The behavior of HeLa-S3 cells under the influence of supranormal temperatures

© 1971 by Academic Press, Inc.

J. ULTRASTRUCTURERESEARCH34, 375-396 (1971)

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The Behavior of HeLa-S3 Cells Under the Influence of Supranormal Temperatures U. HEINE, L. SVERAK, J. KONDRATICK, AND R. A. BONAR

National Cancer Institute, Bethesda, Maryland 20014, and Duke University Medical Center, Department of Surgery, Durham, North Carolina 27706 Received June 15, 1970 HeLa cells exposed to environmental temperatures higher than the usual 37°C undergo marked morphological and biochemical changes which are reversible after return to normal temperatures. Exposures to 41°C had little effect, but at 42 to 43 °C changes were evident by 30 minutes and pronounced at 1-2 hours. At 44-45°C degenerative changes and cell death were frequent. At 42-43°C cell response was characterized by partial loss of the granular component of the nucleolus with retention of its fibrils. Synthesis of the 45 S ribosome precursor RNA was partially inhibited at 43°C as was the conversion to ribosome RNA of precursor molecules formed at 37 ° and then metabolized at 43 °. The number of perichromatin granules was markedly increased in the nuclei of heated cells and threadlike structures of the approximate diameter and density of the perichromatin granules appeared. Mitochondria and Golgi apparatus remained essentially unchanged morphologically but the polysome arrangement of the ribosomes was completely lost and they were distributed as monosomes after heating. Cell recovery from exposure to 2 hours at 43°C was complete within 24 hours, the only alteration remaining being an increase in the number of lysosomes. It has been shown that the exposure of mammalian cells to elevated temperatures provokes specific morphologic changes in both D N A - and RNA-containing structures of the nucleus and the nucleolus in established cell cultures (BHK) and rat fibroblasts in vitro (16). Supranormal temperatures between 41 ° and 45°C, also strongly influence the biochemical and morphologic behavior of nuclei in ascites cells of the hepatoma of Zajdela (1, 15). Retraction of nucleolar chromatin in heated cells was associated with decreased synthesis of nucleolar R N A and a morphologic alteration of the granular component of the nucleolus accompanied the inhibition of cleavage of 45 S ribosomal precursor RNA. Heating also inhibited transfer of ribosomal R N A (rRNA) to the cytoplasm. These changes were reversible and after return to normal temperature recovery of the cells was complete. Furthermore, protein synthesis was found to be limited during exposure of HeLa cells to elevated

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t e m p e r a t u r e s , b u t p a r t i a l recovery t o o k place d u r i n g p r o l o n g e d h e a t t r e a t m e n t depending, p r o b a b l y , on the synthesis of a new R N A (10). H e L a cells are used often for m e t a b o l i c studies b u t seem n o t to have been exa m i n e d for the m o r p h o l o g i c effects of s u p r a n o r m a l t e m p e r a t u r e s . I n this study the results of such e x a m i n a t i o n a p p e a r to differ in s o m e respects f r o m those o b s e r v e d p r e v i o u s l y in o t h e r cells. I n a d d i t i o n , h e a t t r e a t m e n t of the H e L a cells resulted in an a c c u m u l a t i o n of p e r i c h r o m a t i n granules, p r o v i d i n g an o p p o r t u n i t y for s t u d y of the structures which we h o p e m a y c o n t r i b u t e to a better u n d e r s t a n d i n g of their function. Biochemical studies of the H e L a cells were m a d e in p a r a l l e l with the m o r p h o l o g i c i n v e s t i g a t i o n a n d the results are r e p o r t e d here along with a m o r e limited e x a m i n a t i o n of a n o t h e r cell strain ( H E F ) u n d e r similar conditions.

