Cancer Letters, 61 (1991)
15-
15
20
Elsevier Scientific Publishers Ireland Ltd.
Lung adenoma structure among inbred strains of mice: the pulmonary adenoma histologic type (P&I) genes Muriel N. Nesbitta and Alvin M. Malkinsonb
Larry G. Thaete”,
Toxicology and Enoironmental Health Sciences Program, Colorado Cancer Center, School of Pharmacy, University of Colorado, Boulder, CO 80309-0297 and *Department of Biology, University of California at San Diego, La Jolla, CA 92093 (U.S.A.) “Molecular
(Received 27 June 1991) (Revision received 29 August (Accepted 30 August 1991)
1991)
Summary Urethane-induced, lung adenoma multipliciand histologic type oary among mouse strains. We asked whether the Pas genes which control multiplicity also determine adenoma structure. Lung adenomas from inbred mice, Fl hybrids, and recombinant inty
bred mice were classified by growth pattern as either solid or papillary. Since no correlation was obserued between adenoma multiplicity and histologic type, no linkage apparently exists between the Pas genes and adenoma morphology . We propose the name Pah
(Pulmonary Adenoma Histologic genes controlling lung adenoma
type) for the
growth patterns. Genetic analysis indicated dominance of the papillary phenotype, and that two or more
Pah genes determine
adenoma
structure.
Keywords: mouse; lung; adenoma; genetics; recombinant inbred strains Introduction Primary
lung neoplasms
Correspondence Campus
Box
80309-0297,
in mice develop
to: Alvin M. Malkinson, 297,
University
U.S.A.
0304-3835/91/$03.50
0
1991
Published and Printed in Ireland
of
School of Pharmacy,
Colorado,
Boulder,
CO
through multiple stages, from hyperplasia to a benign stage to malignancy [3,11,19]. The adenomas are observed to be of two histologic types. Solid adenomas grow along the alveolar septae as compact masses and do not form distinct borders [ 1,10,111. These adenoma cells possess characteristics of type II alveolar cells, such as surfactant-containing lamellar bodies [5,22]. Papillary adenomas grow as finger-like projections and form a pseudocapsule of compressed normal tissue at their border. Their cells possess some of the morphologic [lo] and biochemical [21] characteristics of bronchiolar non-ciliated Clara cells, but the cellular derivation of these neoplasms is somewhat controversial [ 13,171. Papillary neoplasms have a longer latency [9] and are more frequently associated with malignancy than are solid adenomas [20]. Strains of mice vary in their susceptibility to spontaneous and carcinogen-induced lung tumorigenesis due to the actions of several pulmonary adenoma susceptibility (Pas) genes [12,13,15]. Interestingly, a large subset of human called lung adenocarcinomas, bronchiole-alveolar [6] or papillolepidic carcinomas [4], share both these presumptive cellular origins and a strong genetic basis for susceptibility [ 121. Strains of mice vary in the relative proportion of solid and papillary adenomas which oc-
Elsevier Scientific Publishers Ireland Ltd
16
cur in response to the carcinogen, urethane [ 1,20,23]. Both histologic types of adenomas may be present in lungs of individual mice but the relative proportion of the two types is strain-dependent, with little individual variation within a strain. We sought to determine whether adenoma morphology is also controlled by the Pas genes or by a distinctly different set of genes, This was done by examining tumor structure in the AXB and BXA recombinant inbred (RI) strains, derived from A/J and C57BL/6J (B6) progenitors, whose Pas phenotype had been previously established [ 151. Additionally, crosses between various inbred strains were done to study further the inheritance of adenoma structure. The concept of pulmonary adenoma histologic type (Pah) genes has been briefly introduced previously [13]. This paper presents extensive new data from many inbred and RI strains not previously analyzad to support the existence of Pah genes which determine lung adenoma structure independently of adenoma multiplicity. Materials and Methods Animals
