Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture

  • Kirsten H. Limesand
  • Katherine A. Barzen
  • Linda A. Sanders
  • Robert A. Sclafani
  • Mary V. Raynolds
  • Mary E. Reyland
  • Steven M. Anderson
  • David O. Quissell
Articles Cell Growth/Differentiation/Apoptosis

Summary

Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 μM etoposide or 1.5 μM BFA inducing maximal caspace activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5′-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspace-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8–18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two-to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.

Key words

apoptosis primary culture salivary gland etoposide brefeldin A 

References

  1. Allen, R. T.; Cluck, M. W.; Agrawald D. K. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell. Mol. Life Sci. 54:427–445; 1998.PubMedCrossRefGoogle Scholar
  2. Anderson, S. M.; Reyland, M. E.; Hunter, S.; Deisher, L. M.; Barzen, K. A.; Quissell, D. O. Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells. Cell Death Differ. 6:454–462; 1999.PubMedCrossRefGoogle Scholar
  3. Atkinson, J. C.; Travis, W. D.; Pillemer, S. R.; Bermudez, D.; Wolff, A.; Fox, P. C. Major salivary gland function in primary Sjögren’s syndrome and its relationship to clinical features. J. Rheumatol. 17:318–322; 1990.PubMedGoogle Scholar
  4. Cohen, G. M. Caspases: the executioners of apoptosis. Biochem. J. 326:1–16; 1997.PubMedGoogle Scholar
  5. Decker, P.; Muller, S. Modulating poly (ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress. Curr. Pharm Biotechnol. 3:275–283; 2002.PubMedCrossRefGoogle Scholar
  6. Denning, M. F.; Wang, Y. H.; Nickoloff, B. J.; Wrone-Smith, T. Protein kinase C delta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis in human keratinocytes. J. Biol. Chem. 273:29995–30002; 1998.PubMedCrossRefGoogle Scholar
  7. DeVries, T. A.; Neville, M. C.; Reyland, M. E. Nuclear import of PKC delta is required for apoptosis: identification of a novel nuclear import sequence. EMBO J. 21:6050–6060; 2002.PubMedCrossRefGoogle Scholar
  8. Ek, B.; Antonsson, B.-M. Studies of muscarinic receptor reserve linked to phosphoinositide hydrolysis in parotid gland and cerebral cortex. Acta Physiol. Scand. 147:289–295; 1993.PubMedCrossRefGoogle Scholar
  9. Enari, M.; Sakahira, H.; Yokoyama, H.; Okawa, K.; Iwamatsu, A.; Nagata, S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43–50; 1998.PubMedCrossRefGoogle Scholar
  10. Fox, P. C. Saliva composition and its importance in dental health. Compend. Contin. Educ. Dent. Suppl. 13:S457-S460; 1989.Google Scholar
  11. Green, D. R. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1–4; 2000.PubMedCrossRefGoogle Scholar
  12. Green, D. R.; Evan, G. I. A matter of life and death. Cancer Cell. 1:19–30; 2002.PubMedCrossRefGoogle Scholar
  13. Guo, H.; Tittle, T. V.; Allen, H.; Maziarz, R. T. Brefeldin A-mediated apoptosis requires the activation of caspases and is inhibited by Bcl-2. Exp. Cell Res. 245:57–68; 1998.PubMedCrossRefGoogle Scholar
  14. Hendricks, L. C.; McClanahan, S. L.; Palade, G. E.; Farquhar, M. G. Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells. Proc. Natl. Acad. Sci. USA 89:7242–7246; 1992.PubMedCrossRefGoogle Scholar
  15. Hengartner, M. O. The biochemistry of apoptosis. Nature 407:770–776; 2000.PubMedCrossRefGoogle Scholar
  16. Hofmann, K. The modular nature of apoptotic signaling proteins. Cell. Mol. Life Sci. 55:1113–1128; 1999.PubMedCrossRefGoogle Scholar
  17. Limesand, K. H.; Barzen, K. A.; Quissell, D. O.; Anderson, S. A. Synergistic suppression of apoptosis in salivary acinar cells by IGF1 and EGF. Cell Death Differ. 10:345–355; 2003.PubMedCrossRefGoogle Scholar
  18. Mandel, I. D. The functions of saliva. J. Dent. Res. 66:623–627; 1987a.PubMedGoogle Scholar
  19. Mandel, I. D. Impact of saliva on dental caries. Compend. Contin. Educ. Dent. Suppl. 13:S476-S481; 1987b.Google Scholar
  20. Mandel, I. D. The role of saliva in maintaining oral homeostasis. J. Am. Dent. Assoc. 119:298–304; 1989.PubMedGoogle Scholar
  21. Matassa, A. A.; Carpenter, L.; Biden, T. J.; Humphries, M. J.; Reyland, M. E. PKC delta is required for mitochondrial-dependent apoptosis in salivary epithelial cells. J. Biol. Chem. 276:29719–29728; 2001.PubMedCrossRefGoogle Scholar
