Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 15;323(Pt 2):489–495. doi: 10.1042/bj3230489

Promotion-resistant JB6 mouse epidermal cells exhibit defects in phosphatidylethanolamine synthesis and phorbol ester-induced phosphatidylcholine hydrolysis.

Z Kiss 1, B Guyer 1, Z Dong 1
PMCID: PMC1218346  PMID: 9163343

Abstract

The tumour-promotion-sensitive (P+) and -resistant (P-) variants of mouse JB6 epidermis-derived cells have often been used to study the requirements for the tumour-promoting effect of PMA. As part of an effort to identify the defect(s) in JB6 P- cells that might prevent the promoting effect of PMA, stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PMA as well as the rate of phospholipid synthesis were compared in three P+ variants, two P- variants and a transformed variant of the JB6 cell line. PMA (5-100 nM) had significantly less stimulatory effect on PtdCho hydrolysis in P- cells than in P+ or transformed JB6 cells. The effects of PMA on PtdEtn hydrolysis in the P+ and P- cell lines were similar, whereas in transformed cells PMA had slightly less effect. Each JB6 cell line was found to contain similar amounts of PtdCho. In contrast, P- cells contained significantly less PtdEtn and a correspondingly higher level of ethanolamine phosphate compared with P+ and transformed cells. P- cells also secreted ethanolamine phosphate into the medium; this process was greatly enhanced by PMA. In the two P- variants the synthesis of PtdEtn from [14C]ethanolamine was reduced to various extents, whereas the rate of PtdCho synthesis was comparable in each JB6 cell line. The synthesis of PtdCho, but not PtdEtn, was greatly stimulated by PMA in both the P+ and P- clones. The results indicate that decreased synthesis/level of PtdEtn and suboptimal functioning of a PtdCho-specific PLD are common characteristics of the P- JB6 cells examined so far. The observed alterations in phospholipid metabolism may play a role in the resistance of P- cells to the tumour-promoting action of PMA.

