TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m401078200
ISSN1083-351X
AutoresJunichi Fukuchi, Richard A. Hiipakka, John M. Kokontis, Kazuhiro Nishimura, Kazuei Igarashi, Shutsung Liao,
Tópico(s)Pancreatic function and diabetes
ResumoIdentification 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. 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. Polyamines are ubiquitous cellular components that affect a variety of biochemical processes, especially those involving synthesis of macromolecules (1Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar). The growth of mammalian cells requires polyamines, and the optimal intracellular concentration is regulated by multiple pathways, including synthesis from amino acid precursors, cellular uptake mechanisms, as well as stepwise degradation and efflux.The importance of polyamines for cell proliferation has led to the development of various inhibitors of the biosynthetic pathway or analogs that disrupt normal polyamine functions (2Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar). One inhibitor, methylglyoxal bis(guanylhydrazone) (MGBG), 1The abbreviations used are: MGBG, methylglyoxal bis(guanylhydrazone); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary; CMV, cytomegalovirus; HA, hemagglutinin; JNK, c-Jun NH2-terminal kinase; ORNT2, ornithine transporter 2; RACE, rapid amplification of cDNA ends; RNAi, RNA interference; TAF7, TATA-binding protein-associated factor 7.1The abbreviations used are: MGBG, methylglyoxal bis(guanylhydrazone); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary; CMV, cytomegalovirus; HA, hemagglutinin; JNK, c-Jun NH2-terminal kinase; ORNT2, ornithine transporter 2; RACE, rapid amplification of cDNA ends; RNAi, RNA interference; TAF7, TATA-binding protein-associated factor 7. is a structural analog of the natural polyamine spermidine, and it is a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme that supplies propylamino groups for synthesis of spermidine and spermine (3Williams-Ashman H.G. Schenone A. Biochem. Biophys. Res. Commun. 1972; 46: 288-295Crossref PubMed Scopus (299) Google Scholar). MGBG is transported into cells by the polyamine transporter (4Heaton M.A. Flintoff W.F. J. Cell. Physiol. 1988; 136: 133-139Crossref PubMed Scopus (20) Google Scholar, 5Byers T.L. Pegg A.E. Am. J. Physiol. 1989; 257: C545-C553Crossref PubMed Google Scholar). Accumulation of MGBG in cells induces apoptosis as well as reduction of cellular polyamine levels (6Davidson K. Petit T. Izbicka E. Koester S. Von Hoff D.D. Anticancer Drugs. 1998; 9: 635-640Crossref PubMed Scopus (12) Google Scholar). Because polyamines are important for cell proliferation, MGBG has been recognized as a potential antineoplastic agent. Despite the long-standing availability of MGBG, the exact mechanism for its cytotoxicity is not clear. Mandel and Flintoff (7Mandel J.L. Flintoff W.F. J. Cell. Physiol. 1978; 97: 335-343Crossref PubMed Scopus (107) Google Scholar) reported that MGBG-resistant Chinese hamster ovary (CHO) cells have decreased polyamine transport activity, indicating that an active mammalian polyamine transport system was a critical factor for the intracellular accumulation of this polyamine analog as well as natural polyamines.Polyamine transport activity can be assayed using radioactive polyamines (8Kakinuma Y. Hoshino K. Igarashi K. Eur. J. Biochem. 1988; 176: 409-414Crossref PubMed Scopus (88) Google Scholar), and recently, transport activity was visualized using polyamines conjugated to a fluorescent dye (9Cullis P.M. Green R.E. Merson-Davies L. Travis N. Chem. Biol. 1999; 6: 717-729Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Although bacterial and/or fungal polyamine transporter genes have been characterized (10Kashiwagi K. Hosokawa N. Furuchi T. Kobayashi H. Sasakawa C. Yoshikawa M. Igarashi K. J. Biol. Chem. 1990; 265: 20893-20897Abstract Full Text PDF PubMed Google Scholar, 11Igarashi K. Kashiwagi K. Biochem. Biophys. Res. Commun. 