MATERIALS AND METHODS

Cells and media. HeLa-S3 cells were grown as monolayers in T-60 tissue culture flasks (Falcon Plastics, Los Angles, California) in Eagle's minimum essential medium (4) supplemented with 10 % heat-inactivated calf serum, 3 % glutamine, 100 units of penicillin and 100 #g streptomycin per milliliter. Before the experiments the cells were removed from the culture flasks by treatment for 7 minutes at 37°C with 0.25 % Viokase (Grand Island Biological Co., Grand Island, New York), collected by centrifugation, washed with phosphatebuffered saline (PBS) (3) and sedimented again. About 2.2 × 107 cells were resuspended in 25 ml of Eagle's minimum essential medium adapted for spinner cultures, and were maintained for several hours on a rotary shaker in 125-ml Erlenmeyer flasks. Human embryo fibroblasts (HEF) were grown as monolayers in T-60 plastic flasks. Heat treatment and radioisotopes. The heat treatment of HeLa cells was carried out by partially submerging the Erlenmeyer flasks in a water bath at 41°, 42 °, 43 °, 44 °, or 45°C for times of 15 minutes to 3 hours. The H E F culture flasks were submerged directly in the water bath. Uridine-5-SH, 26 Ci/mmole (New England Nuclear Corp, Boston, Massachusetts) was used as a tracer in "pulse" and "pulse-chase" experiments. To each flask was added 0.5 ml of medium containing 250/zCi uridine-3H. After 15 minutes the cells in the "pulse" experiments were harvested, and actinomycin D was added in "chase" experiments to a concentration of 1/zg/ml of tissue culture fluid for 90 minutes further incubation. After incubation the cell suspension was poured over 10 ml frozen crushed PBS, and the cells were collected by centrifugation. The pellet was resuspended once more in ice-cold PBS, and the cells were sedimented, frozen in a mixture of solid CO2 and ethanol and stored at - 78 °. R N A extraction and gradient sedimentation. Cytoplasmic and nuclear R N A s were extracted separately at 4°C and 40°C (5), respectively, with a solution of 0.5 % sodium dodecyl sulfate in 0.1 M sodium acetate at p H 5 (17) and an equal volume of water-saturated phenol containing 0.1% 8-hydroxyquinoline. The R N A was precipated with ethanol, and purified with two additional ethanol preparations. The purified R N A s were dissolved in a small volume of 0.005 M Tris-HC1, 0.025 M NaC1, 0.001 M EDTA, p H 7.0, layered over a 5 % 20 % sucrose gradient in the same buffer, and centrifuged for 17 hours at 22 500 rpm in a

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Spinco rotor SW 40 (Beckman Instruments Inc., Palo Alto, California). The optical density was determined in a Cary model 15 spectrophotometer recording at 260 nm. Radioactivity was measured in a liquid scintillation spectrophotometer Model 3310 (Packard Instrument Co., Inc., Downers Grove, Illinois) with 0.5 % 2,5-diphenyloxazole and 0.05 % 1,4-bis-2(4-methyl-5-phenyloxazolyl)benzene in toluene containing 30 % Triton X-100 (Rohm and Haas, Philadelphia, Pennsylvania) and 7 % water (13). Size classes of RNA were identified by their position in the gradient in relation to the 28 S and 18 S optical density peaks (9) and by comparison with published values (2). Optical density and radioisotope data were corrected for variable losses during purification by adjusting to a common height of the 28 S peak. Fixation and embedding. The methods for the preparation of specimens for electron microscopy have been described (6), as have the procedures used in the enzyme studies, i.e., digestion with pepsin, RNase, and DNase (7).

RESULTS MORPHOLOGIC FINDINGS

Controls The appearance of HeLa-S3 cells in suspension cultures has been described previously (6). The nuclei are large and round and exhibit one or two deep indentations. The major part of the chromatin is distributed evenly throughout the nucleus, with only small amounts of condensed chromatin found adjacent to the nuclear membrane and throughout the nucleolus. As can be seen in Fig. 1, the large nucleoli consist primarily of granules (component A), and fibriUar material (component B), which is distributed in small clumps or bands throughout the structure. Amorphous, less dense material (component C) can be recognized in a few areas. Interchromatin and perichromatin granules are infrequent and found irregularly scattered in the nucleoplasm. Usually 35 to 40 perichromatin granules can be counted in one thin section cut near the center of the nucleus. The cytoplasm forms a small rim around the nucleus, but is expanded around the Golgi area, giving the nucleus an eccentric position in the cell. As shown in Fig. 2, mitochondria are small, with circular to oval profiles. Lysosomal bodies are few. The cytoplasm is rich in polysomes, whereas the rough endoplasmic reticulum is only poorly developed. Microtubules and fibrils are frequent in the cytoplasm, and microvilli are common at the cell surface. Human embryo fibroblasts (HEF) grown as monolayers have an appearance characteristic of fibroblasts.

