Exp Toxic Pathol 2003; 55: 295–300 URBAN & FISCHER http://www.elsevier-deutschland.de 1
Department of Medical Biology and Genetics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey Department of Biology and, Faculty of Science, Cumhuriyet University, Sivas, Turkey 3 Department of Radiology, Faculty of Medicine, Cumhuriyet University, Sivas,Turkey 4 Department of Pathology, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey 2
Subcutaneous undifferentiated sarcoma induced by N′-ethylN′-nitrosourea in rat: radiology, histopathology and mutagenesis ÖZTÜRK ÖZDEMIR1, FEVZI BARDAKCI 2, HULUSI EGˇ ILMEZ3, and REYHAN EˇGILMEZ4 With 5 figures Received: October 24, 2002; Revised: August 20, 2003; Accepted: September 17, 2003 Address for correspondence: Dr. ÖZTÜRK ÖZDEMIR (PhD), Department of Medical Biology and Genetics, Faculty of Medicine, Cumhuriyet University 58140-Sivas, Turkey; Tel.: 00 90 346 219 10 10/1068, Fax: 00 90 346 219 11 55, e-mail:
[email protected] Key words: Undifferentiated sarcoma; KRAS gene; ENU; Rat.
Summary
Introduction
The aim of the present study was to investigate high dose and long-term effects of a common industrial agent, N′-ethyl-N′-nitrosourea (ENU), on soft tissues in a rat model. ENU, which was dissolved in polyethyleneglycol (PEG) was injected intra-peritoneally once a week (300 mg/kg) in the first experimental group. The second group received only PEG. The control group was free of any agent administration. Only rats treated with ENU for a period of 45 weeks developed large subcutaneous tumours (approximately 5–9 cm in size). Tumoral tissues were examined radiologically, histopathologically and immunohistochemically. There was no bone destruction beneath the soft tumoral tissues in direct X radiograms. Computed tomographic (CT) images showed heterogeneous soft tissue masses with a density ranging from 50 to 65 HU. Macroscopically, all tumors were circumscribed with a graywhite surface in the cross-sections. The histopathological and immunohistochemical examination of the subcutaneous tumoral tissues showed a spindle cell type of sarcoma. Lymphatic and skeletal muscle invasion, atypical mitoses and necroses were determined in all tumoral tissues in the experimental group. A somatic point mutation was detected in exon 2 of KRAS oncogene in sarcoma tissues using the single strand conformational polymorphism (SSCP) analysis. In conclusion, the activated KRAS oncogene might contribute to the progression of subcutaneous sarcoma in experimentally ENU induced rats due to point mutation.
Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of the chemical compounds. Complete carcinogens must possess both initiating and promoting properties. ENU is a potent monofunctional-ethylating agent that has been found to be mutagenic in a wide variety of mutagenicity test systems, from viruses to mammalian germ cell lines (JUSTICE et al. 1997). It has also been shown in different tumors in various organs of mammals by experimental procedures (SASAKI et al. 1997). This monofunctional-alkylating agent is a potent inducer of cellular stress, leading to chromosomal aberrations such as point mutations, translocations, deletions, insertions, and cell death (WILHELM et al. 1997). ENU is also known as a potent rodent cell mutagen due to its alkylating function and therefore an inducer of DNA damage in the cell (TONG et al. 1997). ENU possess the dual action, ethylation and carbonylation in carcinogenesis process in mammals. The ethyl group and carbonyl group can be transferred to nucleophilic sites of cellular constituents and to an amino group of any cellular proteins, respectively. Previous studies showed higher mutational frequencies in post spermatogonial stage in rats after treatment with ENU (KATOH et al. 1997; BROOKS and DEAN 1997). LEIGEBEL and SCHMEZER (1997) investigated the mutagenic effects of ENU in the mouse spermatagonia together 0940-2993/03/55/04-295 $ 15.00/0