2000; 271: 559-564Crossref PubMed Scopus (718) Google Scholar), the mammalian polyamine transporter gene has not been identified, even though the entire mouse and human genomes have been sequenced. Genetic approaches to identify the mammalian polyamine transporter gene have been tried by several research groups. Adair et al. (12Adair G.M. Siciliano M.J. Mol. Cell. Biol. 1985; 5: 109-113Crossref PubMed Scopus (10) Google Scholar) mapped the MGBG resistance locus to part of chromosome Z3 in Chinese hamster ovary cells, and Byers et al. (13Byers T.L. Wechter R. Nuttall M.E. Pegg A.E. Biochem. J. 1989; 263: 745-752Crossref PubMed Scopus (51) Google Scholar) demonstrated that a human DNA fragment could restore MGBG sensitivity to MGBG-resistant cells. Most recently, using a plasmid base gene targeting vector, Shao et al. (14Shao D. Xiao L. Ha H.C. Casero Jr., R.A. J. Cell. Physiol. 1996; 166: 43-48Crossref PubMed Scopus (10) Google Scholar) reported a decrease in polyamine transport activity in the human non-small cell lung carcinoma line NCI H157. Despite these efforts, genes responsible for the transport activity have not been identified. Here we report the identification of a gene involved in polyamine transport activity using retroviral gene trap screening and the characterization of its physiological function.EXPERIMENTAL PROCEDURESMaterials—Hygromycin B was purchased from Calbiochem. MGBG, spermidine, spermine, ornithine, and Hoechst 33258 were obtained from Sigma. d-JNKI-1 and SP600125 were purchased from Alexis Biochemicals (San Diego). [α-32P]dCTP (111 TBq/mmol), [14C]spermidine (4.29 GBq/mmol), [14C]spermine (4.07 GBq/mmol), [1-14C] acetyl-CoA (1.89 GBq/mmol), and [1-14C]ornithine (2.05 GBq/mmol) were purchased from Amersham Biosciences. Monoclonal antibody against the HA tag was obtained from Covance (Princeton, NJ). Polyclonal antibodies against cyclin A and eIF-4E were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Cell Culture—Cells were cultured at 37 °C in the indicated media and in the presence of 5% CO2. The Chinese hamster ovary cell line CHO-Cl22 (CHO) was cloned by limiting dilution from wild type CHO-K1 (ATCC, Manassas, VA) in our laboratory. For convenience, the parent cells, CHO-Cl22 are described as “CHO cells” in this paper. CHO cells and derivatives were maintained in minimum essential medium-α (Invitrogen) supplemented with 10% fetal bovine serum (Gemini BioProducts, Woodland, CA), 100 units/ml penicillin G, and 0.1 mg/ml streptomycin sulfate. Gene trap virus-producing cells, ψ2/U3Hygro (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar), were grown in Dulbecco's modified Eagle medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin sulfate. The human prostate cancer cell line LNCaP104-S and 104-R1 cells were passaged and maintained as described previously (16Kokontis J. Takakura K. Hay N. Liao S. Cancer Res. 1994; 54: 1566-1573PubMed Google Scholar).Gene Trap—The retroviral gene trap method was performed as described previously (17von Melchner H. Ruley H.E. J. Virol. 1989; 63: 3227-3233Crossref PubMed Google Scholar). Briefly, ψ2/U3Hygro cells were seeded at 2 × 106 cells/10-cm dish. After 18 h, the culture medium was removed, and 2 ml of fresh medium was added. After 2 h, the culture medium was collected and then filtered through a 0.22-μm pore size membrane. This medium containing virus was added to 5 × 105 CHO cells in a 10-cm dish. Polybrene (Aldrich) was added to a final concentration of 8 μg/ml, and after incubation for 1 h at 37 °C 10 ml of fresh medium was added. After 18 h, the medium was changed, and hygromycin B was added to 0.6 mm. Hygromycin selection was continued for 10 days, and then hygromycin-resistant colonies were cultured in the presence of 10 μm MGBG. Surviving colonies were picked after a week and expanded in the absence of MGBG.Cell Proliferation Assay—Assays were performed based on the method of Rago et al. (18Rago R. Mitchen J. Wilding G. Anal. Biochem. 1990; 191: 31-34Crossref PubMed Scopus (386) Google Scholar). Cells were seeded at 3,000–4,000 cells/well in 96-well tissue culture plates. After 18 h, MGBG was added without changing the medium. At the indicated time, cells were lysed in distilled water and frozen. Cell lysates were incubated with 10 μg/ml Hoechst 33258 in 5 mm Tris-HCl, pH 7.4, 0.5 mm EDTA, and 1 m NaCl. Fluorescence was measured using a Wallac 1420 Multilabel plate reader (PerkinElmer Life Sciences) with excitation at 355 nm and emission at 460 nm. A standard curve was generated using CHO cells counted using a hemocytometer.Polyamine Transport Assay—CHO cells were plated at 4 × 104 cells/well in a 24-well plate. LNCaP cells were plated at 5 × 105 cells/well in a 6-well plate. The growth medium was aspirated, and transport assay buffer consisting of 135 mm NaCl, 1 mm MgCl2, 2 mm CaCl2, 20 mm Hepes/Tris, pH 7.2, and 10 mm glucose (8Kakinuma Y. Hoshino K. Igarashi K. Eur. J. Biochem. 