Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells

Experimental

Cell Research 182 (1989) 61-74

Induction of Heat Shock Protein Transcripts and 82 Transcripts by Various Stresses in Chinese Hamster Cells ALBERT J. FORNACE, Jr.,’ ISAAC ALAMO, Jr., M. CHRISTINE HOLLANDER, and ETIENNE LAMOREAUX Radiation Oncology Branch, NIH, Bethesda, Maryland 20892 We have investigated the induction of known hsp (heat shock protein) RNA and other heat shock (HS) inducible transcripts in Chinese hamster cells by various stresses including DNA damaging agents. cDNA clones coding for at least 14 different HS-inducible transcripts were isolated. By DNA sequence analysis and homology with cDNA clones of other species, some of these cDNA clones were identified as coding for hsp27, hsp89a, hsp89/.l, two different hsp70s, ubiquitin, and the HS-inducible RNA polymerase III transcript B2. In addition, hsprelated cDNA clones, hsp60 and four with hsp70 homology, were isolated which coded for transcripts which were not induced by HS or other stresses in two different Chinese hamster cell tines. After HS or treatment with the HS-mimetic agent ethanol, there was coordinate induction of all 14 transcripts. With severe HS treatments which produced substantial cytotoxicity, the increase in all transcripts except B2 RNA was delayed and, in some cases, suppressed. The only DNA damaging agent, which induced many HS-inducible transcripts, was high-dose methyhnethane sulfonate (MMS). However, induction by MMS was not coordinate for all transcripts as it was for HS, and B2 RNA was not induced. hsp27 RNA induction differed from the others in several respects including induction by irradiation and other agents which produce high levels of DNA damage repaired by nucleotide excision repair. The implications of these fmdings in cellular events such as cytotoxicity, thermotolerance, and regulation of stress responses will be discussed. @ 1989 Academic RCSS. IIIC.

A variety of cellular stresses induce the heat shock (HS) response in most organisms. Although the function(s) of the heat shock proteins (hsp) is unknown, a role(s) for certain heat shock proteins (hsp) has been suggested in processes such as cell transformation, cell replication, development, and differentiation El, 21. A possible role(s) for hsp in stresses other than HS is indicated by their induction by many different agents including oxidizing agents, heavy metals, sulfbydryl reagents, amino acid analogs, steroid hormones, virus infection, teratogens, ethanol, and other organic solvents (reviewed in [3]). Many inducers of the HS response can damage DNA and chromatin, which raises the possibility that hsp may play a role in the response to genotoxic stress. In Tetrahymena, hsp were induced by an alkylating agent [3]; in rat liver hepatocarcinogens were found to induce hsp [4]. In both yeast [5] and Drosophila [6], genes have been identified which are induced by either HS or DNA damaging agents. We have recently isolated a cDNA clone, DDZAZS, from Chinese hamster cells which codes for a transcript which is induced also by either DNA damaging agents or ’ To whom reprint requests should be addressed at Building 10, Room B3B69. 61

Copyright 0 1989 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/89 $03.00

62

Fornace et al.

