Travel and risk of venous thrombosis.
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Aircraft↗
Biomedical subjects
M Waltham
Publications and source records attributed to M Waltham.
Recruitment of labelled monocytes by experimental venous thrombi.
OBJECTIVE: Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease. DESIGN AND METHODS: Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction, Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval). RESULTS: Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter. CONCLUSION: The structure of thrombi produced by this model was comparable to that of human venous thrombi. Endogenous and injected monocytes migrated into the thrombus during natural resolution, possibly via the vein wall. Monocyte targeting could therefore be used to develop novel treatments for venous thrombosis, with the aim of reducing post-thrombotic complications.
Animals↗
Identification of gel-separated tumor marker proteins by mass spectrometry.
Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.
Biomarkers, Tumor↗
Systematic variation in gene expression patterns in human cancer cell lines.
We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.
Breast↗
A gene expression database for the molecular pharmacology of cancer.
We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.
Antineoplastic Agents↗
Vascular endothelial growth factor and basic fibroblast growth factor are found in resolving venous thrombi.
OBJECTIVE: Resolution of venous thrombi is accompanied by an ingrowth of capillaries, which appears to be analogous to angiogenesis in other tissues. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are major regulators of angiogenesis. The aim of this study was to determine the temporal changes and the location of VEGF and bFGF expression in a rat model of venous thrombus resolution. DESIGN AND METHODS: Thrombi were induced in the inferior venae cavae of rats by creating a stenosis to reduce blood flow by 80% to 90%. Thrombi, adjacent inferior vena cava wall, and systemic blood were collected at 1, 3, 7, 14, 21, and 28 days after thrombus generation (n = 9). Sham operations were performed (n = 5), and blood was collected at 1, 3, and 7 days. VEGF and bFGF were measured with specific immunoassays, and levels in tissues were expressed as picogram per milligram soluble protein. Tissues from two animals that were humanely killed 7 days after thrombus formation were prepared for histological examination. Immunohistochemistry was performed by the use of antibodies against VEGF, bFGF, and ED-1 (a monocyte/macrophage marker). RESULTS: Laminated thrombi were reliably produced with a median weight at 1 day of 39 mg (range, 23-63 mg). There was a significant increase in thrombus VEGF concentration between 1 day (median, 247; range, 0-514) and 7 days (median, 556; range, 254-1741) (P =.02). There was no difference between the seventh day and subsequent days. There was a positive linear correlation between thrombus bFGF concentration and time (R = 0.74, P <.0001), with a more than 300-fold increase in bFGF concentration over the 28 days of the study. VEGF and bFGF concentrations in the adjacent vena caval wall did not change significantly with time. The serum VEGF was significantly raised at 1 day (median, 5520 pg/mL; range, 4040-7912 pg/mL) and 3 days (median, 3880 pg/mL; range, 2564-7232 pg/mL) compared with 7 days (median, 1790 pg/mL; range, 232-3228 pg/mL) (P <.0001). Similar changes in the serum VEGF also developed in the sham-operated animals. The serum bFGF (day 1 median, 15.5 pg/mL; range, 1-42 pg/mL) did not change with time. Immunohistochemistry showed that the VEGF antigen was localized to monocytes, endothelial cells, and spindle-shaped cells within the thrombus. The bFGF antigen was localized to mononuclear cells and spindle-shaped cells and was also present in the extracellular matrix. CONCLUSION: VEGF and bFGF are found in organizing thrombi and have characteristic temporal expression patterns, which suggest that they have a role in thrombus resolution. Augmenting angiogenic growth factor expression may enhance thrombus recanalization, thus reducing long-term complications.
Animals↗
Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi.
