Transgenic Mice Expressing the Sh Ble Bleomycin Resistance Gene Are Protected Against Bleomycin-Induced Pulmonary Fibrosis1

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[CANCERRESEARCH56, 5659-5665. December 15, 1996] Transgenic Mice Expressing the Sh ble Bleomycin Resistance Gene Are Protected against Bleomycin-induced Pulmonary Fibrosis1

JÃ©rÃ´meWeinbach, Anne Camus, Jacqueline Barra, Patrick Dumont, Monique Julian, Suzy Cros, Charles Babinet, and Gerard Tirab? Laboratoire de GÃ©nÃ©tiqueetMicrobiologie, 118 Route de Narbonne, UniversitÃ©Paul Sabatier, 31062 Toulouse Cedex (J. W., G. TI; Unite de Biologie du DÃ©veloppement. Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15 (A. C., J. B., C. B.]; and Laboratoire de Toxicologie et Pharmacologie Fondamentales du Centre National de Ia Recherche Scientifique, 205 Route de Narbonne, 31077 Toulouse Cedex (P. D., M. J., S. CI, France

ABSTRACT transgenic mouse strains susceptible to BLM-induced lung fibrosis (11â€”13).After initial biochemicallesions,aperiodof inflammation Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic occurs, including infiltration of alveolar macrophages and lympho agent against various carcinomas, the potentially lethal and chronic fl cytes ( 14), cellular proliferation, and dedifferentiation ( 15). Other brotic response of the lung is a major dose-limiting side effect. Here, we explore the possibility ofa direct inhibition oflung tissue injury by in vivo changes have been noticed during this period; NAD@ and ATP expression of the actinomycetes BLM resistance protein Sh ble. Tram depletion correlated with the poly(ADP-ribose) polymerase activation genic mice expressing the Sh ble gene under the control of a composite (1 1) and platelet trapping to the alveolar endothelium (16). Further viral promoter were produced after introduction of the transgene into D3 more, release of cellular mediators occurs; cytokines such as trans ES cells. The protein was detected at high level In lungs, spleen, and forming growth factor /3 (17), tumor necrosis factor a (18), interleu kidney. We then assessed its ability to modulate the BLM-induced fibrotic kins 1â€”6(19,20), and chemokines such as macrophage inflammatory response in the transgenic mice in comparison with CS7BLI6 and 129/Sv protein 1 (21) and monocyte chemoattractant protein 1 (22) mediate parental mice. Cumulative doses of 300, 400, or 500 mg/kg BLM were the initiation and maintenance of inflammatory lesions and the re administered either by i.p. or s.c. repeated injections in the different strains. Trausgenic mice were shown to be clearly less sensitive to BLM cruitment of specific leukocyte populations. Finally, fibroblasts dem toxicity, as assessed by lung histology. The pulmonary hydroxyproline onstrate an increase in steady-state levels of mRNA encoding base content in the treated transgenic mice was close to its baseline level, ment membrane collagens of different types (23â€”26).Therefore, whereas it was up to 50% higher than the control level in C57BL/6 and poly(ADP-ribose) polymerase inhibitors or a NAD@ precursor such as 129/Sv parental mke. These observations are consistent with the hypoth niacin, antagonists of the platelet accumulation or activation (27, 28), esis that a resistance gene specifically expressed In lungs may prevent the anti-transforming growth factor @3antibodies (29), and human recom BLM-induced inflammation. binant soluble tumor necrosis factor receptor (30) have been tested in mice to prevent lung injury. All of these provide potentialities for INTRODUCTION intervening in the fibrotic process in the lung. As an alternative to prevent inflammatory reactions, we investigate Despite its potential great efficacy against various human malig in the present study the possibility of a direct inhibition of the BLM nancies and its relative lack of myelosuppression, the use of the activity that injures the lung tissue. Indeed, it has been reported that antitumor antibiotic BLM3 is often limited by pulmonary fibrosis (1, 2). This dose-dependent, cumulative, and irreversible side effect (3) is the bacterial strain producers of the BLM family type of antibiotics increased by radiation therapy and may develop into fatal hypoxemia contain genes that render them resistant to BLM. An example of such if chemotherapy combining BLM administration with other antineo genes is the one isolated from the chromosomal DNA of the tallyso plastic agents is not immediately stopped (4). Variations among organ mycin-producing actinomycete Streptoalloteichus hindustanus, the SI, sensitivity to BLM toxicity have been correlated to the level of a ble gene used in this study (31). The Sh ble gene encodes a small cytosolic-inactivating enzyme named BLM hydrolase (5â€”7),which is (14-kilodalton)acidicproteinthat protectsagainstBLM-induced lowest in pulmonary tissues and skin of animal species that are DNA cleavage in vitro, owing to its capacity to bind to Fe (II)-BLM sensitive to BLM-induced pulmonary fibrosis (8, 9). with 1:1 stoichiometry (32). This stable protein appears nontoxic for Development of new strategies to prevent BLM-related pulmonary a variety of eukaryotic organisms in which Sh ble has been expressed toxicity is closely connected to progress in understanding the com (33, 34). These observations lead to the following question: Is it plex, multistep process leading to the final fibrotic state. The sequen possible to transfer this system into an animal model? To elucidate the tial pulmonary events induced by the drug are well described (10). role of Sh ble gene expression in vivo to confer BLM resistance in the Fibrosing alveolitis results from an inflammatory response to an initial lung, transgenic mice with the Sh ble gene were produced. This strain injury to the alveolar epithelium. The oxygen radical generated by the was obtained using the D3 ES cell line (35) transfected by the degradative action of BLM on DNA is assumed to induce lipid linearized plasmid pUT 526 carrying the Sh ble gene under the control peroxidation, leading to pulmonary inflammation. Most of our in of the polyoma enhancer-TK promoter (kindly provided by CAYLA, sights on the biochemical and histological changes associated with Toulouse, France). This in situ expression model allowed us to test the fibrosis originated from numerous studies conducted with normal or ability of this protein to modulate BLM-induced pulmonary fibrosis, in comparison with BLM-treated C57BU6 mice. Murine strain Received6/10/96;accepted10/14/96. C57BL/6waschosenfor the presentinvestigationbecauseofits The costs of publication of this article were defrayed in part by the payment of page characterization as a good model for BLM-induced pulmonary fibro charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sis, as evidenced by morphometric analysis of histological lesions and I This work was supported by grants from the Centre National de la Recherche increased pulmonary OH-proline content, a convenient index of col Scientifique, La Ligue Nationale Contra Ic Cancer ComitÃ©HauteGaronne, the Associa lagen deposition, after s.c. BLM administration (36). Fibrosis was tion pour la Recherche sur Ic Cancer (Grant ARC 1063), and the Institut Pasteur. A. C. was supported by a fellowship from the Association pour la Recherche sur Ic Cancer. further diagnosed at the end of our experiment by conventional 2 To whom requests for reprints should be addressed. Phone: 33-61-55-67-35; Fax: determination of pulmonary collagen and histology. The present study 33-61-55-60-00; E-mail: [email protected]. demonstrates the ability of the Sh ble protein to prevent lung toxicity 3 The abbreviations used are: BLM, bleomycin; ES, embryonic stem; TK, thymidine kinase; NA, numerical aperture; OH-proline, hydroxyproline. of BLM. 5659