HS [7]. The purpose of our current study was to study the induction of known hsp RNA and other HS-inducible transcripts in Chinese hamster cells by various stressesincluding DNA damaging agents. The HS response in mammalian cells is complex probably involving many different genes. In prokaryotes, there are many HS-inducible genes; e.g., the htpR regulon in Escherichia coli contains at least 15 genes. In addition to the 7 major hsp, 29 new putative candidates for hsp were found in mouse cells by computer analyzed two-dimensional gel analysis where 1500different spots could be distinguished [8]. Rodent cells also contain HS-inducible genes other than the classical hsp genes; for instance, RNA polymerase III transcripts of the rodent B2 repetitive element have been shown to be rapidly induced by HS [9]. We have isolated a large number of different cDNA clones coding for H&inducible transcripts in Chinese hamster cells. Given that Chinese hamster lines have been used in a large portion of mammalian HS cell biology and experimental hyperthermia, our analysis of the HS response at the level of transcription in such cell lines can be correlated with these previous studies. MATERIALS AND METHODS Cells and cell treatments. Chinese hamster lines, V79 lung fibroblasts, and CHO-Kl ovarian cells, were grown as previously described [9]. All experiments were performed with exponentially growing cells at a density 2.0 to 2.5x 10’ per 175cm2 flask or 15cm dish, unless otherwise noted. To study nongrowing Go phase cells, CHO cells were grown to approximately 5 X 10’ cells per 15icm dish and incubated for 2 days without medium change in medium containing 15 m&f Hepes buffer. Go phase cells were also studied by incubating CHO cells in medium which contained reduced serum (0.5 %). For heat shock experiments, cells were heated in 175-cm* flasks containing 35 ml of medium in a water bath at the specified temperature as previously described [9]. For treatment with chemical agents, a stock solution was added directly to the tissue culture medium for the indicated time, and a portion of the original medium without the chemical agent was then added or the cells were harvested. Cells were uv-irradiated and X-irradiated as previously described [12]. For near uv irradiation, cells in 15-cm dishes were rinsed with Dulbecco’s phosphate-buffered saline and irradiated in 5 ml of the same buffer with Westinghouse Sunlamp fluorescent bulbs at a fluence of 3 Jm-* s-’ (measured at 320 nm); the buffer was then removed, the original medium WBSreplaced, and the cells were incubated at 37°C for 4 h. cDNA. Chinese hamster cDNA clones were isolated from a library described previously [9]. Briefly, the library was constructed with poly(A) RNA from V79 cells which had been heated to 455°C for 17 min and incubated at 37°C for 4 h. cDNA was synthesized by the Gubler and Hoffman procedure [13]. Double-strand cDNA was size selected on Sepharose 2B and inserted into the PstI site of pBR322 by standard GC tailing [14]. Bacteria, E. coli strain HBlOl, were transformed with this plasmid DNA; and 4600 colonies were individually picked and grown in microtiter plates [lS]. Ninetyeight percent of the cDNA inserts from clones of this library were greater than 0.5 kb in length; the average insert size was 1.2 kb. pA2, which contained a 1 .l-kb insert, was isolated by screening this library with a chicken j3-actin cDNA probe [Hi]; pA2 hybridized, at high stringency, with the same size Chinese hamster RNA as the chicken pactin probe (data not shown). pH162 was isolated from this library by screening with a human hsp60 cDNA probe [17]; pH162 hybridized with the same size Chinese hamster RNA as the human hsp60 probe (see Results). DDZAlS was isolated from a CHO cDNA library which had been enriched by hybridization subtraction for cDNA coding for DNA damage-inducible transcripts [7] and will be described in more detail elsewhere, DDZAZS (referred to as Al5 in Fig. 1 and Tables 2 and 3) was found to code for a transcript which was also induced by HS and has been included in the current study for this reason. pM1.8 (provided by R. Morimoto) is a 1.7-kb mouse hsp70 genomic clone which contains 1.5 kb of the transcribed region of this HE&inducible gene (Morimoto, personal communication). pHS208, pHS601, pHS709, pHS801, and pHS811 (provided by L. Weber) are cDNA clones which code for human hsp27, hsp60, hsp70, hsp89a. and hsp89,!? mRNA, respectively 1171.

Induction

of hsp RNA and B2 RNA in Chinese hamster cells

63

Colony blot hybridization. cDNA clones were transferred from microtiter plates to nitrocellulose membranes on LB agar, grown for 16 h, and amplified with chloramphenicol [IS]. Hybridization was as described previously [14, IS]; following hybridization, the final most stringent rinse was 1XSSC (saline citrate) at 65°C. cDNA inserts, which had been excised from their plasmid vector, were labeled 1181and used as probes in some experiments. cDNA clones coding for H&induced transcripts were initially identified by differential hybridization [14,15]. High specific activity (109 dpm/pg) cDNA probes were synthesized [9] with MMLV reverse transcriptase (BRL) using poly(A) RNA from untreated V79 cells, or cells heated to 45.5”C for 17 min and incubated at 37°C for 5 h. For colony hybridization, complex cDNA probes synthesized with reverse transcriptase often result in high background. For this reason, the labelled cDNA, 0.2 pg/ml, was hybridized with 1 mg/ml single-stranded E. coli DNA, E. coli RNA, and pBR322 DNA for 1.5 h at 65°C in 0.3 it4 sodium phosphate/O.2 % SDS; single stranded cDNA was separated from duplex on hydroxylapatite at 62°C in 0.12 M sodium phosphate as described previously [9]. From 2 to 4% of the cDNA eluted in the double-stranded fraction and was discarded. Replicate colony blots were hybridized with 106 dpm/ml of the remaining cDNA for 20 h; following hybridization, the final most stringent rinse was lx SSC at 65°C. With this approach, the signal-to-noise ratio on colony blot hybridization was substantially reduced compared to cDNA probes which had not been preadsorbed with bacterial and plasmid nucleic acids. RNA isolation and analysis. Cells were lysed by addition of guanidine thiocyanate directly to the tissue culture flasks or dishes and RNA was isolated as described previously [9]. Poly(A) or whole cell RNA was bound to nylon falters for RNA dot blots or was size-separated in denaturing gels prior to transfer, as previously described [9]. For hybridization, labeled [17] cDNA, which had been excised from its plasmid vector, was used at 3x lo6 dpm ml-‘; hybridization conditions were the same as in [9]. The poly(A) content of all RNA samples was estimated using a labeled polythymidylic acid probe [9]; this correction was small and usually varied from the RNA content measured by OD, by less than 25 %. Hybridization was quantified by densitometry measurements of autoradiographs. Relative hybridization was determined by normalizing to the result with RNA from untreated cells isolated in the same experiment on the same day. Hybridization was nearly linear when the signal varied by less than b&fold, but deviated from pseudo-linear [19] when the signal was much greater. For this reason a computer program (RNA Analysis) was written in TurboPascal for the Macintosh computer which generated a standard curve for dilutions of RNA from untreated cells. Values for experimental samples were then compared to this standard curve: the amount of RNA from control cells was divided by the amount of RNA from treated cells, which gave the same densitometry reading; this result was defined as induction. Each value under Results represents the mean of four dot blot determinations at different dilutions [9]; the SD usually varied by less than 25 % of the mean. Nucfeur runoffs. Nuclei were isolated as described previously [9] but the isolated nuclei were treated with RNase A [20]. Nuclei (2x 10’) were incubated in 0.1 ml of 70 mM KCl/S mM MgClrI2 mM dithiothreitoUO.1 mM EDTA/O.4 mM ATP, CTP, and GTP/0.24 mCi of [“PIUTP (800 Ci mmol-I)/200 units RNasin (Promega)/O.S% bovine serum albumin/O.5 mM MnCl#O% glycerol00 mM ‘Iris, pH 8.0, for 20 min at 30°C. Labeled RNA was isolated as described previously [9]. DNA dot blots were hybridized with 3.5~ 10’ dpm ml-’ for 48 h at 66°C. Blots were washed extensively including 15 min in 2x SSC with 1 ug ml-’ RNase A; the fmal most stringent wash was with 40 mM sodium phosphate containing 1% SDS and 1 m&f EDTA [9] at 66°C.

