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1.
BMC Genomics ; 6: 125, 2005 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-16162288

RESUMO

BACKGROUND: The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSalpha and rTSbeta. The rTSbeta isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. RESULTS: Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSbeta. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSgamma, which is absent in rTSbeta. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSgamma and rTSbeta proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. CONCLUSION: The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.


Assuntos
Biologia Computacional/métodos , Genômica/métodos , Mitocôndrias/metabolismo , Timidilato Sintase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Códon , Genoma , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas , Estrutura Terciária de Proteína , Frações Subcelulares , Timidilato Sintase/metabolismo
2.
Cancer Res ; 65(13): 5917-24, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15994970

RESUMO

The rTSbeta protein has been hypothesized to synthesize signaling molecules that can down-regulate thymidylate synthase. These molecules share biological and chemical properties with acyl-homoserine lactones (AHL), suggesting some AHLs might act as rTS signaling mimics and down-regulate thymidylate synthase. We have determined that the AHL, 3-oxododecanoyl homoserine lactone (3-oxo-C12-(L)-HSL) can down-regulate thymidylate synthase protein at 10 micromol/L and reduce H630 (human colorectal cancer) growth by 50% at 23 micromol/L (IC50) in cell culture. At its IC50 concentration, 3-oxo-C12-(L)-HSL reduces the apparent IC50 of 5-fluorouracil (5-FU) from 1 micromol/L to 80 nmol/L (12-fold) in a colony formation assay. 3-Oxo-C12-(L)-HSL enhances the activity of 5-fluorodeoxyuridine, tomudex, and taxol but not the activity of 5-fluorouridine, methotrexate or Adriamycin. The unexpected interaction with taxol probably results from effects of the AHL on tubulin expression. Differences in taxol sensitivity, tubulin, and cellular morphology between H630 and the thymidylate synthase and rTSbeta-overproducing, 5-FU-resistant H630-1 cell line as determined by colony formation assays, Western analysis of one-dimensional and two-dimensional gels, and photomicroscopy confirm that cytoskeletal changes are induced by the AHL or by rTS signaling. Isozyme differences in thymidylate synthase and rTSbeta also exist in the two cell lines. Phosphorylation of rTSbeta amino acid S121 is shown to occur and is decreased at least 10-fold in the drug-resistant cells. The data presented provide support for further investigations of rTS signaling mimics as enhancers to thymidylate synthase-directed chemotherapy, evidence that the phosphorylation state of rTSbeta may be a marker for 5-FU resistance and a previously unrealized relationship between rTS signaling and the cytoskeleton.


Assuntos
4-Butirolactona/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Fluoruracila/farmacologia , Homosserina/análogos & derivados , Timidilato Sintase/metabolismo , 4-Butirolactona/administração & dosagem , 4-Butirolactona/farmacologia , Materiais Biomiméticos/administração & dosagem , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Homosserina/administração & dosagem , Homosserina/farmacologia , Humanos , Isoenzimas , Fosforilação , Isoformas de Proteínas , RNA Antissenso/biossíntese , RNA Antissenso/genética , Transdução de Sinais/fisiologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/biossíntese , Timidilato Sintase/genética , Tubulina (Proteína)/metabolismo
3.
Clin Colorectal Cancer ; 5(1): 57-60, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15929807

RESUMO

The rTS gene was discovered because it codes for a complementary (antisense) RNA to the messenger RNA for thymidylate synthase (TS). It was later shown that rTS also encodes 2 proteins, rTSa and rTSb. Recently, it has become apparent that rTSb overexpression can cause the downregulation of TS protein in a colon cancer cell line through the production of > or = 1 previously unknown signaling molecules. This observation signified the presence of a previously unidentified signaling pathway. The existence of a signaling pathway that can regulate TS protein levels and the widespread expression of the rTSb protein suggests that a new target for drug development may be on the horizon. This review describes the relationship between the rTS and TS genes and the known and potential effects of rTS RNAs and rTS proteins. We also present the structure of an identified TS downregulatory compound that may serve as a lead compound for development.


Assuntos
RNA Antissenso , Timidilato Sintase/biossíntese , Timidilato Sintase/genética , Antineoplásicos/farmacologia , Regulação para Baixo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/fisiopatologia , RNA Mensageiro , Proteínas Recombinantes , Transdução de Sinais , Timidilato Sintase/metabolismo
4.
Cancer Biol Ther ; 2(4): 364-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14508106

RESUMO

The rTS gene codes for a naturally occurring antisense RNA to thymidylate synthase (TS) mRNA and two proteins (rTSalpha and rTSbeta). The role of the major protein product of rTS, rTSbeta has been linked to alterations in TS protein expression, but the precise function of rTSbeta is unknown. In this report we demonstrate that increased expression of rTSbeta is associated with the decrease in TS protein expression due to production of novel, diffusible signal molecules. These signal molecules are produced more abundantly when rTSbeta amounts are elevated. This hypothesis is supported by the demonstration that the rTSbeta-overproducing cell line H630-1 can downregulate TS protein in other cells without direct cellular contact. These cells are shown to secrete significant amounts of lipophilic metabolites derived from methionine, in contrast to cells that do not overproduce rTSbeta. In support of the hypothesis that rTSbeta is essential for the generation of these compounds, we demonstrate that rTSbeta can catalyze the transfer of the carboxyl carbon of methionine from S-adenosylmethionine to a lipophilic acceptor molecule in vitro. We propose rTS is involved in regulation of TS through a novel methionine-based signaling pathway.


