ABSTRACT
Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.
Subject(s)
Antineoplastic Agents/pharmacology , Biogenic Polyamines/metabolism , Acetyltransferases/biosynthesis , Drug Design , Gene Expression Regulation/drug effects , Humans , Oxidoreductases Acting on CH-NH Group Donors/biosynthesis , Polyamine OxidaseABSTRACT
The natural polyamines and their analogues have been implicated in transcriptional regulation of specific genes. Human polyamine-modulated factor-1 (hPMF-1) was the first polyamine-responsive transcription factor identified. Here the mouse homologue of the hPMF-1 gene is described. Interestingly, the mouse gene (mPMF-1) codes for two alternatively spliced mRNAs. Both of the mouse splice variants, mPMF-1S and mPMF-1L, possess C-terminal coiled-coil domains nearly identical to that found in hPMF-1 and are highly homologous with the human protein. The C-terminal coiled-coil structure is necessary for transcriptional activation. However, the shorter protein, mPMF-1S, does not contain an N-terminal coiled-coil region as do both hPMF-1 and the longer mPMF-1L. mPMF-1L mRNA codes for a protein of 202 amino acids, 37 amino acids longer than the human protein. By contrast, mPMF-1S codes for only 133 amino acids, as a result of two exons being omitted compared with mPMF-1L. Both mouse transcription factors can interact with Nrf-2 (nuclear factor-E2-related factor 2), the normal partner of hPMF-1, substantiating the importance of the C-terminal coiled-coil region responsible for this interaction. Finally, the expression of mPMF-1 is induced when mouse M1 myeloid leukaemia cells are exposed to polyamine analogues, suggesting control similar to that observed for the hPMF-1.
Subject(s)
Transcription Factors/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Biogenic Polyamines/metabolism , Biogenic Polyamines/pharmacology , Cell Line , Cloning, Molecular , DNA, Complementary/genetics , DNA-Binding Proteins/metabolism , GA-Binding Protein Transcription Factor , Gene Expression/drug effects , Humans , In Vitro Techniques , Liver/metabolism , Mice , Molecular Sequence Data , Protein Biosynthesis , Protein Structure, Tertiary , RNA, Messenger/genetics , Rabbits , Sequence Homology, Amino Acid , Species Specificity , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Mammalian polyamine catabolism is under the control of two enzymes, spermidine/spermine N1-acetyltransferase and the flavin adenine dinucleotide-dependent polyamine oxidase (PAO). In this study, the cloning and initial characterization of human PAO is reported. A 1894-bp cDNA with an open reading frame of 1668-bp codes for a protein of 555 amino acids. In vitro transcription/translation of this cDNA clone produces the expected M(r) 61,900 protein with PAO activity. The PAO activity of this clone is inhibited by MDL 72,527, a specific inhibitor of mammalian PAO. However, neither pargyline, a specific monoamine oxidase inhibitor, nor semicarbazide, a specific diamine oxidase inhibitor, inhibits the PAO activity of this clone. PAO has been referred to as being constitutively expressed. However, 24-h exposure of a non-small cell lung carcinoma cell line, NCI H157, to 10 microM of N1,N"-bis(ethyl)norspermine results in approximately 5-fold induction of PAO mRNA and a >3-fold induction of PAO activity. These results demonstrate that in at least one cell type, PAO is up-regulated in response to polyamine analogue exposure. The PAO clone described here should provide a useful tool, which will facilitate the dissection of the role of polyamine catabolism in normal growth and in response to the antitumor polyamine analogues.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/genetics , Polyamines/pharmacology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Enzymologic/drug effects , Genes/genetics , Humans , Kinetics , Molecular Sequence Data , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Polyamines/chemistry , Protein Biosynthesis , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Transcription, Genetic , Tumor Cells, Cultured , Polyamine OxidaseABSTRACT
Polyamines and polyamine analogues have been demonstrated to modulate the transcription of various genes. Spermidine/spermine N(1)-acetyltransferase (SSAT) is transcriptionally regulated through the interaction of at least two trans-acting transcription factors, NF-E2-related factor 2 (Nrf-2) and PMF-1 (polyamine modulated factor-1). Nrf-2 has previously been shown to regulate transcription of other genes through interactions between its C-terminal leucine zipper and the leucine-zipper region of other members of the small Maf protein family (the term "Maf" is derived from MusculoAponeurotic-Fibrosarcoma virus). Here it is demonstrated that the interaction between Nrf-2 and PMF-1 is mediated through the binding of the leucine-zipper region of Nrf-2 and a C-terminal coiled-coil region of PMF-1 that does not contain a leucine zipper. Mutations that interrupt either the leucine zipper of Nrf-2 or the coiled-coil region of PMF-1 are demonstrated to alter the ability of these factors to interact, thus their ability to regulate the transcription of the SSAT gene is lost.
Subject(s)
Acetyltransferases/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Acetyltransferases/chemistry , Amino Acid Sequence , Base Sequence , DNA, Recombinant , Humans , Leucine Zippers , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-E2-Related Factor 2 , Two-Hybrid System TechniquesABSTRACT
The increased transcription and ultimate superinduction of the spermidine/spermine N(1)-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of approximately 1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of approximately 20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.