Effects of supranormal temperatures After incubation at 41 ° for various times, HeLa cells exhibited little morphologic change. At 42-43°C deviations from normal appeared after 30 minutes and were

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FI~. 3. Nucleolus of a HeLa cell after 3 hours incubation at 43°C. The nucleolus appears slightly smaller than in controls. Only a few of the nucleolar granules are still recognizable (arrow), most of them having been lost. x 35 000.

p r o n o u n c e d after 1-2 hours. I n c u b a t i o n for 3 h o u r s at 43°C p r o d u c e d , in a d d i t i o n , m i n o r degenerative changes in the cells. A f t e r e x p o s u r e to higher temperatures, 44 ° or 45°C, for different times degenerative changes were frequent. F o r this r e a s o n m o s t of o u r experiments were c a r r i e d out at 43°C. Changes in the nucleus. A s illustrated in Fig. 3, after a 3-hour i n c u b a t i o n at 43°C the irregular shape of the nucleolus h a d n o t c h a n g e d p r o f o u n d l y . It a p p e a r e d to be smaller t h a n in controls, due to the loss of the m a j o r p a r t of its g r a n u l a r c o m p o n e n t , a l t h o u g h a few granules were still a p p a r e n t t h r o u g h o u t the structure (arrow). A r e a s of lightly stained m a t e r i a l similar to those in the adjacent p a r t of the nucleus were present in the nucleolus. FIG. 1. Part of the nucleolus from a HeLa cell kept at 37°C consisting primarily of granules (A) and fibrillar material (B). Amorphous, less dense material (C) can be recognized in few areas. Arrow points to perichromatin granules, x 30 000. Fro. 2. Section through cytoplasm of a HeLa cell kept at 37°C showing small, round mitochondria, little rough endoplasmic reticulum and an abundance of polysomes, x 25 000.

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The increased number of intensely stained perichromatin granules (Figs. 4 and 5) was one of the most pronounced alterations in the nuclei of cells treated at 43°C. The accumulation of granules was seen in about one of 15 to 20 thin sections. They were clustered in areas containing condensed chromatin, either throughout the interior of the nucleus (Fig. 4) or adjacent to the nuclear membrane (Fig. 5). Often 30 to 50 granules were contained in one area. Their average diameter was 420 A (400500 A) and they were always surrounded by a clear halo, a characteristic feature of perichromatin granules. Sometimes two or more granules were connected by less dense material forming threads of short but variable length (Fig. 5). This phenomenon was especially frequent in heat-exposed H E F cells (Figs. 6 and 7). Some of the dense particles were continuous through 3 serial sections (Fig. 8). Assuming a section thickness of 600-800 A (silver-gray sections) the length of these structures was at least 2 000 A. Cytochemical investigations confirmed previous observations (11, 12) that the application of pepsin, RNase, or DNase or a combination of these enzymes had no pronounced digestive effect on the granules (Figs. 9-12). Another feature frequently observed during heat treatment at 43°C as well as at 44°C was the accumulation of interchromatin granules in different areas of the nucleus (Fig. 13). It is of interest that only a few perichromatin granules were observed in H e L a cells exposed to 44°C or 45°C (Fig. 13, PG), but, instead, another type of granule was seen frequently. These were of irregular shape and size, very electron dense, and apparently composed of smaller units as illustrated in Fig. 14. They were distributed throughout the nucleus in areas free of condensed chromatin. Changes in the cytoplasm. Mitochondria, endoplasmic reticulum, and the Golgi apparatus did not exhibit any major changes in number or form due to the influence of supranormal temperatures; only lysosomes were seen more frequently than in control cells. After exposure to 43 °, however, the normal grouping of ribosomes into polysomes characteristic of untreated cells (Fig. 2) was no longer evident and only monosomes were seen (Fig. 15). This phenomenon was observed as early as 30 minutes after transfer to supranormal temperatures.

Recovery after heat treatment Even after a long exposure (3 hours) to 43°C the majority of the cells recovered completely within 24 hours after return to a normal temperature of 37°C. One of the first changes, seen 8 hours after return to 37°C, was the rearrangement of the nucleolar FIG. 4. Perichromatin granules are abundant in nuclei of heat-treated HeLa cells (3 hours, 43°C) in areas containing condensed chromatin, x 18 000. F~a. 5. Cluster of perichromatin granules and short rodqike structures of similar electron density near the nuclear membrane of a HeLa cell kept at 43°C for 2 hours, x 45 000.

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FIGS. 6 and 7. Nuclei of H E F cells (2 hours, 43°C) containing clusters of perichromatin granules and short threads with comparable staining qualities. One of the threads is in direct contact with the condensed chromatin (Fig. 6, arrow). Fig. 6, x 50 000; Fig. 7, x 35 000.

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FIG. 8. Serial sections through the nucleus of a H E F cell kept 2 hours at 43°C revealing the continuity of some of the perichromatin granules through 3 sections (2 and 3). Another granule (1) can be recognized in only one picture (center). x 50 000.

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fibrils into an intricate open network interspersed with areas of condensed and diffuse chromatin (Fig. 16). A n enlargement of the open spaces was c o m m o n during recovery (Fig. 17), and nucleolar granules were seen frequently at the edges of the fibrillar c o m p o n e n t bordering the open areas (Fig. 18). Twenty-four hours after return to n o r m a l temperature the nucleoli were very large (Fig. 19), and the arrangement of their c o m p o n e n t s was comparable to that of control cells (Fig. 20). The accumulation of perichromatin granules was still evident 8 hours after heat treatment (Fig. 16), but 16 hours later their n u m b e r and distribution were similar to those f o u n d in the cells kept at 37°C. At the same time the m o n o s o m e s in the cytoplasm were reassembled into polysomes. A n unusual morphologic feature in the cytoplasm present 24 hours after return to n o r m a l temperature was the large n u m b e r of lysosomes (Fig. 19). BIOCHEMICAL FINDINGS

R i b o s o m a l R N A is synthesized in the nucleolus as a large precursor molecule of about 45 S, which is then cleaved nonconservatively t h r o u g h a series of intermediate steps to 28 S and 18 S R N A s . These, combined with protein, move to the cytoplasm and constitute the ribosome subunits (2, 14, 18). Some aspects of this process can be studied by short-term ("pulse") treatment of cells with radioisotope-labeled R N A precursors, followed by further incubation ("chase") in the presence of actinomycin D, which inhibits additional r R N A synthesis but does not interfere with the conversion of the nucleolar R N A . To evaluate the effect of different temperatures on r R N A synthesis H e L a cells were incubated with uridine-3H for 15 minutes at: (a) 37°C, (b) 43°C, and (c) at 43°C after preincubation at 43°C for 60 minutes. The cells were then collected, and the R N A was extracted and analysed. As shown in Fig. 21, the separation of nuclear (40°C) and cytoplasmic (4°C) R N A s was satisfactory, and essentially all the 45 S was f o u n d in the 40°C extract. W h e n the temperature of incubation of the cells with uridine-3H was increased f r o m 37°C to 43°C, synthesis of 45 S nucleolar R N A was reduced (Fig. 21), although substantial a m o u n t s were still formed, and there was a FIGs. 9-12. Heat-treated HeLa cells (3 hours, 43°C) with clusters of perichromatin granules and few elongate structures were subjected to different enzyme treatments. Cells were fixed with 1.5 % glutaraldehyde for 10 minutes, washed in PBS, and resuspended in appropriate enzyme solutions at 37°C. Dehydration and embedding in Epon-Araldite was carried out after the enzyme treatment. The perichromatin granules and the rodlike structures are resistant to the enzyme treatment. FIG. 9. Control, kept for 1 hour in PBS at 37°C. x 50 000. FIG. 10. 0.01 mg/ml pepsin in 0.1 N HC1 for 30 minutes at 37°C. x 50 000. FIG. 11. Pepsin treatment as in Fig. 10, then washed 3 times with PBS and resuspended in 0.5 mg/ ml RNase (pH 6.5) for 4 hours at 37°C. x 50 000. FIG. 12. Pepsin treatment as in Fig. 10, washed 3 times with PBS and resuspended in 1.0 mg/ml DNase (pH 6.4) for 4 hours at 37°C. × 50 000.