1988; 176: 409-414Crossref PubMed Scopus (88) Google Scholar) was added. After incubation at 37 °C for 10 min, the uptake assay was started by the addition of transport assay buffer containing 3.7 kBq of 14C-labeled polyamine so that the final concentration of polyamine was 5 μm. After the indicated incubation time, the buffer was aspirated, and the cells were washed three times with transport assay buffer containing 10 mm unlabeled substrate. Washed cells were lysed in 0.1 n NaOH. Aliquots of lysed cells were used for protein determination with the Bradford reagent (Bio-Rad). After acidification with HCl, radioactivity was determined by scintillation counting. Uptake measurements were performed under conditions in which uptake was known to be linear with respect to time.Ornithine Decarboxylase, S-Adenosylmethionine Decarboxylase, and Spermidine/Spermine N1-Acetyltransferase Assay—Growing cells were harvested, lysed with ornithine decarboxylase buffer (19Kakinuma Y. Sakamaki Y. Ito K. Cragoe Jr., E.J. Igarashi K. Arch. Biochem. Biophys. 1987; 259: 171-178Crossref PubMed Scopus (25) Google Scholar), and dialyzed against 500 ml of ornithine decarboxylase buffer to remove endogenous small molecules. Ornithine decarboxylase (19Kakinuma Y. Sakamaki Y. Ito K. Cragoe Jr., E.J. Igarashi K. Arch. Biochem. Biophys. 1987; 259: 171-178Crossref PubMed Scopus (25) Google Scholar), S-adenosylmethionine decarboxylase (7Mandel J.L. Flintoff W.F. J. Cell. Physiol. 1978; 97: 335-343Crossref PubMed Scopus (107) Google Scholar), and spermidine/spermine N1-acetyltransferase (20Matsui I. Pegg A.E. Biochim. Biophys. Acta. 1980; 633: 87-94Crossref PubMed Scopus (90) Google Scholar) activities were measured according to published methods.Measurement of the Cellular Content of Polyamines—Polyamines were extracted from the cells with 5% trichloroacetic acid. Polyamine content was analyzed by high performance liquid chromatography as described previously (21Igarashi K. Kashiwagi K. Hamasaki H. Miura A. Kakegawa T. Hirose S. Matsuzaki S. J. Bacteriol. 1986; 166: 128-134Crossref PubMed Scopus (155) Google Scholar). The cellular content of polyamines was normalized to the amount of total cellular protein determined with the Bradford reagent.Determination of MGBG in CHO Cells—MGBG content was measured according to the method of Seppanen et al. (22Seppanen P. Alhonen-Hongisto L. Poso H. Janne J. FEBS Lett. 1980; 111: 99-103Crossref PubMed Scopus (46) Google Scholar). CHO cells were washed three times with the transport assay buffer and then lysed with 0.1% Nonidet P-40. Before the determination of MGBG, the cell lysates were boiled for 10 min to inactivate endogenous S-adenosylmethionine decarboxylase. A homogenate of LNCaP104-S cells was used as a S-adenosylmethionine decarboxylase source. The boiled CHO lysates were added to the S-adenosylmethionine decarboxylase assay system as described. A series of standards containing known amounts of MGBG was run in parallel, and a standard curve was obtained by plotting MGBG concentration versus the reciprocal of initial velocity. The MGBG content in unknown samples was calculated from the standard curve. The MGBG content was normalized to cellular protein content.Southern Blot Analysis—Genomic DNA of CHO cells was isolated as described (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), digested with appropriate restriction enzymes, fractionated in 0.7% agarose gels, and transferred to a Zetaprobe nylon membrane (Bio-Rad). A 32P-labeled DNA probe, containing the EcoRI-XbaI fragment of the TAF7 3′-rapid amplification of cDNA ends (RACE) product isolated from pSTBlue-1, was generated by the random priming method using the Prime-It Labeling Kit (Stratagene). Probe hybridization and detection were performed as described previously (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).RACE-PCR—Total RNA was extracted using the TRIzol Reagent (Invitrogen). 5′-RACE was performed with 1 μg of total RNA using a 5′-RACE system kit (Invitrogen), following the manufacturer's instructions. The specific hygromycin resistance gene primers were 5′-AGCGGGCAGTTCGGTTTCA-3′ for cDNA synthesis and 5′-ACCATCGGCGCAGCTATTTA-3′ for PCR. Using HotStarTaq polymerase (Qiagen, Valencia, CA), the following program was used for amplification: preamplification at 95 °C for 15 min, 35 cycles of 95 °C for 30 s, 58 °C for 1 min, 72 °C for 1 min followed by 72 °C for 8 min. The PCR product was cloned into pBluescript (Stratagene) and sequenced. The sequence was then used for primer design for 3′-RACE. mRNA, isolated from CHO-Cl22 cells by using an oligo(dT)-cellulose column, was used for 3′-RACE using a 3′-RACE system kit and Advantage-2 polymerase (Clontech), following the manufacturer's instructions. The sequence of specific trapped gene primer and 3′-counterprimer were 5′-GCTCCGCCCGCGAGAAGCCGGTTATAG-3′ and 5′-GGCCACGCGTCGACTAGTAC-3′, respectively. The PCR cycle parameters were: preheating at 95 °C for 1 min, 30 cycles of 95 °C for 15 s and 68 °C for 2 min, followed by 68 °C for 3 min. The PCR product was cloned into the pSTBlue-1 cloning vector (Novagen, Madison, WI), and the sequence was determined.Northern Blot Analysis—mRNA (5 μg/lane) was size fractionated by electrophoresis on 1% formaldehyde-agarose gels, transferred to Zetaprobe nylon membranes, and probed with 32P-labeled cDNA, as described for Southern blot analysis.Expression Plasmid Construction—Full-length hamster TAF7 was amplified with 5′-GGATCCGAAGGAACCACCATGAGTAAGAGCAAAGA-3′ (T55-F) and 5′-CTCGAGTCTAAGGACCGAAATCAACAT-3′. After verification by sequencing, the DNA fragment was subcloned into the mammalian expression vector pcDNA3 (Invitrogen). Cloning vectors pSG5HA(N-) for amino-terminal HA tagging and pSG5HA(C-) for carboxyl-terminal HA tagging were generated from the pSG5 vector (Stratagene) with the following oligonucleotide DNA pairs: 5′-GATCCATGTACCCATACGACGTGCCAGACTACGCTA-3′ and 5′-GATCTAGCGTAGTCTGGCACGTCGTATGGGTACATG-3′ were for (N-), and 5′-AATTCTATCCATATGACGTCCCAGACTACGCTTAAG-3′ and 5′-GATCCTTAAGGGTAGTCTGGGACGTCATATGGATAG-3′ were for (C-). TAF7 was amplified with 5′-GGATCCATGAGTAAGAGCAAAGATGAT-3′ and 5′-GGATCCTCTAAGGACCGAAATCAACAT-3′ and cloned into pSG5(N-) so that the HA tag was fused to the amino terminus of TAF7 (TAF7-N). TAF7 was also amplified using T55-F and 5′-GAATTCCTTCTCTAGAAGTGATTCTAGCCCT-3′ and cloned into pSG5(C-) so that the HA tag was fused to the carboxyl terminus of TAF7 (TAF7-C). Finally, these HA-tagged TAF7 genes were subcloned into pcDNA3. Cells stably transformed with these plasmids were generated using the cell transfection agent, Effectene (Qiagen), following the manufacturer's instructions and selection with 0.8 mg/ml G418.Western Blot Analysis—Protein extracts were obtained by lysing phosphate-buffered saline-washed cells on the dish with 2× Laemmli gel loading buffer without bromphenol blue dye (16Kokontis J. Takakura K. Hay N. Liao S. Cancer Res. 1994; 54: 1566-1573PubMed Google Scholar). The protein concentration was determined with Bradford reagent using bovine serum albumin standards as described for the polyamine transport assay. Electrophoresis and blotting were performed essentially as described previously (16Kokontis J. Takakura K. Hay N. Liao S. Cancer Res. 1994; 54: 1566-1573PubMed Google Scholar). All antibodies were used at a concentration of 0.5 μg/ml.Reporter Gene Assay—An AP-1-responsive luciferase reporter construct (pGL3-73coll.) was generated from Coll.-73CAT (24Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (583) Google Scholar), which contains the human collagenase promoter region. Briefly, Coll.