Cancer
Treatment
Reviews
(1990)
The molecular fibrosis caused John
S. Lazo
17, 165-167
basis of interstitial by antineoplastic
and Dale
pulmonary agents
G. Hoyt
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.
E- 1346 Biomedical
Science Tower,
Pulmonary toxicity is an important, proliferation-independent, undesirable aspect of anticancer chemotherapy, which can ultimately develop into lethal interstitial pulmonary fibrosis. The loss of lung function, which defines pulmonary fibrosis, is due to pathological alterations in the content or composition of extracellular matrix components such as collagen, fibronectin, glycosaminoglycans and elastin (1). Biochemical studies suggest that the alteration in matrix is due primarily to an increase in extracellular matrix deposition. The altered synthesis of matrix may reflect an expansion of a cell population (e.g. fibroblasts) (15, 20). New matrix-producing cells also may be recruited into the lungs by the chemotactic activities of polypeptides (15). I n addition, the constitutive levels of matrix synthesis by the resident cell population could be altered. There is increasing evidence that endogenous growth factors and cytokines participate in the remodeling of the interstitial regions of the lungs (20). One focus of our research has been to identify these factors and the mechanisms that regulate altered collagen and extracellular matrix deposition. We have studied the pulmonary toxicity of the DNA interactive anticancer agents, bleomycin (BLM) and cyclophosphamide (CYC). Wh en administered to animals, they cause a reproducible fibrosis that has great similarity to the final fibrotic lesion seen in humans (4, 8, 16). We and others (3, 4, 7, 8, 16) have identified inbred murine strain differences in the pulmonary responsiveness to both BLM and CYC. Thus, C57B1/6 mice are much more sensitive to BLM-induced fibrosis compared to BALB/c mice. Conversely, BALB/c mice are more sensitive to CYC-induced fibrosis than C57B1/6 mice. The differences in strain sensitivity to these fibrogenic agents may reflect differences in drug metabolizing enzymes in the lungs (3, 7). In the case of BLM, the resistant strains have higher levels of the inactivating enzyme, bleomycin hydrolase, which has recently been identified as a member of the thiol protease family (17). Th e availability of murine strains with different sensitivities to fibrogenic drugs provides an important tool for identifying essential prefibrotic factors of fibrosis. The accumulation of pulmonary collagen in C57B1/6N mice after S.C. BLM infusion is preceded by sequential increases in total pulmonary fibronectin, ~(~111 procollagen and sl,I procollagen mRNA (7), as well as increases in a,IV procollagen and a,IV procollagen mRNA levels. Other laboratories have observed similar results with fibronectin, a,111 procollagen and a,1 procollagen mRNA in rats after BLM (2, 9, 13, 14). In our studies, however. these increases are largely confined to the sensitive C57B1/6N mice and are 0305-7372/90/2&3165+03
ii” 1990 Acadmnic
9603.00/o 165
Pwss
I.imitrd
166
J. S. LAZO
AND
D.
G.
HOYT
delayed and/or reduced in the resistant BALB/cN mice (7). Furthermore, after i.p. CYC treatment, increases in steady-state pulmonary ~(~111, CQI, a,IV and a,IV procollagen mRNA levels are seen only in the responding BALB/cN mice (8). The increase in mRNA levels for matrix components seen in responsive mice may be due to an increase in the rate of transcription ( 14). Degradation of collagen may be reduced also, since after exposure of lungs to BLM, a decrease in pulmonary collagenase activity has been observed (9). Although BLM may have some limited direct effects on fibroblast proliferation and collagen production (2, IO), pulmonary fibrosis is a multicellular event and endogenous polypeptide factors appear to control normal matrix deposition. One candidate fibrogenic factor is transforming growth factor B, (TGF-/?,), w h ic h is a multifunctional homodimeric peptide that belongs to a family of potent growth factors (18). TGF-/?, stimulates the expression of multiple matrix genes (11). TGF-PI m . t eracts with a nuclear factor protein (NF-I) to increase transcription of