[CANCER RESEARCH56, 5659-5665. December 15, 1996]
Transgenic Mice Expressing the Sh ble Bleomycin Resistance Gene Are Protected against Bleomycin-induced Pulmonary Fibrosis1 JérômeWeinbach,
Anne Camus,
Jacqueline
Barra,
Patrick
Dumont,
Monique
Julian,
Suzy Cros, Charles
Babinet,
and Gerard Tirab? Laboratoire de Génétique et Microbiologie, 118 Route de Narbonne, UniversitéPaul Sabatier, 31062 Toulouse Cedex (J. W., G. TI; Unite de Biologie du Développement. 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
Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fl 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 expression
of the actinomycetes
BLM
resistance
protein
Sh ble. Tram
genic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3
ES cells. The protein was detected at high level In lungs, spleen, and kidney. We then assessed its ability to modulate the BLM-induced fibrotic response
in the transgenic
parental
mice.
Cumulative
mice in comparison doses
with CS7BLI6 and 129/Sv
of 300, 400, or 500 mg/kg
BLM
were
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
toxicity, as assessed by lung histology. The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mke. These observations are consistent with the hypoth esis that a resistance gene specifically BLM-induced inflammation.
expressed
In lungs may prevent the
INTRODUCTION Despite its potential great efficacy against various human malig nancies and its relative lack of myelosuppression, the use of the antitumor antibiotic BLM3 is often limited by pulmonary fibrosis (1, 2). This dose-dependent, cumulative, and irreversible side effect (3) is increased by radiation therapy and may develop into fatal hypoxemia if chemotherapy combining BLM administration with other antineo plastic agents is not immediately stopped (4). Variations among organ sensitivity to BLM toxicity have been correlated to the level of a cytosolic-inactivating enzyme named BLM hydrolase (5—7),which is lowest in pulmonary tissues and skin of animal species that are sensitive to BLM-induced pulmonary fibrosis (8, 9). Development of new strategies to prevent BLM-related pulmonary toxicity is closely connected to progress in understanding the com plex, multistep process leading to the final fibrotic state. The sequen
tial pulmonary events induced by the drug are well described (10). Fibrosing alveolitis results from an inflammatory response to an initial injury to the alveolar epithelium. The oxygen radical generated by the degradative action of BLM on DNA is assumed to induce lipid peroxidation, leading to pulmonary inflammation. Most of our in sights on the biochemical and histological changes associated with fibrosis originated from numerous studies conducted with normal or
work
was
supported
by
grants
from
the
Centre
National
de
la
Recherche
Scientifique, La Ligue Nationale Contra Ic Cancer Comité Haute Garonne, the Associa 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. 2 To whom
33-61-55-60-00; 3 The
requests
for reprints
should
be addressed.
Phone:
33-61-55-67-35;
Fax:
E-mail:
[email protected].
abbreviations
used
are:
BLM,
occurs, including infiltration of alveolar macrophages and lympho cytes ( 14), cellular proliferation, and dedifferentiation ( 15). Other changes have been noticed during this period; NAD@ and ATP depletion correlated with the poly(ADP-ribose) polymerase activation (1 1) and platelet trapping to the alveolar endothelium (16). Further more, release of cellular mediators occurs; cytokines such as trans forming growth factor /3 (17), tumor necrosis factor a (18), interleu kins 1—6(19, 20), and chemokines such as macrophage inflammatory protein 1 (21) and monocyte chemoattractant protein 1 (22) mediate the initiation and maintenance of inflammatory lesions and the re cruitment of specific leukocyte populations. Finally, fibroblasts dem onstrate an increase in steady-state levels of mRNA encoding base ment membrane collagens of different types (23—26). Therefore, poly(ADP-ribose) polymerase inhibitors or a NAD@ precursor such as niacin, antagonists of the platelet accumulation or activation (27, 28), anti-transforming growth factor @3 antibodies (29), and human recom binant soluble tumor necrosis factor receptor (30) have been tested in mice to prevent lung injury. All of these provide potentialities for intervening in the fibrotic process in the lung. As an alternative to prevent inflammatory reactions, we investigate in the present study the possibility of a direct inhibition of the BLM activity that injures the lung tissue. Indeed, it has been reported that the bacterial strain producers of the BLM family type of antibiotics contain genes that render them resistant to BLM. An example of such genes is the one isolated from the chromosomal DNA of the tallyso mycin-producing actinomycete Streptoalloteichus hindustanus, the SI, ble gene used in this study (31). The Sh ble gene encodes a small
