RESUMO
The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.
Assuntos
Estramustina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Compostos de Mostarda Nitrogenada/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Estramustina/farmacologia , Imunofluorescência , Humanos , Masculino , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/análise , Microtúbulos/efeitos dos fármacos , Neoplasias da Próstata , Células Tumorais CultivadasRESUMO
The super family of glutathione S-transferases (GSTs) is composed of multiple isozymes with significant evidence of functional polymorphic variation. Over the last three decades, data from cancer studies have linked aberrant expression of GST isozymes with the development and expression of resistance to a variety of chemicals, including cancer drugs. This review addresses how differences in the human GST isozyme expression patterns influence cancer susceptibility, prognosis and treatment. In addition to the well-characterized catalytic activity, recent evidence has shown that certain GST isozymes can regulate mitogen-activated protein kinases or can facilitate the addition of glutathione to cysteine residues in target proteins (S-glutathionylation). These multiple functionalities have contributed to the recent efforts to target GSTs with novel small molecule therapeutics. Presently, at least two drugs are in late-stage clinical testing. The evolving functions of GST and their divergent expression patterns in individuals make them an attractive target for drug discovery.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Neoplasias/epidemiologia , Neoplasias/terapia , Polimorfismo Genético , Resistencia a Medicamentos Antineoplásicos , Estudos Epidemiológicos , Predisposição Genética para Doença , Humanos , Isoenzimas , Neoplasias/genética , Prognóstico , Resultado do TratamentoRESUMO
ATP-binding cassette (ABC) transporters are a family of proteins that translocate molecules across cellular membranes. Substrates can include lipids, cholesterol and drugs. Mutations in ABC transporter genes can cause human pathologies and drug resistance phenotypes in cancer cells. ABCA2, the second member the A sub-family to be identified, was found at high levels in ovarian carcinoma cells resistant to the anti-cancer agent, estramustine (EM). In vitro models with elevated levels of ABCA2 are resistant to a variety of compounds, including estradiol, mitoxantrone and a free radical initiator, 2,2'-azobis-(2-amidinopropane) (AAPH). ABCA2 is most abundant in the central nervous system (CNS), ovary and macrophages. Enhanced expression of ABCA2 and related proteins, including ABCA1, ABCA4 and ABCA7, is found in human macrophages upon bolus cholesterol treatment. ABCA2 also plays a role in the trafficking of low-density lipoprotein (LDL)-derived free cholesterol and is coordinately expressed with genes involved in cholesterol homeostasis. Additionally, ABCA2 expression has been linked with gene cluster patterns consistent with pathologies including Alzheimer's disease (AD). A single-nucleotide polymorphism (SNP) in exon 14 of the ABCA2 gene was shown to be linked to early onset AD in humans, supporting the observation that ABCA2 expression influences levels of beta-amyloid peptide (Abeta), the primary component of senile plaques. ABCA2 may play a role in cholesterol transport and affect a cellular phenotype conducive to the pathogenesis of a variety of human diseases including AD, atherosclerosis and cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Doença de Alzheimer/etiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Colesterol/metabolismo , Resistencia a Medicamentos Antineoplásicos , Homeostase , Humanos , Dados de Sequência MolecularRESUMO
Amplified differential gene expression (ADGE) is a novel technique, designed to profile gene expression of the whole transcriptome or to compare expression of a set of genes between two samples. ADGE employs hybridization to quadratically amplify the ratio of an expressed gene between control and tester samples before displaying. The subtle structures of adapters and primers are designed for displaying the amplified ratio of an expressed gene between two samples. Four selective nucleotides at the 3' end of primers are used to increase PCR efficiency for targeted molecules and to improve detection of PCR products. Double PCR with the same pair of primers expands the detection range, especially for genes of low abundance. Integration of these steps makes ADGE sensitive and accurate. Application to drug resistant human tumor cell lines showed that ADGE accurately profiled expression levels for induced, repressed or unchanged genes. The qualitative expression patterns for ADGE were verified with RT-PCR.
