Garry J. Smith

Development and Characterization of Type 2 Pneumocyte-Related Cell Lines from Normal Adult Mouse Lung

&NA; Cell lines which exhibit epithelial morphology with surface microvilli and inclusion bodies characteristic of type 2 pneumocytes have been derived from normal adult mouse lung by a simple procedure involving enzymatic dispersal and mechanical elimination of other cell types. One of these cell lines designated NAL 1A, examined in detail, shows features consistent with its being related to type 2 pneumocytes of mouse lung. These features include desmosomes, dense lamellar bodies as well as phospholipid profiles related to immature surface active material, the inhibition of cell growth rate by dexamethasone, and the close similarity of the cytoskeletal protein patterns of this cell line to those of a metastatic type 2 pneumocyte‐related cell line of mouse lung. The cell line from normal lung demonstrated near diploid chromosome number at low passage number with some evidence of karyotype instability at high passage number.

Short–term diethylnitrosamine–induced oval cell responses in three strains of mice

&NA; The oval cells of the liver have been identified as target cells of chemical carcinogens during rat hepatocarcinogenesis and are believed to act as liver stem cells. In this study mice (strains C3H/EJ (C3H), C57/BL6J (C57) and hybrid B6C3F1 (F1)) were sacrified at 1, 3 and 7 days after administration of a single dose of the carcinogen diethyInitrosamine (DEN), and histopathological studies of oval cells were evaluated using Haematoxylin and Eosin (H&E), Picro‐Mallory (P‐M), α‐fetoprotein (A‐FP) and glutathione S‐transferase placental form (GST‐π) staining techniques and electron microscopy (EM). Increased oval cell proliferation was observed as soon as one day following exposure of the mice to DEN, in a manner consistent with C3H and C57 mice exhibiting high and low susceptibility to DEN respectively, with hybrid F1 mice being intermediate in DEN sensitivity. This analysis indicates that, in mice, oval cells are target cells at very early stages of liver carcinogenesis and supports the notion that oval cells are potential liver stem cells.

Establishment of epithelial cell strains from normal adult mouse lung resembling a urethane-induced lung adenoma cell strain and a metastasizing mouse lung carcinoma cell line

Epithelial cell strains, designated NAL, have been established from normal lungs of adult female Balb/c mice. The ultrastructural characteristics, effect of dexamethasone on cellular morphology and proliferation rate, and the cytoskeletal protein pattern of NAL suggests that these cell strains may be related to a urethane-induced mouse lung adenoma cell strain NUL and to a metastasizing mouse lung tumour cell line CMT. NAL exhibited no anchorage-independent growth under normal conditions, however extensive passaging in the presence of 5 X 10(-6)M dexamethasone resulted in a colony forming efficiency in soft agar of 8.4% at passage number 30.

Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.

Immunohistochemical identification of type 2 pneumocytes by an antibody to a lamellar body-enriched fraction of lung homogenate

An antibody for immunohistochemical identification of alveolar type 2 pneumoyctes was prepared by immunization of rabbits with a lamellar-body-enriched fraction of mouse lung homogenate. Following absorption with mouse serum and tissue membrane fractions the antibody preparation was shown to be monospecific by immunodiffusion against delipidated lamellar body proteins. Immunoperoxidase staining of tissues revealed specific labeling of alveolar type 2 pneumoyctes, best demonstrated in acetone-fixed frozen sections, together with staining of the surfactant layer lining most small and medium-sized airways. Immunospecific staining was seen in sections of mouse, rat, and human lung, and clearly demonstrated hypertrophic and hyperplastic type 2 pneumocytes in human fibrosing alveolitis (interstitial pulmonary fibrosis). The antibody also stained cells of a type 2 pneumocyte-related strain derived from normal adult mouse lung. No cross-reactivity with cells of other tissues or in vitro cell lines was observed. The immunostaining pattern is consistent with the notion that the antibody recognizes a surfactant apoprotein. The antibody may usefully be employed as an immunologic marker of the in vivo and in vitro responses of type 2 pneumocytes.