(14-kilodalton)acidic proteinthat protectsagainstBLM-induced DNA cleavage in vitro, owing to its capacity to bind to Fe (II)-BLM with 1:1 stoichiometry (32). This stable protein appears nontoxic for a variety of eukaryotic organisms in which Sh ble has been expressed (33, 34). These observations lead to the following question: Is it possible to transfer this system into an animal model? To elucidate the role of Sh ble gene expression in vivo to confer BLM resistance in the lung, transgenic mice with the Sh ble gene were produced. This strain was obtained using the D3 ES cell line (35) transfected by the linearized plasmid pUT 526 carrying the Sh ble gene under the control of the polyoma enhancer-TK promoter (kindly provided by CAYLA, Toulouse, France). This in situ expression model allowed us to test the ability of this protein to modulate BLM-induced pulmonary fibrosis, in comparison with BLM-treated C57BU6 mice. Murine strain
C57BL/6waschosenfor the presentinvestigationbecauseof its
Received6/10/96; accepted10/14/96.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This
lung fibrosis
(11—1 3). After initial biochemicallesions,a periodof inflammation
bleomycin;
ES,
embryonic
kinase; NA, numerical aperture; OH-proline, hydroxyproline.
stem;
TK,
thymidine
characterization as a good model for BLM-induced pulmonary fibro sis, as evidenced by morphometric analysis of histological lesions and increased pulmonary OH-proline content, a convenient index of col lagen deposition, after s.c. BLM administration (36). Fibrosis was further diagnosed at the end of our experiment by conventional determination of pulmonary collagen and histology. The present study demonstrates the ability of the Sh ble protein to prevent lung toxicity of BLM.
5659
BLEOMYCIN LUNG TOXICITY PREVENTED IN Sh ble TRANSGENIC MICE
BstEIL Bas@HL
Bai@HL HpaL 1612 BgIIL
XhoL
1481
BstElL
T
1621
* Polyoma Enh.ITK prom
Sh ble
SV4O splice
PolyA Opi ColEl
ampr
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 Production
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 constant voltage (1 V/cm). DNA fragments were transferred to a nylon
AND METHODS of Transgenic
Mice. Transgenic
mice carrying
the Sh ble
gene, which confers BLM resistance, were generated by the use of the ES cells strategy as described previously line (35), expressing
(37). For this purpose, the D3 ES cell
the Sit ble gene under
the control
of the TK promoter
enhancer-promoter as a probe. Hybridization was carried out for 16—20 h at 42°C with 5 x 106cpm/ml heat-denatured, 32P-labeledprobe (specific activity, 1.0-2.0 x l0@cpm4@g).The membrane was washed, exposed to Kodak XRP X-ray film, and kept for 3 days at —80°C. PCR was performed using a Thermojet machine (Eurogentec, Seraing,
and polyoma enhancer (38), has been established as follows. BstEII linearized plasmid pUT 526 (CAYLA) was electroporated into D3 ES cells (isolated from 129/Sv blastocysts). We preferred indeed to use the entire linearized pUT526 plasmid rather than only the enhancer-TK-Sh ble se
quence, protecting the Sh ble gene sequence from exonuclease activity during D3 ES cell transfection. Cells from one of the resistant clones recovered
after selection with 5 ,.tg/ml phleomycin
membrane. Southern blot analysis were performed using a BamHl fragment from pUT 526 containing the Sh ble gene under the dependence of the viral
(CAYLA) were micro
Belgium). PCRs were cycled 30 times (one cycle consisting of 1 mm at 94°C, 1 mm at 55°C,and 2 mm at 72°C)with Tfl DNA polymerase (Epicentre 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 C57BL/6 female mated with one chimeric male gave birth to transgenic
pUT 526 sequences: the 5' and 3' primers were 5'-ATGGCCAAGTTGAC
progeny, allowing the establishment
sponding to the 326—345and 1658—1677nucleotide sequences of the pUT
of the transgenic
mouse strain TKPH.