  22. Nakamura, S.; Ikebe-Hiroki, A.; Shinohara, M.; Ohyama, Y.; Mouri, T.; Sasaki, M.; Shirasuna, K.; Nomoto, K. An association between salivary gland disease and serological abnormalities in Sjogren’s syndrome. J. Oral Pathol. Med. 26:426–430; 1997.PubMedCrossRefGoogle Scholar
  23. Nicholson, D. W.; Thornberry, N. A. Caspases: killer proteases. Trends Biochem. Sci. 22:299–306; 1997.PubMedCrossRefGoogle Scholar
  24. Pilihronis, M.; Tapinos, N. I.; Theocharis, S. E.; Economou, A.; Kittas, C.; Moutsopoulos, H. M. Modes of epithelial cell death and repair in Sjögren’s syndrome (SS). Clin. Exp. Immunol. 114:485–490; 1998.CrossRefGoogle Scholar
  25. Quissell, D. O.; Barzen, K. A.; Redman, R. S.; Camden, J. M.; Turner, J. T. Development and characterization of SV40 immortalized rat parotid acinar cell lines. In Vitro Cell. Dev. Biol. 34:58–67; 1998.Google Scholar
  26. Quissell, D. O.; Redman, R. S.; Mark, M. R. Short-term primary culture of acinar-intercalated duct complexes from rat submandibular glands. In Vitro Cell. Dev. Biol. 22:469–480; 1986.PubMedGoogle Scholar
  27. Redman, R. S.; Quissell, D. O.; Barzen, K. A. Effects of dexamethasone, epidermal growth factor, and retinoic acid on rat submandibular acinar-intercalated duct complexes in primary culture. In Vitro Cell. Dev. Biol. 24:734–742; 1988.PubMedGoogle Scholar
  28. Redman, R. S.; Quissell, D. O.; Barzen, K. A. Effects of oxygen, insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture. In Vitro Cell. Dev. Biol. 30:833–842; 1994.Google Scholar
  29. Reyland, M. E.; Anderson, S. M.; Matassa, A. A.; Barzen, K. A.; Quissell, D. O. Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells. J. Biol. Chem. 274:19115–19123; 1999.PubMedCrossRefGoogle Scholar
  30. Reyland, M. E.; Barzen, K. A.; Anderson, S. M.; Quissell, D. O.; Matassa, A. A. Activation of PKC is sufficient to induced an apoptotic program in salivary gland acinar cells. Cell Death Differ. 7:1200–1209; 2000.PubMedCrossRefGoogle Scholar
  31. Rich, T.; Allen, R. L.; Wyllie, A. H. Defying death after DNA damage. Nature 407:777–783; 2000.PubMedCrossRefGoogle Scholar
  32. Sakahira, H.; Enari, M.; Nagata, S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96–99; 1998.PubMedCrossRefGoogle Scholar
  33. Salvesen, G. S. Programmed cell death and the caspases. APMIS 107:73–79; 1999.PubMedCrossRefGoogle Scholar
  34. Tabak, L. A.; Bowen, W. H. Roles of saliva (pellicle), diet, and nutrition of plaque formation. J. Dent. Res. 68:1560–1566; 1989.Google Scholar
  35. Tamaki, H.; Yamashina, S. Structural integrity of the Golgi stack is essential for normal secretory function of rat parotid acinar cells: effects of brefeldin A and okadaic acid. J. Histochem. Cytochem. 50:1611–1623; 2002.PubMedGoogle Scholar
  36. Thornberry, N. A. Caspases: key mediators of apoptosis. Chem. Biol. 5:R97-R103; 1998.PubMedCrossRefGoogle Scholar
  37. Thornberry, N. A.; Lazabnik, Y. Caspases: enemies within. Science 281:1312–1316; 1998.PubMedCrossRefGoogle Scholar
  38. Van Maanen, J. M.; Retel, J.; de Vries, J.; Pinedo, H. M. Mechanism of action of antitumor drug etoposide: a review. J. Natl. Cancer Inst. 80:1526–1533; 1988.PubMedCrossRefGoogle Scholar
  39. Widmann, C.; Gibson, S.; Johnson, G. L. Caspase-dependent cleavage of signaling proteins during apoptosis. A turn-off mechanism for anti-apoptotic signals. J. Biol. Chem. 273:7141–7147; 1998.PubMedCrossRefGoogle Scholar
  40. Zimmermann, K. C.; Green, D. R. How cells die: apoptosis pathways. J. Allergy Clin. Immunol. 108:S99-S103; 2001.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 2003

Authors and Affiliations

  • Kirsten H. Limesand
    • 1
  • Katherine A. Barzen
    • 4
  • Linda A. Sanders
    • 4
  • Robert A. Sclafani
    • 3
  • Mary V. Raynolds
    • 2
  • Mary E. Reyland
    • 4
  • Steven M. Anderson
    • 1
  • David O. Quissell
    • 4
  1. 1.Department of Pathology School of MedicineUniversity of Colorado Health Sciences CenterDenver
  2. 2.Department of Medicine School of MedicineUniversity of Colorado Health Sciences CenterDenver
  3. 3.Department of Biochemistry and Molecular School of MedicineUniversity of Colorado Health Sciences CenterDenver
  4. 4.Department of Craniofacial Biology, School of DentistryUniversity of Colorado Health Sciences CenterDenver

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