-73CAT was digested with HindIII, treated with the Klenow-fragment of DNA polymerase and then cut with BamHI. The resulting DNA fragment was cloned into the BglII/SmaI site of pGL3-Basic (Promega, Madison, WI). Cells were plated at 3 × 104 cells/well on a 24-well plate and grown overnight. Cells were transiently transfected with 0.3 μg of pGL3-73coll. and 0.5 ng of pRL-CMV (normalization reporter plasmid from Promega) using PolyFect (Qiagen), according to the manufacturer's instruction. After 6 h the medium was changed, and MGBG was added. After a 24–36-h incubation, cells were harvested, and luciferase activity was measured with a commercial kit (Dual-Luciferase, Promega) on a Monolight Luminometer (Pharmingen, San Diego), and their relative activities were compared.Caspase-3 Assay—Cells were treated as indicated, harvested, and lysed in a buffer containing 25 mm Hepes/NaOH, pH 7.4, 5 mm EDTA, 10% CHAPS, 2 mm dithiothreitol, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Cells were lysed by freezing and thawing one time. Total cell protein in the cell lysate was measured using Bradford reagent. The caspase-3 substrate, DEVD-AFC (ApoAlert, Clontech) was added to the cellular lysate and incubated for 2 h at 37 °C. Fluorescence was measured using a CytoFluor II fluorescence plate reader (Applied Biosystems, Foster City, CA) with excitation at 440 nm and emission at 490 nm. The activity was normalized to cellular protein content.RNA Interference (RNAi) Experiments—An RNAi expression vector pH1RP was constructed as follows. The human H1 RNA promoter was amplified from LNCaP104-S genomic DNA using primers 5′-CCATGGAATTCGAACGCTGACGTC-3′ and 5′-GCAAGCTTAGATCTGTGGTCTCATACAGAACTTATAAGATTCCC-3′. The PCR product was cloned into pcDNA3 from which the CMV promoter was removed by BamHI-BglII digestion and religation. The RNAi sequence was designed according to the method of Brummelkamp et al. (25Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar) using the design program from OligoEngine (Seattle, WA). The sequences of RNAi for human TAF7 were 5′-GATCCCCTTAGTAGACCTGCCCTGTGTTCAAGAGACACAGGGCAGGTCTACTAATTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAATTAGTAGACCTGCCCTGTGTCTCTTGAACACAGGGCAGGTCTACTAAGGG-3′. These 64-mer oligonucleotides were annealed and ligated into the pH1RP vector, and the construct was verified by sequencing. The TAF7-RNAi expression plasmid was stably transfected into LNCaP104-R1 cells using Effectene as described above.Real Time Quantitative PCR—Total RNA was isolated using the TRIzol Reagent and was treated with DNase I (DNA-free, Ambion, Austin, TX). Reverse transcription was performed with random hexamers and Moloney murine leukemia virus reverse transcriptase (Omniscript, Qiagen). The TaqMan primer/probe was designed using Primer Express (Applied Biosystems). The probe for the TAF7 gene was labeled with the fluorescent dye, FAM. The sequence of the forward primer, reverse primer, and TaqMan probe were 5′-CCGTGCAGTCTGGACACGT-3′, 5′-CAATTCCATGACGCCCATC-3′, and 6FAM-5′-AACCTGAAAGACAGACTGACCATTGAGTTACACC-3′-TAMRA, respectively. Real time PCR was performed on an ABI Prism 7700 system (Applied Biosystems) using the QuantiTect Probe real time PCR protocol (Qiagen). The Ribosomal RNA Control Kit (Applied Biosystems) was used to normalize transcript levels between samples.RESULTSScreening for MGBG Resistance in CHO Cells Transfected with Gene Trap Retrovirus—Gene trap screening was performed with U3Hygro (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar) to attempt to identify the gene responsible for MGBG resistance and polyamine transport activity. CHO cells were infected with U3Hygro and selected with hygromycin B. CHO cells are functionally hemizygous at a number of loci (26Siminovitch L. Cell. 1976; 7: 1-11Abstract Full Text PDF PubMed Scopus (338) Google Scholar), and so a single integration event may result in loss of gene function. The expression of the viral hygromycin resistance gene (hygromycin B phosphotransferase) relies on the activity of the trapped promoter because the hygromycin resistance gene lacks a promoter. Viral gene insertion can disrupt critical controlling elements that can disrupt expression of genes. In theory, a library of 104–105 hygromycin-resistant clones containing proviruses would cover all genes (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar). In our gene trap experiment (Fig. 1), we incubated ∼2 × 107 cells with the U3Hygro retrovirus, from which 1 × 105 hygromycin-resistant CHO cell colonies were selected. Chang et al. (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar) found a similar frequency of hygromycin-resistant colonies using the same virus and CHO cells. After further selection with 10 μm MGBG, a concentration that will kill all uninfected CHO cells in 4–5 days (data not shown), a total of 38 colonies survived. Those