Homozygous mice for the S/i ble transgene did not display any abnormal phenotype
and reproduced
normally.
Analysis of Tail DNA. Tail segments of 1—1.5 cm were reduced to powder under liquid N2. DNA was extracted by digesting tissues overnight at 37°Cin
3 ml of lysis buffer containing50 mMTris-HC1(pH 7.5), 0.5% SDS, 0.1 M NaCI, 5 mM EDTA, and 100 @tg/mlproteinase K. DNA was then extracted with phenol-chloroform and precipitated with ethanol. The DNA pellet was col
lected, washed with 70% ethanol, and then resuspended in 200 @tl of distilled water. Transgenic mice were identified either by Southern blot or PCR.
4.072 Kb 3,054 Kb
@
2,036 Kb
@
1,636Kb
@
1.018Kb
526, giving rise to an amplification
respectively, corre
product of 1350 bp.
Immunohistochemical Localization of the Sh ble Protein. Organs from sacrificed animals were mounted in ornithine carbamyl transferase compound (Miles Laboratories, Inc., Napperville, IL) and flash frozen in isopentane cooled by liquid N2 for cryosectioning. Sections (10 @sm thick) were fsxed for 30 mm in 4% p-formaldehyde in PBS (pH 7.4). Subsequently, the cells were permeabilized by incubation in 2% p-formaldehyde, 0.1% Triton X-l00, and 2% BSA in PBS for 15 mm at room temperature. Tissue sections were washed 3 X for 5 mm each with PBS, and nonspecific antibody binding was blocked
mented with 5% normal goat serum. Tissue sections were then incubated with
the primary polyclonal antibody anti-Sh (dilution, 1:100; obtained from CAYLA) overnightat room temperature.Afterthreewashingswith bufferA, sections were incubated with the secondary biotinylated goat antirabbit IgG for 1 h at room temperature. Tissue sections were washed and incubated with fluorescein-conjugated streptavidin during 30 mm at room temperature. After three final washings with buffer A, sections were processed for microscopic examination (moviol embedded). Light and fluorescence microscopy of the 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
396 Kb
and 5'-ACCGTAUACCGCC1Tl@GAG-3',
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 25°Cwith buffer A supple
1. 2. 3. 4. 5. @ @
CAGTGC-3'
NA, 0.5; X40 Apofluo; NA, 0.75; and an oil immersion objective, X63 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 food (Usine d'Alimentation Rationnelle type A03) and water. To obtain maximal BLM-induced lung lesions, several protocols for treat ment of mice with various BLM concentrations were designed. BLM sulfate
@.
Fig. 2. Southern blot analysis of transgenic mice. Tall DNA (10 sag) from transgenic mice was digested with appropriate restriction enzymes Bgl and Bst ElI, followed by
electrophoresis on a 0.8% agarose gel for 12h. DNA was transferred to a nylon membrane and hybridized with a 32P-labeledprobe, the Barn Hl fragment from pUTS26 containing (BELLON, Neuilly-sur-Seine, the Sh ble gene under the dependence of the Polyoma enhancer and TK promoter. 10 @.sg of digested tail DNA from C57BL16 mouse containing
suitable
50 pg of the 883 bp Barn
HI-fragment from pUTS26@containing the Sb ble gene was used for a positive control. Digested tail DNA from C57BU6 mouse was used for a negative control. Lanes 1—2: Bglll-Bst ElI digested tall DNA from a fourth generation transgenic mouse; Lane 3: 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 5: Negative control: C57BL/6 mouse DNA.
concentration
France) dissolved in sterile saline solution at the
was administered
by either
the i.p. or s.c. route
(0.1
mI/lO g of body weight) as five doses of 20 mg/kg twice weekly for 3 weeks
(cumulative dose, 100 mg/kg) or 10 doses of 30, 40, or 50 mg/kg twice weekly for 5 weeks (cumulative dose, respectively, 300, 400, or 500 mg/kg). Control mice received equivalent volumes of saline. Mice were weighed twice weekly, and mortality was recorded daily. At the
5660
BLEOMYCIN LUNG TOXICITY PREVENTED IN Sh ble TRANSGENIC MICE
Fig. 3. Immunohistochemical
A
B
C
D
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.