procollagen cr,I gene; it also increases steady-state levtls of fibronectin mRNA and has been shown to stabilize the mRNA for fibronectin in some cells (11). TGF-8, has b een found in lungs and results from our laboratory (7, 8) demonstrate that mRNA encoding TGF-P, increases prior to drug-induced pulmonary fibrosis only in sensitive murine strains independent of the fibrogenic agent. This observation has lead to the hypothesis that transient increases in TGF-/3, mediate drug-induced interstitial pulmonary fibrosis, perhaps in conjunction with other cytokines, such as interleukin- 1 or tumor necrosis factor. It is unclear what triggers the increase in TGF-P, mRNA levels in the lungs. Druginduced effects on gene structure may affect cytokine gene expression. The most prominent pharmacologic properties of BLM and CYC are their actions on DNA. Bleomycin cleaves DNA; cyclophosphamide alkylates DNA. DNA interactive agents have been found to increase some mRNA transcripts in cultured cells and pulmonary tissue (5, 6). We have found that subsequent to the initial DNA damage, the lungs of a sensitive murine strain sustain a more prolonged DNA damage than a resistant strain after a single iv. injection of BLM (5). Whether similar strain-selective DNA damage occurs after CYC treatment has not been determined. Information from mechanistic studies with anticancer drugs in animal models have lead to pharmacological attempts to inhibit the initiation and progression ofpulmonary fibrosis. Unfortunately few of these approaches have found their way into common clinical usage. This is due in part to their adverse effects on other tissues or to the transient nature of the blockade (2). Furthermore, these agents are generally oflittle usefulness in the later stages of the fibrotic process. One approach that may help prevent the pulmonary fibrosis associated with BLM is to develop novel analogs of the drug that lack this serious untoward effect. Liblomycin, a lipophilic analog ofBLM, has recently been identified as one potential candidate (19). Further studies on the mechanisms by which current antineoplastic agents produce pulmonary toxicity may provide information useful in the design of less toxic agents and could also lead to better diagnosis and therapy for this serious disorder.
References 1. Adamson, I. Y. R. (1984) Drug-induced pulmonary fibrosis. Environ. Health. Perspec. 55: 25-36. 2. Cutroneo, K. R. & Sterling, K. M. Jr. (1988) The biochemical and molecular bases of bleomycin-induced pulmonary fibrosis. In: Hollinger, M., ed., Focus on Pulmonary Pharmacology and Toxicolou, Vol. 1, pp. l-22. Boca Raton, FL: CRC Press.
PULMONARY 3. Ekimoto, Different
FIBROSIS
H., Takada, K., Ohnuki, T., sensitivity to bleomycin-induced
Nutr. 2: 25-3 1. 4. Harrison, J. H. & Laze,
J. S. (1987)
AND
Takahashi, pulmonary
High
M. C. & Fornace,
49: 1687-1692. 7. Hoyt, D. G. & Laze, transforming
Thu.
J. S. (1988)
growth
246: 765-77
A. J. Jr.
factor-b
(1989)
infusion
Thu.
of bleomycin
in mice:
pulmonary and -resistant
offis
in pulmonary
RNA
a new model
of bleomycin: of mice. Mol.
by DNA
mRNA
bleomycin-induced
toxicity strains
damaging
encoding
pulmonary
for
DNA
scission
Pharmacol.
fibronectin
and
J. Pharmacol.
in mice.
36:
Cancer Res.
agents.
procollagens,
fibrosis
Exp.
1.
8. Hoyt, D. G. & Laze, J. S. (1989) Early increases in pulmonary mRNA transforming growth factor-/I in mice sensitive to cyclophosphamide-induced
macol. Ex$
167
242: 118551194.
Acute
Induction
Alterations
precede
AGENTS
K., Matsuda, A., Takita, T. & Umezawa, H. (1987) fibrosis among various strains of mice. J. Clin. B&hem.
dose continuous
drug-induced pulmonary fibrosis. J. Pharmacol. Exp. 5. Harrison, J. H. Jr., Hoyt, D. G. & Laze, J. S. (1989) and matrix protein mRNA levels in bleomycin-sensitive 231-238. 6. Hollander,
ANTICANCER
encoding pulmonary
procollagens and fibrosis. J. Phar-
Ther. 249: 38-43.
9. Kellcy, J., Chrin, mRNA levels for 131: 836-843. 10. Moseley, bleomycin.
L., Shull, procollagen
S., Rowe, D. W. & Cutroneo, K. R. (1985) and fibronectin following acute lung injury.
P. L., Hemken, C. & Hunninghake, J. Clin. Invest. 78: 1150-l 154.
G. W.
(1986)
Bleomycin
selectively
Biochem. Biophys.
Augmentation
of fibroblast
elevates
Rex. Comnun.
proliferation
by
11. Pcnttincn, R. P., Kobayashi, S. & Bornstein, P. (1988) Transforming growth factor p increases mRNA for matrix proteins both in the presence and absence of changes in mRNA stability. Pm. Natl. Acad. Sci. USA 85: 110551108. 12. Phan, S. H., Varani, pulmonary 13. Raghow,
J. & Smith,
in cultured
hamster
Rat lung
fibroblast
Phenotypic
collagen
plasticity
metabolism
ofextracellular
in bleomycin-induced matrix
lung libroblasts: regulation of type I procollagen and fibronrctin 15. S., Seyer, J. M. & Kang, A. H. (1985) Profiles ofsteady-state levels
Chem. 262: 8409-84 14. Raghow,
D. (1985)
J. C/in. Inuest. 76: 241-247. A. H. & Pidikiti, D. (1987)
fibrosis. R., Kang,
R., Lurie,
gene expression synthesis.
J. Biol.
of messenger
RNAs
coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis. ./. Clin. Inuest. 76: 173331739. 15. Rennard, S. I., Hunninghake, G. W., Bitterman, P. B. & Crystal, R. G. (1981) Mechanism offibrosis. Pmt. ,Vatl. Acad. Sci. USA 78: 7 14777 15 1. 16. Schrier, induced
D. J., Kunkel, R. pulmonary fibrosis.
G. & Ph an, S. H. Am. Reu. Resp. Dir.
(1983) The 127: 63-66.
role
of strain
variation
in murine
bleomycin-
17. S&i, S. M., Mignano, J. E., Jani, J. P., Srimatkandada, S. & Laze, J. S. (1989) Bleomycin hydrolase: molecular cloning, sequencing and biochemical studies reveals membership in the cyst&e proteinase family.
Bimhemist7y 18. Sporn,
28: 6544-6548.
.X4. B. & Roberts,
19. Takakita,
A. B. (1988)
7‘. & Ogino, Pharmacother. 41: 219-226.
20. Lt’itschi,
I:und. A#l.
H. P., ‘I‘ryka,
Toxicol.
T.
(1987)
Peptide
growth
Peplomycin
and
A. F. & Lindenschmidt,
5: 240-250.
factors
are multifunctional.
liblomycin,
R. C. (1985)
a new
Th e many
analogues
Nature 332: 217-219. of bleomycin. Biomed.
faces of an increase
in lung collagen.
Treatment
Reviews
(1990)
The molecular fibrosis caused John
S. Lazo
17, 165-167
basis of interstitial by antineoplastic
and Dale
pulmonary agents
G. Hoyt
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.
E- 1346 Biomedical
Science Tower,
Pulmonary toxicity is an important, proliferation-independent, undesirable aspect of anticancer chemotherapy, which can ultimately develop into lethal interstitial pulmonary fibrosis. The loss of lung function, which defines pulmonary fibrosis, is due to pathological alterations in the content or composition of extracellular matrix components such as collagen, fibronectin, glycosaminoglycans and elastin (1). Biochemical studies suggest that the alteration in matrix is due primarily to an increase in extracellular matrix deposition. The altered synthesis of matrix may reflect an expansion of a cell population (e.g. fibroblasts) (15, 20). New matrix-producing cells also may be recruited into the lungs by the chemotactic activities of polypeptides (15). I n addition, the constitutive levels of matrix synthesis by the resident cell population could be altered. There is increasing evidence that endogenous growth factors and cytokines participate in the remodeling of the interstitial regions of the lungs (20). One focus of our research has been to identify these factors and the mechanisms that regulate altered collagen and extracellular matrix deposition. We have studied the pulmonary toxicity of the DNA interactive anticancer agents, bleomycin (BLM) and cyclophosphamide (CYC). Wh en administered to animals, they cause a reproducible fibrosis that has great similarity to the final fibrotic lesion seen in humans (4, 8, 16). We and others (3, 4, 7, 8, 16) have identified inbred murine strain differences in the pulmonary responsiveness to both BLM and CYC. Thus, C57B1/6 mice are much more sensitive to BLM-induced fibrosis compared to BALB/c mice. Conversely, BALB/c mice are more sensitive to CYC-induced fibrosis than C57B1/6 mice. The differences in strain sensitivity to these fibrogenic agents may reflect differences in drug metabolizing enzymes in the lungs (3, 7). In the case of BLM, the resistant strains have higher levels of the inactivating enzyme, bleomycin hydrolase, which has recently been identified as a member of the thiol protease family (17). Th e availability of murine strains with different sensitivities to fibrogenic drugs provides an important tool for identifying essential prefibrotic factors of fibrosis. The accumulation of pulmonary collagen in C57B1/6N mice after S.C. BLM infusion is preceded by sequential increases in total pulmonary fibronectin, ~(~111 procollagen and sl,I procollagen mRNA (7), as well as increases in a,IV procollagen and a,IV procollagen mRNA levels. Other laboratories have observed similar results with fibronectin, a,111 procollagen and a,1 procollagen mRNA in rats after BLM (2, 9, 13, 14). In our studies, however. these increases are largely confined to the sensitive C57B1/6N mice and are 0305-7372/90/2&3165+03
ii” 1990 Acadmnic
9603.00/o 165
Pwss
I.imitrd
166
J. S. LAZO
AND
D.
G.
HOYT
delayed and/or reduced in the resistant BALB/cN mice (7). Furthermore, after i.p. CYC treatment, increases in steady-state pulmonary ~(~111, CQI, a,IV and a,IV procollagen mRNA levels are seen only in the responding BALB/cN mice (8). The increase in mRNA levels for matrix components seen in responsive mice may be due to an increase in the rate of transcription ( 14). Degradation of collagen may be reduced also, since after exposure of lungs to BLM, a decrease in pulmonary collagenase activity has been observed (9). Although BLM may have some limited direct effects on fibroblast proliferation and collagen production (2, IO), pulmonary fibrosis is a multicellular event and endogenous polypeptide factors appear to control normal matrix deposition. One candidate fibrogenic factor is transforming growth factor B, (TGF-/?,), w h ic h is a multifunctional homodimeric peptide that belongs to a family of potent growth factors (18). TGF-/?, stimulates the expression of multiple matrix genes (11). TGF-PI m . t eracts with a nuclear factor protein (NF-I) to increase transcription of procollagen cr,I gene; it also increases steady-state levtls of fibronectin mRNA and has been shown to stabilize the mRNA for fibronectin in some cells (11). TGF-8, has b een found in lungs and results from our laboratory (7, 8) demonstrate that mRNA encoding TGF-P, increases prior to drug-induced pulmonary fibrosis only in sensitive murine strains independent of the fibrogenic agent. This observation has lead to the hypothesis that transient increases in TGF-/3, mediate drug-induced interstitial pulmonary fibrosis, perhaps in conjunction with other cytokines, such as interleukin- 1 or tumor necrosis factor. It is unclear what triggers the increase in TGF-P, mRNA levels in the lungs. Druginduced effects on gene structure may affect cytokine gene expression. The most prominent pharmacologic properties of BLM and CYC are their actions on DNA. Bleomycin cleaves DNA; cyclophosphamide alkylates DNA. DNA interactive agents have been found to increase some mRNA transcripts in cultured cells and pulmonary tissue (5, 6). We have found that subsequent to the initial DNA damage, the lungs of a sensitive murine strain sustain a more prolonged DNA damage than a resistant strain after a single iv. injection of BLM (5). Whether similar strain-selective DNA damage occurs after CYC treatment has not been determined. Information from mechanistic studies with anticancer drugs in animal models have lead to pharmacological attempts to inhibit the initiation and progression ofpulmonary fibrosis. Unfortunately few of these approaches have found their way into common clinical usage. This is due in part to their adverse effects on other tissues or to the transient nature of the blockade (2). Furthermore, these agents are generally oflittle usefulness in the later stages of the fibrotic process. One approach that may help prevent the pulmonary fibrosis associated with BLM is to develop novel analogs of the drug that lack this serious untoward effect. Liblomycin, a lipophilic analog ofBLM, has recently been identified as one potential candidate (19). Further studies on the mechanisms by which current antineoplastic agents produce pulmonary toxicity may provide information useful in the design of less toxic agents and could also lead to better diagnosis and therapy for this serious disorder.
References 1. Adamson, I. Y. R. (1984) Drug-induced pulmonary fibrosis. Environ. Health. Perspec. 55: 25-36. 2. Cutroneo, K. R. & Sterling, K. M. Jr. (1988) The biochemical and molecular bases of bleomycin-induced pulmonary fibrosis. In: Hollinger, M., ed., Focus on Pulmonary Pharmacology and Toxicolou, Vol. 1, pp. l-22. Boca Raton, FL: CRC Press.
PULMONARY 3. Ekimoto, Different
FIBROSIS
H., Takada, K., Ohnuki, T., sensitivity to bleomycin-induced
Nutr. 2: 25-3 1. 4. Harrison, J. H. & Laze,
J. S. (1987)
AND
Takahashi, pulmonary
High
M. C. & Fornace,
49: 1687-1692. 7. Hoyt, D. G. & Laze, transforming
Thu.