PURPOSE: Organization, recanalization, and contraction are common to wound healing and thrombus resolution. Monocytes are essential to wound healing and are also found in venous thrombi. We measured endogenous levels of the monocyte chemotactic protein-1 (MCP-1) in naturally resolving venous thrombi and determined the effect of injecting MCP-1 into newly formed thrombus. METHODS: Endogenous MCP-1 levels were estimated in rat blood, thrombi, and the adjacent vessel wall after thrombus formation, in cohorts of eight animals at 1, 7, and 14 days. In another group (n = 10), 1 microgram of MCP-1 was injected into newly formed thrombi. Carrier was injected into the thrombi of control animals (n = 10). Thrombi and adjacent vein walls were obtained for histology at 7 days. Thrombi were given an arbitrary organization score based on erythrocyte and extracellular matrix content, which was assessed by means of computerized and observer analysis. Specimen weight, thrombus area, and cellular and monocyte content were measured. RESULTS: Endogenous MCP-1 increased between days 1 and 7 in the thrombus (1-day median, 1.1 ng/g wet wt; 1-day range, 0.8 to 1.4 ng/g wet wt; 7-day median, 5.4 ng/g wet wt; 7-day range, 1.5 to 7.4 ng/g wet wt; P <.0001) and vein wall (1-day median, 1.5 ng/g wet wt; 1-day range, 0.8 to 4.3 ng/g wet wt; 7-day median, 3.3 ng/g wet wt; 7-day range, 2.7 to 8.3 ng/g wet wt; P <. 05). At 14 days, thrombus was incorporated in the vein wall, and total MCP-1 levels remained high (median, 3.9 ng/g wet wt; range, 1.1 to 7.4 ng/g wet wt). Less MCP-1 was found in the thrombus than the adjacent vessel wall at day 1 (P <.05), but there was no difference at day 7. MCP-1 could not be detected in the blood. MCP-1 injection into thrombus increased the computer (P =.016) and observer (P =.004) organization scores, reduced the thrombus area (from median, 3. 4 mm(2), and range, 1.5 to 5.7 mm(2), to median, 0.2 mm(2), and range, 0.02 to 2.6 mm(2); P =.048), and increased the surrounding vessel wall monocyte content (P =.008). Specimen weights of treated animals were lower than those of control animals (P <.02). CONCLUSION: Venous thrombus MCP-1 levels increase during natural resolution. MCP-1 treatment increased the organization and resolution of thrombi. MCP-1 may therefore be of therapeutic use.
Animals↗
Intrinsic and acquired resistance to methotrexate in acute leukemia.
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Acute Disease↗
Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations.
Although dihydrofolate reductase (DHFR) gene amplification is a common mechanism of resistance to methotrexate (MTX) in tumor cell lines, with the exception of a few case reports, the incidence of this phenomenon as a mechanism of MTX resistance in the clinic has not been reported. We studied 38 untreated patients and 29 patients in relapse with acute lymphoblastic leukemia (ALL) for gene amplification and p53 gene mutations. Three patients were studied both at diagnosis and at each of two relapses after treatment with MTX. Nine of 29 relapsed patients (31%) had low-level DHFR gene amplification (two to four gene copies) associated with increased levels of DHFR mRNA and enzyme activity. Of significance was a correlation of gene amplification with p53 mutations in seven of nine relapsed patients (P < .001). Low-level DHFR gene amplification may be an important cause of MTX resistance in ALL and strengthens the concept that mutations in the p53 gene may lead to gene amplification as a consequence of defective cell cycle control.
Adolescent↗
2,4-Diamino-5-substituted-quinazolines as inhibitors of a human dihydrofolate reductase with a site-directed mutation at position 22 and of the dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.
2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.
Animals↗
Acute monocytic leukemia: a myeloid leukemia subset that may be sensitive to methotrexate.
Impaired polyglutamylation of methotrexate (MTX) and thus poor retention is believed to be the basis of intrinsic resistance in blasts from patients with acute myeloid leukemia (AML) to MTX. We studied additional samples from patients with this disease, and confirmed that polyglutamylation of MTX was poor in ANLL blast cells. However, in one subset of ANLL, acute monocytic leukemia, (M5) leukemia blasts were found to be capable of accumulating and forming long-chain MTX polyglutamates. An acute monocytic leukemia cell line, THP-1 also was found to accumulate high levels of MTX polyglutamates and was relatively sensitive to MTX, strengthening the concept that M5 blasts may be sensitive to this drug. MTX may be an overlooked drug for the treatment of acute monocytic leukemia.
Adult↗
Molecular mechanisms of resistance to antifolates, a review.