Full Text

The Full Text of this article is available as a PDF (673.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Band V., Sager R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1249–1253. doi: 10.1073/pnas.86.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bazzi M. D., Youakim M. A., Nelsestuen G. L. Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes. Biochemistry. 1992 Feb 4;31(4):1125–1134. doi: 10.1021/bi00119a022. [DOI] [PubMed] [Google Scholar]
  3. Ben-Ari E. T., Bernstein L. R., Colburn N. H. Differential c-jun expression in response to tumor promoters in JB6 cells sensitive or resistant to neoplastic transformation. Mol Carcinog. 1992;5(1):62–74. doi: 10.1002/mc.2940050111. [DOI] [PubMed] [Google Scholar]
  4. Bernstein L. R., Colburn N. H. AP1/jun function is differentially induced in promotion-sensitive and resistant JB6 cells. Science. 1989 May 5;244(4904):566–569. doi: 10.1126/science.2541502. [DOI] [PubMed] [Google Scholar]
  5. Carnero A., Cuadrado A., del Peso L., Lacal J. C. Activation of type D phospholipase by serum stimulation and ras-induced transformation in NIH3T3 cells. Oncogene. 1994 May;9(5):1387–1395. [PubMed] [Google Scholar]
  6. Colburn N. H., Bruegge W. F., Bates J. R., Gray R. H., Rossen J. D., Kelsey W. H., Shimada T. Correlation of anchorage-independent growth with tumorigenicity of chemically transformed mouse epidermal cells. Cancer Res. 1978 Mar;38(3):624–634. [PubMed] [Google Scholar]
  7. Colburn N. H., Former B. F., Nelson K. A., Yuspa S. H. Tumour promoter induces anchorage independence irreversibly. Nature. 1979 Oct 18;281(5732):589–591. doi: 10.1038/281589a0. [DOI] [PubMed] [Google Scholar]
  8. Colburn N. H., Smith B. M. Genes that cooperate with tumor promoters in transformation. J Cell Biochem. 1987 Jun;34(2):129–142. doi: 10.1002/jcb.240340207. [DOI] [PubMed] [Google Scholar]
  9. Colburn N. H., Smith B. M., Wendel E. J., Dowjat W. K., Shimada T. Transfer by pro gene transfection of tumor promoter-sensitive phenotype to promotion-insensitive JB6 cells. Cancer Res. 1988 Mar 1;48(5):1195–1200. [PubMed] [Google Scholar]
  10. Colburn N. H. Tumor promotion and preneoplastic progression. Carcinog Compr Surv. 1980;5:33–56. [PubMed] [Google Scholar]
  11. Colburn N. H., Wendel E. J., Abruzzo G. Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen-resistant variants are sensitive to promotion. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6912–6916. doi: 10.1073/pnas.78.11.6912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dong Z. A., Jeffrey A. M. Mechanisms of organ specificity in chemical carcinogenesis. Cancer Invest. 1990;8(5):523–533. doi: 10.3109/07357909009012077. [DOI] [PubMed] [Google Scholar]
  13. Dong Z., Birrer M. J., Watts R. G., Matrisian L. M., Colburn N. H. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):609–613. doi: 10.1073/pnas.91.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fisk H. A., Kano-Sueoka T. Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine. J Cell Physiol. 1992 Dec;153(3):589–595. doi: 10.1002/jcp.1041530321. [DOI] [PubMed] [Google Scholar]
  15. Gindhart T. D., Nakamura Y., Stevens L. A., Hegameyer G. A., West M. W., Smith B. M., Colburn N. H. Genes and signal transduction in tumor promotion: conclusions from studies with promoter resistant variants of JB-6 mouse epidermal cells. Carcinog Compr Surv. 1985;8:341–367. [PubMed] [Google Scholar]
  16. Humble E., Berglund L. Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids. J Lipid Res. 1991 Nov;32(11):1869–1872. [PubMed] [Google Scholar]
  17. Irvine R. F., Letcher A. J., Dawson R. M. Phosphatidylinositol-4,5-bisphosphate phosphodiesterase and phosphomonoesterase activities of rat brain. Some properties and possible control mechanisms. Biochem J. 1984 Feb 15;218(1):177–185. doi: 10.1042/bj2180177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jiang H., Luo J. Q., Urano T., Frankel P., Lu Z., Foster D. A., Feig L. A. Involvement of Ral GTPase in v-Src-induced phospholipase D activation. Nature. 1995 Nov 23;378(6555):409–412. doi: 10.1038/378409a0. [DOI] [PubMed] [Google Scholar]
  19. Jiang Y. W., Song J., Zang Q., Foster D. A. Phosphatidylcholine-specific phospholipase D activity is elevated in v-Fps-transformed cells. Biochem Biophys Res Commun. 1994 Sep 15;203(2):1195–2003. doi: 10.1006/bbrc.1994.2309. [DOI] [PubMed] [Google Scholar]
  20. Kano-Sueoka T., King D. M. Effects of phosphatidylethanolamine and phosphatidylcholine in membrane phospholipid on binding of phorbol ester in rat mammary carcinoma cells. Cancer Res. 1988 Mar 15;48(6):1528–1532. [PubMed] [Google Scholar]