:
f
E
@
@::
F
. .
@. .@
t@
@
.
‘@5.\
@
.
. .
:
.
@t@:
:
times indicated, mice were sacrificed, and fibrosis was further diagnosed by
appropriate
determination of pulmonary collagen (right lung) and histological examination
@ourrages) program using Student's t test (P < 0.05) as described by Snedecor and Cochran (41).
(left lung). The care and use of animals was in accordance with the guidelines of the Council of European Communities (cancellation 86/C 331/01). Assay for Lung OH-proline Content. Lung OH-proline content, an index of collagen, was determined by the method of Woessner (39) as applied to the lung assay (40). The right lung of each individual mouse was dissected free of major bronchi, and the lobes were minced in 1-mm-thick pieces. They were hydrolyzed in 2 ml of 6 N HC1at 110°C overnight in tightly capped tubes to
groups by a Stat.ITCF (jnstitut Technique
des cereales
et des
Histological Examination. The left lung of each individual mouse was fixed for 24—48 h in a 10% p-formaldehyde solution and then embedded in paraffin. The lungs were then cut into parasagittal slices (5 ,.sm thick) and
stained either with H&E or with Masson's trichrome for the identification of collagen fibrils in the region of the bronchiole-alveolar duct junction over large areas of the lung parenchyma. The fibrotic response was blindly 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
F±,thickening of the alveolar wall, characterized by an alveolar cell
N NaOH
proliferation, i.e., a fibroblastic metaplasia with more or less collagen deposition in the wall; F+, confined dense fibrotic lesions, characterized
and extracted
with
phenol-chloroform-isoamyl
alcohol
to clarify
the
aqueous phase. The extracts were filtered through a 0.45-pm membrane (Minisart; Sartorius), adjusted to pH 6—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 weight (@Lg)of OH-proline/right lung. Purified OH-proline was used as a
regions
are swallowed
fibrotic
areas,
standard. The values are reported as the mean ±SEM and analyzed among
part or the whole pulmonary 5661
by collagen
characterized
fibers;
by wide acellular
lobe.
and F+ +, mutilating collagenic
tissue
large
including
a
BLEOMYCIN LUNG TOXICITY PREVENTED IN Sh ble TRANSGENIC MICE
Table I Evaluation of the pulnionars fibrotic response to various BLM treatments BLM treatments 120, 30, 40, or 50 mg/kg twice weekly for 5 weeks (total, 10 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
bleomycin. For histologic evaluation, the fibrotic response was blindly judged on each section of the left lung stained with Masson's trichrome for identification of collagen fibrils. (@.sgRung)°129/SvControl MiceCumulativedose of BLMInjection routeHistologicevaluationOH-proline (saline) 100 mg/kg 400 mg/kgs.c. 18.7cC57BL16Control
s.c. i.p.N
(saline) 100 mg/kg 300 mg/kg
s.c. i.p. s.c. i.p.N
400 mg/kgs.c. 15.5'TransgenicControl
a Results
for OH-proline
content
(right
±12.1
207.0 ±21.6
F+ (6/7) F+ (4/8)176.0
292.7 ±26.4' 266.4 ±
(0/10) F±(3/10)
±8.5 188.2 ±11.1
F+ F+ F+ F+
235.9 258.1 262.6 279.3
(7/8) (6/8) + (6/7) (6/7)199.1
±19.9' ±9.5' ±24.2c ±
(0/10) N (0/10)
±14.3 161.4 ±12.3
i.p.
F±(1/4)
195.8 ±21.8
i.p.N
N (0/6)170.9
198.4 ±16.5
(saline) 100 mg/kg 400 mg/kg 500 mg/kgs.c.
(O/lO)@'
F±(4/10)
lung)
are mean
± SD.
b Numbers in parentheses are numbers of mice affected/numbers
of mice examined.
( P
control