Methotrexate (MTX) is a clinically important antifolate that has been used in combination with other chemotherapeutic agents in the treatment of malignancies including acute lymphocytic leukemia, osteosarcoma, carcinomas of the breast, head and neck, choriocarcinoma and non-Hodgkin's lymphoma. The primary target of MTX is the enzyme dihydrofolate reductase (DHFR) which catalyzes the reduction of folate and 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Understanding of MTX action has revealed how cells acquire resistance to this drug. The four known mechanisms of MTX resistance are a decrease in the uptake of the drug, a decrease in the retention of the drug due to defective polyglutamylation or an increase in polyglutamate breakdown, an increase in the enzyme activity and a decrease in the binding of MTX to DHFR. The molecular basis for some of these mechanisms has been elucidated in MTX resistant cell lines; in particular the occurrence of gene amplification resulting in increased DHFR and point mutations resulting in altered DHFR with reduced affinity for MTX. Cloning of the human folylpolyglutamate synthase gene and the reduced folate transport gene have been reported recently and should facilitate the identification of the molecular basis of these resistant phenotypes. DHFR protein has been shown to regulate its synthesis by exerting an inhibitory influence on its own translation. Addition of MTX relieves this inhibition thus providing a possible molecular explanation for the rapid rise in DHFR activity noted in some cells after MTX administration. Alterations in genes involved in regulating the cell cycle such as cyclin D1 and the retinoblastoma (Rb) gene have also been shown to influence cellular response to MTX. Overexpression of cyclin D1 in HT1080, a human fibrosarcoma cell line, results in decreased MTX sensitivity. The molecular basis of this observation is under investigation. Abnormalities in the Rb gene may also have profound effects on MTX sensitivity. Rb interacts with the family of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR. When Rb is deleted or rendered nonfunctional levels of "free" or unbound E2F are high resulting in enhanced transcription of genes such as DHFR. This results in increased DHFR protein and may lead to MTX resistance. As the knowledge regarding mechanisms of resistance increases newer approaches to circumvent such resistance or to target resistant cells can be undertaken.
Antineoplastic Agents↗
Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.
Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.
Animals↗
Effect of codon 22 mutations on substrate and inhibitor binding for human dihydrofolate reductase.
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Amino Acid Sequence↗
Translational regulation of the synthesis of dihydrofolate reductase.
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Animals↗
Increased activity of gamma-glutamyl hydrolase in human sarcoma cell lines: a novel mechanism of intrinsic resistance to methotrexate (MTX).
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Cell Line↗
Mutations leading to antifolate resistance in Chinese hamster ovary cells after exposure to the alkylating agent ethylmethanesulfonate.
Chinese hamster ovary cells with a single allele for dihydrofolate reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving colonies were selected at random, and cell lines were established. Cell lines 1-3 were maintained in culture in the presence of 8 x 10(-8) M trimetrexate and were 66-170-fold resistant to the drug. Cell lines 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4-26-fold resistant to the drug, compared with the parental cell line. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.
Alkylating Agents↗
Interactions with hemoglobin: a source of error in measurements of transketolase activity in hemolysates.
Measurements of the activity of transketolase in human erythrocyte lysates by an assay coupled to NADH oxidation indicate that interactions of assay substrates with hemoglobin can give rise to overestimations of transketolase activity. Three potential sources of error are identified. Thus, in lysates containing methemoglobin, NADH oxidation can be due firstly to methemoglobin reductase activity or secondly to the monooxygenase activity of methemoglobin, for which the substrate can be ribose 5-phosphate, a substrate also of transketolase. Thirdly, the addition of high concentrations of the transketolase cofactor, TDP, to an insufficiently buffered reaction mixture can cause the aggregation and precipitation of hemoglobin: a phenomenon that may be misconstrued as an enhanced increase in absorbance at 340 nm and hence as additional transketolase activity. Although the present study concentrates on these potential artefacts in assays of transketolase activity, the findings may well be relevant to the measurement of other enzyme activities in hemolysates by procedures based ultimately on the rate of consumption or production of NAD(P)H.
Chromatography, Gel↗