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FIG. 14. Part of the nucleus of a HeLa cell kept for 3 hours at 45°C. Clusters of very dense granules are apparent in these cells in areas free of condensed chromatin, x 40 000. small increase in the a m o u n t of r a d i o a c t i v i t y in the 30 S to 4 S region. W h e n the cells were k e p t for 60 m i n u t e s at 43°C a n d then i n c u b a t e d with uridine-3H at t h a t t e m p e r a t u r e , the r a d i o a c t i v i t y in the 45 S R N A was a b o u t 50 % of t h a t in the c o n t r o l cells a n d the increase in the 30 S to 4 S region was relatively m o r e p r o n o u n c e d . F o r m a t i o n of r i b o s o m a l R N A f r o m the p r e c u r s o r molecules synthesized at different t e m p e r a t u r e s is s h o w n in Figs. 22 a n d 23. A f t e r a 15-minute " p u l s e " (Fig. 21) f o l l o w e d by a 90-minute " c h a s e " at 37°C in the presence of a c t i n o m y c i n D, a considerable a m o u n t of r a d i o a c t i v i t y r e m a i n s in the nuclear R N A (Fig. 2 2 A ) with a p e a k at a b o u t 30 S b u t m o s t has m o v e d to the c y t o p l a s m (Fig. 22B) with p e a k s at 28 S, a n d 18 S a n d smaller fragments. W h e n the cells were l a b e l e d for 15 minutes at 37°C a n d t h e n " c h a s e d " for 90 minutes at an elevated temlzerature (Fig. 22C), m u c h less uridine-aH was f o u n d in the 28 S a n d 18 S R N A s . FIo. 13. HeLa cell kept at 44°C for 2 hours. Intranucleolar chromatin is present (arrow) and, as illustrated in the inset, nucleolar granules are evident throughout the nucleolus. Interchromatin granules accumulate in different areas of the nucleus (IG). Perichromatin granules are seen only infrequently (PG). × 17 500. Inset, x 40 000.

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FIG. 15. Cytoplasm of a heat-treated HeLa cell. The ribosomes are distributed as monosomes after exposure to 43°C for 2 hours, x 35 000.

T o d e t e r m i n e w h e t h e r the p r e c u r s o r R N A f o r m e d at h i g h t e m p e r a t u r e c o u l d b e c l e a v e d to r i b o s o m a l R N A s , cells w h i c h h a d b e e n p r e i n c u b a t e d at 4 3 ° C f o r 60 m i n u t e s a n d t h e n t r e a t e d w i t h u r i d i n e - 3 H f o r 15 m i n u t e s at 4 3 ° C (Fig. 21), w e r e i n c u b a t e d f o r 90 m i n u t e s at 3 7 ° C in t h e p r e s e n c e of a c t i n o m y c i n D . A n a l y s i s of t h e n u c l e a r (Fig. 23 A ) a n d c y t o p l a s m i c (Fig. 23 B) R N A