Assuntos
Perfilação da Expressão Gênica/métodos , Primers do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Matemática , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
The gross distribution of levels of [32P]cyclophosphamide ([32P]CY) in August female rats was similar in tumor, intestinal mucosa, spleen, bone marrow, and striated muscle tissue, with slightly higher levels in liver and kidney tissue. A triphasic association with nucleic acids was found and actual alkylation of DNA and RNA reached a maximum of 48 hours post treatment. There was no evidence of 32P-label reutilization that could have accounted for prolonged alkylation. We found that 100 mg CY/kg suppressed the incorporation of [3H]thymidine, [3H]deoxyuridine, and [14C]sodium formate into the DNA of the BICR-A15 carcinoma for at least 10 days. This correlated well with the observed regression of tumor volume. Bone marrow and intestinal mucosa, two tissues which limit chemotherapuetic treatment of the tumor, were less affected by 100 mg CY/kg. Bone marrow had regained normal levels of DNA precursor incorporation by 5 days, and intestinal mucosa had regained normal levels by 3 days. Results indicated that this differential in recovery time may assist in the successful scheduling of vital tissue-sparing drug regimens.
Assuntos
Ciclofosfamida/farmacologia , DNA de Neoplasias/biossíntese , DNA/biossíntese , Neoplasias Experimentais/metabolismo , RNA Neoplásico/biossíntese , RNA/biossíntese , Alquilação , Animais , Medula Óssea/metabolismo , Ciclofosfamida/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo , Músculos/metabolismo , Neoplasias Experimentais/patologia , Ratos , Ratos Endogâmicos , Baço/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Previous preclinical studies of combinations of estramustine and vinblastine or paclitaxel (Taxol) have shown that it is possible to achieve a greater than additive cytotoxicity with these antimicrotubule drug combinations. Phase II studies in hormone-refractory prostate cancer have demonstrated clinical antitumor activity of sufficient magnitude to stimulate further laboratory and clinical studies of these drugs combinations. PURPOSE: Our purpose was to characterize the interactions of estramustine with P-glycoprotein and to determine its effects. METHODS: Standard laboratory techniques were used to study the effects of estramustine on intracellular drug concentrations, cytotoxicity, and induction of messenger RNA (mRNA) for the MDR1 (also known as PGY1) gene. Using a photoaffinity analogue of estramustine 17-0-[[2-[3-(4-azido-3-[125I]-iodophenyl) propionamido]ethyl]-carbamyl]estradiol-3-N-bis(2-chloroethyl)ca rba mate ([125I]AIPP-estramustine), binding to the membrane proteins of human ovarian (SKOV3) and their multidrug-resistant counterpart SKVLB1 cells was studied. Southern-blot analysis was performed on DNA extracted from human prostate carcinoma wild-type DU145, estramustine-resistant cell line (E4), and SKVLB1 cells. RESULTS: Membrane fractions from SKOV3 and SKVLB1 cells were analyzed for proteins that could be photoaffinity labeled with [125I]AIPP-estramustine. Competitive inhibition of this binding was achieved with excess concentrations of (in order of efficacy) estramustine, vinblastine, verapamil, progesterone, and to a lesser degree, by paclitaxel but not with estramustine phosphate, estradiol, and estriol. SKVLB1 cells accumulated much less [3H]vinblastine and [3H]paclitaxel than did SKOV3 cells. Estramustine caused a concentration-dependent enhancement of drug accumulation in the SKVLB1 cells to a maximum of approximately 12-fold. No effect of estramustine was apparent for the wild-type SKOV3 cells. In comparison with verapamil, estramustine was less effective as a modulator; however estramustine demonstrated good chemosensitizing activity in combination with actinomycin D and vinblastine. Neither short-term, low-dose no longer-term, higher concentration were found to produce measurable transcript (mRNA for the MDR1 gene levels. Such data suggest that, at least levels. Such data suggest that, at least for two distinct human cell line (SKOV3 and DU145), estramustine does not induce the overexpression of the MDR1 gene. CONCLUSION: It is apparent from the P-glycoprotein data that estramustine interacts with this efflux pump, altering intracellular drug accumulation. Overall, the nonempiric basis for including estramustine in clinical protocols that contain other multidrug-resistant drugs is strengthened by the present data.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Estramustina/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Proteínas de Transporte/metabolismo , Resistência a Medicamentos/genética , Estramustina/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/genética , Fenótipo , Ligação Proteica , RNA Mensageiro , Células Tumorais CultivadasRESUMO
The importance of thiol-mediated detoxification of anticancer drugs that produce toxic electrophiles has been of considerable interest to many investigators. Glutathione and glutathione S-transferases (GST) are the focus of much attention in characterizing drug resistant cells. However, ambiguous and sometimes conflicting data have complicated the field. This article attempts to clarify some of the confusion. The following observations are well established: (a) tumors express high levels of GST, especially GST psi, although the isozyme components vary quite markedly between tissues and the isozymes are inducible; (b) nitrogen mustards are good substrates for the GST alpha family of isozymes which are frequently overexpressed in cells with acquired resistance to these drugs; (c) most drugs of the multidrug-resistant phenotype have not been shown to be GST substrates and although GST psi is frequently overexpressed in multidrug-resistant cells, most indications are that this is an accompaniment to, rather than a cause of, the resistant phenotype; (d) transfection of GST complementary DNAs has produced some lines with increased resistance to alkylating agents. Most studies of the relationships between GST and resistance have overlooked the potential importance of other enzymes involved in the maintenance of cellular glutathione homeostasis, and this has complicated data interpretation. Translational research aimed at applying our knowledge of glutathione pathways has produced preclinical and clinical testing of some glutathione and GST inhibitors, with some encouraging preliminary results. In brief, GSTs are important determinants of drug response for some, not all, anticancer drugs. Caution should be encouraged in assessing cause/effect relationships between GST overexpression and resistance mechanisms.