RESULTS Isolation of Chinese hamster cDNA clones coding for HS-induced transcripts. One hundred and sixty-five cDNA clones were isolated by differential screening of 4600 clones on the basis of preferential hybridization with labeled cDNA synthesized from HZ&treated cells (see Materials and Methods). These cDNA clones were tentatively identified by hybridization with known Chinese hamster cDNA clones, human hsp cDNA clones, and a mouse hsp70 clone (Table 1). The B2 (pHS 18A) and ubiquitin (pH37) Chinese hamster cDNA clones were identified by DNA sequence analysis which will be presented elsewhere. To confirm that our cDNA clones hybridized with the same RNA as the mouse and human hsp 5-898335

64

Fornace et al. hsp70

hsp27 0709

Ml.8

M3

M2

Homology #4

hsp60 -MS

MB

M8

~801

M

Ii182

-4.4-R -2.3 -2.0-R

hsp89or Origin

-

~801

Ii82

hsp898 ~811

Ubiquitin -H40

H37

82 H818A

@actin A2

Others Ii129

H74

W75H122

H104

Al5

HI05

Fig. 1. Induction of various transcripts by heat shock. Equal amounts of poly(A) RNA from untreated (f&t lane of each pair) or heat shock-treated (second lane of each pair) V79 cells were sizeseparated in a 1% formaldehyde agarose gel and hybridized with the indicated labeled probe. DDIAlS is designated A15; the prefm “p” has been deleted from the name of the particular cloned DNA in all cases. The heat shock treatment consisted of heating cells to 45.5”C for 9 min, with a subsequent 4-h incubation at 37°C before harvest. Size markers, designated M, consisted of labeled single-stranded DNA [9].

clones, poly(A) RNA from untreated and HZ&treated cells was analyzed by Northern blots at high stringency. As seen in Fig. 1, many cDNA clones, which had hybridized with known hsp clones, hybridized with the same size transcripts. A representative cDNA clone from each group is shown in this figure. In, most cases, all members of a group, which hybridized to a specific hsp probe, hybridized to the same size transcript; e.g., all four Chinese hamster cDNA clones, which hybridized with the human hsp27 clone (Table l), had the same Northern blot profile as pH8 of this group and pHS208. Results with the eight Chinese hamster cDNA clones, which had homology to the mouse hsp70 clone, were more complicated. Two clones (pM3 in Fig. 1) hybridized with the same size HSinducible transcripts as the human and mouse hsp probes; two clones (PM4 in Fig. 1) hybridized with a transcript of 2.6 kb; pM2, pM5, pM6, and pM8 hybridized to different size transcripts. At high stringency. (40 nuV sodium phospate rinse-at ,66X), pM3 colony blots hybridized -stronglywitkmouse and

Induction

of hsp RNA and B2 RNA in Chinese hamster cells

65

TABLE 1 Heat shock cDNA clones isolated by diflerential Identification Ubiquitin B2 hsp27 hsp70 hsp89a hsp89B hs@‘J

screening’

Number of isolates

Homologyb

52 34 4 8 7 13 3’ 47

Chinese hamster ubiquitin Chinese hamster B2 Human hsp27 Mouse hsp70 Human hsp89a Human hsp89jI Human hsp60 No homology with any of above

’ 4600 cDNA clones were hybridized with labeled cDNA synthesized from poly(A) RNA of unheated V79 cells or cells heated to 45.5”C for 17 min followed by 5 h at 37°C. The cDNA clones listed above preferentially hybridized with the cDNA derived from HS-treated cells. b cDNA clones were tentatively identified by homology with these known cDNA clones (see Material and Methods). ’ These three isolates were isolated by screening the cDNA library (4600 clones) with labeled pHS601 [171, human hsp60, cDNA. None of the cDNA clones isolated by differential screening had homology with human hsp60.