J. S. (1988)
growth
246: 765-77
A. J. Jr.
factor-b
(1989)
infusion
Thu.
of bleomycin
in mice:
pulmonary and -resistant
offis
in pulmonary
RNA
a new model
of bleomycin: of mice. Mol.
by DNA
mRNA
bleomycin-induced
toxicity strains
damaging
encoding
pulmonary
for
DNA
scission
Pharmacol.
fibronectin
and
J. Pharmacol.
in mice.
36:
Cancer Res.
agents.
procollagens,
fibrosis
Exp.
1.
8. Hoyt, D. G. & Laze, J. S. (1989) Early increases in pulmonary mRNA transforming growth factor-/I in mice sensitive to cyclophosphamide-induced
macol. Ex$
167
242: 118551194.
Acute
Induction
Alterations
precede
AGENTS
K., Matsuda, A., Takita, T. & Umezawa, H. (1987) fibrosis among various strains of mice. J. Clin. B&hem.
dose continuous
drug-induced pulmonary fibrosis. J. Pharmacol. Exp. 5. Harrison, J. H. Jr., Hoyt, D. G. & Laze, J. S. (1989) and matrix protein mRNA levels in bleomycin-sensitive 231-238. 6. Hollander,
ANTICANCER
encoding pulmonary
procollagens and fibrosis. J. Phar-
Ther. 249: 38-43.
9. Kellcy, J., Chrin, mRNA levels for 131: 836-843. 10. Moseley, bleomycin.
L., Shull, procollagen
S., Rowe, D. W. & Cutroneo, K. R. (1985) and fibronectin following acute lung injury.
P. L., Hemken, C. & Hunninghake, J. Clin. Invest. 78: 1150-l 154.
G. W.
(1986)
Bleomycin
selectively
Biochem. Biophys.
Augmentation
of fibroblast
elevates
Rex. Comnun.
proliferation
by
11. Pcnttincn, R. P., Kobayashi, S. & Bornstein, P. (1988) Transforming growth factor p increases mRNA for matrix proteins both in the presence and absence of changes in mRNA stability. Pm. Natl. Acad. Sci. USA 85: 110551108. 12. Phan, S. H., Varani, pulmonary 13. Raghow,
J. & Smith,
in cultured
hamster
Rat lung
fibroblast
Phenotypic
collagen
plasticity
metabolism
ofextracellular
in bleomycin-induced matrix
lung libroblasts: regulation of type I procollagen and fibronrctin 15. S., Seyer, J. M. & Kang, A. H. (1985) Profiles ofsteady-state levels
Chem. 262: 8409-84 14. Raghow,
D. (1985)
J. C/in. Inuest. 76: 241-247. A. H. & Pidikiti, D. (1987)
fibrosis. R., Kang,
R., Lurie,
gene expression synthesis.
J. Biol.
of messenger
RNAs
coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis. ./. Clin. Inuest. 76: 173331739. 15. Rennard, S. I., Hunninghake, G. W., Bitterman, P. B. & Crystal, R. G. (1981) Mechanism offibrosis. Pmt. ,Vatl. Acad. Sci. USA 78: 7 14777 15 1. 16. Schrier, induced
D. J., Kunkel, R. pulmonary fibrosis.
G. & Ph an, S. H. Am. Reu. Resp. Dir.
(1983) The 127: 63-66.
role
of strain
variation
in murine
bleomycin-
17. S&i, S. M., Mignano, J. E., Jani, J. P., Srimatkandada, S. & Laze, J. S. (1989) Bleomycin hydrolase: molecular cloning, sequencing and biochemical studies reveals membership in the cyst&e proteinase family.
Bimhemist7y 18. Sporn,
28: 6544-6548.
.X4. B. & Roberts,
19. Takakita,
A. B. (1988)
7‘. & Ogino, Pharmacother. 41: 219-226.
20. Lt’itschi,
I:und. A#l.
H. P., ‘I‘ryka,
Toxicol.
T.
(1987)
Peptide
growth
Peplomycin
and
A. F. & Lindenschmidt,
5: 240-250.
factors
are multifunctional.
liblomycin,
R. C. (1985)
a new
Th e many
analogues
Nature 332: 217-219. of bleomycin. Biomed.
faces of an increase
in lung collagen.