Bai@HL BstEIL Bas@HL XhoL HpaL 1612 BgIIL BstElL 1481 1621 * T Polyoma Sh ble SV4O PolyA Opi ampr Enh.ITK splice ColEl prom pUT526 3779bp

Fig. 1. Schematic diagram of BstEIl-linearized plasmid pUTS26 used for D3 ES cells electroporation. Solid line, probe used for Southern blot hybridizations; Enh., enhancer; prom, promoter.

MATERIALS AND METHODS For Southern blot analysis, 10 p.g of Bg1II-and BstEII-digested tail DNA were subjected to electrophoresis in a 0.8% agarose gel overnight under a Production of Transgenic Mice. Transgenic mice carrying the Sh ble constant voltage (1 V/cm). DNA fragments were transferred to a nylon gene, which confers BLM resistance, were generated by the use of the ES membrane. Southern blot analysis were performed using a BamHl fragment cells strategy as described previously (37). For this purpose, the D3 ES cell from pUT 526 containing the Sh ble gene under the dependence of the viral line (35), expressing the Sit ble gene under the control of the TK promoter enhancer-promoter as a probe. Hybridization was carried out for 16â€”20hat and polyoma enhancer (38), has been established as follows. BstEII 42Â°Cwith5 x 106cpm/ml heat-denatured, 32P-labeledprobe (specific activity, linearized plasmid pUT 526 (CAYLA) was electroporated into D3 ES cells 1.0-2.0 x l0@cpm4@g).Themembrane was washed, exposed to Kodak XRP (isolated from 129/Sv blastocysts). We preferred indeed to use the entire X-ray film, and kept for 3 days at â€”80Â°C. linearized pUT526 plasmid rather than only the enhancer-TK-Sh ble se PCR was performed using a Thermojet machine (Eurogentec, Seraing, quence, protecting the Sh ble gene sequence from exonuclease activity Belgium). PCRs were cycled 30 times (one cycle consisting of 1 mm at 94Â°C, during D3 ES cell transfection. Cells from one of the resistant clones 1 mm at 55Â°C,and 2 mm at 72Â°C)with Tfl DNA polymerase (Epicentre recovered after selection with 5 ,.tg/ml phleomycin (CAYLA) were micro Technologies, Madison, WI). The Sh ble primer set was designed based on injected into 3.5-day-old C57BL/6 blastocysts to generate chimeras. A pUT 526 sequences: the 5' and 3' primers were 5'-ATGGCCAAGTTGAC C57BL/6 female mated with one chimeric male gave birth to transgenic CAGTGC-3' and 5'-ACCGTAUACCGCC1Tl@GAG-3', respectively, corre progeny, allowing the establishment of the transgenic mouse strain TKPH. sponding to the 326â€”345and1658â€”1677nucleotidesequences of the pUT Homozygous mice for the S/i ble transgene did not display any abnormal 526, giving rise to an amplification product of 1350 bp. phenotype and reproduced normally. Immunohistochemical Localization of the Sh ble Protein. Organs from Analysis of Tail DNA. Tail segments of 1â€”1.5cmwere reduced to powder sacrificed animals were mounted in ornithine carbamyl transferase compound under liquid N2. DNA was extracted by digesting tissues overnight at 37Â°Cin (Miles Laboratories, Inc., Napperville, IL) and flash frozen in isopentane 3 ml of lysis buffercontaining50 mMTris-HC1(pH7.5), 0.5% SDS, 0.1 M cooled by liquid N2 for cryosectioning. Sections (10 @smthick)were fsxed for NaCI, 5 mM EDTA, and 100 @tg/mlproteinase K. DNA was then extracted with 30 mm in 4% p-formaldehyde in PBS (pH 7.4). Subsequently, the cells were phenol-chloroform and precipitated with ethanol. The DNA pellet was col permeabilized by incubation in 2% p-formaldehyde, 0.1% Triton X-l00, and lected, washed with 70% ethanol, and then resuspended in 200 @tlofdistilled 2% BSA in PBS for 15 mm at room temperature. Tissue sections were washed water. Transgenic mice were identified either by Southern blot or PCR. 3 X for 5 mm each with PBS, and nonspecific antibody binding was blocked by washing twice for 10 mm in PBS containing 0.5% BSA and 0.15% glycine (buffer A) followed by incubation for 30 mm at 25Â°Cwithbuffer A supple 1. 