colonies were picked and amplified for further experimentation. Eventually, only one clone, 432c, showed significant and stable MGBG resistance (Fig. 2A). This frequency of selection (1 MGBG-resistant cell in 105 hygromycin-resistant cells) was similar to the frequency of selection of hemizygous genes in the study by Chang et al. (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar). The MGBG-resistant 432c cells also had a significant decrease in polyamine transport activity (Fig. 2B). Consistent with these observations, intracellular accumulation of MGBG (Fig. 2C) or spermine (Fig. 2D) in 432c cells was less than in CHO cells. U3Hygro was used at a fairly low multiplicity of infection (1:1) to allow predominantly one insertion/cell. Previously the locus of MGBG resistance was mapped to a hemizygous region of the Z3 chromosome (12Adair G.M. Siciliano M.J. Mol. Cell. Biol. 1985; 5: 109-113Crossref PubMed Scopus (10) Google Scholar), indicating that this function was a single-copy gene (4Heaton M.A. Flintoff W.F. J. Cell. Physiol. 1988; 136: 133-139Crossref PubMed Scopus (20) Google Scholar). The copy number of the U3 insertion in 432c cells was determined by Southern blot analysis using the neomycin resistance gene as a probe. U3Hygro harbors this gene as a marker for viral infection and insertion (15Chang W. Hubbard S.C. Friedel C. Ruley H.E. Virology. 1993; 193: 737-747Crossref PubMed Scopus (34) Google Scholar). Genomic DNA of 432c cells was digested with HindIII, SspI, and StuI, which do not cut U3Hygro, followed by electrophoresis and blotting. The results of Southern blots indicated that the insertion of U3Hygro occurred at only one site (data not shown). 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 (27Suzuki T. He Y. Kashiwagi K. Murakami Y. Hayashi S. Igarashi K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8930-8934Crossref PubMed Scopus (138) Google Scholar, 28McCloskey D.E. Pegg A.E. J. Biol. Chem. 2000; 275: 28708-28714Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We measured the enzymatic activities of ornithine decarboxylase and spermidine/spermine N1-acetyltransferase in 432c cells, and their activities were not significantly changed compared with the parent CHO cell line (data not shown).Fig. 2Characterization of the MGBG-resistant clone, 432c. A, effect of MGBG on cell growth of the parent strain, CHO (closed circles) and 432c cells (open circles). Cells were exposed to the indicated concentrations of MGBG for 24 h. The cell number was determined from DNA content/cell using Hoechst 33258. B, spermine uptake activity in CHO (closed circles) and 432c (open circles) cells. Cells were incubated with 5 μm [14C] spermine and uptake measured every 5 min. The activity was normalized to total cellular protein. C, MGBG uptake in CHO (hatched bars) and 432c (open bars) cells. Cells were treated with 3 μm MGBG for the indicated times. Intracellular MGBG content was normalized to total cellular protein. D, effect of additional spermine in media on the intracellular spermine content. Spermine was added to medium at the indicated concentration with 0.5 mm aminoguanidine to inhibit degradation of spermine by serum amine oxidase. After 12 h, cells were collected, and spermine was analyzed using high performance liquid chromatography and normalized to total cellular protein. The intracellular spermine content without additional spermine in the medium was 7.23 and 11.1 nmol/mg protein for CHO and 432c cells, respectively, and the spermine content of polyamine-treated cells is shown as a percentage of these control cells.View Large Image Figure ViewerDownload (PPT)Identification of TAF7 as the Trapped Gene in 432c Cells— Using total RNA from 432c cells, 5′-RACE was performed with priming from the hygromycin resistance gene (Fig. 1). A 495-bp PCR product was obtained, and this was subcloned and sequenced. Sequencing revealed that 226 nucleotides of CHO cDNA (Fig. 3A) were present. The sequence of this DNA did not match any of the currently available genes from the GenBank data base. No open reading frame was found in this sequence. Because we were unable to identify a gene associated with viral insertion based on 5′-RACE data, we designed DNA p
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