Expression of a type 2 pneumocyte-specific antigen by a cell strain from normal adult mouse lung.

The mouse lung epithelial cell strain NAL 1A has been confirmed as type 2 pneumocyte-related by immunostaining with a type 2 pneumocyte-specific antiserum. Cytoplasmic immunoreactivity with the antiserum paralleled the appearance of phospholipid-containing osmiophilic cytoplasmic inclusions, which were much more abundant in confluent cell cultures at low passage number than in exponentially growing cultures. However, phospholipid analysis indicated that NAL 1A cells were impoverished in phosphatidylglycerol as compared to lung type 2 pneumocytes. Confluent cultures of the neoplastic cell lines NAL 1AM and NUL 1 did not reveal any reactivity with the specific antiserum.

Malignant epithelial cell strains cultured from BALB/c mouse lung adenoma

Urethane-induced lung adenomas from adult BALB/c mice were explanted onto a plastic substratum and cultured in order to establish the epithelial cell strain NUL1. The cell strain exhibited a polygonal morphology with high nuclear to cytoplasmic ratio and osmiophilic lamellar bodies characteristic of lung adenoma cells. A reproducible large and small cell heterogeneity was preserved despite multiple cell cloning. NUL1 was malignant at all passage numbers tested exhibiting anchorage-independent growth and subcutaneous formation of carcinomas in immune-suppressed mice. The cell strain was diploid at low passage numbers and became pseudo-diploid with increasing passages.

Decreased 8N3-[γ-32P]GTP photolabeling of Gsα in tumorigenic lung epithelial cell lines: Association with decreased hormone responsiveness and loss of contact-inhibited growth

Abstract The 45-kDa α subunit of the signal transducing G s protein complex, which stimulates receptor-coupled adenylate cyclase, incorporated less of the photoaffinity probe, 8N 3 -[γ- 32 P]GTP, in extracts from tumorigenic cell lines in comparison with nontumorigenic cell lines derived from mouse lung epithelium. Immunoblotting experiments using anti-G s α antibodies demonstrated that tumor cells do not have a decreased amount of G 3 α and photolabeling of tumor cell G 3 α increased when the rate of nucleotide exchange was promoted. Therefore, tumor cell G s α function may be altered. Consistent with this hypothesis is the observation that the tumor cells exhibited decreased responsiveness to the β-adrenergic agonist, isoproterenol. G s α photolabeling in growing nontumorigenic cells was reduced to a level resembling that observed in tumor cells, but photolabeling increased when cells became contact-inhibited. This increase in 8N 3 -[γ- 32 P]GTP incorporation into G s α by normal cells at confluence was not seen in the tumorigenic cells. Since G s α photolabeling was inversely proportional to the percentage of [ 3 H]thymidine-labeled nuclei at confluence, we suggest that the altered G s α in tumor cells is involved in the loss of cell growth regulation.

A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma

Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines. Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice. They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin. These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms. A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma. Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma.

EGF-receptor and extracellular matrix changes in mouse pulmonary carcinogenesis

Malignant Balb/c mouse lung cell clones related to alveologenic carcinoma exhibited low levels of epidermal growth factor (EGF) receptor activity compared to nonmalignant cell clones. Immunoprecipitation of cell homogenates and immunohistochemistry on urethane-induced lung tumors suggest that the absence of activity reflects decreased amounts of EGF receptor protein. Low levels of EGF receptor alone cannot cause neoplastic transformation, since a nonneoplastic cell cone, B5D3, exhibited low levels of EGF receptor despite its nontransformed phenotype. The reduced levels of EGF receptor in malignant clones have been mimicked by long-term (12 h) treatment of a nontransformed cell clone with 200 nM phorbol dibutyrate. The detection of mutated ras oncogene in the transformed cell lines, taken together with the EGF receptor findings, suggests that more than one alteration in the signal transduction pathway may be necessary for transformation in alveologenic adenoma and carcinoma cell systems. A further phenotypic feature of transformation, reduced expression of the extracellular matrix proteins fibronectin and laminin, may be mediated at the transcriptional level.