The Response of Renal Tubular Epithelial Cells to Physiologically and Chemically Induced Growth Arrest

Cells respond to a variety of stresses by activating the transcription of a battery of “acute phase” or “stress response” genes. The nature of this response is tailored to the nature of the stress. The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear. We therefore compared the effects of a physiologically induced (serum and nutrient depletion) and a chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) stress in renal tubular epithelial cells (LLC-PK1). The response to physiological stress, induced by serum depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA, one of the growth arrest and DNA damage inducible genes. In contrast, the chemical-induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153 Chemical-induced changes in histone mRNA inversely correlate to changes in the expression of a stress gene, hsp70, whose expression is dependent upon the maintenance of appropriate nucleosomal structure. Cells respond to a variety of stresses by activating the transcription of a battery of “acute phase” or “stress response” genes. The nature of this response is tailored to the nature of the stress. The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear. We therefore compared the effects of a physiologically induced (serum and nutrient depletion) and a chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) stress in renal tubular epithelial cells (LLC-PK1). The response to physiological stress, induced by serum depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA, one of the growth arrest and DNA damage inducible genes. In contrast, the chemical-induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153 Chemical-induced changes in histone mRNA inversely correlate to changes in the expression of a stress gene, hsp70, whose expression is dependent upon the maintenance of appropriate nucleosomal structure. INTRODUCTIONCells respond to a variety of stresses by activating the transcription of a battery of “acute phase” or “stress response” genes (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4341) Google Scholar, 2Morimoto R.I. Tissieres A. Georgopoulus C. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MA1990Google Scholar). The nature of this response is tailored to the nature of the stress. For example, serum- and nutrient-deprived cells usually enter a quiescent state (G0) that requires both the up-regulation and down-regulation of genes involved in growth control. In contrast, the initial response to DNA-damaging agents includes arrest of cell cycle progression, presumably to facilitate the transcription of a battery of genes involved in the DNA repair process (3Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (644) Google Scholar) prior to DNA replication (G1 arrest) and cell division (G2 arrest). The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear and is the focus of the present studies.The conjugation of ortho-, or para-quinones with GSH results in the formation of conjugates that frequently exhibit more potent toxicological activity than the parent quinone (4Monks T.J. Lau S.S. CRC Crit. Rev. Toxicol. 1992; 22: 243-270Crossref Scopus (124) Google Scholar). GSH conjugates of polyphenolics are also formed as metabolites of a variety of “non-genotoxic” carcinogens (5Hill B.A. Kleiner H.E. Ryan E.A. Dulik D.M. Monks T.J. Lau S.S. Chem. Res. Toxicol. 1993; 6: 459-469Crossref PubMed Scopus (57) Google Scholar, 6Peters M.M. Lau S.S. Dulik D.M. Murphy D. van Ommen B. van Bladeren P.J. Monks T.J. Chem. Res. Toxicol. 1996; 9: 133-139Crossref PubMed Scopus (33) Google Scholar, 7Butterworth M. Lau S.S. Monks T.J. Chem. Res. Toxicol. 1996; 9: 793-799Crossref PubMed Scopus (51) Google Scholar). As a model of quinone-thioether-mediated toxicity we have been investigating the cellular and molecular responses to 2-Br-bis-(glutathion-S-yl)hydroquinone (2-Br-bis-(GSyl)HQ) 1The abbreviations used are: 2-Br-bis-(GSyl)HQ2-bromo-bis-(glutathion-S-yl)hydroquinone2-Br-6-(GSyl)HQ2-bromo-6-(glutathion-S-yl) hydroquinoneDMEMDulbecco's modified Eagle's mediumEBSSEarl's balanced salt solutionEGTA-AMethylene glycol-bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acidrRNAribosomal RNA. and 2-Br-6-(glutathion-S-yl)hydroquinone (2-Br-6-(GSyl)HQ). 2-Br-bis-(GSyl)HQ (30 μmol/kg) causes margination of heterochromatin and loss of chromatin staining when administered to male Fischer 344 rats (8Rivera M.I. Jones T.W. Lau S.S. Monks T.J. Toxicol. Appl. Pharmacol. 1994; 128: 239-250Crossref PubMed Scopus (17) Google Scholar), and in renal tubular epithelial cells (LLCPK1) it causes the formation of single strand breaks in DNA (9Mertens J.J.W.M. Gibson N.W. Lau S.S. Monks T.J. Arch. Biochem. Biophys. 1995; 320: 51-58Crossref PubMed Scopus (42) Google Scholar), rapid inhibition of DNA synthesis (10Jeong J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar), and the induction of the growth arrest and DNA Damage-inducible gene, gadd153 (10Jeong J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar). Quinone-thioethers therefore provide a useful model with which to investigate chemically induced growth arrest and the consequences of chemically induced chromatin disruption. We have therefore compared physiologically induced (serum and nutrient depletion) and chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) growth arrest in renal tubular epithelial cells (LLC-PK1) and determined some of the factors involved in regulating gene expression in response to the chemically induced stress. We report that the response to physiological stress (serum deprivation) involves growth arrest characterized by an inhibition of DNA synthesis in the absence of changes in histone or gadd153 mRNA. In contrast, the chemically induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2A and H2B mRNA, and the induction of gadd153DISCUSSIONDNA synthesis is rapidly inhibited in serum-deprived subconfluent cultures of renal proximal tubular epithelial cells (LLC-PK1) (Fig. 1), but this physiological mode of growth arrest is insufficient to induce gadd153 expression (Fig. 2). However, removal of serum and nutrients, including glucose, from the culture medium is sufficient to induce the expression of gadd153 (Fig. 2). Chen et al (16Chen Q. Yu K. Holbrook N.J. Stevens J.L. J. Biol. Chem. 1992; 267: 8207-8212Abstract Full Text PDF PubMed Google Scholar) also noted that culturing LLC-PK1 cells in EBSS induces the expression of gadd153 The addition of glucose to serum- and nutrient-deprived LLC-PK1 cells did not overcome the induction of gadd153 (Fig. 1). Glucose is not essential for energy production in cultured cells (17Wice B.M. Reitzer L.J. Kennel D. J. Biol. Chem. 1981; 256: 7812-7819Abstract Full Text PDF PubMed Google Scholar), and glutamine, not glucose, is the major energy source in HeLa cells (18Reitzer L.J. Wice B.M. Kennel D. J. Biol. Chem. 1979; 254: 2669-2676Abstract Full Text PDF PubMed Google Scholar). The mitochondrial glutamine to α-ketoglutarate pathway is also the major regulatory site of ammoniagenesis in LLC-PK1 cells (19Cole L.A. Scheid J.M. Tannen R.L. Am. J. Physiol. 1986; 251: C293-C298Crossref PubMed Google Scholar). Increased expression of gadd153 occurs in H4-II-E rat hepatoma cells grown in a limited amino acid medium (20Marten N.W. Burke E.J. Hayden J.M. Straus D.S. FASEB J. 1994; 8: 538-544Crossref PubMed Scopus (114) Google Scholar). Glutamine can also serve as a precursor for pyrimidine nucleotides, via the glutamate-aspartate transamination reaction, and cells grow readily in sugar-free medium supplemented with uridine or cytidine (17Wice B.M. Reitzer L.J. Kennel D. J. Biol. Chem. 1981; 256: 7812-7819Abstract Full Text PDF PubMed Google Scholar). The absence of glutamine, rather than glucose, probably initiates gadd153 expression in LLC-PK1 cells, and changes in the nucleotide pool may be a signal for gadd153 expression. Gadd153 is induced by growth arrest and DNA Damage (3Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (644) Google Scholar), and the nucleotide pool will also decline as DNA repair is initiated in cells experiencing DNA damage, again signaling gadd153 expression. The p53-dependent G0-G1 cell cycle arrest is induced by ribonucleotide depletion (21Linke S.P. Clarkin K.C. Leonardo A.D. Tsou A. Wahl G.M. Genes & Dev. 1996; 10: 934-947Crossref PubMed Scopus (478) Google Scholar), suggesting that p53 may serve as a ribonucleotide sensor. However, although p53 lies upstream of gadd45 (22Zhan Q. Bae I. Kastan M.B. Fornace Jr., A.J. Cancer Res. 1994; 54: 2755-2760PubMed Google Scholar), it does not appear to control gadd153 expression (23Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1994; 71: 587-597Abstract Full Text PDF Scopus (2923) Google Scholar).The expression of histone genes is tightly coupled to DNA replication, but the levels of histone mRNA were unchanged in both serum-, and nutrient-deprived LLC-PK1 cells (Fig. 3), despite significant reductions in DNA synthesis (Fig. 2). Although changes in the level of histone mRNA occur between G1 and S phase cells, the signal for rapid histone mRNA degradation appears to be confined to the S-G2 border (24Harris M.E. Bohni R. Schneiderman M.H. Ramamurthy L. Schumperli D. Marzluff W.F. Mol. Cell. Biol. 1991; 11: 2416-2424Crossref PubMed Scopus (186) Google Scholar, 25Morris T.D. Weber L.A. Hickey E. Stein G.S. Stein J.L. Mol. Cell. Biol. 1991; 11: 533-544Crossref PubMed Scopus (48) Google Scholar), and histone mRNA levels only decline rapidly as cells exit the S phase of the cell cycle. Moreover, the half-life of histone mRNA is comparatively long during S phase, ranging from 110 min (25Morris T.D. Weber L.A. Hickey E. Stein G.S. Stein J.L. Mol. Cell. Biol. 1991; 11: 533-544Crossref PubMed Scopus (48) Google Scholar) to 4-5 h (26DeLisle A.J. Graves R.A. Marzluff W.F. Johnson L.F. Mol. Cell. Biol. 1983; 3: 1920-1929Crossref PubMed Scopus (107) Google Scholar).In contrast to the lack of effects of serum and nutrient deprivation on gadd153 and histone mRNA, chemical-induced growth arrest of LLC-PK1 cells causes a decrease in both core and linker histone mRNAs (Figs. 4, 5, 6) and an increase in gadd153 mRNA (Fig. 8, Fig. 9). Differences in the cellular response to nutrient deprivation and chemical challenge might simply reflect different points on a continuum of a single stress response. We therefore compared the effects of a chemical stress (2-Br-6-(GSyl)HQ exposure in DMEM) and the combination of chemical stress under conditions of nutrient depletion (2-Br-6-[GSyl]HQ exposure in EBSS). However, not only are the two stresses not additive, but decreases in H2B expression are attenuated when the chemical and nutrient stress are combined (Fig. 7). In addition, nutrient deprivation for 2 h causes a 6-fold increase in gadd153 mRNA, and the combination of nutrient deprivation and 2-Br-6-(GSyl)HQ actually decreases the gadd153 response (Fig. 8). The ability of 2-Br-6-(GSyl)HQ to attenuate the gadd153 response is not simply a consequence of a delay in the stress response. Nutrient depletion causes a continual increase in gadd153 mRNA, whereas the combination of nutrient depletion and 2-Br-6-(GSyl)HQ blunts the gadd153 response by 60% (Fig. 9). The chemical challenge, therefore, appears to interfere with pathways utilized by the cell to signal the physiological stress to the nucleus. Whether the same, albeit compromised, pathways are utilized to signal the chemical stress response is unclear, but likely involves the recruitment of alternative signaling pathways. Nonetheless, the genomic response to nutrient depletion and chemically induced growth arrest in LLC-PK1 cells is clearly not additive.Chromatin structure plays an important role in regulating gene expression (27Felsenfeld G. Nature. 1992; 355: 219-224Crossref PubMed Scopus (713) Google Scholar, 28van Holde K.E. Lohr D.E. Robert C.R. J. Biol. Chem. 1992; 267: 2837-2840Abstract Full Text PDF PubMed Google Scholar, 29Stein G.S. van Wijen A.J. Stein J. Lian J.B. Montecino M. Int. Rev. Cytol. 1995; 162A: 251-278PubMed Google Scholar, 30Wolffe A.P. Regulation of Chromatin Structure and Function. R. G. Landes Co., Austin, TX1994Google Scholar). For example, histones play an important role in maintaining chromatin structure and are intimately involved in the regulation of gene transcription (31Grunstein M. Sci. Am. 1992; 267: 68-74Crossref PubMed Scopus (69) Google Scholar, 32Wolffe A.P. Cell. 1994; 77: 13-16Abstract Full Text PDF PubMed Scopus (225) Google Scholar). The molecular mechanisms regulating gene expression in eukaryotes, therefore, include specific roles for the histone proteins. For example, when core histone H2A or H2B synthesis is repressed, the promoter elements of several inducible genes, which are normally repressed because of their incorporation into nucleosomes, become accessible to the basal transcriptional machinery (33Hayes J.J. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1229-1233Crossref PubMed Scopus (100) Google Scholar, 34Tremethick D. Zucker K. Worcel A. J. Biol. Chem. 1990; 265: 5014-5023Abstract Full Text PDF PubMed Google Scholar). Histones are therefore essential for nucleosomal structure and function. Interestingly, core histone H2A and H2B mRNAs are more sensitive to quinone-thioether-induced DNA damage than core histone H3 and H4 mRNA (Fig. 5, Fig. 6). Because the H3/H4 histone tetramer forms the central core of the nucleosome, with H2A/H2B heterodimers capping each end of the core, H2A/H2B dimers are more readily displaced from the nucleosomal core than H3 and H4 (35Jackson V. Biochemistry. 1990; 29: 719-731Crossref PubMed Scopus (209) Google Scholar). Histone mRNAs are rapidly degraded via the activation of a specific mRNA destabilization process (36Marzluff W.F. Hanson R.J. Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, Inc., San Diego1993: 267-290Crossref Google Scholar) that is autoregulated by histone proteins and initiated by increases in cytoplasmic histones (37Peltz S.W. Ross J. Mol. Cell. Biol. 1987; 7: 4345-4356Crossref PubMed Scopus (58) Google Scholar). Both core and linker histones induce the destabilization of histone H4 mRNA (38McLaren R.S. Ross J. J. Biol. Chem. 1993; 268: 14637-14644Abstract Full Text PDF PubMed Google Scholar). 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Res. 1993; PubMed Scopus Google Scholar). or by synthesis and ribonucleotide reductase cell cycle and histone gene expression. H2B histone mRNA levels in cells were by with 2-Br-6-(GSyl)HQ (Fig. which is with the ability of to DNA damage (10Jeong J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar) and (9Mertens J.J.W.M. Gibson N.W. Lau S.S. Monks T.J. Arch. Biochem. Biophys. 1995; 320: 51-58Crossref PubMed Scopus (42) Google Scholar, J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar). also plays an important role in cell and several cell cycle including the phase the phase and exit from J. Physiol. 1996; PubMed Scopus Google Scholar). is also for histone activity M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). will delay cell cycle progression, the for histone synthesis as DNA synthesis is of in LLC-PK1 cells with decreases the expression of histone H2B mRNA and decreases in H2B mRNA (Fig. Although the between and histone gene expression is likely to be a between the renal tubular epithelial cells respond to physiologically and chemically induced stress. The response to physiological stress, induced by a combination of serum and nutrient depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA. In contrast, a chemical-induced stress by exposure of cells to involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153 Chemical-induced expression of mRNA appears to be to decreases in histone mRNA. INTRODUCTIONCells respond to a variety of stresses by activating the transcription of a battery of “acute phase” or “stress response” genes (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4341) Google Scholar, 2Morimoto R.I. Tissieres A. Georgopoulus C. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MA1990Google Scholar). The nature of this response is tailored to the nature of the stress. For example, serum- and nutrient-deprived cells usually enter a quiescent state (G0) that requires both the up-regulation and down-regulation of genes involved in growth control. In contrast, the initial response to DNA-damaging agents includes arrest of cell cycle progression, presumably to facilitate the transcription of a battery of genes involved in the DNA repair process (3Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (644) Google Scholar) prior to DNA replication (G1 arrest) and cell division (G2 arrest). The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear and is the focus of the present studies.The conjugation of ortho-, or para-quinones with GSH results in the formation of conjugates that frequently exhibit more potent toxicological activity than the parent quinone (4Monks T.J. Lau S.S. CRC Crit. Rev. Toxicol. 1992; 22: 243-270Crossref Scopus (124) Google Scholar). GSH conjugates of polyphenolics are also formed as metabolites of a variety of “non-genotoxic” carcinogens (5Hill B.A. Kleiner H.E. Ryan E.A. Dulik D.M. Monks T.J. Lau S.S. Chem. Res. Toxicol. 1993; 6: 459-469Crossref PubMed Scopus (57) Google Scholar, 6Peters M.M. Lau S.S. Dulik D.M. Murphy D. van Ommen B. van Bladeren P.J. Monks T.J. Chem. Res. Toxicol. 1996; 9: 133-139Crossref PubMed Scopus (33) Google Scholar, 7Butterworth M. Lau S.S. Monks T.J. Chem. Res. Toxicol. 1996; 9: 793-799Crossref PubMed Scopus (51) Google Scholar). As a model of quinone-thioether-mediated toxicity we have been investigating the cellular and molecular responses to 2-Br-bis-(glutathion-S-yl)hydroquinone (2-Br-bis-(GSyl)HQ) 1The abbreviations used are: 2-Br-bis-(GSyl)HQ2-bromo-bis-(glutathion-S-yl)hydroquinone2-Br-6-(GSyl)HQ2-bromo-6-(glutathion-S-yl) hydroquinoneDMEMDulbecco's modified Eagle's mediumEBSSEarl's balanced salt solutionEGTA-AMethylene glycol-bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acidrRNAribosomal RNA. and 2-Br-6-(glutathion-S-yl)hydroquinone (2-Br-6-(GSyl)HQ). 2-Br-bis-(GSyl)HQ (30 μmol/kg) causes margination of heterochromatin and loss of chromatin staining when administered to male Fischer 344 rats (8Rivera M.I. Jones T.W. Lau S.S. Monks T.J. Toxicol. Appl. Pharmacol. 1994; 128: 239-250Crossref PubMed Scopus (17) Google Scholar), and in renal tubular epithelial cells (LLCPK1) it causes the formation of single strand breaks in DNA (9Mertens J.J.W.M. Gibson N.W. Lau S.S. Monks T.J. Arch. Biochem. Biophys. 1995; 320: 51-58Crossref PubMed Scopus (42) Google Scholar), rapid inhibition of DNA synthesis (10Jeong J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar), and the induction of the growth arrest and DNA Damage-inducible gene, gadd153 (10Jeong J.K. Stevens J.L. Lau S.S. Monks T.J. Mol. Pharmacol. 1996; 50: 592-598PubMed Google Scholar). Quinone-thioethers therefore provide a useful model with which to investigate chemically induced growth arrest and the consequences of chemically induced chromatin disruption. We have therefore compared physiologically induced (serum and nutrient depletion) and chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) growth arrest in renal tubular epithelial cells (LLC-PK1) and determined some of the factors involved in regulating gene expression in response to the chemically induced stress. We report that the response to physiological stress (serum deprivation) involves growth arrest characterized by an inhibition of DNA synthesis in the absence of changes in histone or gadd153 mRNA. In contrast, the chemically induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2A and H2B mRNA, and the induction of gadd153