Male and female adult mice of the A/J, AKR/J, CBA/J, C57BL/6J (B6), C58/J, DBA/ZJ, LP/J, PL/J, MA/MyJ, SJL/J and SM/J strains were obtained from Jackson Laboratories (Bar Harbor, ME), and strains GRS/SN, NGP/N, and ST/BN were obtained from the National Institutes of Health (Bethesda, MD). F1 hybrid mice were generated by mating A/J females with either MA/My, AKR, or DBA males. Thirty-eight AXB and BXA RI strains developed from A/J and B6 progenitors [ 161 were shipped from La Jolla to Boulder. Mice were maintained on hardwood bedding, kept on a 12-h light/dark cycle, and provided food (Wayne Lab Blox) and water ad libitum, in accordance with National Institutes of Health guidelines. All mice were housed for 2 weeks before being used experimentally. Lung
adenoma
induction
and processing
Mice were injected i.p. with 1 mg urethane
(Sigma, St. Louis, MO) in 0.9% NaCl/g body weight at 8 weeks of age, as described previously [14]. The mice were killed by cervical dislocation 14 weeks post-treatment, lungs were removed and macroscopically detectable (60 x magnification) neoplasms excised and placed into Bouin’s fixative for at least 24 h. Neoplasms were then processed for light microscopy, and paraffin sections stained with hematoxylin and eosin for histologic examination. Although the relative distribution of adenoma histologic patterns is in part a function of the length of time following carcinogen injection [20], we adopted the strategy of using only the 14-week interval to keep this variable constant so that the identity of the strain was the only independent variable examined. Classification
of adenomas
Adenomas were classified as either solid or papillary according to criteria established by Kauffman et al. [lo]. Solid adenomas grow as a uniform mass, do not form a capsule or pseudocapsule at their borders, and their cells contain round nuclei. Papillary adenomas contain finger-like arrangements of cells with pleomorphic nuclei, and compress the surrounding tissue into a pseudocapsule. Statistical
analyses
A standard Pearson’s correlation coefficient [8] was calculated when comparing the two continuous variables of adenoma multiplicity and percentage occurrence of solid adenoma. Results Analysis of the AXB inbred (RI) strains
and BXA recombinant
We analyzed the genetics of lung adenoma histologic type using the AXB and BXA RI strains derived from A/J and B6 progenitors [16]. Although the progenitor strains do not differ significantly in this phenotype (Table I) [20], the RI strains derived from them show a tremendous range (0- 100%) in the relative distribution of adenoma histologic types (Table I). Consistent with previous reports on inbred strains [ 1,201, the mouse-to-mouse variation
17 Table 1.
Histologic type of lung adenomas
in the AXB and BXA RI strains and their inbred progenitors.
Strain a
No. of mice tested
No. of adenomas examined
Percent solid adenomasb
Strain
A/J C57BL/6J AXB 1 AXB 5 AXB 6
53 30 6 5 5 6 6 6 4
1425 45 163 73 18 48 60 83 5 8 93 27 12 60 67
78 67 83 53 56 15 52 67 100 88 58 48 25 78 60’ 100 90 67 88 86
AXB AXB AXB BXA BXA
23 24 25 2 4
BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA
6 8 9 10 11 12 13 14 18 19 20 22
AXB AXB AXB AXB
7 8 9 10
AXB AXB AXB AXB
11 12 13 14
AXB AXB AXB AXB
15 17 18 19
5 11 6 5 5 6
10 3 59 7
AXB 20 AXB 21 AXB 22 ‘Adenoma
multiplicities
(number
BXA 23
BXA 24 BXA 25
No. of mice tested
No. of adenomas examined
Percent solid adenomas
6 12
36 70 81 68 90 2 13 80 63 11 7 7 18 22 17 9 30
14 26
5 5 4 4 5 8 6 3 3 4 3 5 6 2 6 5 1 3
68 10 5
95’ 51 80’ 50 69 61 63 0 71 86’ 67 36 41 67 37 51 50 80
of adenomas/mouse) in these strains in response to urethane treatment have been in this table were derived using new mice and new neoplasms not previously
published [15]. The data presented
examined. bThe relative proportion of solid and papillary adenomas was consistent among individual mice-within a given RI strain. ‘The morphology of some neoplasms in these strains was difficult to classify as they had cytologic features characteristic of both solid and papillary adenomas. Classification was based on the predominant characteristics for a given adenoma (see text).
