Induced differentiation in HT29, a human colon adenocarcinoma cell line.
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The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma. (+info)
Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein.
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We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells. (+info)
Successful peripheral blood stem cell mobilization with etoposide (VP-16) in patients with relapsed or resistant lymphoma who failed cyclophosphamide mobilization.
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High-dose chemotherapy (HDCT) followed by autologous blood stem cell transplantation is considered the treatment of choice for patients with relapsed or resistant aggressive non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). However, several authors report failure of standard mobilization regimens in 29% to 56% of these patients making the completion of HDCT impossible and as a result, negatively influencing long-term outcome. Thus, effective new regimens for patients failing initial mobilization are needed. Here we report the results of using etoposide as a mobilizing agent in 16 patients with primary resistant or relapsed malignant lymphoma who had failed prior mobilization of peripheral blood stem cells (PBSC) with cyclophosphamide (4 g/m2) followed by G-CSF. The use of etoposide 500 mg/m2 (days 1-4) + G-CSF resulted in the successful collection of adequate numbers of PBSC with a median harvest of 3.6 x 10(6)/kg (range 2.2-12.6) CD34+ cells in all 16 patients. In 7/16 (44%) patients, the target yield of at least 2.0 x 10(6) CD34+ cells was harvested by a single apheresis and the maximum number of separations for all patients was two. No excessive toxicities appeared, allowing all patients to proceed to myeloablative chemotherapy. In addition, median peak values of circulating CD34+ cells were significantly higher after etoposide as compared to cyclophosphamide (49.2/microl vs 4.7/microl; P = 0.0004). These results indicate that etoposide + G-CSF is a highly effective mobilization regimen in patients who have failed cyclophosphamide mobilization. (+info)
Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons.
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This study investigates the mechanisms underlying death of cultured embryonic cortical neurons exposed to the DNA-damaging agent camptothecin and in particular the interdependence of the roles of cyclin-dependent kinases (Cdks), caspases, and mitochondrial function. Camptothecin evokes rapid neuronal death that exhibits nuclear features of apoptosis. This death is accompanied by loss of cytochrome c and mitochondrial transmembrane potential as well as by induction of caspase-3-like activity and caspase-2 processing. The Cdk inhibitor flavopiridol provides long-term rescue from death and prevents loss of cytochrome c and mitochondrial transmembrane potential as well as caspase activation and processing. General caspase inhibitors rescue neurons from this rapid apoptotic death but do not prevent them from undergoing delayed death in which nuclear features of apoptosis are absent. Moreover, the caspase inhibitors do not affect early cytochrome c release and delay but do not prevent the loss of transmembrane potential. Agents that directly disrupt mitochondrial function without inducing cytochrome c release lead to a caspase-independent death. These observations favor a model in which (1) DNA damage leads to Cdk activation, which lies upstream of release of cytochrome c and caspase activation; (2) cytochrome c release is caspase-independent and may occur upstream of caspase activation; (3) early apoptotic death requires caspases; and (4) delayed nonapoptotic death that occurs in the presence of caspase inhibitors is a consequence of prolonged loss of mitochondrial function. These findings shed light on the mechanisms by which DNA damage kills neurons and raise questions regarding the general utility of caspase inhibitors as neurotherapeutic agents. (+info)
Water soluble 20(S)-glycinate esters of 10,11-methylenedioxycamptothecins are highly active against human breast cancer xenografts.
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Water-soluble 20(S)-glycinate esters of two highly potent 10,11-methylenedioxy analogues of camptothecin (CPT) have been synthesized and evaluated for their ability to eradicate human breast cancer tumor xenografts. The glycinate ester moiety increases the water solubility of the 10,11-methylenedioxy analogues 4-16-fold. However, in contrast to CPT-11, a water-soluble CPT analogue that was recently approved for second line treatment of colorectal cancer, the 20(S)-glycinate esters do not require carboxylesterase for conversion to their active forms. The glycinate esters are hydrolyzed to their parent, free 20(S)-hydroxyl active analogues in phosphate buffer (pH 7.5) and in mouse and human plasma. The glycinate esters are also 20-40-fold less potent than CPT-11 in inhibiting human acetylcholinesterase. In vivo, we examined 20(S)-glycinate-10,11-methylenedioxycamptothecin, 20(S)-glycinate-7-chloromethyl-10,11-methylenedioxycamptothecin, and CPT-11. We found that the two 10,11-methylenedioxy analogues had antitumor activity against breast cancer xenografts that was comparable to that of CPT-11. Our results indicate that water-soluble 20(S)-glycinate esters of highly potent CPT analogues provide compounds that maintain biological activity, do not require interactions with carboxylesterases, and do not inhibit human acetylcholinesterase. (+info)
Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro.
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MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity. (+info)
The use of human hepatocyte cultures to study the induction of cytochrome P-450.
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We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively. (+info)
Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.
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The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression. (+info)