Clinical Cancer Research The Science of Cancer Health Disparities
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gabrielson, E. W.
Right arrow Articles by Casero, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gabrielson, E. W.
Right arrow Articles by Casero, R. A., Jr.
Clinical Cancer Research Vol. 5, 1638-1641, July 1999
© 1999 American Association for Cancer Research

Clinical Trials

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)norspermine1

Edward W. Gabrielson, Anthony E. Pegg and Robert A. Casero, Jr.2

Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21231 [E. W. G., R. A. C.], and The Department of Cellular and Molecular Physiology and Pharmacology, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033 [A. E. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Several new polyamine analogues have been developed for the treatment of human solid tumors. The phenotype-specific activity of some of these analogues has been associated with the superinduction of the rate-limiting enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Using immunohistochemistry, we found a majority (64%) of human primary lung cancer explants to exhibit high expression of SSAT after treatment with 10 µMN1,N11-bis(ethyl)norspermine, an agent currently undergoing Phase II clinical trials against several important human solid tumors. The staining of SSAT was found specifically in the tumor tissue and not in the neighboring normal lung tissue. These results demonstrate the ability to detect induction of SSAT in clinical specimens and suggest the potential of this test as a prognostic indicator of drug response.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The polyamine metabolic pathway has been investigated as a target for antineoplastic therapy for several years (1, 2, 3) . Recently, several new antitumor polyamine analogues have demonstrated impressive activity both in vitro and in vivo against a variety of important human solid tumors (4, 5, 6, 7, 8, 9, 10, 11) . These results have been so encouraging that multiple compounds are now in human clinical trials (12) . Among these newer agents, the symmetrically substituted bis(ethyl)polyamine analogues and a series of unsymmetrically substituted polyamine analogues have demonstrated great promise (13, 14, 15, 16, 17) . With several of the most promising agents, there has been an association demonstrated between the superinduction of the rate-limiting enzyme SSAT3 and cellular response (5 , 7) . This association was first demonstrated using models representing the major forms of human lung cancer. Specifically, it was demonstrated that the non-SCLCs typically responded to analogue treatment with a significant induction of SSAT and subsequent cytotoxicity. In contrast, the human SCLCs did not respond with significant induction of SSAT and were generally only moderately growth-inhibited (4, 5, 6) . These results were subsequently confirmed in other systems, including human melanomas and pancreatic carcinomas (7, 8, 9) . In at least some systems, there is a clear role for the induction of SSAT and the resultant cytotoxic response. The present study was undertaken with the objective of determining how frequently SSAT is significantly induced in primary lung cancers, tumor phenotypes for which we are currently conducting human Phase II clinical trials.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals and Tissue Samples.
BENSpm was kindly provided by Parke-Davis Pharmaceuticals (Ann Arbor, MI). Primary lung tumor samples (stages I and II) were obtained at the time of scheduled surgical resection in accordance with institutional policy and were in excess of tissue needed for diagnosis.

Exposure of Tumor Tissue to BENSpm and Immunohistochemical Analysis.
Thin slices (~1 mm in thickness) of tumor tissue were incubated in RPMI 1640 supplemented with 10% fetal bovine serum, with or without 10 µM BENSpm, for 22 h. Samples were then fixed in 10% buffered formalin and routinely embedded in paraffin. The concentration of BENSpm was chosen based on past experience in both in vitro and in vivo studies and seems to be an attainable concentration with minimal toxicity, as observed in Phase I clinical trials (5 , 18) .

Sections of the paraffin-embedded samples were prepared for immunohistochemical analysis using a modification of our previously described technique. Sections (5 µm-thick) were treated by the high pH antigen retrieval technique, using an antiserum raised against a recombinant human SSAT protein (19) . Specifically, slides were incubated in xylene to remove paraffin and then hydrated in ethanol/water solutions. Slides were then placed in Dako Target Retrieval Solution, high pH (DAKO, Inc., Carpinteria, CA) prewarmed to 95°C for 30 min. After antigen retrieval, staining was performed on the DAKO Autostainer by incubating slides with SSAT antiserum (1:1000 dilution) for 2 h at room temperature and then developing the slides using the DAKO LSAB 2 kit (which includes secondary antibody).

The scoring system used for analysis of the antibody-stained samples is indicated in Table 1Citation . This antiserum has worked well in both tissue and cell culture samples.


View this table:
[in this window]
[in a new window]
  		Table 1 Immunohistochemical analysis of human primary lung tumor explants exposed to 10 µM BENSpm for 22 h in vitro

 

   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The phenotype-specific response to the new antitumor polyamine analogues has led to an intense investigation into the underlying mechanisms responsible for their activity. Although the induction of SSAT is not the only mechanism associated with analogue response, the correlation between high SSAT induction and tumor response in a number of important human solid tumor models suggests it can have a significant role in determining drug response (7 , 20 , 21) . Additionally, recent transgenic studies overexpressing SSAT in mouse tissue has clearly demonstrated a significant effect of SSAT overexpression in response to polyamine analogues (22 , 23) .

In light of these data, the current study was undertaken to determine the frequency of SSAT induction in primary human lung tumors treated with the analogue. We analyzed a total of 34 paired patient samples obtained from 22 males and 12 females. Overall, 21 of 34 (62%) fresh tumor samples stained 2+ or greater after a 22-h exposure to BENSpm; whereas, 4 tumor samples demonstrated weak staining, with only 9 tumor samples demonstrating no staining at all. Representative staining for SSAT in lung cancer samples is shown in Fig. 1Citation . All tumors tested were from patients with stage I or stage II disease, and there was no apparent association between disease stage and staining for SSAT.



View larger version (122K):
[in this window]
[in a new window]
  		Fig. 1. Immunohistochemical stain-ing of human lung tumor samples. Samples were prepared as detailed in "Materials and Methods." A, untreated squamous cell carcinoma. B, squamous tumor from the same patient treated with 10 µM BENSpm; C, untreated adenocarcinoma; adenocarcinoma from the same patient treated with 10 µM. Note that the antibody staining is limited to the tumor tissue and does not extend into the normal tissue, indicating the specificity of SSAT induction to the tumor tissue.

 
By histological phenotype, we studied 19 squamous cancers, 11 adenocarcinomas, and 3 large cell carcinomas. (Table 1)Citation . Only a single small cell cancer sample was available for analysis because this variant of lung cancer is not typically treated by surgical resection (Table 1)Citation . Ten of 19 squamous carcinomas exhibited 2+ or greater staining. Although previous studies have suggested the potential of increased expression of SSAT in female cells because SSAT is an X-linked gene (24) , there was no apparent difference between the staining of tumor tissue derived from males or females. However, it should be noted that 5 squamous carcinomas exhibited no significant staining. Six of 11 adenocarcinomas stained 2+ or greater, with 3 demonstrating no significant staining. As expected, the large cell carcinoma samples demonstrated strong staining and the single small cell carcinoma sample did not stain for induced SSAT (5 , 20) . It should be noted that although not directly measured in the current study, the susceptibility to BENSpm and differences in analogue-induced SSAT have not been attributable to differences in the ability to accumulate the analogue (5 , 7 , 20) .

It is highly significant that the staining by the SSAT-specific antibody occurs only in tumor tissue, indicating that the high induction of SSAT by BENSpm is a tumor-specific phenomenon. This observation is consistent with preliminary cell culture and nude mouse studies demonstrating the SSAT antibody specificity for induced tumor tissue (25) . However, it is important to note that this is the first study examining the response of SSAT induction of primary human tumors to BENSpm exposure.

Although there have only have been a few studies directly linking high SSAT induction and cellular response (21, 22, 23) , the tumor-specific induction of SSAT in response to BENSpm has potentially other clinically relevant significance. Specifically, a pro-drug strategy using a combination of agents; the first agent to specifically induce SSAT in tumor tissue, and a second agent provided as a pro-drug that would specifically be activated by the highly induced enzyme into a cytotoxic agent at the desired site.

Previous studies have clearly demonstrated a direct correlation between the amount of SSAT protein and activity (26 , 27) and, in limited settings, between SSAT induction and tumor response. Furthermore, there have been no studies demonstrating an increase in SSAT protein without a concomitant proportional increase in activity (28) . Thus, although we have not directly measured SSAT activity in this study, it is reasonable to hypothesize that induction of SSAT protein, as indicated by antiserum staining, correlates with increased enzyme activity in response to polyamine analogue exposure.

In summary, the in vitro exposure of human primary lung tumor samples clearly demonstrates that a specific SSAT antiserum can be used to label SSAT from polyamine analogue-induced cells. It also confirms our previously published results using cells lines that indicated that the non-SCLCs frequently respond to analogue treatment with high induction of SSAT (5 , 20) . As clinical trials progress, these data will be useful in designing strategies to evaluate both tumor site drug delivery and potential tumor response. Furthermore, in those instances where tumor tissue is obtainable through minimally invasive techniques before treatment, the measurement of SSAT induction after in vitro exposure to drug may prove to be a useful prognostic indicator.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA51085, CA58184, and GM26290. Back

2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Center Research Laboratories, 424 North Bond Street, Baltimore, MD 21231. E-mail: casero{at}welchlink.welch.jhu.edu Back

3 The abbreviations used are: SSAT, N1-acetyltransferase; SCLC, small cell lung carcinoma; BENSpm, N1,N11-bis(ethyl)norspermine. Back

Received 1/29/99; revised 3/19/99; accepted 3/22/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. McCann P. P., Pegg A. E. Ornithine decarboxylase as an enzyme target for therapy. Pharmacol. Ther., 54: 195-215, 1992.[Medline]
  2. Marton L. J., Pegg A. E. Polyamines as targets for therapeutic intervention. Ann. Rev. Pharmacol., 35: 55-91, 1995.[Medline]
  3. Porter C. W., Regenass U., Bergeron R. J. Polyamine inhibitors and analogues as potential anticancer agents Dowling R. H. Folsch U. R. Loser C. eds. . Polyamines in the Gastrointestinal Tract, : 301-322, Academic Publishers Dordrecht 1992.
  4. Casero R. A., Ervin S. J., Celano P., Baylin S. B., Bergeron R. J. Differential response to treatment with bis(ethyl)polyamine analogues between human small cell lung carcinoma and undifferentiated large cell lung carcinoma in culture. Cancer Res., 49: 639-643, 1989.[Abstract/Free Full Text]
  5. Casero R. A., 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., 49: 3829-3833, 1989.[Abstract/Free Full Text]
  6. Casero R. A., Mank A. R., Saab N. H., Wu R., Dyer W. J., Woster P. M. Growth and biochemical effects of asymmetrically substituted polyamine analogues in human lung tumor cells. Cancer Chemother. Pharmacol., 36: 69-74, 1995.[Medline]
  7. 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., 51: 3715-3720, 1991.[Abstract/Free Full Text]
  8. 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., 53: 581-586, 1993.[Abstract/Free Full Text]
  9. 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., 30: 179-182, 1992.[Medline]
  10. Bernacki R. J., Bergeron R. J., Porter C. W. Antitumor activity of N,N'-bis(ethyl)spermine homologues against human MALME-3 melanoma xenografts. Cancer Res., 52: 2424-2430, 1992.[Abstract/Free Full Text]
  11. Bernacki R. J., Oberman E. J., Seweryniak K. E., Atwood A., Bergeron R. J., Porter C. W. Preclinical antitumor efficacy of the polyamine analogue N1,N11-diethylnorspermine administered by multiple injection or continuous infusion. Clin. Cancer Res., 1: 847-857, 1995.[Abstract]
  12. Ettinger D. S., Bowling K., Hoker B., Chen T. L., Zabelina Y., Casero R. A. Phase I study of N1,N11-diethylnorspermine (DENSPM) in patients with non-small cell lung cancer (NSCLC). Proc. Am. Soc. Clin. Oncol., 17: 733 1998.
  13. Bergeron R. J., Neims A. H., McManis J., Hawthorne J. S., Vinson J. R. T., Bortell R., Ingeno M. J. Synthetic polyamine analogues as antineoplastics. J. Med. Chem., 31: 1183-1190, 1988.[Medline]
  14. Bergeron R. J., Hawthorne T. R., Vinson J. R. T., Beck D. E., Jr., Ingeno M. J. Role of the methylene backbone in the antiproliferative activity of polyamine analogues of L1210 cells. Cancer Res., 49: 2959-2964, 1989.[Abstract/Free Full Text]
  15. Woster P. M. S-Adenosylmethionine decarboxylase and spermidine/spermine-N1-acetyltransferase: emerging targets for rational inhibitor design Casero A. eds. . Molecular Biology Intelligence Unit—Polyamines: Regulation and Molecular Interaction, : 171-204, R. G. Landes Company Austin, TX 1996.
  16. Wu R., Saab N. H., Huang H., Wiest L., Pegg A. E., Casero R. A., Jr., Woster P. M. Synthesis and evaluation of polyamine phosphinates and phosphonamidates as transition state analogue inhibitors of spermidine/spermine-N1-acetyltransferase. Bioorg. Med. Chem., 4: 825-836, 1996.[Medline]
  17. Saab N. H., West E. E., Bieszk N. C., Preuss C. V., Mank A. R., Casero R. A., Woster P. M. Synthesis and evaluation of unsymmetrically substituted polyamine analogs as inhibitors of human spermidine/spermine N1-acetyltransferase (SSAT) and potential antitumor agents. J. Med. Chem., 36: 2998-3004, 1993.[Medline]
  18. 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., 12: 1083-1090, 1992.[Medline]
  19. Casero R. A., Gabrielson E. W., Pegg A. E. Immunohistochemical staining of human spermidine/spermine N1-acetyltransferase super-induced in response to treatment with antitumor polyamine analogues. Cancer Res., 54: 3955-3958, 1992.[Abstract/Free Full Text]
  20. Casero R. A., 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., 52: 5359-5363, 1992.[Abstract/Free Full Text]
  21. Ha H. C., Woster P. M., Yager J. D., Casero R. A., Jr. The role of polyamine catabolism in polyamine analogue-induced programmed cell death. Proc. Natl. Acad. Sci. USA, 94: 11557-11562, 1997.[Abstract/Free Full Text]
  22. Pietila M., Alhonen L., Halmekyto M., Kanter P., Janne 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., 272: 18746-18751, 1997.[Abstract/Free Full Text]
  23. Alhonen L., Karppinen A., Uusi-Oukari M., Vujcic S., Korhonen V-P., Halmekyto M., Kramer D. L., Hines R., Janne 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., 273: 1964-1969, 1998.[Abstract/Free Full Text]
  24. Mank-Seymour A. R., Murray T. R., Berkey K. A., Xiao L., Kern S., Casero R. A. The reactivation of the normally inactive copy of the X-linked gene, spermidine/spermine N1-acetyltransferase (SSAT) is associated with an increase in sensitivity to an antitumor polyamine analogue. Clin. Cancer Res., 4: 2003-2008, 1998.[Abstract]
  25. Casero R. A., Gabrielson E. W., Pegg A. E. Immunohistochemical staining of human spermidine/spermine N1-acetyltransferase super-induced in response to treatment with antitumor polyamine analogues. Cancer Res., 54: 3955-3958, 1994.
  26. Della Ragione F., Pegg A. E. Purification and characterization of spermidine/spermine N1-acetyltransferase from rat liver. Biochemistry, 21: 6152-6158, 1982.[Medline]
  27. Casero R. A., 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., 270: 615-620, 1990.[Medline]
  28. Casero R. A., Pegg A. E. Spermidine/spermine N1-acetyltransferase-the turning point in polyamine metabolism. FASEB J., 7: 653-661, 1993.[Abstract]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
W. L. Allen, E. G. McLean, J. Boyer, A. McCulla, P. M. Wilson, V. Coyle, D. B. Longley, R. A. Casero Jr., and P. G. Johnston
The role of spermidine/spermine N1-acetyltransferase in determining response to chemotherapeutic agents in colorectal cancer cells
Mol. Cancer Ther., January 1, 2007; 6(1): 128 - 137.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Pledgie, Y. Huang, A. Hacker, Z. Zhang, P. M. Woster, N. E. Davidson, and R. A. Casero Jr.
Spermine Oxidase SMO(PAOh1), Not N1-Acetylpolyamine Oxidase PAO, Is the Primary Source of Cytotoxic H2O2 in Polyamine Analogue-treated Human Breast Cancer Cell Lines
J. Biol. Chem., December 2, 2005; 280(48): 39843 - 39851.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
N. Martinez, M. Sanchez-Beato, A. Carnero, V. Moneo, J. C. Tercero, I. Fernandez, M. Navarrete, J. Jimeno, and M. A. Piris
Transcriptional signature of Ecteinascidin 743 (Yondelis, Trabectedin) in human sarcoma cells explanted from chemo-naive patients
Mol. Cancer Ther., May 1, 2005; 4(5): 814 - 823.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
J. J.W. Chen, Y.-C. Lin, P.-L. Yao, A. Yuan, H.-Y. Chen, C.-T. Shun, M.-F. Tsai, C.-H. Chen, and P.-C. Yang
Tumor-Associated Macrophages: The Double-Edged Sword in Cancer Progression
J. Clin. Oncol., February 10, 2005; 23(5): 953 - 964.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
Y. Huang, E. R. Hager, D. L. Phillips, V. R. Dunn, A. Hacker, B. Frydman, J. A. Kink, A. L. Valasinas, V. K. Reddy, L. J. Marton, et al.
A Novel Polyamine Analog Inhibits Growth and Induces Apoptosis in Human Breast Cancer Cells
Clin. Cancer Res., July 1, 2003; 9(7): 2769 - 2777.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
K. R. Lawson, S. Marek, J. A. Linehan, P. M. Woster, R. A. Casero Jr., C. M. Payne, and E. W. Gerner
Detoxification of the Polyamine Analogue N1-Ethyl-N11-[(cycloheptyl)methy]-4,8-diazaundecane (CHENSpm) by Polyamine Oxidase
Clin. Cancer Res., May 1, 2002; 8(5): 1241 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. Moffatt, M. Hashimoto, A. Kojima, D. O. Kennedy, A. Murakami, K. Koshimizu, H. Ohigashi, and I. Matsui-Yuasa
Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation
Carcinogenesis, December 1, 2000; 21(12): 2151 - 2157.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gabrielson, E. W.
Right arrow Articles by Casero, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gabrielson, E. W.
Right arrow Articles by Casero, R. A., Jr.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online