Assuntos
Antineoplásicos/metabolismo , Glutationa Transferase/fisiologia , Glutationa/fisiologia , Apoptose/efeitos dos fármacos , Resistência a Medicamentos , Indução Enzimática , Glutationa/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Homeostase , Humanos , Pesquisa , Relação Estrutura-Atividade , Especificidade por Substrato , Células Tumorais Cultivadas/metabolismoRESUMO
Using polyclonal antibodies raised against a rat liver nuclear envelope protein, lamin protein A, the nuclear matrix proteins of a Walker 256 rat mammary carcinoma wild-type (WS) and a selected cell line with acquired resistance to nitrogen mustards (WR) were found to possess antigenic determinants which were recognized by the antibodies. In one-dimensional immunoblotting analysis, the nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these proteins showed that this Mr 70,000 WS protein had a pI of approximately 7.5. Immunoprecipitation analysis revealed that the altered mobility of this protein could be a function of phosphorylation. The nuclear matrix proteins from both WS and WR cells were shown to bind 3':5'-cyclic adenylic acid (cAMP), as judged by photoaffinity labeling and gel electrophoresis studies. The WS nuclear matrix proteins showed a quantitatively greater level of cAMP binding compared to WR, with predominant binding to proteins with molecular weights of 45,000, 55,000, and 70,000. In WR cells, there was no cAMP binding in the Mr 70,000 region. These data indicate that the Mr 70,000 nuclear matrix lamin proteins are antigenically similar in WS and WR but differ in that the WR protein is hypophosphorylated and does not bind cAMP.
Assuntos
Núcleo Celular/metabolismo , Neoplasias Mamárias Experimentais/patologia , Mecloretamina/farmacologia , Nucleoproteínas/metabolismo , Animais , Núcleo Celular/ultraestrutura , AMP Cíclico/metabolismo , Resistência a Medicamentos , Feminino , Ponto Isoelétrico , Laminas , Peso Molecular , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , RatosRESUMO
Estramustine [17 beta-estradiol 3 N bis(2-chloroethyl)carbamate; EM] is a stable conjugate of estradiol and nor-nitrogen mustard that is used for the treatment of human prostatic carcinoma. We have studied the cytotoxic effects of EM on the cytoskeletal organization of squirrelfish pigment cells (erythrophores) and human prostatic tumor cells (DU 145) in culture. Light and whole-mount electron microscopy studies reveal that, at microM levels (60 to 120 microM), EM has a dose-dependent disruptive effect on cell shape, cytoskeletal organization, and intracellular transport. Upon removal of the drug, the cytological effects of EM are rapidly reversible in fish cells but not DU 145s. Immunofluorescent studies reveal that EM produces microtubule disassembly in fish erythrophores and DU 145 cells. A concomitant disruption of actin-microfilament arrays also occurs in DU 145 cells. These morphological data suggest that EM, in contradistinction to its constituent estradiol: nitrogen mustard species, induces cytotoxicity as an antimicrotubule drug. The observed disruption of the microtubules and cytomatrix of interphase cells is not reversible in the prostatic carcinoma cells. The disruptive action of EM on the cytoskeleton could ultimately produce a cytotoxic antimitotic effect in dividing cells.