  21. Kano-Sueoka T., Nicks M. E. Abnormal function of protein kinase C in cells having phosphatidylethanolamine-deficient and phosphatidylcholine-excess membranes. Cell Growth Differ. 1993 Jul;4(7):533–537. [PubMed] [Google Scholar]
  22. Kiss Z., Anderson W. B. Phorbol ester stimulates the hydrolysis of phosphatidylethanolamine in leukemic HL-60, NIH 3T3, and baby hamster kidney cells. J Biol Chem. 1989 Jan 25;264(3):1483–1487. [PubMed] [Google Scholar]
  23. Kiss Z., Anderson W. H. Selective down-regulation of protein kinase c-epsilon by carcinogens does not prevent stimulation of phospholipase D by phorbol ester and platelet-derived growth factor. Biochem J. 1994 Jun 15;300(Pt 3):751–756. doi: 10.1042/bj3000751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kiss Z., Crilly K. S., Anderson W. B. Protein kinase C inhibitors enhance the synergistic mitogenic effects of ethanolamine analogues and insulin in NIH 3T3 fibroblasts. Biochem Biophys Res Commun. 1996 Mar 7;220(1):125–130. doi: 10.1006/bbrc.1996.0368. [DOI] [PubMed] [Google Scholar]
  25. Kiss Z. Effects of phorbol ester on phospholipid metabolism. Prog Lipid Res. 1990;29(3):141–166. doi: 10.1016/0163-7827(90)90001-2. [DOI] [PubMed] [Google Scholar]
  26. Kiss Z. Possible phospholipid precursor for phosphatidylserine in rat heart. Eur J Biochem. 1976 Aug 16;67(2):557–561. doi: 10.1111/j.1432-1033.1976.tb10721.x. [DOI] [PubMed] [Google Scholar]
  27. Kiss Z. Regulation of phospholipase D by protein kinase C. Chem Phys Lipids. 1996 May 24;80(1-2):81–102. doi: 10.1016/0009-3084(96)02547-9. [DOI] [PubMed] [Google Scholar]
  28. Kiss Z., Tomono M., Anderson W. B. Phorbol ester selectively stimulates the phospholipase D-mediated hydrolysis of phosphatidylethanolamine in multidrug-resistant MCF-7 human breast carcinoma cells. Biochem J. 1994 Sep 15;302(Pt 3):649–654. doi: 10.1042/bj3020649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Low M. G., Carroll R. C., Cox A. C. Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets. Biochem J. 1986 Jul 1;237(1):139–145. doi: 10.1042/bj2370139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moreau P., Cassagne C. Phospholipid trafficking and membrane biogenesis. Biochim Biophys Acta. 1994 Dec 9;1197(3):257–290. doi: 10.1016/0304-4157(94)90010-8. [DOI] [PubMed] [Google Scholar]
  31. Nakamura S., Kiyohara Y., Jinnai H., Hitomi T., Ogino C., Yoshida K., Nishizuka Y. Mammalian phospholipase D: phosphatidylethanolamine as an essential component. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4300–4304. doi: 10.1073/pnas.93.9.4300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nishizuka Y. The Albert Lasker Medical Awards. The family of protein kinase C for signal transduction. JAMA. 1989 Oct 6;262(13):1826–1833. [PubMed] [Google Scholar]
  33. Seddon J. M. Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochim Biophys Acta. 1990 Feb 28;1031(1):1–69. doi: 10.1016/0304-4157(90)90002-t. [DOI] [PubMed] [Google Scholar]
  34. Simek S. L., Kligman D., Patel J., Colburn N. H. Differential expression of an 80-kDa protein kinase C substrate in preneoplastic and neoplastic mouse JB6 cells. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7410–7414. doi: 10.1073/pnas.86.19.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Slater S. J., Kelly M. B., Taddeo F. J., Ho C., Rubin E., Stubbs C. D. The modulation of protein kinase C activity by membrane lipid bilayer structure. J Biol Chem. 1994 Feb 18;269(7):4866–4871. [PubMed] [Google Scholar]
  36. Slater S. J., Kelly M. B., Taddeo F. J., Larkin J. D., Yeager M. D., McLane J. A., Ho C., Stubbs C. D. Direct activation of protein kinase C by 1 alpha,25-dihydroxyvitamin D3. J Biol Chem. 1995 Mar 24;270(12):6639–6643. doi: 10.1074/jbc.270.12.6639. [DOI] [PubMed] [Google Scholar]
  37. Slater S. J., Kelly M. B., Taddeo F. J., Rubin E., Stubbs C. D. Evidence for discrete diacylglycerol and phorbol ester activator sites on protein kinase C. Differences in effects of 1-alkanol inhibition, activation by phosphatidylethanolamine and calcium chelation. J Biol Chem. 1994 Jun 24;269(25):17160–17165. [PubMed] [Google Scholar]
  38. Smith B. M., Colburn N. H. Protein kinase C and its substrates in tumor promoter-sensitive and -resistant cells. J Biol Chem. 1988 May 5;263(13):6424–6431. [PubMed] [Google Scholar]
  39. Taylor S. J., Exton J. H. Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements. Biochem J. 1987 Dec 15;248(3):791–799. doi: 10.1042/bj2480791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weinstein I. B. The origins of human cancer: molecular mechanisms of carcinogenesis and their implications for cancer prevention and treatment--twenty-seventh G.H.A. Clowes memorial award lecture. Cancer Res. 1988 Aug 1;48(15):4135–4143. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES