Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jul 15;373(Pt 2):629–634. doi: 10.1042/BJ20021895

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

Tracy Murray-Stewart 1, Nancy B Applegren 1, Wendy Devereux 1, Amy Hacker 1, Renee Smith 1, Yanlin Wang 1, Robert A Casero Jr 1
PMCID: PMC1223504  PMID: 12697027

Abstract

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

Full Text

The Full Text of this article is available as a PDF (137.5 KB).

Selected References

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

  1. Alhonen L., Karppinen A., Uusi-Oukari M., Vujcic S., Korhonen V. P., Halmekytö M., Kramer D. L., Hines R., Jänne J., Porter C. W. Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem. 1998 Jan 23;273(4):1964–1969. doi: 10.1074/jbc.273.4.1964. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Casero R. A., Jr, Gabrielson E. W., Pegg A. E. Immunohistochemical staining of human spermidine/spermine N1-acetyltransferase superinduced in response to treatment with antitumor polyamine analogues. Cancer Res. 1994 Aug 1;54(15):3955–3958. [PubMed] [Google Scholar]
  7. 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]
  8. Coleman C. S., Huang H., Pegg A. E. Role of the carboxyl terminal MATEE sequence of spermidine/spermine N1-acetyltransferase in the activity and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine. Biochemistry. 1995 Oct 17;34(41):13423–13430. doi: 10.1021/bi00041a020. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Fogel-Petrovic M., Kramer D. L., Ganis B., Casero R. A., Jr, Porter C. W. Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N1-acetyltransferase--a gene uniquely regulated by polyamines and their analogs. Biochim Biophys Acta. 1993 Nov 16;1216(2):255–264. doi: 10.1016/0167-4781(93)90152-4. [DOI] [PubMed] [Google Scholar]
  12. Fogel-Petrovic M., Vujcic S., Miller J., Porter C. W. Differential post-transcriptional control of ornithine decarboxylase and spermidine-spermine N1-acetyltransferase by polyamines. FEBS Lett. 1996 Aug 5;391(1-2):89–94. doi: 10.1016/0014-5793(96)00710-7. [DOI] [PubMed] [Google Scholar]
  13. Gabrielson E. W., Pegg A. E., Casero R. A., Jr The induction of spermidine/spermine N1-acetyltransferase (SSAT) is a common event in the response of human primary non-small cell lung carcinomas to exposure to the new antitumor polyamine analogue N1,N11-bis(ethyl)norspermine. Clin Cancer Res. 1999 Jul;5(7):1638–1641. [PubMed] [Google Scholar]
  14. Kabra P. M., Lee H. K., Lubich W. P., Marton L. J. Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue. J Chromatogr. 1986 Jul 11;380(1):19–32. doi: 10.1016/s0378-4347(00)83621-x. [DOI] [PubMed] [Google Scholar]
  15. Libby P. R., Bergeron R. J., Porter C. W. Structure-function correlations of polyamine analog-induced increases in spermidine/spermine acetyltransferase activity. Biochem Pharmacol. 1989 May 1;38(9):1435–1442. doi: 10.1016/0006-2952(89)90182-2. [DOI] [PubMed] [Google Scholar]
  16. Lu L., Berkey K. A., Casero R. A., Jr RGFGIGS is an amino acid sequence required for acetyl coenzyme A binding and activity of human spermidine/spermine N1acetyltransferase. J Biol Chem. 1996 Aug 2;271(31):18920–18924. doi: 10.1074/jbc.271.31.18920. [DOI] [PubMed] [Google Scholar]
  17. Mank-Seymour A. R., Murray T. R., Berkey K. A., Xiao L., Kern S., Casero R. A., Jr Two active copies of the X-linked gene spermidine/spermine N1-acetyltransferase (SSAT) in a female lung cancer cell line are associated with an increase in sensitivity to an antitumor polyamine analogue. Clin Cancer Res. 1998 Aug;4(8):2003–2008. [PubMed] [Google Scholar]
  18. McCloskey D. E., Coleman C. S., Pegg A. E. Properties and regulation of human spermidine/spermine N1-acetyltransferase stably expressed in Chinese hamster ovary cells. J Biol Chem. 1999 Mar 5;274(10):6175–6182. doi: 10.1074/jbc.274.10.6175. [DOI] [PubMed] [Google Scholar]
  19. McCloskey D. E., Pegg A. E. Altered spermidine/spermine N1-acetyltransferase activity as a mechanism of cellular resistance to bis(ethyl)polyamine analogues. J Biol Chem. 2000 Sep 15;275(37):28708–28714. doi: 10.1074/jbc.M004120200. [DOI] [PubMed] [Google Scholar]
  20. Niiranen Kirsi, Pietilä Marko, Pirttilä Terhi J., Järvinen Aki, Halmekytö Maria, Korhonen Veli-Pekka, Keinänen Tuomo A., Alhonen Leena, Jänne Juhani. Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues. J Biol Chem. 2002 May 8;277(28):25323–25328. doi: 10.1074/jbc.M203599200. [DOI] [PubMed] [Google Scholar]
  21. Parry L., Balaña Fouce R., Pegg A. E. Post-transcriptional regulation of the content of spermidine/spermine N1-acetyltransferase by N1N12-bis(ethyl)spermine. Biochem J. 1995 Jan 15;305(Pt 2):451–458. doi: 10.1042/bj3050451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pegg A. E., Hu R. H. Effect of polyamine analogues and inhibition of polyamine oxidase on spermidine/spermine N1-acetyltransferase activity and cell proliferation. Cancer Lett. 1995 Aug 16;95(1-2):247–252. doi: 10.1016/0304-3835(95)03897-6. [DOI] [PubMed] [Google Scholar]
  23. Pietilä M., Alhonen L., Halmekytö M., Kanter P., Jänne J., Porter C. W. Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem. 1997 Jul 25;272(30):18746–18751. doi: 10.1074/jbc.272.30.18746. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Suppola S., Pietilä M., Parkkinen J. J., Korhonen V. P., Alhonen L., Halmekytö M., Porter C. W., Jänne J. Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue. Biochem J. 1999 Mar 1;338(Pt 2):311–316. [PMC free article] [PubMed] [Google Scholar]
  27. Vujcic S., Halmekyto M., Diegelman P., Gan G., Kramer D. L., Janne J., Porter C. W. Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. J Biol Chem. 2000 Dec 8;275(49):38319–38328. doi: 10.1074/jbc.M003270200. [DOI] [PubMed] [Google Scholar]
  28. Wang Y., Devereux W., Stewart T. M., Casero R. A., Jr Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene. J Biol Chem. 1999 Jul 30;274(31):22095–22101. doi: 10.1074/jbc.274.31.22095. [DOI] [PubMed] [Google Scholar]
  29. Wang Y., Xiao L., Thiagalingam A., Nelkin B. D., Casero R. A., Jr The identification of a cis-element and a trans-acting factor involved in the response to polyamines and polyamine analogues in the regulation of the human spermidine/spermine N1-acetyltransferase gene transcription. J Biol Chem. 1998 Dec 18;273(51):34623–34630. doi: 10.1074/jbc.273.51.34623. [DOI] [PubMed] [Google Scholar]
  30. Xiao L., Casero R. A., Jr Differential transcription of the human spermidine/spermine N1-acetyltransferase (SSAT) gene in human lung carcinoma cells. Biochem J. 1996 Jan 15;313(Pt 2):691–696. doi: 10.1042/bj3130691. [DOI] [PMC free article] [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