Assuntos
Estramustina/farmacologia , Microtúbulos/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/farmacologia , Linhagem Celular , Peixes , Humanos , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Mitose/efeitos dos fármacos , Pigmentos Biológicos/metabolismo , Neoplasias da Próstata/ultraestruturaRESUMO
Ethacrynic acid and piriprost (6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1) have been shown to potentiate the cytotoxic activity of chlorambucil in rat and human tumor cell lines. Walker 256 rat breast carcinoma cells (WS), with acquired resistance to nitrogen mustards (WR), and two human colon carcinoma cell lines, HT 29 and BE, were sensitized to chlorambucil when either ethacrynic acid or piriprost was administered at the same time as the alkylating agent. Both as single agents and in combination with chlorambucil, there was inhibition of glutathione S-transferase activity as measured with 1-chloro-2,4-dinitrobenzene as a substrate. A depletion in intracellular glutathione was also evident following ethacrynic acid alone or in combination with chlorambucil. Thus, diuretic plant phenols or prostaglandin analogues may have potential therapeutic utility in combination with alkylating agents.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Clorambucila/administração & dosagem , Epoprostenol/administração & dosagem , Ácido Etacrínico/administração & dosagem , Animais , Carcinoma 256 de Walker , Resistência a Medicamentos , Sinergismo Farmacológico , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Técnicas In Vitro , Ratos , Células Tumorais CultivadasRESUMO
Estramustine (EM) and taxol, two antimicrotubule agents with distinct and apparently opposing mechanisms of action, were found to be effective in combination in the preclinical treatment of EM-resistant and sensitive, wild-type human prostatic carcinoma cell lines. Estramustine combined with 1 nM taxol (concentration 100-fold less than that measured in plasma of patients treated with taxol) produced greater than additive effects on the inhibition of cell survival of both wild-type and EM-resistant cells. When taxol was used with another microtubule-destabilizing drug, vinblastine, no significantly increased cytotoxicity was observed. Other effects on wild-type and EM-resistant cells produced by the combination of EM and taxol included (a) an increased proportion of the cells in the S phase of the cell cycle; (b) no mitotic block; and (c) an increase in the percentage of micronucleated cells from a control value of less than 1% to greater than 20% after drug treatment. Immunofluorescent microscopic analysis of the effect of this drug combination on the mitotic spindle apparatus revealed specific examples of aberrant mitotic figures, including multiple asters, cells with two distinct spindles, and tripolar spindles able to traverse mitosis and complete cytokinesis. These data provide supportive preclinical evidence for the potential development of an EM/taxol combination clinical regimen either for prostate or other cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Carcinoma/ultraestrutura , Ciclo Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estramustina/farmacologia , Citometria de Fluxo , Humanos , Masculino , Testes para Micronúcleos , Microtúbulos/ultraestrutura , Paclitaxel , Neoplasias da Próstata/ultraestrutura , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The stimulation of poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase activity at the nuclear level after damage of HeLa cells by 1-methyl-1-nitrosourea has been previously reported. We have observed a similar activation of the enzyme after treatment of cells with MNU at the nucleosomal level of chromatin (greater than 1N). This stimulation of enzyme activity did not occur through an inhibition of the glycohydrolase enzyme which cleaves poly(ADP-ribose), or elongation of poly(ADP-ribose) chains, or an increased biosynthesis of enzyme protein. The increased activity appears to be a consequence of the generation of more acceptor sites on nuclear proteins for initiation of poly(ADP-ribose) synthesis. The data indicate that MNU increased the accessibility of nucleosome core histones for modification by poly(ADP) ribosylation.
Assuntos
Cromatina/efeitos dos fármacos , Metilnitrosoureia/farmacologia , NAD+ Nucleosidase/metabolismo , Compostos de Nitrosoureia/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Cromatina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Histonas/metabolismo , HumanosRESUMO
A Walker 256 rat carcinoma cell line (WR) with acquired resistance to nitrogen mustards has been found to lack cross-resistance to nitrosoureas. Although total cellular glutathione pools were similar in the parent (WS) and resistant cell lines (WS, 2.5 X 10(-6); WR, 2.0 X 10(-6) mol/mg protein), glutathione reductase activity was 3.98 in WR compared to 8.67 nmol reduced nicotinamide adenine dinucleotide phosphate oxidized per microgram protein per min in WS cells. Treatment of cells with a carbamoylating nitrosourea, N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea, produced a dose-dependent inhibition of glutathione reductase and depletion of thiols in both cell lines. The drug caused no direct DNA strand breakage, but a differential mitotic spindle-chromosome stain showed that spindle formation was inhibited in WR cells at N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea concentrations of greater than 50 microM. In WS cells, mitotic figures were still visible at 100 microM. Chromosomal damage was expressed in both cell lines at concentrations of 25 microM. The number and extent of these aberrations were greater in WR than WS. Observed karyotypic abnormalities included polyploidy, chromosome decondensation, and endoreduplication. In interphase cells, transmission electron microscopy showed that the most prevalent drug-induced lesions included (a) disappearance of plasma membrane filopodia, (b) appearance of membrane blebbing, and (c) development of irregular crescent-shaped nuclei. These morphological and cytogenetic changes correlate with cytotoxic responses of these cell lines to N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea and would be consistent with drug-induced inhibition of glutathione reductase.
