Skip to main content
SpringerLink
Log in

Establishment of immortalized alveolar type II epithelial cell lines from adult rats

  • Cellular Models
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ahlstrom, U. Chromosomes of primary carcinomas induced by 7, 12 dimethylbenzanthracene in the rat. Hereditas 78:235–244; 1974.

    PubMed  CAS  Google Scholar 

  2. Breneman, J. W.; Ramsey, M. J.; Lee, D. A., et al. The development of chromosome specific composite probes for the mouse and their application to chromosome painting. Chromosoma 102:591–598; 1993.

    Article  PubMed  CAS  Google Scholar 

  3. Castranova, V.; Bowman, L.; Wright, J. R., et al. Toxicity of metallic ions in the lung: effects on alveolar macrophages and alveolar Type II cells. J. Toxicol. Environ. Health 13:845–856; 1984.

    Article  PubMed  CAS  Google Scholar 

  4. Committee for a Standardized Karyotype ofRattus norvegicus, Standard Karyotype of the Norway Rat,Rattus norvegicus. Cytogenet. Cell Genet. 12:199–205; 1973.

  5. Dobbs, L. G. Isolation and culture of alveolar Type II cells. Am. J. Physiol. 258:L134–147; 1990.

    PubMed  CAS  Google Scholar 

  6. Dobbs, L. G.; Gonzalez, R.; Williams, M. C. An improved method for isolating Type II cells in high yield and purity. Am. Rev. Respir. Dis. 134:141–145; 1986.

    PubMed  CAS  Google Scholar 

  7. Douglas, W. H. J.; Kaighn, M. E. Clonal isolation of differentiated rat lung cells. In Vitro 10:230–237; 1974.

    Article  PubMed  CAS  Google Scholar 

  8. Edelson, J. D.; Shannon, J. M.; Mason, R. J. Alkaline phosphatase: a marker of alveolar Type II cell differentiation. Am. Rev. Respir. Dis. 138:1268–1275; 1988.

    PubMed  CAS  Google Scholar 

  9. Evans, M. J.; Stephens, R. J.; Freeman, G. Transformation of alveolar Type 2 cells to Type 1 cells following exposure to NO2. Exp. Mol. Pathol. 22:142–150; 1975.

    Article  PubMed  CAS  Google Scholar 

  10. Fraslon, C.; Rolland, G.; Bourbon, J. R., et al. Culture of fetal alveolar epithelial Type II cells in serum-free medium. In Vitro Cell. Dev. Biol. 27A:843–852; 1991.

    PubMed  CAS  Google Scholar 

  11. Fiers, W.; Contreras, R.; Haegeman, G., et al. Complete nucleotide sequence of SV40 DNA. Nature 273:113–120; 1978.

    Article  PubMed  CAS  Google Scholar 

  12. Funaki, K.; Everitt, J.; Bermudez, E., et al. Trisomy of rat chromosome 1 associated with mesothelial cell transformation. Cancer Res. 51:4059–4066; 1991.

    PubMed  CAS  Google Scholar 

  13. Gollahon, L. S.; Aldaz, C. M. Chromosomal localization of the rat Harvey ras-1 gene (HRAS) by in situ hybridization. Cytogenet. Cell Genet. 61:123–124; 1992.

    PubMed  CAS  Google Scholar 

  14. Haagsman, H. P.; Schuurmans, E. A. J. M.; Battenburg, J. J., et al. Phospholipid synthesis in isolated alveolar Type II cells exposedin vitro to paraquat and hyperoxia. Biochemistry. 245:119–126; 1987.

    CAS  Google Scholar 

  15. Ham, R. G. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci. USA. 53:288–293; 1965.

    Article  PubMed  CAS  Google Scholar 

  16. Herring, A. S.; Raychaudhuri, R.; Kelley, S. P., et al. Repeated establishment of diploid epithelial cell cultures from normal and partially hepatectomized rats. In Vitro 19:576–588; 1983.

    Article  Google Scholar 

  17. Hoebee, B.; deStoppelaar, J. M.; Suijkerbuijk, R. F., et al. Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed PCR. Cytogenet. Cell Genet. 66:277–282; 1994.

    PubMed  CAS  Google Scholar 

  18. Hsieh, J.-T.; Zhau, H. E.; Wang, X.-H., et al. Regulation of basal and luminal cell-specific cytokeratin expression in rat accessory sex organs. J. Biol. Chem. 267:2303–2310; 1992.

    PubMed  CAS  Google Scholar 

  19. Ichinose, Y.; Hashido, K.; Miyamoto, H., et al. Molecular cloning and characterization of cDNA encoding mouse cytokeratin no. 19. Gene 80:315–323; 1989.

    Article  PubMed  CAS  Google Scholar 

  20. ISCN guidelines for cancer cytogenetics, supplement to an international system for human cytogenetic nomenclature. Mitelman, F., ed. Basel, Switzerland: S. Karger; 1991.

    Google Scholar 

  21. Jones, G. S.; Miles, P. R.; Lantz, R. C., et al. Ionic content and regulation of cellular volume in rat alveolar Type II cells. J. Appl. Physiol. Respir. Environ. Exercise Physiol. 53(1):258–266; 1982.