in the distribution of the solid and papillary phenotypes within a given RI strain was minimal. In some RI strains only a few neoplasms were available for analysis, so more reliance was placed on the morphologic results established in those strains where several neoplasms were examined. The adenoma morphologies in four strains, AXB17, AXB25, BXA4 and BXA13, were difficult to classify because the cells in some adenomas had cytologic features and growth patterns characteristic of both adenoma types (e.g. uniformly round nuclei and a papillary pattern or pleomorphic nuclei in cells arrayed in a solid pattern). These were classified on the basis of
morwhat constituted the predominant phologic characteristics for a given adenoma. These adenomas were a uniform ‘composite’ of morphologic characteristics of solid and papillary adenomas as opposed to ‘mixed’ adenomas (which were not observed in these strains at the 14-week post-urethane interval used for this study) containing both regions of distinctly solid and regions of distinctly papillary morphology. While most of the neoplasms in these four strains exhibited the composite morphology, there were also adenomas in the same individuals which were clearly solid or papillary. In contrast, each neoplasm in the other RI strains was readily
18
assignable to a single phenotype. There was no correlation between adenoma multiplicity and adenoma morphology. We also compared the distribution in the RI strains of adenoma histologic type with that of the restriction fragment length polymorphism in the protooncogene, K-t-as, which is the only identified Pas gene [12,18], and found no correlation (data not shown). Development in the RI strains of solid and papillary distributions distinct from those in the progenitor strains indicates that A/J and B6 mice have at least two loci controlling adenoma morphology, and that these loci interact with each other. We suggest that the genes determining the growth pattern of mouse lung adenomas be designated Pulmonary Adenoma Histologic type (Pah) genes. Analysis
of inbred
strains
The proportion of urethane-induced lung adenomas having a solid morphology is summarized in Table II for several inbred strains. There was no discernible difference in the size of adenomas present in mice in which predominantly solid adenomas developed as compared with mice having mainly papillary adenomas. Only A/J and NGP mice exhibit high sensitivity to tumorigenesis. The proportion of adenomas classified as solid, however, ranged widely from 11% to 100% among the strains. The lack of any correlation between adenoma multiplicity and percent solid adenomas again indicated no linkage between genes having a major effect on susceptibility and those determining adenoma morphology. To test the nature of the genetic influence on adenoma type, A/J and MA/My mice were mated, and the structure of the urethaneinduced lung adenomas in their progeny was analyzed. The MA/My strain was chosen in contrast to A/J, it displays because, predominantly papillary adenomas and yields a sufficiently large adenoma multiplicity for a reliable analysis. (A X MA)FI mice had an intermediate almost exactly number of adenomas (19 adenomas/mouse) as compared to their respective parents. In marked contrast, the predominantly papillary histologic
Table 11. Histologic type of lung adenomas various strains Strain a
No. of mice tested
SensitiueC (>
No. of adenomas examined
Percent solid adenomasb
10 adenomas/mouse)
A/J
53
1425
NGP/N
11
253
Intermediate
in mice of
78 70
(1 - 10 adenomas/mouse)
GRS/SN
16
131
MA/MyJ ST/BN PL/J LP/J CBA/J
33 11 6 6 7
213 33 14 7 9
48 31 70 43 100 11
1 adenomas/mouse) 30 45 5 7 5 5 2 2
67 71 100 100
Resistantd C57BL/6J C58/J SM/J SJL/J
(
15-
15
20
Elsevier Scientific Publishers Ireland Ltd.
Lung adenoma structure among inbred strains of mice: the pulmonary adenoma histologic type (P&I) genes Muriel N. Nesbitta and Alvin M. Malkinsonb
Larry G. Thaete”,
Toxicology and Enoironmental Health Sciences Program, Colorado Cancer Center, School of Pharmacy, University of Colorado, Boulder, CO 80309-0297 and *Department of Biology, University of California at San Diego, La Jolla, CA 92093 (U.S.A.) “Molecular
(Received 27 June 1991) (Revision received 29 August (Accepted 30 August 1991)
1991)
Summary Urethane-induced, lung adenoma multipliciand histologic type oary among mouse strains. We asked whether the Pas genes which control multiplicity also determine adenoma structure. Lung adenomas from inbred mice, Fl hybrids, and recombinant inty
bred mice were classified by growth pattern as either solid or papillary. Since no correlation was obserued between adenoma multiplicity and histologic type, no linkage apparently exists between the Pas genes and adenoma morphology . We propose the name Pah
(Pulmonary Adenoma Histologic genes controlling lung adenoma
type) for the
growth patterns. Genetic analysis indicated dominance of the papillary phenotype, and that two or more
Pah genes determine
adenoma
structure.
Keywords: mouse; lung; adenoma; genetics; recombinant inbred strains Introduction Primary
lung neoplasms
Correspondence Campus
Box
80309-0297,
in mice develop
to: Alvin M. Malkinson, 297,
University
U.S.A.
0304-3835/91/$03.50
0
1991
Published and Printed in Ireland
of
School of Pharmacy,
Colorado,
Boulder,
CO
through multiple stages, from hyperplasia to a benign stage to malignancy [3,11,19]. The adenomas are observed to be of two histologic types. Solid adenomas grow along the alveolar septae as compact masses and do not form distinct borders [ 1,10,111. These adenoma cells possess characteristics of type II alveolar cells, such as surfactant-containing lamellar bodies [5,22]. Papillary adenomas grow as finger-like projections and form a pseudocapsule of compressed normal tissue at their border. Their cells possess some of the morphologic [lo] and biochemical [21] characteristics of bronchiolar non-ciliated Clara cells, but the cellular derivation of these neoplasms is somewhat controversial [ 13,171. Papillary neoplasms have a longer latency [9] and are more frequently associated with malignancy than are solid adenomas [20]. Strains of mice vary in their susceptibility to spontaneous and carcinogen-induced lung tumorigenesis due to the actions of several pulmonary adenoma susceptibility (Pas) genes [12,13,15]. Interestingly, a large subset of human called lung adenocarcinomas, bronchiole-alveolar [6] or papillolepidic carcinomas [4], share both these presumptive cellular origins and a strong genetic basis for susceptibility [ 121. Strains of mice vary in the relative proportion of solid and papillary adenomas which oc-
Elsevier Scientific Publishers Ireland Ltd
16
cur in response to the carcinogen, urethane [ 1,20,23]. Both histologic types of adenomas may be present in lungs of individual mice but the relative proportion of the two types is strain-dependent, with little individual variation within a strain. We sought to determine whether adenoma morphology is also controlled by the Pas genes or by a distinctly different set of genes, This was done by examining tumor structure in the AXB and BXA recombinant inbred (RI) strains, derived from A/J and C57BL/6J (B6) progenitors, whose Pas phenotype had been previously established [ 151. Additionally, crosses between various inbred strains were done to study further the inheritance of adenoma structure. The concept of pulmonary adenoma histologic type (Pah) genes has been briefly introduced previously [13]. This paper presents extensive new data from many inbred and RI strains not previously analyzad to support the existence of Pah genes which determine lung adenoma structure independently of adenoma multiplicity. Materials and Methods Animals