Assuntos
Carcinoma 256 de Walker/enzimologia , Aberrações Cromossômicas , Glutationa Redutase/metabolismo , Compostos de Mostarda Nitrogenada/farmacologia , Compostos de Nitrosoureia/farmacologia , Animais , Carcinoma 256 de Walker/genética , Carcinoma 256 de Walker/patologia , Linhagem Celular , Resistência a Medicamentos , Feminino , Glutationa/análise , Glutationa Redutase/antagonistas & inibidores , RatosRESUMO
The removal of DNA adducts is an essential step of DNA repair following exposure to chloroethylnitrosoureas. Adduct removal was evaluated in both L1210 and murine bone marrow DNA for lesions induced by three chloroethylnitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea, a marrow-toxic agent with high carbamoylating activity, was not removed in either system for at least 6 to 12 hr. These results were compared with those obtained with two glucose-linked chloroethylnitrosoureas, chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea. Both of these agents have low marrow toxicity at therapeutic doses. Chlorozotocin, which has very low chemical carbamoylating activity, was found to permit approximately 40% removal of drug-derived DNA adducts in both systems within the first 6 hr and approximately 50% by 18 hr. The second glucose-linked analog, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, has relatively high carbamoylating activity and was found to inhibit early removal of DNA adducts as effectively as does 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. It would thus appear that the selective marrow-sparing property of the sugar-linked chloroethylnitrosoureas is not dependent upon carbamoylation-mediated differences in the rate and extent of DNA adduct removal. In view of the comparable therapeutic activity of the three drugs for L1210 leukemia, therapeutic efficacy does not appear to be impaired by the increased rate of adduct removal observed with chlorozotocin in this system.
Assuntos
Medula Óssea/metabolismo , DNA de Neoplasias/metabolismo , DNA/metabolismo , Leucemia L1210/metabolismo , Compostos de Nitrosoureia/farmacologia , Alquilação , Animais , Reparo do DNA/efeitos dos fármacos , Técnicas In Vitro , Lomustina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Estreptozocina/análogos & derivados , Estreptozocina/farmacologiaRESUMO
Chronic lymphocytic leukemia (CLL) is a disease state which frequently responds to alkylating agent chemotherapy but ultimately becomes refractory through acquired resistance mechanisms. In the present study, we have examined the expression of glutathione S-transferases (GST) in both CLL and normal control lymphocytes, as these enzymes have been implicated in mechanisms of natural and acquired resistance. Lymphocyte GST was purified from samples by high-pressure liquid affinity chromatography, and subunits were identified by two-dimensional gel electrophoresis and immunoblotting by using polyclonal antibodies specific for individual subunits. Analysis of CLL lymphocyte GST activity using the general substrate 1-chloro-2,4-dinitrobenzene showed a statistically significant 2-fold increase in cells from chlorambucil-resistant patients over those from untreated patients and normal individuals. Furthermore, chlorambucil therapy was seen to cause a 1.3- to 1.5-fold elevation of enzyme activity in three previously drug-naive patients. Analysis of GST isozyme subunits indicated that 95% of the CLL patients examined were positive for the pi isozyme, and this appeared quantitatively to be the major isozyme present. The alpha and mu isozymes were also expressed in 63 and 53% of the patients, respectively. Examination of control lymphocytes, as well as separated B- and T-cell subpopulations, yielded similar results. The present study indicates that a high degree of interindividual variation occurs and that the pattern of CLL lymphocyte GST expression differs from that of other tumor tissues. While there were no obvious correlations between the disease state or stage and isozymes expressed, the quantitative increase in GST activity in chlorambucil-resistant CLL patients may be of relevance to the overall resistant phenotype.
