Isolation and characterization of a cDNA clone that codes for human spermidine/spermine N1-acetyltransferase.

Spermidine/spermine N1-acetyltransferase (Spd/Spm acetyltransferase) is the rate-limiting enzyme in the catabolism of polyamines. This enzyme is highly inducible by several stimuli, including the natural polyamines and their structural analogues. To investigate the underlying mechanism responsible for the control of this enzyme a cDNA which codes for an active human Spd/Spm acetyltransferase has been isolated from a random primed cDNA library constructed from mRNA of a polyamine analogue treated large cell lung carcinoma line, NCI H157. The 972-base pair cDNA was identified using a 32-fold degenerate, 20-base oligomer probe to a 7-amino acid polypeptide sequence derived from the purified protein. The cDNA has a 513-base open reading frame that codes for a protein of 171 amino acids with a predicted molecular weight of 20,023. In vitro translation studies demonstrated the protein product of this cDNA to be a biologically active enzyme. The cDNA recognizes a 1.5-kilobase transcript in human cells which is highly induced in the human large cell lung carcinoma NCI H157 line following treatment with the polyamine analogue. The unusually high expression of Spd/Spm acetyltransferase mRNA by the NCI H157 cells in response to treatment does not appear to be a result of an amplification of the Spd/Spm acetyltransferase gene.

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The cytotoxic response of the human large cell lung carcinoma line NCI H157 to exposure to the polyamine analogue N1 N12-bis(ethyl)spermine (BESpm) is preceded by an extremely high induction of spermidine/spermine N1-acetyltransferase (SSAT). The human enzyme has now been purified greater than 300-fold to apparent homogeneity and found to cross-react with antisera raised against rat liver SSAT. Although other acetylases are capable of acetylating polyamines using acetyl-CoA as the acetyl donor, the greater than 600-fold induction within 24 h was found to be specifically SSAT, since essentially all activity was precipitable by the specific antisera. The human enzyme appears to be similar to the rat enzyme in subunit size under reducing conditions (approximately 20 kDa), substrate specificity and kinetic parameters. Preliminary results using actinomycin D and cycloheximide suggested that the unusually high induction by N1 N12-bis(ethyl)spermine in the human lung cancer line result from new mRNA and protein synthesis. This hypothesis is further substantiated here by 'in vitro' translation experiments comparing poly(A) mRNA from control and treated cells. The large cell lung carcinoma line NCI H157 represents a useful system to produce large amounts of the SSAT protein and to study the molecular events responsible for the induction and control of this important polyamine-metabolic enzyme. By using this rich source of SSAT protein, a partial amino acid sequence was determined by N-terminal sequencing of endoproteinase Lys-C digestion fragments. Further, this system should be useful in determining whether there is an association between the unusually high induction of the acetylase and the observed cytotoxicity in the NCI H157 cells.

Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues.

We have investigated the induction of an important polyamine metabolic enzyme, spermidine/spermine N1-acetyltransferase, in two human lung cancer cell lines which respond differently to treatment with the bis(ethyl)polyamine analogues. The human small cell lung carcinoma line NCI H82 has previously been shown to be minimally affected by treatment with these analogues, whereas the large cell undifferentiated lung carcinoma line, NCI H157, responds in a rapid cytotoxic manner (R.A. Casero, Jr., S. J. Ervin, P. Celano, S. B. Baylin, and R. J. Bergeron, Cancer Res., 49:639-643, 1989). The mechanisms underlying the differential response are unknown. In the responsive NCI H157 cells, the bis(ethyl)polyamines were found to induce spermidine/spermine N1-acetyltransferase in a time- and dose-dependent manner to maximum levels greater than 1700-fold over baseline. By contrast, the unresponsive NCI H82 cells exhibit minimal induction of spermidine/spermine N1-acetyltransferase to less than 7-fold increase after bis(ethyl)polyamine treatment, regardless of time or concentration examined. The results of the current study suggest that the differential induction of this key enzyme, which is rate limiting in the back conversion pathway of polyamine metabolism, may play a role in determining cell specific to the bis(ethyl)polyamine analogues.

Differential response to treatment with the bis(ethyl)polyamine analogues between human small cell lung carcinoma and undifferentiated large cell lung carcinoma in culture.

We have compared the effects of treatment with each of three bis(ethyl)polyamine analogues on a human small cell lung carcinoma (SCLC) line, NCI H82, and a non-small cell line, NCI H157, an undifferentiated large cell lung carcinoma. The bis(ethyl)polyamines have been shown to interfere with polyamine metabolism, presumably by regulation of the polyamine biosynthetic pathway in a manner similar to the natural polyamines, in contrast to direct inhibition of specific enzymes, such as ornithine decarboxylase. Each of these compounds was found to be relatively inactive in reducing growth rate, polyamine levels, or polyamine biosynthetic enzyme activity in the SCLC cells, a line which we have previously shown to be particularly sensitive to inhibition of polyamine biosynthesis by the direct ornithine decarboxylase inhibitor difluoromethylornithine. By contrast, each of the bis(ethyl)polyamines tested was found to be markedly cytotoxic (at concentrations of only 10 microM) to the non-SCLC line, NCI H157. Interestingly, the non-SCLC line has previously been demonstrated to be resistant to polyamine depletion by difluoromethylornithine. For each bis(ethyl)polyamine, cytotoxicity was accompanied by nearly complete depletion of all intracellular polyamines and a decrease in ornithine decarboxylase activity to undetectable levels. The current study emphasizes the phenotypic variability which can exist in response to inhibitors of polyamine biosynthesis and suggests a class of agents which may have clinical utility against the treatment-resistant non-SCLC lung cancers.

Comparison of the effects of treatment with the polyamine analogue N1,N8 bis(ethyl)spermidine (BESpd) or difluoromethylornithine (DFMO) on the Topoisomerase II mediated formation of 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex in the human lung carcinoma line NCI H157.

The positively charged polyamines putrescine, spermidine, and spermine are thought to be important in the maintenance of chromosomal structure. Polyamine depletion by the ornithine decarboxylase inhibitor, 2-difluoromethyl-ornithine (DFMO) is known to alter the effect of several DNA active agents, presumably resulting from the altered conformation of the polyamine depleted DNA. Here we compare the polyamine depletion effects of DFMO and the spermidine analogue N1,N8 bis(ethyl)spermidine (BESpd) on the formation of Topoisomerase II mediated, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex formation in human large cell undifferentiated lung carcinoma NCI H157 cells. This human cell line responds in the normal cytostatic manner to DFMO, whereas it responds in an unusual cytotoxic manner to treatment with BESpd. Here we report that neither DFMO nor BESpd alone affects the formation of cleavable complex. However, both compounds significantly enhance the m-AMSA induced formation of cleavable complex, each by approximately 1.6 fold. These results indicate that both DFMO and BESpd lead to a similar depletion of nuclear polyamines. Additionally, although BESpd closely resembles the natural polyamine spermidine, it appears that it cannot substitute for Spd at the level of DNA.