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FIG. 16. Nucleus of a HeLa cell 8 hours after an exposure to 43°C for 2 hours. Rearrangement of nucleolar fibrils into an open network containing areas of condensed and diffuse chromatin. Perichromatin granules are still frequent (arrow). × 24 000. FIGs. 17 and 18. Appearance of the nucleolus during recovery after heat treatment. The cells were kept for 2 hours at 43°C, then transferred to 37°C; the specimen was taken 8 hours later. Large open spaces (Fig. 17) are frequent throughout the nucleoli (S). × 30 000. At higher magnification (Fig. 18) nucleolar granules (A) are seen at the edges of the fibrillar component (B) near the open areas. × 60 000. FIG. 19. Nucleus of a heat-treated HeLa cell (2 hours at 43°(2) 24 hours after the return to normal temperature (37°C). The nucleolus is very large and lysosomes are frequent (arrows). x 10 000. FIG. 20. Part of the nucleolus in a HeLa cell after recovery from the heat treatment (compare with Fig. 19). The arrangement of the nucleolar components is comparable to that found in controls (Fig. 1). A, nucleolar granules; B, nucleolar fibrils; C, amorphous material, x 35 000.

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Fro. 21. Formation of ribosomal RNA precursors in HeLa cells at different temperatures. After incubation for 15 minutes with 10 #Ci of uridine-SH per milliliter, the cells were harvested and the extracted and purified RNAs were separated on a 5 % to 20 % sucrose gradient. Optical density and radioactivity values were corrected as described. , absorbance at 260 nm; 0 0, uridine-3H incorporation (cpm) into nuclear RNA under normal conditions (37°C); zx zx uridine-SH incorporation into nuclear RNA at 43°C; [] •, incorporation of uridine-SH into nuclear RNA of cells which had been kept for 60 minutes at 43°C and then treated at that temperature with uridine-3H for 15 minutes; y v , incorporation of uridine-3H (cpm) into cytoplasmic RNA of the cells at 37°C. peared i n the cytoplasm as 28 S a n d 18 S forms, b u t that a substantial p o r t i o n rem a i n e d i n the nucleus with a b r o a d peak c o r r e s p o n d i n g in s e d i m e n t a t i o n rate to the 28 S a n d smaller R N A s . DISCUSSION Several types of m a m m a l i a n cells when g r o w n in vitro have been shown to r e s p o n d to the stress of s u p r a n o r m a l temperature with striking nucleolar changes (1, 15, 16). The nucleolar changes b o t h m o r p h o l o g i c a l a n d metabolic become p r o n o u n c e d at temperatures r a n g i n g from 41°C to 45°C a n d are expressed by a retraction of intranucleolar c h r o m a t i n paralleled by the i n h i b i t i o n of nucleolar R N A synthesis in the exposed cells. There was also a loss of the g r a n u l a r r i b o n u c l e o p r o t e i n c o m p o n e n t of the nucleolus which was t h o u g h t to revert to the fibrillar f o r m (16). Perhaps in con-

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sequence of the latter change, nucleolar R N A already synthesized at 37°C did not move to the cytoplasm when the cells were transferred to supranormal temperature (44.5°C). Exposure of HeLa cells to elevated temperatures resulted in changes similar in some respects to those reported (1, 15, 16) for B H K cells, rat fibroblasts, and ascites A.

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FIG. 23. Conversion of nucleolar R N A synthesized at supranormal temperature. Cells were kept for 60 minutes at 43°C and then treated at 43°C with 10 #Ci/ml of uridine-3H for 15 minutes. Actinomycin D was added to a concentration of 1 #g/ml, and the cells were incubated for 90 minutes at the normal temperature (37°C). (A) Distribution of label in the nuclear RNA. (B) Radioactivity in the cytoplasmic RNA. , Absorbance at 260 nm; o ©, radioactivity.

