RESUMO
The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum™ assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15 months p = 0.02) and OS (21 vs. 38 months, p < 0.0001), respectively. After adjustment for age, metastatic site and study treatment TK1 showed a trend as predictor of PFS (p = 0.059) and was an independent prognostic factor for OS, (HR 1.81, 95 % confidence interval (CI) 1.26-2.61, p = 0.001). There was a trend of shortened OS for women with high CA 15-3 (p = 0.054) in univariate analysis, but not after adjustment for the above mentioned covariates. Both TK1 (p = 0.0011) and CA 15-3 (p = 0.0004) predicted response to treatment. There were statistically different distributions of TK1 and CA 15-3 in relation to the site of metastases. TK1 activity measured by DiviTum™ predicted therapy response, PFS and OS in loco regional inoperable or disseminated breast cancer. These results suggest that this factor is a useful serum marker. In the present material, a prognostic value of CA 15-3 could not be proven.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Mucina-1/sangue , Timidina Quinase/sangue , Adulto , Idoso , Neoplasias da Mama/terapia , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
A non-radioactive reverse transcriptase (RT) assay, reported as useful for lentivirus RTs, was optimized for the measurement of Moloney murine leukemia virus (MMuLV) RT. The optimized assay could detect 0.3 microU of MMuLV RT. The specificities of the MMuLV and lenti RT assays were demonstrated using the RTs of human immunodeficiency virus type 1, simian immunodeficiency virus, feline immunodeficiency virus (FIV), visna virus, human T-cell lymphotropic virus type 1, MMuLV and feline leukemia virus (FeLV). An RT activity blocking antibody (RTb-ab) assay was standardized for Mn2+ dependent MuLV-related RTs. The assay was used to demonstrate the distinct antigenic properties of RTs from mammalian MuLV-related retroviruses and lentiviruses. Cross-reactivity between MMuLV RTb-ab and FeLV RT but not between MMuLV RTb-ab and e.g. FIV RT was demonstrated. An RT activity found in the murine myeloma cell line SP2/0 was found to have similar assay preferences as MMuLV RT, and the MMuLV-RT hyperimmune sera reacted strongly against this RT, indicating the RT to be of MuLV-related etiology. The use of the RT and RTb-ab assays for detection and characterization of RTs of known or unknown identity is discussed.
Assuntos
Colorimetria/métodos , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/análise , Células 3T3 , Animais , Gatos , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Isoenzimas , Lentivirus/enzimologia , Camundongos , Vírus da Leucemia Murina de Moloney/isolamento & purificação , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Células VeroRESUMO
A nonradioactive reverse transcriptase (RT) assay was used to measure RT activity in serum during the viremia peak associated with primary infection and for measuring the generation and maintenance of RT activity-blocking antibody (RTb-ab) titers during and after seroconversion in SIV-infected macaques. The RT assay was compared to an antigen capture immunoassay designed for HIV-2/SIVsm and was found to be approximately 40 times more sensitive in detecting SIVsm in serum from infected macaques. The RT assay detected RT activity in serum corresponding to levels from 3 pg/ml. Earliest detection of viral replication using the RT assay was on day 6-8, with a peak at day 10 (up to 8000 pg/ml). The earliest detection of RTb-ab was seen on day 17-23, with established RTb-ab titers by day 29, followed by increasing titers of 15,000-120,000 by day 62-77. The usefulness of RT and RTb-ab for monitoring the course of SIV infection in monkey models is discussed.
Assuntos
Anticorpos Bloqueadores/análise , Anticorpos Bloqueadores/imunologia , DNA Polimerase Dirigida por RNA/análise , DNA Polimerase Dirigida por RNA/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Antivirais/análise , Produtos do Gene gag/análise , Produtos do Gene gag/imunologia , Antígenos HIV/análise , Antígenos HIV/imunologia , HIV-2/imunologia , Imunoensaio , Isoenzimas/análise , Isoenzimas/metabolismo , Macaca fascicularis , Camundongos , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Viremia/virologia , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms of primary infection. In addition, HIV-1 RTb-Ab was detected in the same recently infected individuals in most cases within 1 month and in some cases as early as 10 to 12 days after the onset of symptoms. A cross-reactivity study involving HIV-1 and HIV-2 RTb-Abs and their homologous RT showed HIV-1 RTb-Ab to be highly type specific. None of 10 serum samples from HIV-1-infected individuals showed cross-reacting RTb-Ab toward HIV-2 RT, whereas 4 of 10 serum samples from HIV-2-infected patients showed cross-reactivity toward HIV-1 RT; however, the cross-reactivity toward HIV-1 RT was 3,000 times lower than that toward its homologous RT. Future uses for the assay with reference to the recent World Health Organization proposal for other methods instead of Western blotting (immunoblotting) for confirming HIV-1 infection and for methods for the diagnosis of infection as follow-up in vaccine trials are also discussed.
Assuntos
Colorimetria/métodos , Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , Transcriptase Reversa do HIV/sangue , HIV-1/imunologia , Feminino , Humanos , Masculino , GravidezRESUMO
A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine-leukaemia-virus RT. The assay was found to be useful with various types of cell-culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications.
Assuntos
DNA/análise , Nucleotídeos de Desoxiuracil , HIV/isolamento & purificação , DNA Polimerase Dirigida por RNA/análise , Células Cultivadas , Infecções por HIV/enzimologia , Humanos , Padrões de Referência , Sensibilidade e Especificidade , Frações Subcelulares/enzimologia , Moldes Genéticos , Nucleotídeos de Timina , TitulometriaRESUMO
The levels of deoxythymidine kinase in tumour cells (C-TK) and in serum (S-TK) were investigated and the tumour volume calculated in 89 patients with non-Hodgkin lymphoma (NHL), in order to ascertain the importance of C-TK and tumour burden as regards the S-TK levels. Among all patients, a correlation was seen between S-TK and tumour volume but not between S-TK and C-TK. However, within different tumour volume categories (small, medium-sized and large), there was a correlation between S-TK and C-TK. Multiple regression analysis supported this notion. C-TK correlated with the proliferation-associated parameters, S-phase fraction and mitotic index. As already known, S-TK was found to have a strong prognostic value. C-TK and tumour burden were also of prognostic value. In multivariate analyses, C-TK and tumour volume did not provide prognostic information in addition to S-TK, whereas, in the absence of S-TK, C-TK and tumour volume did provide additional information. It is concluded that the serum level of TK depends on both the tumour burden and the tumour cell proliferation rate. Based upon estimations of S-TK in patients assessed shortly after chemotherapy, we also suggest that S-TK reflects the number of proliferating cells that have died during the period immediately before sampling.
Assuntos
Linfoma não Hodgkin/enzimologia , Timidina Quinase/metabolismo , Divisão Celular/fisiologia , Humanos , Linfoma não Hodgkin/patologia , Índice Mitótico , Estadiamento de Neoplasias , Prognóstico , Fase S/fisiologia , Timidina Quinase/sangueRESUMO
Inhibitory effects of snuff extract and the tobacco chemicals nicotine, anabasine, diethyl-N-nitrosamine (DEN), and the tobacco-specific nitrosamines (TSNA), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on herpes simplex virus type 1 (HSV-1) replication in vitro and on HSV-1 protein synthesis in infected cells were analysed. Snuff extract and nicotine caused a significant reduction of HSV-1 attachment to cell membranes whereas anabasine, DEN, NNN and NNK did not affect adsorption of HSV-1. Virus production assays in the presence of snuff added after virus adsorption resulted in a significantly reduced production of virus at low multiplicities of infection (MOI), but at high MOI the inhibitory effect of snuff extract was less pronounced. DEN, NNN and NNK only affected virus production at toxic concentrations. Nicotine and anabasine reduced virus production in non-toxic doses but not at the concentrations present in snuff extract. In HSV-infected cells exposed to snuff extract, the immediate early (alpha-) infected cell proteins (ICPs) 4 and 27 (as well as the early (beta-) ICPs 6 and 8) were markedly increased, whereas the late (gamma-) ICPs 5, 11 and 29 were reduced. Nicotine had a less pronounced stimulating effect on the production of alpha-proteins but no detectable effect on production of beta- or gamma-proteins. Anabasine, DEN, NNN and NNK did not affect HSV protein synthesis at non-toxic concentrations. Synthesis of thymidine kinase and DNA polymerase was significantly reduced by snuff extract. Also nicotine and anabasine affected thymidine kinase and DNA polymerase but only at toxic concentrations. The production of the cellular protein actin, which almost disappears a few hours after HSV-1 infection, remained at a significant level in HSV-infected cells exposed to snuff. Thus snuff extract blocks the replicative cycle of HSV at an early stage, which results in an increased production of alpha-proteins in the infected cells and in prolonged maintenance of cellular functions. This may be of importance for HSV-induced transformation and the development of HSV-associated tumours.
Assuntos
Alcaloides/farmacologia , Nicotiana , Nitrosaminas/farmacologia , Plantas Tóxicas , Simplexvirus/efeitos dos fármacos , Simplexvirus/fisiologia , Proteínas Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Anabasina/farmacologia , Animais , Carcinógenos/farmacologia , Células Cultivadas , DNA Polimerase Dirigida por DNA/biossíntese , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Dietilnitrosamina/farmacologia , Humanos , Nicotina/farmacologia , Extratos Vegetais , Timidina Quinase/biossíntese , Timidina Quinase/efeitos dos fármacos , Tabaco sem Fumaça , Células Tumorais Cultivadas , Proteínas Virais/biossínteseRESUMO
The effect of alpha-interferon (alpha-IFN) on spontaneous or induced proliferation of isolated peripheral blood mononuclear cells from 5 hairy cell leukemia patients was studied. alpha-IFN inhibited the low spontaneous proliferation of B-ly7 positive hairy cells (HCs) and also the proliferation induced by tumour necrosis factor (TNF). Interleukin-2 did not affect HCs, but induced CD4 positive T cells to proliferate, an effect which alpha-IFN antagonized. The stimulatory effect of TNF on the growth of HCs proved to be reversible and was partially blocked with either anti-TNF receptor or anti-lymphotoxin antibodies. Cellular or secreted thymidine kinase levels reflected the proliferative state of HCs in response to different in vitro treatments, as confirmed by thymidine incorporation and cell cycle studies.
Assuntos
DNA de Neoplasias/biossíntese , Interferon-alfa/farmacologia , Leucemia de Células Pilosas/imunologia , Linfócitos/fisiologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA de Neoplasias/antagonistas & inibidores , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Interleucina-2/farmacologia , Leucemia de Células Pilosas/sangue , Leucemia de Células Pilosas/terapia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Proteínas Recombinantes/farmacologia , Timidina Quinase/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A new assay for detecting inhibition of reverse transcriptase activity (the RT-i REA) was developed. This assay was standardized for screening serum samples for reverse transcriptase inhibiting antibodies (RT-iAb). High specificity (100%) and sensitivity (greater than 98%) were achieved with samples from HIV-negative individuals and HIV-infected individuals. The RT-i REA was also used in a study of the titers of RT-iAb in serum samples obtained from 33 HIV-infected homosexual men. The results confirmed the relation between decreasing RT-iAb levels and progression to late stages of the disease. Furthermore, a falling RT-iAb titer was observed in 14 of 15 individuals experiencing periods of severe clinical symptoms attributed to HIV-activity. In 7 of the patients the decline in RT-iAb titer began prior to severe clinical symptoms. The fall in RT-iAb titer also correlated with a reduction in core Ab level. The core Ab level has previously been reported to be a disease progression marker with considerable prognostic value. However, whereas all patients were positive for RT-iAb, 8 of the 33 patients did not have detectable core Ab. The use of RT-iAb titer as a marker of disease progression is discussed.
Assuntos
Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , DNA Polimerase Dirigida por RNA/imunologia , Proteínas do Core Viral/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Transcriptase Reversa do HIV , HIV-1/enzimologia , Humanos , Estudos Longitudinais , Masculino , Gravidez , ProibitinasRESUMO
A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.
Assuntos
Sorodiagnóstico da AIDS , Anticorpos Anti-HIV/análise , HIV-1/imunologia , DNA Polimerase Dirigida por RNA/imunologia , Western Blotting , Nucleotídeos de Desoxiuracil , Produtos do Gene gag/imunologia , Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/enzimologia , Humanos , Proteínas do Core Viral/imunologiaRESUMO
Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies.
Assuntos
HIV/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Adenosina , Humanos , Linfócitos/fisiologia , Microesferas , Polímeros , DNA Polimerase Dirigida por RNA/sangue , DNA Polimerase Dirigida por RNA/isolamento & purificação , Solubilidade , Moldes GenéticosRESUMO
The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer (SCLC): lactic dehydrogenase (LDH), serum thymidine kinase (S-TK), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and tissue polypeptide antigen (TPA). More traditional clinical and serologic markers were also evaluated. Univariate analysis showed that all of the biochemical markers mentioned above, the Karnofsky index (KI) and the patient's sex were related to both the stage of disease (limited/extensive disease: LD/ED) and to survival. The strongest marker for the clinical stage was S-TK, whereas TPA showed the strongest relationship with survival. Multivariate analyses produced a model consisting of S-TK, CEA, NSE, and the patient's sex for determining the clinical stage. To compare the prognostic capacity of easily determined biochemical and simple clinical variables to the more resource-demanding variable of the clinical stage, three multivariate analyses in relation to survival were performed: (1) biochemical markers and simple clinical variables; (2) LD/ED and simple clinical variables; and (3) all available variables. The model obtained from the first analysis included TPA, KI, age, and the patient's sex; the model from the second analyses included LD/ED, patient's age, and KI; and the model from the third analysis, TPA, KI, age, sex, and LD/ED. Indices based on these three multivariate models were calculated for each patient and the prognostic capacity of these indices was compared. Pretreatment serum marker levels also had the capacity to predict both the grade and the duration of the response to therapy.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Pequenas/sangue , Neoplasias Pulmonares/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno Carcinoembrionário/sangue , Carcinoma de Células Pequenas/mortalidade , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/terapia , Feminino , Humanos , L-Lactato Desidrogenase/sangue , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Peptídeos/sangue , Fosfopiruvato Hidratase/sangue , Prognóstico , Indução de Remissão , Albumina Sérica/metabolismo , Taxa de Sobrevida , Timidina Quinase/sangue , Antígeno Polipeptídico TecidualRESUMO
A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
Assuntos
Anticorpos Antivirais/análise , DNA Polimerase Dirigida por DNA/sangue , Nucleotídeos de Desoxiuracil/metabolismo , HIV/enzimologia , DNA Polimerase Dirigida por RNA/imunologia , Linhagem Celular , DNA/biossíntese , DNA Polimerase Dirigida por DNA/análise , Nucleotídeos de Desoxiuracil/biossíntese , Nucleotídeos de Desoxiuracil/isolamento & purificação , Infecções por HIV/imunologia , Células HeLa , Humanos , Radioisótopos do Iodo , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes , Simplexvirus/fisiologia , Especificidade por Substrato , Nucleotídeos de Timina/metabolismoRESUMO
Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalovirus (CMV) infection, B12-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (greater than 1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation. The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity. The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.
Assuntos
DNA Polimerase Dirigida por DNA/sangue , Timidina Quinase/sangue , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Ditioeritritol/farmacologia , Células HeLa , Humanos , Cinética , Precursores de Ácido Nucleico/biossíntese , Núcleosídeo-Fosfato Quinase/sangue , Desnaturação Proteica/efeitos dos fármacos , Especificidade por Substrato , Timidina Quinase/metabolismoRESUMO
DNA polymerase activity was demonstrated in sera from patients with diseases affecting DNA metabolism in different ways, i.e. malignant, viral and vitamin B12-deficiency disease. Using the current procedure, such activity was only detected in sera with pathological levels of thymidine kinase, i.e. no reference level of DNA polymerase activity in healthy individuals could be established. The activity detected for all three types of disease was similar to that of proliferation-associated DNA polymerase alpha, both with respect to sensitivity to different chemical inhibitors and to inhibition by monoclonal antibody. The levels of activity of DNA polymerase and thymidine kinase showed a wide variation and were not significantly correlated when all DNA polymerase-positive sera were included in the analysis. The variation in the ratio of polymerase to kinase activity within a given disease was smaller and the distributions of the enzyme ratios induced by the three types of disease differed significantly. Considering that DNA polymerase activity can be quantitated directly in crude sera, and that such analyses seems to give biological and clinical information, the development of an assay with improved sensitivity for extensive studies is justified.
Assuntos
Infecções por Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/sangue , Leucemia/enzimologia , Neoplasias da Próstata/enzimologia , Deficiência de Vitamina B 12/enzimologia , Biomarcadores/sangue , Biomarcadores Tumorais/sangue , Infecções por Citomegalovirus/sangue , Humanos , Cinética , Leucemia/sangue , Masculino , Neoplasias da Próstata/sangue , Vitamina B 12/sangue , Deficiência de Vitamina B 12/sangueRESUMO
The analysis of individual biochemical and clinical variables in 121 patients with multiple myeloma showed that serum beta 2-microglobulin (S-beta 2m) had the most significant relation to survival. Other variables such as serum thymidine kinase (S-TK), serum lactate dehydrogenase (S-LDH), S-creatinine, haemoglobin (Hb), ESR, S-albumin, age and clinical stage were also significant. No such relationship was found with M-component, presence of light chains in urine, type of secreted immunoglobulin or S-calcium. The exclusion of clinical stage in the first multivariate analysis resulted in a model consisting of S-beta 2m, age and S-TK, none of the other variables gave additional information. When in the second multivariate analysis the basic variables involved in staging procedure were excluded and clinical stage included, stage III, but not stage II, was found to give additional information to the model described above. Individual analysis of the variables showed that Hb had the most significant relation to effect of initial therapy. Other significant variables were S-TK, S-beta 2m and age. When using the multivariate approach, Hb alone was found to contain all the relevant information.
