Transcription of the human hsp 7 O gene is induced by serum stimulation ( heat shock / transcriptional regulation / DNA synthesis / human cells ) BARBARA

We have examined the expression of the heat shock protein (hsp7O) gene in human cells. The transcription of the hsp7O gene and accumulation of cytoplasmic hsp70 mRNA is induced by serum stimulation. Populations of HeLa cells and human embryonic kidney cells (cell line 293) were serum starved. Upon serum stimulation, the level of hsp70 mRNA transiently increases between 12 and 18 hr to a 10-fold higher level. The increased levels ofhsp70 mRNA can be accounted for by a 10to 15-fold increase in the rate of transcription of the hsp7O gene. When cells were serum-stimulated in the presence of an inhibitor of DNA synthesis, 1-p-D-arabinofuranosylcytosine (araC), the levels of hsp70 mRNA were induced to only 20% of the maximal level detected in the absence of the inhibitor. This suggests that the expression of the hsp7O gene is coupled to DNA synthesis. The cloned human hsp7O gene contains regulatory sequences that confer serum-stimulated transcriptional control. The endogenous hsp7O gene and the transfected chimeric gene containing sequences upstream of the hsp7O gene fused to bacterial chloramphenicol acetyltransferase are both temporally expressed in stable transfectants of cell line 293 cells. The endogenous hsp70 mRNA and the chimeric mRNA reach maximum levels 12-18 hr after serum stimulation. The recent discoveries of different functional classes of viral oncogenes and their cellular counterparts, the protooncogenes, have provided insights into the events that lead to cellular transformation. One class of viral oncogenes that does not appear to have cellular analogs is the transforming genes of DNA tumor viruses. The process of DNA tumor virus-initiated cellular transformation can be revealed by identifying cellular genes whose expression is directly affected by viral transforming gene products. The transforming gene products of adenovirus serotype 5 (AdS) ElA and polyoma large T antigen have been implicated in the immortalization of primary cells in culture (1-6). The mechanism(s) by which primary cells become immortalized is not understood. Yet, it would seem likely that some of the events are mediated through altered expression of cellular genes. Indeed, adenovirus ElA has been shown to be both a positive transcription activator (7-11) and a repressor (12, 13) of viral and cellular gene expression. One cellular gene that is transcriptionally activated by two different oncogene products, adenovirus ElA (14, 15) and mouse rearranged c-myc (16), is the gene that encodes the major 70,000-Da heat shock protein (hsp70). We have isolated a human hsp70 gene that is induced early in Ad5 infection ofHeLa cells and is constitutively expressed in AdS-transformed human embryonic kidney cells (cell line 293 cells) (17). The high levels of hsp70 mRNA in cell line 293 cells was expected because these cells express the AdS EJA gene (18), whose product has been shown to induce the synthesis of hsp70. HeLa cells also express hsp7O at low but detectable levels (15, 17). The constitutive synthesis of hsp70 mRNA in HeLa and cell line 293 cells maintained at normal growth temperatures suggests that the expression of the hsp70 gene can be regulated by mechanisms independent of heat shock. In this study, we show that transcription of the hsp70 gene and levels of hsp70 mRNA are induced by serum stimulation in two human cell lines, HeLa and 293 cells. The increase in hsp70 mRNA after serum stimulation is blocked by 1-p-Darabinofuranosylcytosine (araC), an inhibitor of DNA synthesis. The DNA sequences that respond to serum-stimulated expression reside in the 5' upstream regions. A chimeric gene, containing the heat shock promoter fused to the bacterial gene encoding chloramphenicol