Male and female adult mice of the A/J, AKR/J, CBA/J, C57BL/6J (B6), C58/J, DBA/ZJ, LP/J, PL/J, MA/MyJ, SJL/J and SM/J strains were obtained from Jackson Laboratories (Bar Harbor, ME), and strains GRS/SN, NGP/N, and ST/BN were obtained from the National Institutes of Health (Bethesda, MD). F1 hybrid mice were generated by mating A/J females with either MA/My, AKR, or DBA males. Thirty-eight AXB and BXA RI strains developed from A/J and B6 progenitors [ 161 were shipped from La Jolla to Boulder. Mice were maintained on hardwood bedding, kept on a 12-h light/dark cycle, and provided food (Wayne Lab Blox) and water ad libitum, in accordance with National Institutes of Health guidelines. All mice were housed for 2 weeks before being used experimentally. Lung
adenoma
induction
and processing
Mice were injected i.p. with 1 mg urethane
(Sigma, St. Louis, MO) in 0.9% NaCl/g body weight at 8 weeks of age, as described previously [14]. The mice were killed by cervical dislocation 14 weeks post-treatment, lungs were removed and macroscopically detectable (60 x magnification) neoplasms excised and placed into Bouin’s fixative for at least 24 h. Neoplasms were then processed for light microscopy, and paraffin sections stained with hematoxylin and eosin for histologic examination. Although the relative distribution of adenoma histologic patterns is in part a function of the length of time following carcinogen injection [20], we adopted the strategy of using only the 14-week interval to keep this variable constant so that the identity of the strain was the only independent variable examined. Classification
of adenomas
Adenomas were classified as either solid or papillary according to criteria established by Kauffman et al. [lo]. Solid adenomas grow as a uniform mass, do not form a capsule or pseudocapsule at their borders, and their cells contain round nuclei. Papillary adenomas contain finger-like arrangements of cells with pleomorphic nuclei, and compress the surrounding tissue into a pseudocapsule. Statistical
analyses
A standard Pearson’s correlation coefficient [8] was calculated when comparing the two continuous variables of adenoma multiplicity and percentage occurrence of solid adenoma. Results Analysis of the AXB inbred (RI) strains
and BXA recombinant
We analyzed the genetics of lung adenoma histologic type using the AXB and BXA RI strains derived from A/J and B6 progenitors [16]. Although the progenitor strains do not differ significantly in this phenotype (Table I) [20], the RI strains derived from them show a tremendous range (0- 100%) in the relative distribution of adenoma histologic types (Table I). Consistent with previous reports on inbred strains [ 1,201, the mouse-to-mouse variation
17 Table 1.
Histologic type of lung adenomas
in the AXB and BXA RI strains and their inbred progenitors.
Strain a
No. of mice tested
No. of adenomas examined
Percent solid adenomasb
Strain
A/J C57BL/6J AXB 1 AXB 5 AXB 6
53 30 6 5 5 6 6 6 4
1425 45 163 73 18 48 60 83 5 8 93 27 12 60 67
78 67 83 53 56 15 52 67 100 88 58 48 25 78 60’ 100 90 67 88 86
AXB AXB AXB BXA BXA
23 24 25 2 4
BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA BXA
6 8 9 10 11 12 13 14 18 19 20 22
AXB AXB AXB AXB
7 8 9 10
AXB AXB AXB AXB
11 12 13 14
AXB AXB AXB AXB
15 17 18 19
5 11 6 5 5 6
10 3 59 7
AXB 20 AXB 21 AXB 22 ‘Adenoma
multiplicities
(number
BXA 23
BXA 24 BXA 25
No. of mice tested
No. of adenomas examined
Percent solid adenomas
6 12
36 70 81 68 90 2 13 80 63 11 7 7 18 22 17 9 30
14 26
5 5 4 4 5 8 6 3 3 4 3 5 6 2 6 5 1 3
68 10 5
95’ 51 80’ 50 69 61 63 0 71 86’ 67 36 41 67 37 51 50 80
of adenomas/mouse) in these strains in response to urethane treatment have been in this table were derived using new mice and new neoplasms not previously
published [15]. The data presented
examined. bThe relative proportion of solid and papillary adenomas was consistent among individual mice-within a given RI strain. ‘The morphology of some neoplasms in these strains was difficult to classify as they had cytologic features characteristic of both solid and papillary adenomas. Classification was based on the predominant characteristics for a given adenoma (see text).
