METHODSThe rats used were adult albinos (150-250 g.) of the Wistar strain, and the mice were adult albino females (2C25 g.) of the Strong A strain. Tritiated thymidine (3HT), specific activity 5c per mM, was obtained from the Radiochemical Centre, Amersham. 3H labelling of white cells was achieved by means of a course of 3 injections, each of 0.5 pc per g., as previously described (Spector et al.). To secure adequate labelling of small lymphocytes, a course of 28 daily intraperitoneal injections, each of 0.5 pc per g., was given to the mice. Colloidal carbon was injected intravenously as before (Spector et al.).Granulomatous inflammation was produced by injecting Complete Freund's adjuvant subcutaneously and intradermally into the dorsum of the feet. The adjuvant was emulsified before use with an equal volume of sterile isotonic saline in an ultra-turrax homogeniser; the quantity of emulsion injected was 0.05 ml. per Present address:

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The epithelial cell strain NAL1A cultured from normal adult mouse lung has been transformed by culturing in dexamethasone into an invasive neoplastic cell strain. The criteria for neoplastic transformation include the capacity for anchorage independent growth in soft agar as well as the formation of invasive neoplastic nodules after subcutaneous transplantation in thymectomized irradiated newborn mice. The cells of the invasive neoplastic nodules induced by dexamethasone culturing of NAL1A were indistinguishable histopathologically and by electron microscopy from invasive nodules evoked by the subcutaneous inoculation of CMT64, a cell line cultured from a metastasizing mouse lung tumour and cell strain NUL1 derived from mouse pulmonary adenomata induced by urethane. Cells of the nodules derived from all three cultured strains possessed desmosomes, surface microvilli and phospholipid lamellar bodies characteristic of the type 2 pneumocyte. It is concluded that cultured cell strains NAL1A, cultured in dexamethasone, NUL1 and CMT64 evoke invasive subcutaneous neoplasms derived from a common ancestor, presumably a type 2 pneumocyte related stem cell.

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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.