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with two postmeiotic germ cell mutagens (MMS and IPMS) in germ cells, using a transgenic mouse mutation assay (lacZ/Muta Mouse, positive selection system). They injected the agent intra-peritoneally into ICR female mice (50 mg/kg) on day 8, 10, 12, or 16 of gestation. Among the embryonic stages tested, the 10th embryonic day was detected as the most susceptible day for ENU treatment as it is the same day of the postnatal development of testes and epididymides. While the fertility of a male offspring was drastically impaired by the prenatal ENU treatment on the 10th embryonic day, the fertility was not affected in male offsprings on embryonic days of 8 and 16. In another study, body weight of sibs were significantly lower in all groups treated with ENU on postnatal day of 21 and at 12 weeks of age compared to the control group (NAGAO et al. 1996). On the other hand, RSM-PCR assay was successfully applied for the detection of ENU-induced mutations in codon 12 of c-H-ras1 (MspI site 1695–1698) (CERUTTI et al. 1994), and codon 248 of p53 tumor suppressor gene (MspI site 14067–14070) in human skin fibroblasts (POURZAND and CERUTTI 1993). The aim of the present study was to investigate the long term and high dose effects of ENU in a rat model. The role of ENU in tumorigenesis was followed by the radiological, histopathological and immunohistochemical examinations. In the same time, tumoral tissues were analysed for a possible KRAS mutation, using the single strand conformational polymorphism (SSCP) method.
Materials and methods Animals: Animals used in the present study were nontransgenic and were bred and fed in optimal laboratory conditions. Forty male 7 to 8-week-old Wistar albino rats (Rattus norvegicus), obtained from “The Experimental Animal Laboratory of Cumhuriyet University, Sivas, Turkey”, were used in the present study. Experimental design: ENU (CASRN: 759-73-9, Sigma Chemical Company, MO, USA) was dissolved in PEG (Sigma Chemical Company, USA) and stored at –70 °C. While rats from the first experimental group received intraperitoneal injection of 300 mg per kilogram of body weight of ENU once a week, only PEG was administered in the second group (11 male rats). Control group animals received no agent administration. Following 45 weeks of experimental period, 10 rats per experimental group were killed by cervical dislocation and the tumoral tissues were obtained surgically. Radiology: All rats from the experimental group that have malignant tumoral lesions were examined with plain radiograms and CT. Prior to radiological examinations, rats were anaesthetised with an intramuscular injection of 50 mg/kg of Ketamin Hydrochloride (Ketalar Eczacıbas¸ı A.S¸. Istanbul/Turkey). The conventional radiograms (Hitachi, D-L 62) were obtained with an anode film distance of 76 cm in order to take the whole body radiogram of a rat. CT examinations were accomplished with Picker PQS model computed tomography system. 296
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Histopathology and immunohistochemistry: Tissue samples were fixed in 10% buffered formalin, dehydrated and embedded in paraffin for light microscopy. Tissue sections were then stained with hematoxylin-eosin for the histopathological examination. The streptavidin-biotin method was used for the immunohistochemical analysis of tumoral tissues. Primary antibodies: murine monoclonal antibody, low–molecular-weight cytokeratin (Dako Co., N1560-028/1), high-molecular-weight cytokeratin (Dako Co., N1560-058/2), vimentin (Dako Co., N1583-066/2), desmin (Biogenex, A310B), muscle specific actin (Dako Co., N1584-088/1), neuron-specific enolase (NSE) (Biogenex, A310B), S-100 protein (Dako Co., N1573-058/2), HMB-45 (Biogenex, CA 95583 USA, A310) and myo D1 (Neomarkers Lab. vision Co. CA 94539) were used. Isolation of genomic DNA: Genomic DNA was extracted from both tumoral and non-tumoral control tissue samples (cut into small pieces) according to the basic DNA isolation protocol (SAMBROOK et al. 1998) with some modification (ÖZDEMIR at al. 2000). Approximately 20 to 50 mg of tissue samples were homogenised in 