human hsp70 probes while pM2, pM4, pM5, pM6, and pM8 did not. The identification of the other Chinese hamster hsp cDNA clones of Fig. 1 were contirmed in similar experiments. Forty-seven cDNA clones did not hybridize with any known probe; results with several of these different cDNA are shown also in Fig. 1. As seen in Fig. 1, most of the Chinese hamster cDNA clones isolated by differential screening coded for transcripts which were induced (increased in abundance) in HS-treated cells. Equal amounts of poly(A) RNA (see Materials and Methods) from untreated and heated cells were used in each lane, and the abundance of @-a&in RNA was equivalent. The HS treatment used was found to optimally induce both B2 RNA [9] and various hsp RNA (see below). In most cases, only a single size RNA hybridized with each cDNA probe. The hsp70 probe, pM3, hybridized with a 2.7-kb RNA, which was detectable in unheated cells and induced by HS, and a 3.3-kb RNA which was seen only after HS. Results of ubiquitin will be presented in more detail elsewhere, but are included here for completeness. Briefly, there were two HS-inducible bands of 1.7 and 2.7 kb plus a faint band at 0.8 kb which was not induced. The two major ubiquitin transcripts were both induced 5- to W-fold after HS. The HS-inducible RNA polymerase III transcripts of the B2 repetitive gene varied in length from 0.2 to 0.6 kb. Several hsp-related transcripts were not HZ&inducible. hsp60 RNA was not induced in Chinese hamster cells using either the human or the Chinese hamster cDNA probes. pM2, pM5, pM6, and pM8, which had homology with the mouse hsp70 probe at moderate stringency, hybridized to transcripts which were not H&inducible. In addition to the cDNA clones isolated from our V79 cell library, a CHO cDNA clone, DDZAZS, was found also to code for a HS-inducible RNA. RNA hybridization tit% DDZA15 was much fainter than with the other

66

Fornace et al.

TABLE 2 Induction

of HS-inducible

transcripts

in V79 cells by difierent

cell treatmentsa

cDNA probe M2 Cell treatment’

45.5oc, 9 mill’ Ethanol (6 %) 45S”C, 9 mind MMS (100 mg/ml) Hz02 (400 WI

54 23 30 2.1 1.2

0.9 0.9 1.5 1.2 1.3

9.8 4.4 4.1 2.3 1.2

1.8 1.5 2.5 1.7 1.2

72 25 10.8 2.1 0.5

1.0 1.4 0.9 0.8 0.9

M4 M5 M6 MS (hsp70-homology)

7.7 1.9 4.0 1.4 4.6 1.2 2.5 1.0 0.7 0.4

1.2 1.7 1.0 1.0 1.0

0.4 0.9 1.0 1.0

’ Cells were harvested 4 h after heat shock or the addition of chemical agents; relative abundance was determined by RNA dot-blot hybridization and was normalized to untreated cells isolated at the same time. b The treatment time was 1 h for H202 and ethanol, and 4 h for methylmethane sulfonate (MMS). ’ The first two samples were prepared at the same time as those of Fig. 2A; whole-cell RNA was used. d The last three samples were prepared on the same day; poly(A) RNA was used.

probes which probably indicates that the DDIAlS dance.

transcript was in lower abun-

The induction of various transcripts by HS in V79 cells was quantified by dot blot hybridization (Fig. 2). These results represent four (panels A-D, E-H, Z-L, and M-P) independent experiments done on different days. In the experiment of panels A to D, cells were heated to 455°C for the indicated times and then incubated at 37°C for 4 h to allow sufficient time for the induced RNA to accumulate in the cells. For all the HZ&inducible transcripts, maximum induction was always seen with the 9-min HS treatment in Fig. 2 and similar experiments which are not shown. The magnitude of induction varied from over 90-fold (for pH105) to 2-fold for pH40. With a more mild HS (5 min at 45.5”C), induction was usually substantially less; a striking exception was observed with pH8 (hsp27) where induction was nearly the same after either HS dose (Sl-fold vs 54-fold). In panels E-H, the time course for induction with the 9-min145.5”C HS dose demonstrates that the maximal levels were reached 2 to 4 h after heating. By 8 h, the level of most HZ&inducible transcripts decreased and approached that of unheated cells by 16 h. hsp27 RNA was again an exception; in all three time course experiments, the increase in hsp27 RNA abundance was more prolonged after HS than the other HS transcripts. In panels Z-L, the HS dose was increased to 17min at 45.5”C and induction (as measured by increased RNA abundance) was clearly delayed for most transcripts until 12 h after HS. The only exception was for B2 RNA where induction was not delayed at the earliest time point of 4 h in panel H and 4 h in panel C. With this more severe HS exposure, induction was more prolonged; e.g., the levels of many RNA were still elevated after 24 h while most Dose response and kinetics

of induction

of HS-inducible

transcripts.

