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 N 1-Acetyltransferase Gene Transcription

Abstract

The superinduction of spermidine/spermine N 1 -acetyltransferase (SSAT) gene has been associated with a cytotoxic response to a new class of antineoplastic polyamine analogues. The initial mechanism of SSAT superinduction is an increase in transcription in response to analogue exposure. This increased transcription appears to be modulated through the association between a nuclear protein factor and a cis-element described here as the polyamine-responsive element (PRE). The PRE was identified as a 9-base pair sequence, 5-TATGACTAA-3, in the context of a 31-base pair stretch from -1522 to -1492 base pairs with respect to the SSAT transcriptional start site. This element binds a nuclear factor from polyamine analogue-responsive cells, but not from polyamine analogue-insensitive cells. The labeled PRE was used to clone and identify the transcription factor, Nrf-2, that binds constitutively to the PRE sequence. Although the PRE sequence shares homology to the originally identified Nrf-2 recognition sequence, the two sequences are not identical. The Nrf-2 transcription factor appears only to be present in cell types that are capable of expressing high amounts of SSAT. The results of these studies suggest that Nrf-2, bound to the PRE, plays an important regulatory role of expression of the human SSAT gene.The requirement for polyamines in growth and development is absolute in eukaryotic cells (1). Although this requirement has been well established, some of the precise molecular functions of the polyamines have only recently been elucidated. Several newly synthesized polyamine analogues have been examined for their potential as antitumor agents (2-4). These compounds have been designed to alter the regulation of polyamine metabolism and interfere with the normal functions of polyamines in tumor cells. Some of these compounds appear to act in an association with an ability to superinduce the expression of spermidine/spermine N 1 -acetyltransferase (SSAT), 1 the rate-limiting enzyme in polyamine catabolism (5-7). The superinduction of SSAT has been associated with a phenotypespecific sensitivity to some of the antitumor polyamine analogues, and there are several reports demonstrating superinduction of SSAT and tumor cell toxicity (8 -13). Therefore, there is considerable interest in elucidating the mechanisms that differentiate sensitive tumors from analogue-insensitive tumors and normal tissue.Control of the analogue-mediated superinduction of SSAT is initiated at the level of transcription (14, 15). The -fold induction of SSAT transcription by analogue exposure is relatively small (2-7-fold) compared with the ultimate induction of enzyme activity; however, this increased transcription is necessary for the downstream events, which can result in Ͼ1000-fold increases in SSAT activity (8,16,17). Here we identify a cis-element in the 5Ј regulatory region of the human SSAT and define it as a polyamine-responsive element (PRE). Further, electrophoretic mobility shift assay (EMSA) results demonstrate that the PRE is bound by a...

Tumor Necrosis Factor α Induces Spermidine/Spermine N1-Acetyltransferase through Nuclear Factor κBin Non-small Cell Lung Cancer Cells

“…Thus, TNF␣ stimulation of SSAT may, in some cases, allow cells to reduce their growth rate when exposed to a stressful environment, and the observed cytotoxic drug responses may be an exaggerated extension of this normal response. Although more studies will be necessary to test this possibility, it is intriguing to consider the potential cross-talk that may occur between the polyamine response element (7,45), which is thought to mediate SSAT induction produced by cytotoxic polyamine analogues, and NFB and how such cross-talk might be exploited for therapeutic advantage.…”

“…SSAT activity is highly regulated and is induced rapidly in response to a number of stimuli, including polyamines, polyamine analogues, hormones, physiological stimuli, drugs, and toxic agents (1). It has been shown that the regulation of SSAT by the natural polyamines and the anti-tumor polyamine analogues is through the polyamine response element (7). Further, the superinduction of SSAT by polyamine analogues has been implicated in the cell type-specific cytotoxic response of several important human tumors (8 -13).…”

Polyamines as anabolic growth regulators revealed by transcriptome analysis and metabolite profiles of tomato fruits engineered to accumulate spermidine and spermine

“…One of the mechanisms of PA-mediated effects on transcription leading to protein synthesis involves binding of trans-acting factors to PA-responsive, ciselement (PRE) identified in human Spd/Spm N 1 -acetyltransferase (SSAT) gene (Chen et al 1997;Wang et al 1998Wang et al , 2001). The sequence 5Ј-TATGACTAA-3Ј represents the PRE in human SSAT.…”

Polyamine analogues inhibit the ubiquitination of spermidine/spermine N1-acetyltransferase and prevent its targeting to the proteasome for degradation

“…Endogenous SSAT activity is normally very low in cultured cells and tissues. Regulation of enzyme activity occurs via changes in the amount of enzyme protein, and these alterations are mediated through effects at several levels, including gene transcription, mRNA translation and protein turnover [3][4][5][6]. In the uninduced state, SSAT turns over very rapidly, although accurate measurements of the half-life are difficult to obtain because of the low level of the protein.…”