    CAS  Google Scholar 

  22. Kaighn, M. E.; Reddel, R. R.; Lechner, J. F., et al. Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes. Cancer Res. 49:3050–3056; 1989.

    PubMed  CAS  Google Scholar 

  23. Kalina, M.; Riklis, S.; Blau, H. Pulmonary epithelial cell proliferation in primary culture of alveolar Type II cells. Exp. Lung Res. 19:153–175; 1993.

    PubMed  CAS  Google Scholar 

  24. Korthoff, G. An improved fixation method for chromosome preparation of Chinese hamster, Chinese hamster hybrids and mouse cell lines. Cytogenet. Cell Genet. 41:181–184; 1986.

    Google Scholar 

  25. Lacagnin, L. B.; Bowman, L.; Ma, J. Y. C., et al. Metabolic changes in alveolar Type II cells after exposure to hydrogen peroxide. Am. J. Physiol. 3:L57–65; 1990.

    Google Scholar 

  26. Leheup, B. P.; Federspiel, S. J.; Guerry-Force, M. L., et al. Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. I. Characterization of the clone and the major genetic types of collagen produced. Lab Invest. 60:791–807; 1989.

    PubMed  CAS  Google Scholar 

  27. Levan, G. The detailed chromosome constitution of a benzpyrene-induced rat sarcoma. A tentative model for G-band analysis in solid tumors. Hereditas 78:273–290; 1974.

    Article  PubMed  CAS  Google Scholar 

  28. Li, A. P.; Hahn, F. F.; Zamora, P. O., et al. Characterization of a lung epithelial cell strain with potential application in toxicological studies. Toxicology 27:257–272; 1983.

    Article  PubMed  CAS  Google Scholar 

  29. Mallampalli, R. K.; Floerchinger, C. S.; Hunninghake, G. W. Isolation and immortalization of rat pre-Type II cell lines. In Vitro Cell. Dev. Biol. 28A:181–187; 1992.

    PubMed  CAS  Google Scholar 

  30. Messmer, T. O.; Armour, R.; Holley, R. W. Factors influencing the growth of alveolar Type II epithelial cells isolated from rat lungs. Exp. Cell Res 142:417–426; 1982.

    Article  PubMed  CAS  Google Scholar 

  31. Paine III, R.; Ben-Ze’ev, A. B.; Farmer, S. R., et al. The pattern of cytokeratin synthesis by alveolar type II cells in primary culture. J. Biol. Chem. 255:5101–5107; 1988.

    Google Scholar 

  32. Paine III, R.; Joyce-Brady, M.; Clement, A., et al. Serum accelerates the loss of Type II cell differentiation In Vitro. Am. J. Respir. Cell Mol. Biol. 3:311–323; 1990.

    PubMed  Google Scholar 

  33. Parda, D. S.; Thraves, P. J.; Kuettel, M. R., et al. Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene. Prostate 23:91–98; 1993.

    Article  PubMed  CAS  Google Scholar 

  34. Reddel, R. R.; Ke, Y.; Gerwin, B. I., et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48:1904–1909; 1988.

    PubMed  CAS  Google Scholar 

  35. Reddel, R. R.; Ke, Y.; Kaign, M. E., et al. Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal differentiation. Oncogene Res. 3:401–408; 1988.

    PubMed  CAS  Google Scholar 

  36. Singer, P. A.; Trevor, K.; Oshima, R. G. Molecular cloning and characterization of the endo B cytokeratin expressed in preimplantation mouse embryos. J. Biol. Chem. 261:538–547; 1986.

    PubMed  CAS  Google Scholar 

  37. Steele, M. P.; Levine, R. A.; Joyce-Brady, M., et al. A rat alveolar Type II cell line developed by adenovirus 12SE1A gene transfer. Am. J. Respir. Cell Mol. Biol. 6:50–56; 1992.

    PubMed  CAS  Google Scholar 

  38. Stoner, G. D.; Kaighn, M. E.; Reddel, R. R., et al. Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells. Cancer Res. 51:365–371; 1991.

    PubMed  CAS  Google Scholar 

  39. Thomassen, D. G.; Saffiotti, U.; Kaighn, M. E. Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens. Carcinogenesis 7:2033–2039; 1986.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Miami Valley Laboratories, The Procter and Gamble Company, P.O. Box 538707, 45253-8707, Cincinnati, Ohio

    K. E. Driscoll, J. M. Carter, L. L. Crosby, M. J. Aardema, R. J. Isfort, D. Cody, M. H. Chestnut, J. L. Burns & R. A. LeBoeuf

  2. Biological Research Faculty and Facility, Inc., 10075-20 Tyler Place, 21754, Ijamsville, Maryland

    P. T. Iype & H. L. Kumari

Authors
  1. K. E. Driscoll
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. M. Carter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. T. Iype
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H. L. Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. L. Crosby
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. J. Aardema
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R. J. Isfort
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D. Cody
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M. H. Chestnut
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J. L. Burns
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. R. A. LeBoeuf
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Driscoll, K.E., Carter, J.M., Iype, P.T. et al. Establishment of immortalized alveolar type II epithelial cell lines from adult rats. In Vitro Cell Dev Biol - Animal 31, 516–527 (1995). https://doi.org/10.1007/BF02634029

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02634029

Key words

Search

Navigation