Assuntos
Mieloma Múltiplo/sangue , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Estadiamento de Neoplasias , Probabilidade , Timidina Quinase/sangue , Microglobulina beta-2/análiseRESUMO
The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.
Assuntos
Nucleotídeos de Desoxiuracil/biossíntese , Núcleosídeo-Fosfato Quinase/análise , Fosfotransferases/análise , Simplexvirus/enzimologia , Timidina Quinase/análise , Trifosfato de Adenosina , Animais , Anticorpos/análise , Neoplasias Encefálicas/enzimologia , Catálise , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Núcleosídeo-Fosfato Quinase/imunologia , Fosforilação , Timidina Quinase/imunologiaRESUMO
The serum thymidine kinase (S-TK) and proliferative activity of the leukemic cells were determined in 27 untreated patients with chronic lymphocytic leukemia (CLL). A significant positive correlation between S-TK and proliferation expressed as a proliferative index (PI) was found (r = 0.70, p less than 0.001). Additionally, PI (r = 0.59, p less than 0.01) and S-TK (r = 0.47, p less than 0.05) correlated to peripheral blood lymphocyte count. When different variables and combinations of variables were studied in order to define their capacity for discriminating between progressive and indolent CLL, S-TK activity and PI proved to be powerful indications. In longitudinal studies, both S-TK and PI paralleled disease activity. A model where a combination of S-TK and PI gives information of the degree of localized disease is proposed.
Assuntos
Leucemia Linfoide/diagnóstico , Linfócitos/metabolismo , Timidina Quinase/sangue , Timidina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Divisão Celular , Feminino , Humanos , Leucemia Linfoide/terapia , Contagem de Leucócitos , Estudos Longitudinais , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , EsplenectomiaRESUMO
Thymidine kinase (s-TK), lactate dehydrogenase (LDH), and carcinoembryonic antigen (CEA) were determined in pretreatment serum from 125 patients with small cell carcinoma of the lung. The distribution of marker levels into three ranges, when including all patients were as follows: s-TK less than 5 units 49%, 5-less than 10 units 25%, greater than or equal to 10 units 26%; LDH less than 6.7 mukat 31%, 6.7-less than 13.4 mukat 48%, greater than or equal to 13.4 mukat 21%; CEA less than 7.5 micrograms/l 51%, 7.5-less than 15 micrograms/l 25%, greater than or equal to 15 micrograms/l 24%. The percentages of patients with limited and with extensive disease within each range were s-TK less than 5 82/18, 5-less than 10 29/71, greater than or equal to 10 9/91; LDH less than 6.7 76/24, 6.7-less than 13.4 51/49, greater than or equal to 13.4 21/79; CEA less than 7.5 70/30, 7.5-less than 15 39/61, greater than or equal to 15 23/77. Analyses in relation to metastases present showed that patients with skeletal and bone marrow metastases had significantly higher s-TK and LDH than those without, while this was not the case for CEA. A strong correlation between s-TK and LDH level, a weaker correlation between CEA and s-TK, and no correlation between CEA and LDH level, was found. Both the level of s-TK and LDH correlated to the patients' performance, as defined by the Karnofsky index. These correlations were mainly confined to the patients with extensive disease. Analyses of the prognostic capacity of variables showed that s-TK, stage, and Karnofsky index could divide the patients into groups with highly significant difference in survival time, while LDH and CEA were of less value. Longitudinal studies showed that the serum markers mirrored the disease activity, with the exception that highly increased s-TK was found during remission induction for some patients. It was concluded that the expression of pathologic levels for the serum markers were dependent on different biological parameters. Of the serum markers, only s-TK was judged useful for estimation of disease spread and prognosis of the individual patient.
Assuntos
Carcinoma de Células Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Timidina Quinase/sangue , Análise Atuarial , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno Carcinoembrionário/análise , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Humanos , L-Lactato Desidrogenase/sangue , Estudos Longitudinais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias/métodos , PrognósticoRESUMO
Measurement of deoxythymidine kinase activity (S-TK) in serum is used as a marker for cytomegalovirus (CMV) infection following transplant surgery. A case is presented where a kidney transplant patient suffered a fatal CMV infection. The new antiviral drug Foscarnet was used to treat the infection. Retrospective analysis of S-TK showed a good correlation with the course of the disease, its treatment, recurrence and final outcome.