Induction

67

of hsp RNA and B2 RNA in Chinese hamster cells

Table 2-Continued cDNA probe

Cell treatment’ 45.5”C, 9 mill’ Ethanol (6%) 45.5”C, 9 mind MMS (1OOmghnl) H202 (400 PW

H37 HSllA (hi(B2) quitin) 7.5 3.2 9.6 3.9 1.6

12.4 4.4 6.9 1.1 1.2

H74

H75

H104

HlO5

H122

H129

15.8 5.3

6.2 2.7 2.9 1.3 1.2

8.2 4.2 4.2 2.2 0.5

92 37

7.9 5.1 5.2 3.0 0.7

24 10.2 14.8 2.3 1.1

Al5

2.8 2.9 1.6

A2 @-actin)

0.6 1.1 1.1 1.5 1.2

had returned nearly to untreated levels by 16 h after the 9-min/45.X dose. This most severe HS treatment decreased induction substantially for H62, H74, H75, H104, HlOS, H122, H129, and M4 transcripts compared to milder HS treatments. For several transcripts, particularly H104, H122, and M4, hardly any induction occurred at any time in panel J. In contrast, maximum induction was similar for M3 (hsp70), HS18A (B2), and H37 (ubiquitin) after 17 min compared to 9 min at 45.5”C. In many studies with mammalian cells, a 42°C HS exposure has been used. As seen in panels M-P, a l-h exposure at this temperature resulted in induction similar to that seen with the 45.5”C/9-min treatment. Maximum induction occurred 2 to 4 h after the start of heating and, for most, returned to control by 13h. Maximum induction in the four experiments of Fig. 2 varied particularly for some transcripts after the most severe HS treatment described above, but there were also examples where induction was similar. For example, the maximum for pH8 (hsp27) RNA was always more than 20-fold; with the same treatment (4 h after 9 min at 45.5’C) pH8 RNA increased 54- or 22-fold in panels A and E, respectively, and 30- or 33-fold in Tables 2 and 3. No evidence of induction was seen using the cDNA probes pH162 (hsp60), pM2, pM5, pM6, pM8, or pA2 @-actin) in Figs. 1 and 2 (all data not shown). In vitro transcription analysis. To demonstrate Ehat induction was due to increased transcription, nuclear runoff determinations were performed with representative cDNA clones in Fig. 3. Compared to the results for /Lactin, transcription was lower for the other RNA in nuclei from unheated cells, although this is only a qualitative estimate since the length of the cDNA inserts varied and less /Iactin DNA was used. After HS, transcription was markedly increased for all the HZ&inducible RNA. Under conditions where the filter bound DNA is in excess, hybridization of labeled RNA should be proportional to the initial concentration of RNA [ 191, and thus the ratio of the relative abundance of induced RNA compared to control should be proportional to the hybridization signal in the HS (induced) compared to the control runoff experiments (where the same amount of DNA was dotted on the filter). By densitometry measurements after various autoradiograph exposure times, hybridization was consistently more than 40-fold

68

Fornace et al.

60

6

4

"0

6

12

DOSE (min)

li

ow

16

TIME (hr)

II 0

12

24

TIME (hr)

36

46

I 0

I 4

6

12

TIME (hr)

Fig. 2. Dose-response and time course for induction of various transcripts by HS in V79 cells. Induction, as measured by the relative RNA abundance normalized to unheated samples prepared on the same day, was determined by dot blot hybridization using various cDNA probes. Equal amounts of whole cell RNA (see Materials and Methods) were used. In A-D, CHO cells were heated to 455°C for the indicated times and then incubated at 37°C for 4 h. In E-H, cells were heated to 45.X! for 9 min and then incubated at 37°C for the indicated times; the earliest time point represents cells which were harvested immediately after heat shock. In I-L, cells were heated to 45.5”C for 17 mitt and then incubated at 37°C for the indicated times. In M-P, cells were heated to 42T for 1 h then incubated at 37°C. In GP, the time axis represents the time from the start of heating; the 0 h values represent untreated samples. In the first row (panels A, E, I, M) cDNA probes are designated: 0, pH8 (hsp27); 0, pM3 (hsp70); 0, pH105; n , pH129. In the second row (panels B, F, J, N) probes are n , pH74; 0, pH104; 0, pH122; c1, pM4. In the third row (panels C, G, K, 0) probes are 0, pHS18A (B2); 0, pH37 (ubiquitin); 0, pH62 (hsp89o); A, pH75; M, pM5. In the fourth row (panels D, H, L, P) probes are n , pH40 (hsp898); A, pH162 (hsp60); 0, pM2; 0, pM6; 0, pM8.

transcripts cDNA probe

in CHO cells by dlperent

33 1.3 1.5 1.8 10.0 0.8 2.8 5.4 2.1 0.9 1.1 1.5 1.1 1.5 1.1 1.2 1.3 1.8 0.9 0.8 1.9 1.0 0.4 0.6