Assuntos
Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Metabolismo dos Lipídeos , RNA Antissenso/genética , S-Adenosilmetionina/metabolismo , Timidilato Sintase/genética , Regiões 3' não Traduzidas , Processamento Alternativo , Ciclo Celular , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação para Baixo , Humanos , Lactonas/química , Luciferases/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Células Tumorais Cultivadas
5.
Mol Pharmacol ; 63(1): 167-73, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12488549

RESUMO

Thymidylate synthase (TS), a key cancer chemotherapeutic target, catalyzes the conversion of deoxyuridylate to thymidylate. TS can serve as a repressor of its own synthesis by binding to its own mRNA through TS-specific binding elements (TBEs). In this report, we describe the use of a luciferase reporter plasmid containing two TBEs that can be used as a tool for the identification and initial profiling of compounds that modulate TS activity, levels, or ability to bind mRNA. To validate this model, we evaluated several groups of drugs. Thus, cells were exposed to the pyrimidine analogs 5-fluorouracil (5-FU), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FUdR), trifluorothymidine (TFT); to the nonpyrimidine TS-inhibitors AG-331, nolatrexed (AG337), and raltitrexed (ZD1694); or to drugs with other primary sites of action (methotrexate, actinomycin D, 5-azacytidine, 8-thioguanosine). Except for 5-azacytidine and 8-thioguanosine, all compounds examined induced luciferase activity compared with untreated cells. Effects of luciferase activity inducing drugs through TS-affected translation were confirmed by examinations of TS protein and mRNA levels. Treatment of H630-C6 cells with 5-FU, FUrd, FUdR, TFT, AG331, AG337, ZD1694, and methotrexate up-regulated TS levels as determined by Western blot analysis, although TS mRNA levels remained unchanged as determined by reverse transcription-polymerase chain reaction. Our studies demonstrate a novel application of a TBE-dependent reporter plasmid that could be used for the high-throughput identification of potential chemotherapeutic agents that modulate TS RNA-binding activity, either directly or indirectly.


Assuntos
Proteínas de Ligação a DNA/análise , Proteínas Imediatamente Precoces , Luciferases/biossíntese , Timidilato Sintase/metabolismo , Fatores de Transcrição/análise , Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Indução Enzimática/efeitos dos fármacos , Floxuridina/farmacologia , Fluoruracila/farmacologia , Humanos , Regiões Promotoras Genéticas , Timidilato Sintase/antagonistas & inibidores , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Regulação para Cima
6.
Biochim Biophys Acta ; 1587(2-3): 183-93, 2002 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12084460

RESUMO

The 3' untranslated region (UTR) of rTSalpha RNA is complementary (i.e., antisense) to human thymidylate synthase (TS) RNA. When HEp2 cells (human epidermoid carcinoma) progressed from late-log to plateau phase growth, ribonuclease protection assay (RPA) revealed an inverse correlation between the levels of rTSalpha RNA and TS mRNA, suggesting a possible effect of rTSalpha RNA on TS mRNA levels. HEp2 cells expressing a Tet-On transactivator were transiently co-transfected with pHook-1 and a construct containing rTSalpha (protein and antisense RNA), rTSalphaDelta3' (rTSalpha protein only), rTSalpha-3' (antisense RNA-luciferase) or luciferase. Transfected cells were selected and evaluated for the effects of induced transgene expression on TS mRNA. Induced expression of transfected rTSalpha or rTSalpha-3', but not rTSalphaDelta3' or luciferase, resulted in decreased TS mRNA levels as measured by RPA. These results demonstrated that the antisense region of rTSalpha RNA is necessary and sufficient for this down-regulation of TS mRNA. RPA for TS mRNA also showed the enhanced appearance of two partial-length protected fragments in rTSalpha or rTSalpha-3' transfected cells. RPA stringency evaluations and primer extension assays indicated that TS mRNA is cleaved in vivo in a site-specific manner. These data demonstrate that rTS gene expression likely plays a role in down-regulating TS through a natural RNA-based antisense mechanism.


Assuntos
RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timidilato Sintase/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , Divisão Celular , Linhagem Celular , Regulação para Baixo , Humanos , Luciferases/genética , Edição de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleases , Especificidade por Substrato , Transfecção
7.
Clin Colorectal Cancer ; 1(3): 174, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12450431

RESUMO

No Abstract

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