RESUMO
The clinical use of cytotoxic deoxynucleoside analogues is often limited by resistance mechanisms due to enzymatic deficiency, or high toxicity in nontumor tissues. To improve the use of these drugs, gene therapy approaches have been proposed and studied, associating clinically used deoxynucleoside analogues such as araC and gemcitabine and suicide genes or myeloprotective genes. In this review, we provide an update of recent results in this area, with particular emphasis on human deoxycytidine kinase, the deoxyribonucleoside kinase from Drosophila melanogaster, purine nucleoside phosphorylase from Escherichia coli, and human cytidine deaminase. Data from literature clearly show the feasibility of these systems, and clinical trials are warranted to conclude on their use in the treatment of cancer patients.
Assuntos
Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Terapia Genética/métodos , Animais , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/fisiologia , Drosophila melanogaster/enzimologia , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/fisiologiaRESUMO
Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.
Assuntos
DNA Viral/análise , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática/métodos , HIV-1/genética , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Síndrome da Imunodeficiência Adquirida/virologia , Sequência de Bases , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Estudos de Avaliação como Assunto , Genes nef , Infecções por HIV/virologia , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/estatística & dados numéricos , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
An enzyme-linked oligosorbent assay (ELOSA) was developed for the detection on microtiter plates of polymerase chain reaction (PCR)-amplified human immunodeficiency virus type 1 (HIV-1) DNA. The denatured PCR product was hybridized with a passively adsorbed oligonucleotide capture probe and a horseradish peroxidase-labeled oligonucleotide detection probe. The sensitivity and specificity of the PCR-ELOSA technique depended to some extent on the nucleotide sequences of the oligonucleotide primer and probe quartet used in the amplification and detection. We evaluated five oligonucleotide quartets located in the gag, pol, vpr, env, and nef regions of HIV-1. DNAs from 39 HIV-1-seropositive individuals and 27 healthy HIV-1-seronegative controls were amplified by the PCR procedure, and the products were detected by ELOSA. Ten copies of HIV-1 DNA against a background of 1 microgram of human DNA were specifically detected by PCR-ELOSA. Specificities and sensitivities were, respectively, 100 and 95% for the gag system, 100 and 97% for the pol system, 100 and 85% for the vpr system, 96 and 95% for the env system, and 100 and 95% for the nef system. The simplicity of ELOSA makes it suitable for automation and applicable to genetic testing and detection of viral and bacterial DNAs or RNAs in most routine laboratories.