acetyltrarisferase, retains the serum-stimulated expression observed for the endogenous hsp70 gene. MATERIALS AND METHODS Cells and Plasmids. HeLa and 293 cells (from P. Sharp, Massachusetts Institute of Technology) were grown in Dulbecco's modified Eagle's medium (DME medium) with 5% fetal calf serum at 370C. Plasmid pH2.3 [a 2.3-kilobase (kb) BamHI/HindIII fragment] contains the entire hsp70 coding region (17). Plasmid pHBCAT, constructed by R. Kingston, contains 2.4 kb of 5' sequences of the human hsp70 gene fused with the bacterial chloramphenicol acetyltransferase (CAT) gene (17). Plasmid pSV2-ECOGPT contains the bacterial xanthine/guanine phosphoribosyltransferase gene (19). Plasmid pJOL contains 0-7.8 map units of Ad5 (20). Isolation of RNA, S1 Nuclease Protection, and Run-on Transcription. Cytoplasmic RNAs were isolated and the levels of hsp70 mRNA were determined by S1 nuclease protection using plasmid pH2.3 3'-end-labeled at the BamHI site (17). The S1 nuclease-protected fragments were analyzed by alkaline/agarose or polyacrylamide gel electrophoresis. The relative amounts of hsp70 mRNA were established by quantitation of densitometric scans of the autoradiographic exposures or by liquid scintillation counting of the excised S1 nuclease-protected fragments. Nuclei were isolated from cells in 10 mM Tris'HCl, pH 7.4/10 mM NaCl/3 mM MgCl2/0.5% Nonidet P-40. The nuclear pellet was frozen in an equal vol of 40% glycerol/50 mM Tris HCl, pH 8.3/5 mM MgCl2/0.1 mM EDTA at 2 X 109 nuclei per ml. [32P]UTP-labeled nascent transcripts were prepared by in vitro runoff transcription using isolated nuclei and were hybridized to plasmid DNAs (21). Serum Stimulation. HeLa and 293 cells were plated in DME medium containing 5% fetal calf serum at 10% confluence and Abbreviations: hsp, heat shock protein; araC, 1-,8-D-arabinofuranosylcytosine; AdS, adenovirus serotype 5; kb, kilobase(s); CAT, chloramphenicol acetyltransferase. *To whom reprint requests should be addressed at: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60201. 6070 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 82 (1985) 6071 grown to 80% confluence. The cells were washed with warmed phosphate-buffered saline, incubated for 48 hr in DME medium without serum, and stimulated with fresh medium containing 20% fetal calf serum. At the indicated times after stimulation, the cells were processed and nuclei and cytoplasmic RNA were isolated. Suspension cultures of 293 cells were maintained at 2 x 105 cells per ml in Joklik's medium containing 5% fetal calf serum. For serum starvation, the cells were grown to a density of 6 X 105 cells per ml, collected by centrifugation, and returned to Joklik's medium containing 0.5% fetal calf serum for 72 hr. The cells were stimulated by addition of 20% fetal calf serum. At the indicated times, 1-ml aliquots were labeled with [3H]thymidine (2 ACi/ml; 1 Ci = 37 GBq), lysed in 0.1 M NaCl/10 mM Tris HCl, pH 7.4/1 mM EDTA/0.5% NaDodSO4, diluted 1:3.5 with H20 and brought to a final concentration of 10% (vol/vol) trichloroacetic acid. DNA Transfection and Colony Selection. Cell line 293 cells were cotransfected with 10 ,tg each of pHBCAT and pSV2ECOGPT. Cells were plated at a density of 106 cells per 10-cm2 dish and were incubated for 24 hr with DNA-calcium phosphate coprecipitates (22). Cells were washed twice with warmed phosphate-buffered saline, and fresh medium was added. After 2 days, the cells were trypsinized and plated at i05 cells per 10-cm2 dish and incubated in DME medium containing 25 ,ug of mycophenolic acid per ml (Eli Lilly), 250 ,ug of xanthine per ml, 10 ,ug of thymidine per ml, 25 ,g of hypoxanthine per ml, 2 ,g of aminopterin per ml, 150 ,ug of L-glutamine per ml, and 5% fetal calf serum (19). Approximately 10-20 colonies per dish were visible by day 10, and individual clones were isolated and expanded.