1.0

0.7 0.8 1.0 1.0 1.1 0.8 0.8 1.0

1.1 1.7 1.0 1.0 1.2

5.2 1.3 1.6 2.3 3.1 0.6 1.4 1.7 1.2 0.9 1.1 1.2 1.1 1.3 1.0 1.1 0.9

1.7

1.2 0.4 0.6

2.4 1.4 1.5 1.9 1.9 0.9 0.9 1.0 0.6 0.9 1.1 1.4 1.0 1.2 1.1 1.0 1.0

26 1.0 1.0 1.3 1.0 1.5 2.5 1.7 3.0 1.5 0.3 1.3 0.4 1.1 0.4 1.0 0.9 1.0 0.9 0.9 1.0 0.9 1.1 A.2 1.0 1.0 1.0 1.4 0.7 1.0 0.5 1.1 0.7 0.9 0.9 0.9 0.9 0.2 1.3
7.0 1.1 0.8 2.6 3.8 0.2 0.3 0.4 0.3 0.7 1.0 1.1 0.9 1.0 0.6 0.3 0.6 0.8 0.7 1.0

0.5 1.3 1.6 1.6 1.6 0.7 0.7 0.7 0.7 0.9 1.0 1.0 1.1 1.2 0.9 1.0 0.8

20 i.3 1.5 4.9 8.7 1.7 1.2 1.5 1.2 1.1 1.6 2.4 1.1 2.0 1.1 1.1 1.3 0.9 1.2 1.2 1.1 1.2 1.1 1.2

1.2 1.7 1.0 1.1 1.0 1.1 1.2

1.0 1.1

1.2

2.4 2.0

0.7 0.2 0.3

1.0

0.6

1.1 1.1 0.4 1.3

1.0 0.9

0.8 1.1 1.0 1.0

0.7 1.3 1.5 1.3 1.1 0.9 1.0 1.2 0.8 0.9

A2 @-actin)

(DDP), nitrogen (MNNG).

0.8

0.7 1.0 1.5

1.2

0.9 1.4 0.8 2.9

2.3

1.0

0.5

1.2

1.3

0.9 1.1 1.2

1.1 0.8

1.3

1.0 1.1

7.2 4.6

4.7 1.4 2.3 2.4 5.5 1.0 1.3

Al5

0.7 0.4 0.5

0.9 1.1 0.7

1.0 0.9 1.0 1.4

0.6 0.9 0.6

0.9

2.7 0.8 0.8 0.5

1.1 1.0

0.9

0.9 0.7

0.2 0.3 0.2

0.9 0.9 0.7

1.0 1.4 0.8 1.1

0.9 1.1

2.4 2.2 5.3 3.6 0.9 0.2

3.9 2.8 0.2

1.0 1.2

1.0 0.9

H129

6.2 >lOO

>lOO 1.3 1.5

H104

1.2 1.4 1.5 1.3 0.8 1.8 1.6

3.6

H75

6.2

H74

cell treatments’

’ Cells were treated as in Table 2 and relative abundance was determined as in Table 2 using poly(A) RNA b The treatment time was 1 h for H202 and adriamycin, and 4 h for methylmethane sulfonate (MMS), cis-Pt(II) diamminedichloride mustard (HNZ), 12-O-tetradecanoylphorboL13acetate (TPA), IV-acetoxy-2-acetylaminofluorene (AAAP), and N-methyl-N’nitrosoguanidine ’ Cells were grown to higb density and then incubated for 2 days without medium change. d Cells were incubated in medium containing reduced serum, 0.5%, for 1 day.

45.X 9 mill MMS (6 &ml) (25 ccg/ml) (100 c1B/ml) (200PlJm H202 (400 M.0 254 nm uv radiation (14 Jm-*) 320 nm uv (300 Jm-*) AAAF (20 pM) DDP (3 &nl) (15 wm (45 ida HN2 (8 p&f) w Pm MNNG (10 @f) (30 Pm Adriamycin (0.4 )&ml) X-rays (40 Gy) TPA (20 rig/ml) Go phase, depleted medium’ Go phase, 0.5% serumd

Cell treatmentb

of HS-inducible

3

M3 M2 M4 M6 H37 HSllA H8 H162 H62 H40 (hsp27) (hsp60) (hsp89a) (hsp89/9) (hsp70) (hsp70(ubiB2 homology) quitin)

Induction

TABLE

$

52

3

3 2

2’ g g

3

E 5’

% q z

E

2 R, fi a 0 : cc, $ a

70

Fornace et al.

1

2

3

4

5

6

7

8

9 Control

HS Fig. 3. Nuclear runoff transcription assay. Replicate DNA dot blots were hybridized with labeled RNA synthesized from isolated nuclei of either untreated cells (designated “Control”), or cells heated to 455°C for 9 min and incubated at 37°C for 1 h (designated “HS”). In lane 1, pM3 (hsp70) plasmid DNA was dotted; lane 2, pM4; lane 3, pH8 (hsp27); lane 4, pH40 (hsp89b); lane 5, pH37 (ubiquitin); lane 6 pH74; lane 7, pH122; lane 8, pA2 @ktctin); and lane 9, pBR322 (plasmid vector alone). Two micrograms of plasmid DNA was used for all except pA2 (0.4 ug).