450 µl STE buffer (0.1 M NaCl, 0.05 M Tris-HCl, 0.001 M EDTA). Twentyfive µl proteinase K (10 mg/ml), 75 µl SDS (20% solution of sodium lauryl sulfate in distilled water) were added into it and incubated at 55 °C for 2 hours. Following incubation, DNA was isolated using the standard phenol-chloroform procedure. Isolated DNA was concentrated by cold absolute ethanol and redissolved in distilled water. PCR and SSCP analysis of exon 2 of KRAS gene: Primers for exon 2 of rat KRAS oncogene were obtained from MWG-Biotech CimbH Paris/FRANCE. The sequences of primers were; Sense: 5′-CTC CTA CAG GAA ACA AGT AG-3′ Antisense: 5′-GGT GAA TAT CTT CAA ATG ATT-3′ Amplifications of exon 2 of KRAS gene were performed in a volume of 50 µl reaction mixtures containing 200 µM dNTPs (MBI, Fermentas), 0.5 M primers, 30 ng template DNA, 10× Taq DNA polymerase buffer, 1.5 U µl Taq DNA polymerase (Boehringer, Mannheim) for 35 cycles in Amplitron I DNA Thermal Cycler (Thermolyne) under the following conditions: denaturation at 96 °C for 30 sec, annealing at 58 °C for 30 sec and extention at 72 °C for 30 sec. PCR product was resolved in 1.5% agarose gel and stained with ethidium bromide (4 µg/ml). After agarose electrophoresis, exon 2 of KRAS gene from control and ENU treated subcutaneous sarcoma tissues were used for SSCP analyses. Three microliters of the amplification product were added into 2–3 µl of denaturing loading solution (95% formamide, 100 mM NaOH, 0.25% bromophenol blue, 0.25% xylencyanol) and denatured at 95 °C for 10 minutes. Electrophoresis were carried out using 10% vertical nondenaturing polyacrylamide gel (37.5:1 acrylamide to bis-acrylamide cross-linking) in TBE buffer (0.089 M Tris, 0.089 M Boric acid, 0.001 M disodium EDTA). Gels were pre-run at 300 v (38 volts/centimetres) for 30 minutes. A thermostatically controlled refrigerated circulator (Grant Instrument Limited, Cambridge) was used to maintain a constant temperature of 10 °C during the electrophoresis. Following the pre-run, approximately 5 µl of denatured mixture prepared above were loaded and run at the pre-run electrophoretic conditions until the bromophenol blue marker reached to
the bottom of the gel. Gels were fixed with 10% ethanol, and 0.5% acetic acid solution twice for 3 min; stained with 0.1% silver nitrate solution for 10 min; rinsed twice with distilled water, then developed in an alkaline solution (1.5% NaOH, 0.1% NaBH4 and 0.15% CH2O).
Results The PEG treated group was used as a second experimental group in order to distinguish the effects of PEG and PEG + ENU administrations. Control rats and PEG treated group demonstrated regular appearance without tumors. The ENU treated rats exhibited large (approxi-
mately 5–9 cm in size) subcutaneous tumors in conventional radiograms (fig. 1). The radiological imaging modalities disclosed no bone destruction beneath the soft tumoral tissues (figs. 1, 2). CT images revealed subcutaneous mass at various locations in soft tissue densities (50–65 HU) with heterogeneous parenchymal pattern (fig. 2). Macroscopically, all tumors were fairly well circumscribed with a gray-white surface in their cross-sections. Radiological examination of tumors showed heterogeneous parenchymal pattern due to necrosis and hypervascularity. The histopathological examination of these subcutaneous tumoral tissues showed a spindle cell type of sarcoma. Tumors were entrapped by degenerated muscle fibers and composed of fascicles and showed a
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Fig. 1. One of the rat’s plain radiogram from experimental group. There was a mass on its left lower neck and left hemithorax. The mass on the left hemithorax was also compressing the left lung. There was not any bone destruction around the mass. Arrow (→) indicates this dorsal subcutaneous tumor. Fig. 2. The CT image of a rat from the experimental group. The mass was seen on the left posterior side of the hemithorax, located subcutaneously. It has soft tissue densities (50–65 HU) and it was in heterogeneous parenchymal pattern.
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storiform pattern (fig. 3). Varying degrees of small undifferentiated, hyperchromatic round or spindle-shaped cells and a few differentiated cells with eosinophilic cytoplasm reminiscent of rhabdomyoblasts were also observed in subcutaneous sarcoma tissues (fig. 4). In those cells, the nuclei varied in size and shape, but no crossstriations were seen. All tumors contained pleomorphic giant cells with eosinophilic cytoplasm admixed with more uniform-appearing spindle-shaped and round cells. While, tumoral cells were positive for vimentin, muscle specific actin, desmin, S100 protein and myo D1, they were negative for NSE, HMB-45, low–molecular and high-molecular-weight cytokeratins. Lymphatic and
skeletal muscle invasions were also seen. Mitotic figures were four mitoses per ten high power fields. We also studied the exon 2 (167 bp) of KRAS oncogene by SSCP analysis in experimental and control groups. Although no mutation was detected in KRAS oncogene in PEG and control groups, three mutations were found in exon 2 of KRAS oncogene from tumoral tissues of ENU treated rats. Comparison of the SSCP profiles of oncogene in control and experimental groups showed different band profiles (fig. 5). While, oncogene from rats in the experimental group has showed an extra band (arrow head), (fig. 5, lanes 5–7), it was not presented in the control group rat (fig. 5, lane 2).
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Fig. 3. Subcutaneous tumour from experimental group rat composed of fascicles of cells and shows storiform pattern (H& E, × 90). Fig. 4. Figure shows a few differentiated strap-shaped cells (arrows) from tumoral tissue belonging to a rat of the experimental group (H& E, × 180).
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Fig. 5. SSCP mutation screening of exon 2 of KRAS oncogene in ENU treated sarcoma tissues. Arrow indicates mutated single strand (ss) of three different USS tissues of experimental group. ds: double strand. Lanes: Lane 1: Marker, λ DNA EcoRI/Hind III. Lane 2: Double stranded DNA of unterated and non-denatured PCR product of control rat tissue. Lane 3: SSCP profile of denatured PCR product of control rat tissue. No mutation was detected. Lane 4: Double stranded DNA of ENU treated and non-denatured PCR product of experimental group rat tissue. Shows the same in size as non-denatured control PCR product. Lanes 5–7: SSCP profile of PCR product of SRMS tissues from ENU treated experimental group rats. Show distinct mutation of exon 2 of KRAS gene in all tumoral tissues.
Discussion Most of the N-nitrosocompounds are mutagens but, only some of them are considered to be tumor initiators in carcinogenesis. Qualitative and quantitative differences in tumorgenesis indicate the complexities of mutation induction in vivo and emphasize no single in vitro test system can adequately represent the in vivo situation (BRUSTLE et al. 1993). There is a wide variation of AGT levels between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced human parenchymal cells, WILHELM et al. (1997) examined the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in 116 mutants derived from two ENU treated normal human skin keratinocyte cell lines. SHOEMAKER et al. (1997) used two mutation detection methods to identify APC mutations in at least 12% of the tumors from ENU-
treated B6 Min/+ mice. They did not find any H- or Kras- mutations in intestinal tumors from either untreated or ENU-treated Min/+ mice. The molecular genetic data obtained from ENU-induced mutants on various species suggest that ENU produces mainly GC-AT transitions and, to a small extent, AT-GC, AT-CG, AT-TA, GC-CG and GC-TA base substitutions (SHIBUYA and MORIMOTO 1993). A probable reason for the ENU-induced AT-TA transversions has been related to the O2-ethylthymine, which was found to be mispair with thymine (OP-HETVELD et al. 1997). Loss of wild-type allele results in a mutator phenotype, accelerating tumorigenesis. Tumorigenesis specifically occurs in the gastrointestinal and genitourinary tracts; the cause of this tissue specificity is elusive (DE-WIND et al. 1998). We investigated ethylnitrosourea-induced subcutaneous tumoral tissue development in rats radiologically, histopathologically and immunohistochemically. Tumoral tissues were also investigated at molecular level by SSCP analysis of KRAS oncogene. No tumors were detected in control and PEG treated groups. However, various kind of tumors emerged primarily subcutaneously in the ENU treated group. Sequential intraperitoneal injections of ENU (300 mg/kg) induced gliomas and colorectal carcinoma after 45 weeks of the treatment (data not shown). ENU-induced tumors were soft tissue masses of unstable densities of 50–65 HU and heterogeneous parenchymal pattern in radiological imaging. No bone destruction was seen on plain radiograms and computed tomographical sections. Meanwhile, DALDRUP et al. (1998) reported the disruption of the endothelial integrity of microvessels in ENU-induced malignant tumors in mammary soft tissues of rats, detected by dynamic magnetic resonance imaging. OKIMATO et al. (1997) suggested that ENU-induced gliomas may be regulated and proliferate by multiple angiogenic growth factors of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Histopathologically, the tumors exhibited a fasciculated arrangement of the tumor cells, sometimes showing a storiform growth pattern. Cells were immunopositive for vimentin, muscle specific actin, desmin, S100 protein and myo D1, but they were negative for NSE, HMB-45, low–molecular and high-molecular-weight cytokeratins. So, we diagnosed the long term ENU- induced subcutaneous soft tissue tumors as undifferentiated sarcomas in the present study. WEISS and GOLDBLUM reported that the intensity of immunostaining for myo-D1 and desmin were proportional to the degree of rhabdomyoblastic differentiation (WEISS and GOLDBLUM 2001). We determined the SSCP profiles of KRAS (exon 2) oncogene in USS tissues of ENU treated rats. Three KRAS exon 2 (encompassing the hotspot codons 59, 61), (BENHATTAR et al. 1993) mutations were detected in the SSCP analysis of 10 tumoral tissues from ENU induced experimental rats. SITHANANDAM et al. (1998) reported that N-ethylnitrosourea caused shift mutation codon from 12 to 61 of K-ras oncogene during fetal mouse lung matuExp Toxic Pathol 55 (2003) 4
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ration. The mutation profiles of activated KRAS oncogenes were also reported in chemically induced mouse lung tumors (REYNOLDS and ANDERSON 1991). These findings suggest that the observed mutations in exon 2 of KRAS oncogene were caused by the genotoxic effects of the agent in question, i.e ENU. It is well known that KRAS protein modulates cell cycle via both positive and negative regulatory pathways (FAN and BERTINO 1997). Somatic cell mutations of the KRAS gene were also reported in nonpolypoid colorectal adenomas by VAN WYK et al. (2000). The mutation detected in the present study might be the first report of an activated KRAS oncogene in experimentally ENU induced sarcoma in rats. According to present study findings and in view of the previous study results we speculate that the mutation in KRAS oncogene might contribute to the progression of subcutaneous sarcoma in experimentally ENU induced rats. Acknowledgement. Authors thank to “TÜB˙ITAKNATO Organisation of B2 – 2000 Science Fellowship Programme” for financial support.
References BENHATTAR E, LOSI L, CHAUBERT P, et al.: Prognostic significance of K-ras mutations in colorectal carcinoma. Gastroenterol 1993; 104:1044–1048. BROOKS TM, DEAN SW: The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea. Mutat Res 1997; 388: 219–22. BRUSTLE O, PETERSEN I, RADNER H, et al.: Complementary tumor induction in neural grafts exposed to N′-ethyl-N′nitrosourea and an activated myc gene. Carcinogenesis 1993; 14: 1715–1718. CERUTTI P, HUSSAIN P, POURZAND C, et al.: Mutagenesis of the H-ras protooncogene and the p53 tumor suppressor gene. Cancer Res 1994; 1: 1934–1938. DALDRUP H, SHAMES DM, WENDLAND M, et al.: Correlation of dynamic contrast-enhanced magnetic resonance imaging with histologic tumor grade: comparison of macromolecular and small-molecular contrast media. Pediatr Radiol 1998; 28: 67–78. DE-WIND N, DEKKER M, VAN-ROSSUM A, et al.: Mouse models for hereditary nonpolyposis colorectal cancer. Cancer Res 1998; 15: 248–255. FAN J, BERTINO JR: K-ras modulates the cell cycle via both positive and negative regulatory pathways. Oncogene 1997; 14: 2595–2607. JUSTICE MJ, ZENG B, WOYCHIK RP, et al.: Using targeted large deletions and high-efficiency N′-ethyl-N′-nitrosourea mutagenesis for functional analyses of the mammalian genome. Method 1997; 13: 423- 436. KATOH M, HORIYA N, VALDIVIA RP: Mutations induced in male germ cells after treatment of transgenic mice with ethylnitrosourea. Mutat Res 1997; 14: 229 –237.
300
Exp Toxic Pathol 55 (2003) 4
LIEGIBEL UM, SCHMEZER P: Detection of the two germ cell mutagens ENU and iPMS using the LacZ/transgenic mouse mutation assay. Mutat Res 1997; 14: 213–8. NAGAO T, SATO M, MARUMO H, et al.: Testicular development and fertility of mice treated prenatally with N′-nitroso-N′-ethylurea at various gestational stages. Teratog Carcino Mutagen 1996; 16: 183–98. OKIMATO T, SHIMOKAWA I, HIGAMI Y, et al.: VEGF and bFGF mRNA are expressed in ethylnitrosoure-induced experimental rat gliomas. Cell Mol Neurobiol 1997; 17: 141–50. OP-HET-VELD CW, VAN-HEES-STUIVENBERG S, VAN-ZEELAND AA, et al.: Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents. Mutagenesis 1997; 12: 417–424. ÖZDEMIR Ö, BULUT HE, KORKMAZ M, et al.: Increased cell proliferation and R.Msp1 fragmentation induced by 5aza–2′-deoxycytidine in rat teste related to the gene imprinting mechanism. Exp Toxic Pathol 2000; 52: 317–322. POURZAND C, CERUTTI P: Genotypic mutation analysis by RFLP/PCR. Mutat Res 1993; 288: 113–12. REYNOLD SH, ANDERSON MW: Activation of proto-oncogenes in human and mouse lung tumors. Environ Health Perspect 199; 93: 145–148. SAMBROOK J, FRITSCH EF, MANIATIS T: Molecular Cloning. A laboratory manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring: Harbor 1998. SASAKI YF, TSUDA SF, IZUMIYAMA E: Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet). Assay Mutat Res 1997; 15: 33–44. SHIBUYA T, MORIMOTO K: A review of the genotoxicity of 1-ethyl–1-nitrosourea. Mutat Res 1993; 297: 3–38. SHOEMAKER AR, LUONGO C, MOSER AR, et al.: Somatic mutational mechanisms involved in intestinal tumor formation in Min mice. Cancer Res 1997; 15: 1999–2006. SITHANANDAM G, RAMAKRISHNA G, DIWAN BA, et al.: Selective mutation of K-ras by N′-ethyl-N′-nitrosourea shifts from codon 12 to codon 61 during fetal mouse lung maturation. Oncogene 1998; 17: 493–502. TONG HH, PARK JH, BRADY T, et al.: Molecular characterisation of mutations in the hprt gene of normal human skin keratinocytes treated with N′-ethyl-N′-nitrosurea: influence of O6-alkylguanine alkyltransferase. Environ Mol Mutagen 1997; 29:168–179. VAN WYK R, SLEZAK P, HAYES VM, et al.: Somatic mutation of APC, KRAS, and TP53 genes in nonpolypoid colorectal adenomas. Genes Chromo Cancer 2000; 27: 202–208. WEISS SW, GOLDBLUM JR: Enzinger and Weiss’s Soft tissue tumors. 4th ed. Mosby Company: St Louis 2001; p 785–835. WILHELM D, BENDER K, KNEBEL A, et al.: The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents. Mol Cell Biol 1997; 17: 4792–4800.