Assuntos
Linfócitos B/enzimologia , Glutationa Transferase/análise , Isoenzimas/análise , Leucemia Linfocítica Crônica de Células B/enzimologia , Idoso , Idoso de 80 Anos ou mais , Clorambucila/uso terapêutico , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Peso Molecular , Linfócitos T/enzimologiaRESUMO
Estramustine (EM), an antimicrotubule agent, is effective against hormone-refractory prostate cancer when used in combination with vinblastine or paclitaxel. To understand the effect of EM on beta-tubulin, a cellular target for this class of drugs, human prostate carcinoma cells (DU-145) were made resistant to EM, and two cell lines were selected at 12- (EM-12) and 15-microMolar (EM-15) concentrations of the drug. These cell lines exhibited 8- to 9-fold resistance to EM and 2- to 4-fold cross-resistance to paclitaxel. Immunofluorescent staining of the cells with beta-tubulin isotype-specific antibodies showed an approximately 6-fold increase in the beta(III)-tubulin levels and moderate increase in overall beta-tubulin levels in EM-resistant cells when compared to DU-145 cells. This increase of beta(III) isotype was confirmed by Western analysis. A reverse transcriptase-PCR assay was also employed using beta-tubulin isotype-specific primers to quantify beta-tubulin isotype RNA. A 4-fold increase in beta(III) and a 3-fold increase in beta(IV alpha) transcript were seen in both EM-resistant cell lines. These results indicate that overexpression of specific beta-tubulin isotypes may play a role in the cellular defense against EM and other antimicrotubule agents.
Assuntos
Antineoplásicos/farmacologia , Estramustina/farmacologia , Neoplasias da Próstata/metabolismo , Tubulina (Proteína)/genética , Sequência de Bases , Primers do DNA/química , Resistência a Medicamentos , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 microM hydrocortisone for 22 hr; this resulted in depressed DNA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 mM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 microM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the increased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin structure. Electron microscopic examination of HeLa nuclear morphology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.
Assuntos
Cromatina/efeitos dos fármacos , Hidrocortisona/farmacologia , Compostos de Nitrosoureia/metabolismo , Alquilação , Sítios de Ligação , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , DNA/biossíntese , Desoxirribonucleases/metabolismo , Células HeLa , Histocitoquímica , Humanos , Magnésio/farmacologia , Ribonucleases/metabolismoRESUMO
Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonhistone chromosomal protein. The binding of MNU to histones was greater than of CCNU. CCNU mainly affected nonhistone proteins. This difference in the reactivity of methyl and chloroethyl nitrosoureas with chromatin may relate to their differential effect on poly(adenosine diphosphate ribose) polymerase activity, as well as to their carcinogenic and antitumor properties.
Assuntos
Cromatina/efeitos dos fármacos , NAD+ Nucleosidase/metabolismo , Compostos de Nitrosoureia/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Alquilação , Cromatina/metabolismo , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Histonas/metabolismo , Humanos , Lomustina/farmacologia , Metilnitrosoureia/farmacologiaRESUMO
The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácido Etacrínico/administração & dosagem , Glutationa Transferase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Tiotepa/administração & dosagem , Adulto , Idoso , Avaliação de Medicamentos , Tolerância a Medicamentos , Ácido Etacrínico/efeitos adversos , Ácido Etacrínico/antagonistas & inibidores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tiotepa/efeitos adversos , Tiotepa/farmacocinéticaRESUMO
We have isolated the full-length cDNA for human ATP-binding cassette, sub-family A, member 2 transporter (ABCA2). The ORF of this cDNA encodes a protein consisting of 2436 amino acids with apparent molecular weight of M(r) 270,000. Accordingly, ABCA2 is the largest known mammalian ABC transporter described thus far. Analysis of mRNA expression levels indicated that ABCA2 is highest in human brain and has a broad expression pattern in a panel of tumor cell lines. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. In ABCA2-transfected cells, the transporter also colocalized with a fluorescently labeled steroid analogue, estramustine. The sequestration of the steroid into the lysosomal/endosomal compartment indicates a potential substrate specificity for ABCA2. Furthermore, the presence of a lipocalin signature motif in the ABCA2 sequence suggests a possible broad role for this protein in the transport of steroids, lipids, and related molecules.