Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jan 15;313(Pt 2):691–696. doi: 10.1042/bj3130691

Differential transcription of the human spermidine/spermine N1-acetyltransferase (SSAT) gene in human lung carcinoma cells.

L Xiao 1, R A Casero Jr 1
PMCID: PMC1216962  PMID: 8573111

Abstract

The expression of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in the catabolism of polyamines, is highly regulated by a number of factors including the natural polyamines and their analogues. The phenotype-specific cytotoxicity that occurs in response to a class of polyamine analogues, the diethylpolyamines, is associated with a phenotype-specific superinduction of SSAT in human non-small-cell lung carcinomas, whereas in non-responding cell types, including the small-cell lung carcinomas, the superinduction of SSAT does not occur. In this study, we have investigated the molecular basis of this phenotype-specific SSAT induction in human lung carcinoma cells in response to N1,N12-diethylspermine (BESpm). To facilitate the study of transcriptional regulation, we have cloned and characterized 11 kb of the human SSAT locus, including 3500 bp of the 5' promoter region. Nuclear run-on transcription studies suggest that the initial induction of SSAT results from an increase in the rate of gene transcription. Results from Northern blot analysis and ribonuclease protection assays indicate a differential expression of SSAT mRNA between the analogue-responsive H157 and non-responsive H82 cells. There is no detectable SSAT mRNA in H82 cells, even after a 24-h analogue treatment, whereas SSAT mRNA in H157 cells was detectable by Northern blot analysis and increased more than 100-fold following drug exposure. Furthermore, nuclear run-on transcription assays do not detect any active transcription of SSAT gene in either treated or untreated H82 cells. These results indicate that at least one component of the phenotype-specific induction of SSAT appears to be due to differences in transcriptional regulation of the gene. In addition, mapping of DNase I-hypersensitive sites of the SSAT gene suggest that the cell type-specific promoter/enhancer utilization may control the expression of the SSAT gene in differentially sensitive cell types in vivo.

Full Text

The Full Text of this article is available as a PDF (361.9 KB).

Selected References

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

  1. Bellard M., Dretzen G., Giangrande A., Ramain P. Nuclease digestion of transcriptionally active chromatin. Methods Enzymol. 1989;170:317–346. doi: 10.1016/0076-6879(89)70054-9. [DOI] [PubMed] [Google Scholar]
  2. Bergeron R. J., Hawthorne T. R., Vinson J. R., Beck D. E., Jr, Ingeno M. J. Role of the methylene backbone in the antiproliferative activity of polyamine analogues on L1210 cells. Cancer Res. 1989 Jun 1;49(11):2959–2964. [PubMed] [Google Scholar]
  3. Bergeron R. J., Neims A. H., McManis J. S., Hawthorne T. R., Vinson J. R., Bortell R., Ingeno M. J. Synthetic polyamine analogues as antineoplastics. J Med Chem. 1988 Jun;31(6):1183–1190. doi: 10.1021/jm00401a019. [DOI] [PubMed] [Google Scholar]
  4. Casero R. A., Jr, Celano P., Ervin S. J., Applegren N. B., Wiest L., Pegg A. E. Isolation and characterization of a cDNA clone that codes for human spermidine/spermine N1-acetyltransferase. J Biol Chem. 1991 Jan 15;266(2):810–814. [PubMed] [Google Scholar]
  5. Casero R. A., Jr, Celano P., Ervin S. J., Porter C. W., Bergeron R. J., Libby P. R. Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues. Cancer Res. 1989 Jul 15;49(14):3829–3833. [PubMed] [Google Scholar]
  6. Casero R. A., Jr, Celano P., Ervin S. J., Wiest L., Pegg A. E. High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma. Biochem J. 1990 Sep 15;270(3):615–620. doi: 10.1042/bj2700615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Casero R. A., Jr, Ervin S. J., Celano P., Baylin S. B., Bergeron R. J. Differential response to treatment with the bis(ethyl)polyamine analogues between human small cell lung carcinoma and undifferentiated large cell lung carcinoma in culture. Cancer Res. 1989 Feb 1;49(3):639–643. [PubMed] [Google Scholar]
  8. Casero R. A., Jr, Mank A. R., Xiao L., Smith J., Bergeron R. J., Celano P. Steady-state messenger RNA and activity correlates with sensitivity to N1,N12-bis(ethyl)spermine in human cell lines representing the major forms of lung cancer. Cancer Res. 1992 Oct 1;52(19):5359–5363. [PubMed] [Google Scholar]
  9. Casero R. A., Jr, Pegg A. E. Spermidine/spermine N1-acetyltransferase--the turning point in polyamine metabolism. FASEB J. 1993 May;7(8):653–661. [PubMed] [Google Scholar]
  10. Celano P., Baylin S. B., Casero R. A., Jr Polyamines differentially modulate the transcription of growth-associated genes in human colon carcinoma cells. J Biol Chem. 1989 May 25;264(15):8922–8927. [PubMed] [Google Scholar]
  11. Celano P., Baylin S. B., Giardiello F. M., Nelkin B. D., Casero R. A., Jr Effect of polyamine depletion on c-myc expression in human colon carcinoma cells. J Biol Chem. 1988 Apr 25;263(12):5491–5494. [PubMed] [Google Scholar]
  12. Celano P., Berchtold C., Casero R. A., Jr A simplification of the nuclear run-off transcription assay. Biotechniques. 1989 Oct;7(9):942–944. [PubMed] [Google Scholar]
  13. Chang B. K., Bergeron R. J., Porter C. W., Liang Y. Antitumor effects of N-alkylated polyamine analogues in human pancreatic adenocarcinoma models. Cancer Chemother Pharmacol. 1992;30(3):179–182. doi: 10.1007/BF00686308. [DOI] [PubMed] [Google Scholar]
  14. Chang B. K., Bergeron R. J., Porter C. W., Vinson J. R., Liang Y., Libby P. R. Regulatory and antiproliferative effects of N-alkylated polyamine analogues in human and hamster pancreatic adenocarcinoma cell lines. Cancer Chemother Pharmacol. 1992;30(3):183–188. doi: 10.1007/BF00686309. [DOI] [PubMed] [Google Scholar]
  15. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  16. Davidson N. E., Mank A. R., Prestigiacomo L. J., Bergeron R. J., Casero R. A., Jr Growth inhibition of hormone-responsive and -resistant human breast cancer cells in culture by N1, N12-bis(ethyl)spermine. Cancer Res. 1993 May 1;53(9):2071–2075. [PubMed] [Google Scholar]
  17. Elgin S. C. The formation and function of DNase I hypersensitive sites in the process of gene activation. J Biol Chem. 1988 Dec 25;263(36):19259–19262. [PubMed] [Google Scholar]
  18. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  19. Fogel-Petrovic M., Shappell N. W., Bergeron R. J., Porter C. W. Polyamine and polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME-3M human melanoma cells. J Biol Chem. 1993 Sep 5;268(25):19118–19125. [PubMed] [Google Scholar]
  20. Kramer D. L., Khomutov R. M., Bukin Y. V., Khomutov A. R., Porter C. W. Cellular characterization of a new irreversible inhibitor of S-adenosylmethionine decarboxylase and its use in determining the relative abilities of individual polyamines to sustain growth and viability of L1210 cells. Biochem J. 1989 Apr 15;259(2):325–331. doi: 10.1042/bj2590325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marton L. J., Pegg A. E. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol. 1995;35:55–91. doi: 10.1146/annurev.pa.35.040195.000415. [DOI] [PubMed] [Google Scholar]
  22. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Migeon B. R. Insights into X chromosome inactivation from studies of species variation, DNA methylation and replication, and vice versa. Genet Res. 1990 Oct-Dec;56(2-3):91–98. doi: 10.1017/s0016672300035151. [DOI] [PubMed] [Google Scholar]
  24. Pegg A. E. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res. 1988 Feb 15;48(4):759–774. [PubMed] [Google Scholar]
  25. Porter C. W., Bernacki R. J., Miller J., Bergeron R. J. Antitumor activity of N1,N11-bis(ethyl)norspermine against human melanoma xenografts and possible biochemical correlates of drug action. Cancer Res. 1993 Feb 1;53(3):581–586. [PubMed] [Google Scholar]
  26. Porter C. W., Ganis B., Libby P. R., Bergeron R. J. Correlations between polyamine analogue-induced increases in spermidine/spermine N1-acetyltransferase activity, polyamine pool depletion, and growth inhibition in human melanoma cell lines. Cancer Res. 1991 Jul 15;51(14):3715–3720. [PubMed] [Google Scholar]
  27. Porter C. W., McManis J., Casero R. A., Bergeron R. J. Relative abilities of bis(ethyl) derivatives of putrescine, spermidine, and spermine to regulate polyamine biosynthesis and inhibit L1210 leukemia cell growth. Cancer Res. 1987 Jun 1;47(11):2821–2825. [PubMed] [Google Scholar]
  28. Shappell N. W., Miller J. T., Bergeron R. J., Porter C. W. Differential effects of the spermine analog, N1, N12-bis(ethyl)-spermine, on polyamine metabolism and cell growth in human melanoma cell lines and melanocytes. Anticancer Res. 1992 Jul-Aug;12(4):1083–1089. [PubMed] [Google Scholar]
  29. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  30. Tabor C. W., Tabor H. Polyamines. Annu Rev Biochem. 1984;53:749–790. doi: 10.1146/annurev.bi.53.070184.003533. [DOI] [PubMed] [Google Scholar]
  31. Xiao L., Celano P., Mank A. R., Griffin C., Jabs E. W., Hawkins A. L., Casero R. A., Jr Structure of the human spermidine/spermine N1-acetyltransferase gene (exon/intron gene organization and localization to Xp22.1). Biochem Biophys Res Commun. 1992 Sep 30;187(3):1493–1502. doi: 10.1016/0006-291x(92)90471-v. [DOI] [PubMed] [Google Scholar]

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

RESOURCES