2. 3. 4. 5. mented with 5% normal goat serum. Tissue sections were then incubated with the primary polyclonal antibody anti-Sh (dilution, 1:100; obtained from @ 4.072 Kb CAYLA) overnightat roomtemperature.Afterthreewashingswith bufferA, @ 3,054 Kb sections were incubated with the secondary biotinylated goat antirabbit IgG for 1 h at room temperature. Tissue sections were washed and incubated with @ 2,036 Kb fluorescein-conjugated streptavidin during 30 mm at room temperature. After @ 1,636Kb three final washings with buffer A, sections were processed for microscopic examination (moviol embedded). Light and fluorescence microscopy of the @ 1.018Kb labeled sections were carried out on a confocal Zeiss LSM microscope. The excitation wavelength is 495 nm for fluorescein. Samples were examined by epifluorescence illumination with different Zeiss objectives (X20 Apofluo; 506 Kb NA, 0.5; X40 Apofluo; NA, 0.75; and an oil immersion objective, X63 396 Kb @. Apofluo; NA, 1.25).Autofluorescence of the untreated samples was negligible under our experimental conditions. Animal Strains and Treatments. Female C57BLJ6 mice (Charles River, St. Aubin, France), 129/Sv mice (Harlan Sprague Dawley, Bicester, Great Britain), and TKPH transgenic mice were maintained with free access to pellet Fig. 2. Southern blot analysis of transgenic mice. Tall DNA (10 sag) from transgenic food (Usine d'Alimentation Rationnelle type A03) and water. mice was digested with appropriate restriction enzymes Bgl and Bst ElI, followed by To obtain maximal BLM-induced lung lesions, several protocols for treat electrophoresis on a 0.8% agarose gel for 12h. DNA was transferred to a nylon membrane ment of mice with various BLM concentrations were designed. BLM sulfate and hybridized with a 32P-labeledprobe, the Barn Hl fragment from pUTS26 containing (BELLON, Neuilly-sur-Seine, France) dissolved in sterile saline solution at the the Sh ble gene under the dependence of the Polyoma enhancer and TK promoter. 10 @.sg of digested tail DNA from C57BL16 mouse containing 50 pg of the 883 bp Barn suitable concentration was administered by either the i.p. or s.c. route (0.1 HI-fragment from pUTS26@containing the Sb ble gene was used for a positive control. mI/lO g of body weight) as five doses of 20 mg/kg twice weekly for 3 weeks Digested tail DNA from C57BU6 mouse was used for a negative control. Lanes 1â€”2: (cumulative dose, 100mg/kg) or 10doses of 30, 40, or 50 mg/kg twice weekly Bglll-Bst ElI digested tall DNA from a fourth generation transgenic mouse; Lane 3: for 5 weeks (cumulative dose, respectively, 300, 400, or 500 mg/kg). Control Positive control: 883 by BamHl contruct fragment mixed with C57BIJ6 mouse DNA; Lane 4: BglII-Bst ElI] digested tail DNA from a first generation transgenic mouse; Lanes mice received equivalent volumes of saline. 5: Negative control: C57BL/6 mouse DNA. Mice were weighed twice weekly, and mortality was recorded daily. At the 5660

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Fig. 3. Immunohistochemical detection of Sh ble expression in an adult transgenic mouse (3 to 4 months old). Ciyosections of lung (A and B), kidney (C and D), and spleen (E and F) were stained for the Sh ble protein in an indirect immunofluorescence staining procedure. A, C, and E, Sh-ble-positive transgenic mouse tissues; B, D, and F, Sh ble-negative tissues from control C57BIJ6 mouse. Bar, 20 pm.

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times indicated, mice were sacrificed, and fibrosis was further diagnosed by appropriate groups by a Stat.ITCF (jnstitut Technique des cereales et des determination of pulmonary collagen (right lung) and histological examination @ourrages)programusing Student's t test (P < 0.05) as described by Snedecor (left lung). and Cochran (41). The care and use of animals was in accordance with the guidelines of the Histological Examination. The left lung of each individual mouse was Council of European Communities (cancellation 86/C 331/01). fixed for 24â€”48h in a 10% p-formaldehyde solution and then embedded in Assay for Lung OH-proline Content. Lung OH-proline content, an index paraffin. The lungs were then cut into parasagittal slices (5 ,.sm thick) and of collagen, was determined by the method of Woessner (39) as applied to the stained either with H&E or with Masson's trichrome for the identification lung assay (40). The right lung of each individual mouse was dissected free of of collagen fibrils in the region of the bronchiole-alveolar duct junction major bronchi, and the lobes were minced in 1-mm-thick pieces. They were over large areas of the lung parenchyma. The fibrotic response was blindly hydrolyzed in 2 ml of 6 N HC1at 110Â°Covernightin tightly capped tubes to evaluated on each section as the following qualitative units: N, no fibrosis; liberate amino acids. The resultant hydrolysat was neutralized with 2 ml of 6 FÂ±,thickening of the alveolar wall, characterized by an alveolar cell N NaOH and extracted with phenol-chloroform-isoamyl alcohol to clarify the proliferation, i.e., a fibroblastic metaplasia with more or less collagen aqueous phase. The extracts were filtered through a 0.45-pm membrane deposition in the wall; F+, confined dense fibrotic lesions, characterized (Minisart; Sartorius), adjusted to pH 6â€”8,and diluted to 20 ml. The OH by triangular fibrotic areas with a large subpleural base; alveoles of these proline concentration was then determined colonmetrically and expressed as regions are swallowed by collagen fibers; and F+ +, mutilating large weight (@Lg)ofOH-proline/right lung. Purified OH-proline was used as a fibrotic areas, characterized by wide acellular collagenic tissue including a standard. The values are reported as the mean Â±SEM and analyzed among part or the whole pulmonary lobe. 5661

Table I Evaluation of the pulnionars fibrotic response to various BLM treatments being observed with respectto weight loss. In C57BL/6 mice, both i.p. BLM treatments 120, 30, 40, or 50 mg/kg twice weekly for 5 weeks (total, 10 and s.c. injections of a 40-mg/kg dose, twice a week during 5 weeks injections) by p. or s.c. routej were given in the left and right lungs of transgenic, C57BL/6, and 129/Sv mice. Mice were sacrificed 6 weeks after the first injection of (cumulative dose, 400 mg/kg), induced toxicity; the i.p. administra bleomycin. For histologic evaluation, the fibrotic response was blindly judged on each tions resulted in a median 4.9-g weight loss on day 28 versus 5.9 g for section of the left lung stained with Masson's trichrome for identification of collagen s.c. administrations. A cumulative dose of 300 mg/kg was also toxic fibrils. in these mice, as evidenced by a 5.0-g weight loss on day 28, whereas the same treatment given i.p. was better tolerated (a maximal weight (@.sgRung)Â°129/SvControlMiceCumulativedose of BLMInjection routeHistologicevaluationOH-proline loss of 3. 1 g was recorded on day 28). In l29/Sv mice, both routes of (saline) (O/lO)@' Â±12.1 100 mg/kg FÂ±(4/10) 207.0 Â±21.6 administration were heavily toxic; a 7.0-g weight loss on day 28 was 400 mg/kgs.c. s.c. F+ (6/7) 292.7 Â±26.4' recorded after 10 X 40-mg/kg i.p. administrations versus 6.5 g after 18.7cC57BL16Control i.p.N F+ (4/8)176.0 266.4 Â± s.c. administrations. Concerning the transgenic mice, a cumulative (saline) (0/10) Â±8.5 100 mg/kg FÂ±(3/10) 188.2 Â±11.1 toxicity was also observed, but to a lesser extent than in the other 300 mg/kg s.c. F+ (7/8) 235.9 Â±19.9' strains of mice; the 40-mg/kg i.p. treatment (total, 400 mg/kg) pro i.p. F+ (6/8) 258.1 Â±9.5' 400 mg/kgs.c. s.c. F+ + (6/7) 262.6 Â±24.2c duced no effect on the weight of these TKPH transgenic mice, and for 15.5'TransgenicControl i.p.N F+ (6/7)199.1 279.3 Â± 10 i.p. administrations of 50 mg/kg BLM, a 2.7-g weight loss was (saline) (0/10) Â±14.3 recorded on day 28 and stabilized afterward. 100 mg/kg N (0/10) 161.4 Â±12.3 400 mg/kg i.p. FÂ±(1/4) 195.8 Â±21.8 i.p.-treated mice were sacrificed 6 weeks after the first injection of 500 mg/kgs.c. i.p.N N (0/6)170.9 198.4 Â±16.5 BLM, and pulmonary fibrosis was further diagnosed by determination a Results for OH-proline content (right lung) are mean Â± SD. of right lung OH-proline content, an index of collagen, and histolog b Numbers in parentheses are numbers of mice affected/numbers of mice examined. ical examination of the left lung. As reported in Table 1, the rate of ( P < 0.05, significance of the difference with the saline-treated control group. BLM-induced pulmonary fibrosis on day 42 varied among the three strains studied. Both histological examinations of lung sections and RESULTS OH-proline concentrations indicated clearly that the 5 X 20-mg/kg BLM treatment, which had not caused sensible weight loss among the Establishment of Transgenic Mice. To analyze the integration of three strains, was unable after 6 weeks to induce significant fibrotic the Sh ble gene in the host genome, tail DNA from transgenic Fl response in the lungs of mice. In contrast, histological observations progeny was digested with appropriate BglII and BstEII restriction showed that at 10 X 30 and 10 X 40 mg/kg, nearly all lungs of enzymes (Fig. I) and analyzed by Southern blot hybridization. Fig. 2 CS7BIJ6 mice (13 of 16 and 12 of 14, respectively) were affected by shows an autoradiogram obtained by analysis of progeny derived from fibrosis to varying degrees. Most lungs showed subpleural regions one of the transgenic mice. When tail DNA from transgenic mice followed by evidence of fibrosis in the perivascular areas. None of the (Lanes 1, 2, and 4) was digested with both BgIll and BstEII, a 1700-bp control mice exhibited fibrosis. In the l29/Sv strain, 4 of 8 i.p.- and 6 band appeared, demonstrating the presence of a single copy of the Sh of 7 s.c.-inoculated mice developed fibrosis at 10 X 40 mg/kg. In the ble gene in the genome. Positive control was a 883-bp BamHI frag transgenic mice, 1 of 4 mice showed a thickening of the alveolar wall ment from pUT526@ (a derivative of pUT526 lacking the XhoI-HpaI in the subpleural area at 10 X 40 mg/kg i.p., and none showed SV4O splice portion) containing the Sh ble gene mixed with normal thickening at 10 x 50 mg/kg i.p. C57BL/6mousetailDNA.ThenegativecontrolwasC57BU6mouse The OH-proline content was up to 50% higher than in the control tail DNA digested with BgllI-BstEII. Finally, transgenic animals were mice in lungs of 10 X 40-mg/kg-treated C57BL/6 mice and 66% typed usingcontrolPCR performedon tail DNA(not shown). higher in lungs of 10 X 40-mg/kg-treated 129/Sv mice. Interestingly, Expression of the Sh ble transgene was further monitored by the pulmonary OH-proline content was not statistically different in immunofluorescence on various tissue sections derived from 3â€”4- month-old, third-generation, homozygous transgenic mice. As shown in Fig. 3, expression was detected in both lungs, evenly distributed in 100 - bronchial structures and alveolar walls. Optical sections showed clearly at higher magnification that bronchiolar and alveolar epithelial i@ r surfaces expressed the transgene (not shown). Examination of spleen 80 and kidney tissues also revealed positively stained cells compared with tissues from control C57BL/6 mice. In contrast, expression of the transgene was nonexistent or insufficient to be detected in liver, 60 muscle, and heart. Assay of Induction of Pulmonary Fibrosis by BLM Treatments in C57BL/6, 129/Sv, and TKPH Transgenic Mice. The objective of ri@ 40 the first part of our study was to determine the best way to get a perceptible fibrotic response in the lungs after BLM injections and to 20 do this with minimum mortality. Thus, we had to define the optimal dose and route for BLM treatments and the day of sacrifice for histological evaluation. Because protocols were carried out in differ 0 ent strains of mice (C57BL/6, 129/Sv, and transgenic mice), we could 0 @IO 20 30 40 50 60 understand variations in the BLM-induced pulmonary toxicity among the strains. The repeated administration of BLM was indeed differ Days After First Injection ently tolerated by the strains used, as evidenced by weight loss on day Fig. 4. Survival in mice group i.p. treated with BLM at a dose of 30, 40, or 50 mg/kg 28 after the first BLM administration, and drug toxicity was depend twice weekly during 5 weeks (total, 10 doses). Each group included at least 7 animals. U, C57B1J6 mice treated with BLM, 30 mg/kg i.p.; â€¢,C57BL16 mice treated with BLM, 40 ent on the route of administration. As reflected by the toxicity curves mg/kg i.p.; i:, transgenic mice treated with BLM, 40 mg/kg i.p.; 0, transgenic mice (not shown), the effect was cumulative, no stabilization or recovery treated with BLM, 50 mg/kg i.p. 5662

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@ â€˜l/ @ V1;@ Fig. 5. Histological sections from the left lungs of C57BIJ6 and transgenic mice treated with BLM (40 mg/kg i.p., twice weekly during 5 ),. weeks) and sacrificed 8 weeks after the first BLM injection. Sections were stained with Mas son's trichrome for the identification of collagen fibrils. A, control lung from mouse treated with saline (N). B, typical aspect of lung from a BLM treated transgenic mouse showing rare areas of A lib diffuse alveolitis with thickening of the alveolar architecture (arrow) without gross change of the alveolar architecture (FÂ±).C, frequent lesions encountered in lungs from CS7BL/6-treated mice. An area of dense subpleural alveolar re modeling (arrow) is evident that is rich in fibrils and has wiped out the alveolar architecture (F+). D, in the most severely affected animals among C57B1J6 mice, about 90% of the parenchyma area was occupied by fibrosis (F+ +, mutilating fibrosis). Bars, A and C, 50 pm; B, 5 pm; D, 200 pm.

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treated and control transgenic mice (see Table 1). These results thickenings, and rarely, small areas demonstrated an early stage of the suggested that transgenic mice were much more resistant to BLM fibrotic process. induced fibrosis than both parental strains. Correlated with these results, collagen deposition in lungs from 40- In addition, survival curves were performed on C57BL/6 and or 50-mg/kg i.p.-treated transgenic mice appeared comparable with TKPH transgenic mice submitted to 10 X 30-, 40-, or 50-mg/kg i.p. the basal level, whereas OH-proline content in the lungs of C57BLI BLM injections. Results showed clearly that BLM treatment was 6-treated mice was 58% at 30 mg/kg versus 57% at 40 mg/kg above better tolerated by TKPH transgenic than C57BL/6 mice (Fig. 4). the control (Table 2). Taken together, these results established the Surviving animals were sacrificed 8 weeks after the first day of protective role of the Sh ble protein against BLM pulmonary toxicity injection. In that way, we hoped to obtain more significant lesions in in our transgenic model. the lungs oftreated mice. Indeed, at the dose of 40 mg/kg, histological examinations indicated that eight of nine of the C57BL/6 mice had DISCUSSION developed severe lung injury (about 90% of the area of a section was affected by fibrosis) with a dense fibrotic gangue in the subpleural Pulmonary fibrosis is a potentially lethal, chronic response of the region (Fig. 5 and Table 2). In contrast, one of eight transgenic mice lung to injury caused by BLM, a highly useful antineoplastic agent developed comparable mutilating lesions at 40 mg/kg, and one of that lacks substantial bone marrow toxicity. The hallmark of this seven developed lesions at 50 mg/kg; most lungs showed only pleural disorder is characterized by an increased deposition of extracellular matrix proteins in the alveolar wall, notably collagen, which compro mises pulmonary function. Table 2 Analysis of the fibroticmicei.p. response in the lungs of transgenic or CS7BII6 BLMMice treated with We have previously isolated and cloned the bacterial protein Sh (10injections).were treated with BLM at 30, 40 or 50 mg/kg twice weekly for 5 weeks ble gene, the product of which is responsible for both prokaryotic For details6.Cumulative of histologic evaluation (left lung), see Fig. and eukaryotic resistance to phleomycin and more generally to OH-prolineMice Injection Histologic antibiotics of the BLM type family (31). The product of the Sh ble (@sg/lung)UC57B1/6dose of BLM route evaluation gene is a BLM-binding protein, routed to nuclei in mammalian cell Control (saline N I77.0 Â±7.88 lines (42), which prevents DNA strand breaks by forming an 300 mg/kg i.p. F+ + 277.8 Â±9.l6'@ inactive complex with the antibiotic (32). Thus, the objective of the 18.0â€•Transgenic400 mg/kg i.p. F++ 280.5 Â± 24.7400Control (saline) N 190.0 Â± present study was to identify a potential method of antagonizing 8.28500mg/kg i.p. F+ 185.4 Â± the pneumotoxic effects of BLM by the in vivo presence of the Sh mg/kg i.p. FÂ± 194.6 Â±5.22 ble protein. To fulfill this purpose, we produced mice transgenic a Results for OH-proline content (right lung) are mean Â± SD of at least seven mice sacrificed 8 weeks after the first injection. for the Sh ble gene. A homozygous TKPH mouse strain carrying b ,@, < o.os, significance of the difference with the saline-treated control group. one copy of the Sh ble transgene was established from heterozy 5663

