alpha-chymotrypsin and Leukemia--Myeloid--Acute

To determine the safety, target inhibition, and signals of clinical activity of tipifarnib in combination with bortezomib in patients with advanced acute leukemias.. In a "3 + 3" design, patients received escalating doses of tipifarnib (days 1-14) and bortezomib (days 1, 4, 8, 11) every 3 weeks until maximum tolerated dose was reached. Peripheral blood mononuclear cells (PBMC) were collected at days 1, 8, and 22 for measurement of chymotrypsin-like and farnesyltransferase activity. Purified bone marrow leukemic blasts were collected at baseline and at day 8 for measurement of NF-κB activity.. The combination was well-tolerated, and maximum tolerated dose was not reached. Dose-limiting toxicities included diarrhea, fatigue, and sensorimotor neuropathy. Chymotrypsin-like and farnesyltransferase activity within PBMCs were decreased in a majority of patients at day 8. NF-κB activity within leukemic blasts was decreased in a majority of patients at day 8. Complete response with incomplete count recovery was observed in 2 patients, and additional 5 patients had stable disease.. Tipifarnib and bortezomib combination in patients with advanced leukemias was well-tolerated, demonstrated relevant target inhibition, and was associated with signals of clinical activity in patients with advanced and refractory acute leukemias. Future studies of this combination may be warranted in more selected groups of patients in whom these molecular targets are of particular importance.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Chymotrypsin; Farnesyltranstransferase; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Male; Maximum Tolerated Dose; Middle Aged; NF-kappa B; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteasome Inhibitors; Pyrazines; Quinolones

We evaluated whether the proteasomal chymotrypsin-like (ChT-L) activity is increased in plasma of patients with acute lymphoblastic (ALL), acute myeloblastic (AML) and chronic lymphocytic (CLL) leukemias.. The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors (n=15) and ALL (n=15), AML (n=28) and CLL (n=22) patients.. The activity was significantly (P<0.001) higher in the plasma of ALL and AML patients at the diagnosis than in healthy subjects and decreased after therapy or remained unchanged or rose during relapse. By contrast, in CLL patients at the diagnosis, the activity did not differ significantly from the healthy controls. In each group, the activity positively correlated with the serum lactic dehydrogenase activity.. Plasma proteasome ChT-L activity can be a useful bio-marker for patients with acute leukemia at the blast stage.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Chymotrypsin; Female; Humans; Hydrolysis; L-Lactate Dehydrogenase; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid, Acute; Male; Middle Aged; Oligopeptides; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Sodium Dodecyl Sulfate

The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR.. Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs, we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I).. The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs in plasma with added analytes were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays.. The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.

Topics: Biomarkers, Tumor; Chymotrypsin; Cross Reactions; Fluoroimmunoassay; Humans; Leukemia, Myeloid, Acute; Male; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Reproducibility of Results; Sensitivity and Specificity; Urokinase-Type Plasminogen Activator

Blood and bone marrow samples from 20 individuals with reactive conditions and 26 cases of acute and chronic myeloid leukemias were tested for the presence of lysozyme, alpha-1-antitrypsin (alpha-1-AT), and alpha-1-antichymotrypsin (alpha-1-ACT). We compared the reactivity of samples in smears, cytocentrifuge preparations, and paraffin sections. Lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were found only in polymorphonuclear leukocytes and monocytes and their precursors. Lymphocytes, E-rosetting cells, Con A-activated lymphocytes, natural killer (NK) cells, red blood cells, erythroblasts, and megakaryocytes were consistently negative. Leukemic myeloblasts showed definite reactivity for both alpha-1-antitrypsin and alpha-1-ACT, but not for lysozyme. By contrast, lysozyme was present in poorly differentiated leukemic monoblasts, while alpha-1-antitrypsin and alpha-1-antichymotrypsin showed only weak reactivity. More mature myeloid and moncytic cells showed positive staining for all three antigens tested with differences in staining distribution and intensity. In four cases of chronic myeloid leukemia (CML), circulating mature polymorphonuclear leukocytes were deficient in both lysozymne and alpha-1-antitrypsin. The use of lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin identifies normal and leukemic cells of the myeloid-monocytic series at all stages of maturation and is applicable to a variety of sample preparations.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Bone Marrow; Cell Separation; Chymotrypsin; Clinical Enzyme Tests; Flow Cytometry; Hematopoietic Stem Cells; Humans; Immunoenzyme Techniques; Killer Cells, Natural; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Microtomy; Monocytes; Muramidase; Paraffin

Two cytochemical methods for detection of granulocytic elastase and chymotrypsin employing alanine and phenylalanine naphthyl esters were developed. Specificity of reaction with the ester substrates was proven by chloromethyl ketone inhibitors. The results of both staining methods were almost identical with the staining for naphthol AS-D chloroacetate (Cl Ac-O Nap AS-D) esterase, since Cl Ac-O Nap AS-D also reacts with granulocyte elastase and chymotrypsin. Mature neutrophils and myeloid precursors except myeloblasts are stained with all three substrates in peripheral blood and bone marrow. Mast cells, however, only react with Cl Ac-O Nap AS-D and the chymotrypsin substrate and not with the elastase substrate. In acute myeloid leukemia the three esterases appear in parallel at a somewhat later stage of maturation than myeloperoxidase. In blood smears from 380 hospital patients no hereditary elastase or chymotrypsin deficiency could be demonstrated. Staining for elastase and chymotrypsin was also normal in hereditary myeloperoxidase deficiency and chronic granulomatous disease. On the other hand 6% of the hospital patients and about two-thirds of patients with acute myeloid leukemia showed a partial elastase deficiency in more than 25% of the peripheral neutrophils.

Topics: Chymotrypsin; Granulocytes; Granulomatous Disease, Chronic; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Naphthol AS D Esterase; Pancreatic Elastase; Peroxidase; Phenylalanine

Topics: Biological Availability; Cell Division; Cells, Cultured; Chromatography, Thin Layer; Chymotrypsin; Clone Cells; Culture Media; Hematopoietic Stem Cells; Humans; Iodine Radioisotopes; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Molecular Weight; Trypsin