greater for the dot blots of lanes 1 to 7 in the HS compared to the control samples, which indicates that transcription of these genes was increased more than 40-fold. In contrast, transcription of /3-actin decreased somewhat after HS; the same result was obtained also using a chicken #I-actin clone except the signal was much less (data not shown). The assertion that induction was due to increased transcription was supported also by the observation that no increase in RNA abundance after HS was seen in the presence of actinomycin D using any of the cDNA probes of Fig. 2 (data not shown). Induction of HS-inducible RNA by various stresses. V79 and CHO cells were treated with a variety of different agents, and the relative abundance of transcripts coded for by our different cDNA clones was determined 4 h after the start of treatment in Tables 2 and 3. Exposure to the HS-mimetic agent ethanol induced ail the HS-inducible RNA; induction was consistently one-third to onehalf that produced by the optimal dose of HS, 9 min/45S”C. The monofunctional alkylating agent, MMS, was found also to induce many of the same transcripts, but induction was less than that of HS or ethanol in V79 cells with the exception of DDZAlS. Similar results with HS and MMS were found in CHO cells with the exception that pH75 RNA was induced 2.2-fold with the same dose of MMS that was ineffective in V79 cells. At the higher dose of MMS which reduced cell survival to ~0.001 %, induction was greater for many of the RNA. No induction of B2 RNA occurred after MMS or any of the other damaging agents with the exception of the H&mimetic agent ethanol and HN2 (where induction was less than 3-fold). H202 was an ineffective inducing agent for all the H&inducible RNA with the exception DDIAlS; this dose of Hz02 reduced cell survival to 1% and was effective in inducing many other DDZ RNA [7]. Most of the other cell treatments were ineffective inducing agents for all except hsp27, ubiquitin, and DDZAlS. In addition to the results in Table 3, other doses of DDP (3 and 15 ug/ml), HN2 (8 pJ4), MNNG (10 CLM),and TPA (40 to 1500 &ml) were also ineffective. The response of pH8 (hsp27) RNA to DNA damaging agents differed in several

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71

respects from the other hsp transcripts. Induction after the highest dose of MMS was greater than for the other transcripts. In addition, pH8 was the only cDNA clone isolated from our HS library which coded for a transcript induced by uv radiation, near uv radiation, and AAF. These agents share the common characteristic that they produce DNA damage which is repaired by nucleotide excision repair (long patch repair). Expression of H&inducible transcripts in non-growing cells. Since both HS and DNA damaging agents will slow cell growth and at high doses prevent progression through the cell cycle, results with Gophase cells have been included in Table 3. Cell growth was prevented by two types of stress: starvation (depleted medium) or reduced serum. Neither of these treatments inreased any of the HSinducible RNA. In most cases, the RNA levels differed by O. 1% abundance in poly(A) RNA) transcripts, only several hundred of the approximatley 20,000 different mRNA in the typical mammalian cell can be surveyed by this technique (discussed in [14]). When 1500 protein spots (which is still
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Our studies demonstrate a coordinate induction (as measured by increased RNA) by HS and the HS-mimetic agent ethanol for multiple hsp transcripts and other HS-inducible transcripts, including B2. For example, all the HS-inducible transcripts were maximally induced with the same HS treatment in the dose-response experiment (panels A-D) of Fig. 2. The kinetics of induction by HS were similar for many transcripts after heating to either 45 or 42°C. With more severe HS treatments (13 or 17 min at 45°C) induction was delayed for all RNAs except B2. Unlike hsp mRNA which is transcribed by RNA polymerase II, the HSinducible B2 RNA is transcribed by RNA polymerase III since its transcription was shown to be a-amanitin-resistant [9]. Severe heating apparently temporally inhibited RNA polymerase II transcription and processing, while RNA polymerase III dependent transcription of B2 RNA was more resistant. The nuclear runoffs of Fig. 3 indicate that HS markedly increased transcription for all the samples tested. In some cases (i.e., pH40) transcription increased much more than the RNA level; this may be due to differences in RNA stability or other posttranscriptional factors. Transcription of a human hsp70 gene has been shown to be serum and growth responsive [2, 221. Our result indicate that this may be the case for Chinese hamster cells. As seen in Table 3, hsp70 and H104 transcripts were markedly decreased in reduced serum or depleted medium. The same transcripts were decreased also after treatment with some DNA-damaging agents at doses which would be expected to slow cell growth. Other H&inducible transcripts were not markedly affected by these treatments. Several correlations between cellular parameters such as HS-induced toxicity and thermotolerance can be inferred from our results. In the dose-response experiment illustrated in Fig. 2, little cytotoxicity occurred after 5- and Pmin treatment at 45.5”C, while cell survival was reduced to 41 and 18% with the 13and 17-min heating times at 45.5”C respectively [9]. As discussed earlier, in the later cases induction of most HS-inducible RNA was delayed and, in some instances, completely suppressed with heat doses which induced significant cytotoxicity. Maximum induction in the HS dose-response experiment was observed with the highest dose which produced no significant cytotoxicity. This dose, 9 min at 45.5”C, was found to be one of the most effective in inducing thermotolerance (data not shown). Thermotolerance has been reported after exposure to more severe HS treatments [24, 251,which suppressed the induction of some transcripts. If elevated hsp play a role in thermotolerance, one would have to conclude from our results that increased RNA levels for only some hsp are necessary. By protein electrophoresis, Li [25] and others have found that the best correlation between elevated levels of hsp and thermotolerance is with hsp70. However, at the RNA level hsp70 transcripts were coordinately induced with many other HZ&induced RNAs; thus other HS genes or a combination of hsp70 with other hsp may play a role in thermotolerance. Treatment with different DNA damaging agents and other chemical agents led to a varied pattern of induction of HS-inducible transcripts in Chinese hamster cells. Such results were not unexpected given the findings in bacteria and yeast

