Glasgow, Lanark, Great Britain, GI2 8QQ

The Pattern of Protein Synthesis Induced by Heat Shock of HeLa Cells

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