in the distribution of the solid and papillary phenotypes within a given RI strain was minimal. In some RI strains only a few neoplasms were available for analysis, so more reliance was placed on the morphologic results established in those strains where several neoplasms were examined. The adenoma morphologies in four strains, AXB17, AXB25, BXA4 and BXA13, were difficult to classify because the cells in some adenomas had cytologic features and growth patterns characteristic of both adenoma types (e.g. uniformly round nuclei and a papillary pattern or pleomorphic nuclei in cells arrayed in a solid pattern). These were classified on the basis of
morwhat constituted the predominant phologic characteristics for a given adenoma. These adenomas were a uniform ‘composite’ of morphologic characteristics of solid and papillary adenomas as opposed to ‘mixed’ adenomas (which were not observed in these strains at the 14-week post-urethane interval used for this study) containing both regions of distinctly solid and regions of distinctly papillary morphology. While most of the neoplasms in these four strains exhibited the composite morphology, there were also adenomas in the same individuals which were clearly solid or papillary. In contrast, each neoplasm in the other RI strains was readily
18
assignable to a single phenotype. There was no correlation between adenoma multiplicity and adenoma morphology. We also compared the distribution in the RI strains of adenoma histologic type with that of the restriction fragment length polymorphism in the protooncogene, K-t-as, which is the only identified Pas gene [12,18], and found no correlation (data not shown). Development in the RI strains of solid and papillary distributions distinct from those in the progenitor strains indicates that A/J and B6 mice have at least two loci controlling adenoma morphology, and that these loci interact with each other. We suggest that the genes determining the growth pattern of mouse lung adenomas be designated Pulmonary Adenoma Histologic type (Pah) genes. Analysis
of inbred
strains
The proportion of urethane-induced lung adenomas having a solid morphology is summarized in Table II for several inbred strains. There was no discernible difference in the size of adenomas present in mice in which predominantly solid adenomas developed as compared with mice having mainly papillary adenomas. Only A/J and NGP mice exhibit high sensitivity to tumorigenesis. The proportion of adenomas classified as solid, however, ranged widely from 11% to 100% among the strains. The lack of any correlation between adenoma multiplicity and percent solid adenomas again indicated no linkage between genes having a major effect on susceptibility and those determining adenoma morphology. To test the nature of the genetic influence on adenoma type, A/J and MA/My mice were mated, and the structure of the urethaneinduced lung adenomas in their progeny was analyzed. The MA/My strain was chosen in contrast to A/J, it displays because, predominantly papillary adenomas and yields a sufficiently large adenoma multiplicity for a reliable analysis. (A X MA)FI mice had an intermediate almost exactly number of adenomas (19 adenomas/mouse) as compared to their respective parents. In marked contrast, the predominantly papillary histologic
Table 11. Histologic type of lung adenomas various strains Strain a
No. of mice tested
SensitiueC (>
No. of adenomas examined
Percent solid adenomasb
10 adenomas/mouse)
A/J
53
1425
NGP/N
11
253
Intermediate
in mice of
78 70
(1 - 10 adenomas/mouse)
GRS/SN
16
131
MA/MyJ ST/BN PL/J LP/J CBA/J
33 11 6 6 7
213 33 14 7 9
48 31 70 43 100 11
1 adenomas/mouse) 30 45 5 7 5 5 2 2
67 71 100 100
Resistantd C57BL/6J C58/J SM/J SJL/J
(