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73

[ 1, 5, 261. For example, in E. coli 6-amino-7-chloro-5.8dioxoquinoline, HzOz, CdClr, and naldixic acid all induced the SOS regulon and DNA damage; however only 1, 3,5, and 10, respectively, of the 15 htpR HS genes were induced by these agents [26]. In the case of ethanol, which has been shown to be HS-mimetic in many organisms [l], there was a coordinate induction of all our HS-inducible transcripts. However, other chemical agents, with the exception of MMS, did not induce most of the HS-inducible transcripts after a 4 h exposure. The purpose of these studies was to determine if treatment with any of these agents led to induction kinetics similar to those found with HS. Although our studies do not rule a late effect, they clearly demonstrate that most DNA damaging agents do not induce HS genes in a manner similar to HS or ethanol. In rat liver, chronic administration of hepatocarcinogens induced hsp70 and hsp83 slowly [4]; this may have been due to a secondary effect of treatment such as cell injury or inflammation. The only consistent positive effect of DNA damaging agents on HS genes was with 200 pg/ml MMS. This result is in contrast with the induction of 14 DDZ (DNA damage inducible) transcripts including DDIAZS, which were induced maximally at 100 ug/ml MMS [7]. For the HS transcripts, induction at 100 &ml was usually less than at the higher dose. In a second experiment at 100 &ml MMS (data not shown), the induction of HS transcripts was similar to or less than that seen in Table 3 with the same dose, while all of the DDZ transcripts including DDIAIS were induced maximally. Induction by MMS differed from that seen after HS or ethanol in several respects. For example, only hsp27 was strongly @lo-fold) induced by MMS and HS while both hsp27 and others such as hsp70 (M3) and H74 were strongly induced by HS. Another difference was that B2 RNA was not induced by MMS; thus the coordinate induction of RNA polymerase II and III dependent transcripts occurred only with HS and ethanol. In a variety of experiments in both V79 and CHO cells (all data not shown), B2 RNA was not induced by any DNA damaging agent with the exception of 40 w HN2. Unlike many other DNA damaging agents, MMS at the doses used produced substantial damage to cellular proteins, in addition to DNA. In Chinese hamster cells, Roberts et al. [27] found that 110 ug/ml MMS produced approximately 1 alkylation per lo6 Da in both DNA and proteins, while the same dose of MNNG as used in Table 3 produced a similar level of DNA alkylation, but 5- to lo-fold less protein alkylation. Since other DNA damaging agents were generally ineffective in most HS transcripts, the signal for induction of HS transcripts by MMS may not be the DNA damage per se but the damage to other targets such as large proteins or protein complexes. Several chemical agents were found consistently to be ineffective as inducing agents for most HS RNA. TPA was included because Herrlich’s group [28] has shown in human cells that several genes were induced by either TPA or DNA damaging agents. In Chinese hamster cells, we have found that two DDI transcripts were also induced by TPA [7]. In the case of Hz02, this agent is often considered a HS-mimetic agent based in Drosophila [29]. In isolated nuclei 20 mM Hz02 induced hsp chromosome puffing, but more biologically relevant doses were ineffective in r&o. As discussed earlier, H202 did not induce many bacterial

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HS genes. In Chinese hamster cells, Hr02 induced only DDZAZS RNA, which was isolated on the basis of DNA damage inducibihty. These results indicate that Hz02 was not a HS-mimetic agent in our study and that regulation of the DDZAZS gene differed in important respects from other HS genes. There were several important differences between the induction of hsp27 RNA and other HS transcripts. With HS, there was a lower threshold for the maximum induction of hsp27 RNA and a more prolonged induction.- Induction by 200 pg/ml MMS was higher for hsp27 than other hsp transcripts. In addition, hsp27 RNA was induced several-fold by agents at doses which produced high levels of DNA damage which is repaired by nucleotide excision repair. These results indicate that the regulation of the hsp27 gene differs in some respects from other HS genes. We thank Dr. J. B. Mitchell and W. G. DeGrafffor their support of this work, and also Drs. A. Laszlo and T. S. Nowak, Jr., for their valuable comments in reviewing this manuscript.

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28. Angel, P., Poting, A., Mallick, U., Rahmsdorf, H. J., Schorpp, M., and Herrlich, P. (1986) Mol. Cell. Biol. 6, 1760-1766. 29. Compton, J. L., and McCarthy, B. J. (1978) Cell 14, 191-201. Received October 7, 1988 Revised version received December 12, 1988 F’rinted

in Sweden