Abstract
Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.
Highlights
Polyamines are ubiquitous cellular components that affect a variety of biochemical processes, especially those involving synthesis of macromolecules [1]
Decreased TATA-binding protein-associated factor 7 (TAF7) expression, by RNA interference, in androgen-independent human prostate cancer LNCaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to methylglyoxal bis(guanylhydrazone) (MGBG)-induced growth arrest. These results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis
It has been reported that the overproduction of ornithine decarboxylase or spermidine/spermine N1-acetyltransferase altered cellular resistance to polyamine analogs that are accumulated through the polyamine transport system, like MGBG [27, 28]
Summary
After 2 h, the culture medium was collected and filtered through a 0.22-m pore size membrane This medium containing virus was added to 5 ϫ 105 CHO cells in a 10-cm dish. CHO cells were washed three times with the transport assay buffer and lysed with 0.1% Nonidet P-40. Southern Blot Analysis—Genomic DNA of CHO cells was isolated as described [23], digested with appropriate restriction enzymes, fractionated in 0.7% agarose gels, and transferred to a Zetaprobe nylon membrane (Bio-Rad). The protein concentration was determined with Bradford reagent using bovine serum albumin standards as described for the polyamine transport assay. The TAF7-RNAi expression plasmid was stably transfected into LNCaP104-R1 cells using Effectene as described above. The Ribosomal RNA Control Kit (Applied Biosystems) was used to normalize transcript levels between samples
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