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. BLEOMYCIN LUNG TOXICITY PREVENTED IN Sh ble TRAN5GENIC MICE gous mice. The expression of the Sh ble gene was studied in hydrolase: molecular cloning, sequencing, and biochemical studies reveal member various organs of adult TKPH mice by immunohistology using an ship in the cysteine proteinase family. Biochemistry, 28: 6544â€”6548, 1989. 6. Ferrando, A. A., Velasco, G., Campo, E., and Lopez-Otin, C. Cloning and expression anti-Sh antibody. Results suggested that the expression level of the analysis of human bleomycin hydrolase, a cysteine proteinase involved in chemo transgene differs depending on tissue types; the Sh ble protein was therapy resistance. Cancer Res., 56: 1746â€”1750,1996. 7. Bromme, D., Rossi, A. B., Smeekens, S. P., Anderson D. C., and Payan, D. G. Human detectable in spleen, kidney, and lung, but it was below a detect bleomycin hydrolase: molecular cloning, sequencing, functional expression, and able level in muscle, liver, and heart. It is not obvious whether this enzymatic characterization. Biochemistry, 35: 6706â€”6714,1996. is due to influence of the integration site of the transgene (position 8. Lazo, J. S., and Humpbreys, C. J. Lack of metabolism as the biochemical basis of bleomycin-induced pulmonary toxicity. Proc. Nail. Aced. Sci. USA, 80: 3064â€”3068, effect; Ref. 43) or to the specificity of the TK promoter driving the 1983. Sh ble gene expression in our construct. It is important to note that 9. Lazo, J. S., Merill, W. W., Pham, E. T., Lynch, T. J., McCallister, J., and Ingbar, D. H. no apparent phenotypic abnormalities and no developmental retar Bleomycin hydrolase activity in pulmonary cells. J. Pharmacol. Exp. Ther., 231: 583â€”588,1984. dation were found, suggesting that the transgene itself did not 10. Hay, J., Shahzeidi, S., and Laurent, G. Mechanisms of bleomycin-induced lung interfere with normal development. damage. Arch. Toxicol., 65: 81â€”94,1991. 11. Hoyt, D. G., and Lazo J. S. Murine strain differences in acute lung injury and The experiments on the induction of fibrosis by BLM treatments activation of poly(ADP-ribose) polymerase by in vitro exposure of lung slices to have shown that fibrosis was induced by i.p. injections of moderate bleomycin. Am. J. Respir. Cell. Mol. Biol., 7: 645â€”651,1992. doses of BLM (cumulative dose, 300 or 400 mg/kg) in C57BL/6 12. Eitzman, D. T., McCoy, R. D., Zheng, X., Fay, W. P., Shen, T., Ginsburg, D., and Simon, R. H. Bleomycin-induced pulmonary fibrosis in transgenic mice that either and 129/Sv mice. In contrast, transgenic mice clearly tolerated lack or overexpress the murine plasminogen activator inhibitor-l gene. J. Clin. higher i.p.-inoculated doses of BLM (50 or 40 mg/kg twice weekly Invest., 97: 232â€”237,1996. during 5 weeks) than the other mice, as evidenced by histological 13. Miyazaki, Y., Amid, K., Vesin, C., Garcia, I., Kapanci, Y., Whitsett, J. A., Piguet, P. F., and Vassalli, P. Expression of a tumor necrosis factor-a transgene in murine examination and lung collagen content. This result could be cor lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive related directly with the transgene expression, because parental pulmonary fibrosis. J. Clin. Invest, 96: 250â€”259,1995. strains of the transgenic mice showed high BLM sensitivity. We 14. Slosman, D. 0., Costabella, P. M., Roth, M., Werlen, G., and Polls, B. S. Bleomycin primes monocytes-macrophages for superoxide production. Eur. Respir. J., 3: 772â€” assumed that Sh ble gene expression in lungs resulted in a suffi 778,1990. cient dose of the binding protein to entrap circulating BLM, thus 15. Zhang, K., Rekhter, M. D., Gordon, D., and Phan, S. H. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined preventing or limiting the accumulation of the antibiotic into lung immunohistochemical and in situ hybridization study. Am. J. Pathol., 145: 114â€” parenchyma and alveolar structures. Because it has been reported 125,1994. that the initial morphological damage seen after systemic injec 16. Piguet, P. F., and Vesin, C. Pulmonary platelet trapping induced by bleomycin: correlation with fibrosis and involvement of the @32integrins. lot. J. Exp. Pathol., 75: tions of BLM is on endothelial cells of the pulmonary capillary bed 321â€”328,1994. (1), biochemical studies will be required to confirm whether BLM 17. Santana, A., Saxena, B., Noble, N. A., Gold, L. I., and Marshall, B. C. increased inactivation occurs de facto in endothelial cells. However, we expression of transforming growth factor @3isoforms(f3 1, f32, @33)in bleomycin induced pulmonary fibrosis. Am. J. Respir. Cell. Mol. Biol., 13: 34â€”44,1995. cannot exclude the possibility that the expression of the Sh ble 18. Piguet, P. F. Is â€œtumornecrosisfactorâ€•the major effector of pulmonary fibrosis? Eur. gene in other organs (spleen and kidney) contributes to the mac Cytokine Netw., 1: 257â€”258,1990. tivation of BLM in the TKPH transgenic mice. It will be very 19. Scheule, R. K., Perkins, R. C., Hamilton, R., and Holian, A. Bleomycin stimulation of cytokine secretion by the human alveolar macrophage. Am. J. Physiol., 262: important to obtain transgenic mice using a specific promoter to L386â€”L39l,1992. target the expression of Sh ble exclusively to the lungs. Such a 20. Baecher, A. C., and Barth, R. K. PCR analysis of cytokine induction profiles associated with mouse strain variation in susceptibility to pulmonary fibrosis. Reg. promoter could be the human surfactant protein C gene promoter, Immunol.,5:207â€”217,1993. because it has already been shown that it confers pulmonary 21. Smith, R. E., Stricter, R. M., Zhang, K., Phan, S. H., Standiford, T. J., Lukacs, N. W., specific gene expression in transgenic mice (44). and Kunkel, S. L. A role for C-C chemokines in fibrotic lung disease. J. Leukocyte Biol., 57: 782â€”787,1995. We have demonstrated that the Sh ble protein expressed in vivo 22. Zhang, K., Gharaee, K. M., Jones, M. L., Warren, J. S., and Phan, S. H. Lung has a suppressive limiting effect on BLM-induced pulmonary monocyte chemoattractant protein-i gene expression in bleomycin-induced puhno fibrosis. Experiments performed with B16 BL/6 melanoma s.c. nary fibrosis. J. Immunol., 153: 4733â€”4741, 1994. 23. Specks, U., Nerlich, A., Colby, T. V., Wiest, I., and Timpl, R. Increased expression transplanted into the TKPH transgenic mice are now being carried oftype VI collagen in lung fibrosis. Am. J. Respir. Crit. Care Med., 151: 1956â€”1964, out to study whether the Sh ble protein expressed in vivo affects the 1995. 24. Hoyt, D. G., and Lazo, J. S. Bleomycin and cyclophosphamide increase pulmonary antitumor activity of BLM and, according to their resistance to type IV procollagen mRNA in mice. Am. J. Physiol., 259: L47â€”L52,1990. BLM-induced lung injury, whether higher BLM doses will prove 25. Shahzeidi, S., Mulier, B., de Crombrugghe, B., Jeffery, P. K., McAnulty, R. J., and more efficacious than current antitumor treatments against B16 Laurent, G. J. Enhanced type ifi collagen gene expression during bleomycin induced lung fibrosis. Thorax, 48: 622â€”628,1993. BL/6 melanoma. 26. Shahzeidi, S., Jeffery, P. K., Laurent, G. J., and McAnulty, R. J. Increased type I procollagen mRNA transcripts in the lungs of mice during the development of bleomycin-induced fibrosis. Eur. Respir. J., 7: 1938â€”1943, 1994. ACKNOWLEDGMENTS 27. Piguet, P. F., Vesin, C., and Thomas, F. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. Transgenic Mice Expressing the Sh ble Bleomycin Resistance Gene Are Protected against Bleomycin-induced Pulmonary Fibrosis

Jérôme Weinbach, Anne Camus, Jacqueline Barra, et al.

Cancer Res 1996;56:5659-5665.

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