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cells of the hepatoma of Zajdela. During a 15-minute exposure to uridine-aH at 43°C, synthesis of 45 S ribosome precursor R N A was reduced compared to that in the control cells at 37°C (Fig. 21). The inhibition was much greater when the cells were kept at the higher temperature for 60 minutes before the labeling period. However, there was an increase in the amount of tritium in more slowly sedimenting R N A s of about 28 S and less relative to the amount found in the 45 S R N A synthesized during the same time. In addition to inhibition of ribosome precursor R N A synthesis, elevated temperatures had an adverse effect on the subsequent conversion of 45 S R N A to yield the 28 S and 18 S R N A s (Figs. 22B and 22C). The conversion to 28 S and 18 S ribosome R N A s of the small amount of 45 S precursor R N A formed at 43°C which takes place during subsequent incubation at 37 ° was much below normal. It appears, therefore, that the synthesis and the subsequent conversion of the 45 S ribosome precursor R N A in H e L a cells are each inhibited to a certain degree at elevated temperatures, but that the small number of precursor molecules formed at high temperature can be at least partially utilized when the cells are then incubated at 37°C. The morphologic observations are consistent with the biochemical findings, but are similar only in part to those made (1, 15, 16) on other cell types. The reduction of the synthesis of 45 S R N A as observed at 43°C and aberrations in its subsequent metabolism are accompanied by a partial loss of the granular component of the nucleolus with retention of the nucleolar fibrils. The retraction of nucleolar chromatin and the essentially complete loss of nucleolar granules due to their conversion into fibrils (16) at a "critical temperature" which has been observed with other cells (1, 15) were not seen with H e L a cells. At temperatures above 43 ° the ultrastructural appearance of H e L a cell nucleoli was highly variable and a substantial number of dead cells and cell ghosts were observed. The nucleoli of intact cells usually retained to some degree their granular component and intranucleolar chromatin. Both forms, the condensed and the diffuse chromatin, were observed. The H e L a cells thus appeared more variable in their response to supranormal temperatures than the other cells studied, and the population as a whole was more sensitive in terms of cell death. An interesting feature of H e L a cell response to supranormal temperatures was the marked increase in the number of perichromatin granules in different areas of the nuclei. Likewise, elongate rodlike structures with an electron density characteristic of perichromatin granules and of comparable diameter were frequently observed in close proximity or attached to condensed chromatin. Perichromatin granules have been known to be abundant in nuclei of cells with interrupted ribosomal R N A synthesis at a time when other rapidly labeled R N A , mostly messenger R N A , were still synthesized. This observation suggested that perichromatin granules may represent the morphologic entities containing messenger R N A (12). The coincidence of

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the accumulation of perichromatin granules and the increase of nucleoplasmic R N A (28 S and smaller components) as observed in this study supports this assumption further. The morphologic similarity of the elongated, rodlike structures reported here to perichromatin granules and the fact that they, too, appear in large numbers in the immediate vicinity of condensed chromatin suggests that they have a similar origin. Their frequency under these experimental conditions provides new opportunities for their further study. A recent study (8) in which the distribution of D N A complementary to different cellular R N A was investigated, revealed the confinement of ribosomal R N A to those chromosomes--the small ones--containing nucleolar organizers, whereas D N A complementary to cytoplasmic messenger RNA is found among a wide range of chromosomes of different sizes. Some alterations in messenger R N A behavior in heated cells is also suggested by the marked change of cytoplasmic ribosomes from polysome to monosome arrangement. Different explanations might be given for this morphologic change. Among others are (a) an interruption of m R N A production, (b) an acceleration in the breakdown of m R N A not supplemented by a more intense synthesis, (c) the destruction of the attachment sides for m R N A on the ribosome, or (d) the arrest of m R N A transfer from the nucleus into the cytoplasm. Although a detailed investigation in the behavior of m R N A under our experimental conditions was not carried out, observations made thus far in this study favor the last explanation given above. As in the case of other cell types (1, 15, 16) HeLa cells returned to 37°C recovered within 24 hours. The number of perichromatin granules remained high for about 8 hours, but later declined to normal level. In the cytoplasm the distribution of ribosomes changed from monosomal back to polysomal. The only persistent abnormality was a relatively large number of lysosomes. The authors are very indebted to Drs. J. W. Beard and A. J. Dalton for their support during this work. They are grateful for the skilled technical help of Messrs. B. Elliott, R. Moore, and R. Steinberg, and Mrs. E. Barber.

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