Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G-->A hypermutation is a remarkable phenomenon resulting from retroviral reverse transcription in the presence of highly biased dNTP concentrations. Of the three reverse transcriptases (RTases) available, those of human immunodeficiency virus type 1 (HIV-1), avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MoMLV), the HIV-1 enzyme showed the greatest sensitivity to biased [dCTP]/[dTTP] ratios. The HIV-1 RTase was able to discriminate between dUTP, dITP and the four DNA precursors and was insensitive to pH. There was little preference for nucleotide contexts. A few exceptionally modified sequences were found presumably resulting from G-->A hypermutation and multiple strand transfer. This particular predilection of the HIV-1 and, by extrapolation, the lentiviral RTases towards G-->A hypermutation suggests that the phenomenon may have contributed to the remarkably elevated A content of these retroviral genomes.
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PMID:Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction. 754 58

Damage to DNA can be assessed using a technique for labeling nicks in DNA by incubating paraformaldehyde-fixed cells in a mixture of biotin-labeled dUTP, dATP with dNTP and DNA polymerase I. The addition of labeled nucleotides can then be identified by fluorescence by their reaction with streptavidin. We have used this method to examine damage to the DNA of OCI/AML-2 cells caused by cytosine arabinoside (ara-C) and the effects of hydrocortisone and retinoic acid on this damage (regulated drug sensitivity). Concurrent measurements of clonogenic cells were used to allow a comparison of damage as shown by labeled nicks in DNA with loss of colony-forming capacity. Both methods gave comparable ara-C dose-response curves, for cells incubated with the drug for 24 h. Both methods showed that exposure of OCI/AML-2 cells to hydrocortisone before ara-C greatly reduced the toxicity of the drug; and that retinoic acid given after ara-C increased both its lethal effects on colony formation and the extent of DNA damage as assessed by labeled nicks. Clonogenic assays required for colony formation are not readily adapted to the study of development and repair of damage. The labeled nick assay is suitable for such kinetic studies. OCI/AML-2 cells were exposed in suspension to either hydrocortisone before ara-C or retinoic acid after ara-C. At 24 h intervals thereafter, cells were harvested, assayed by both methods, and recultured after dilution to the original cell concentration. In cultures exposed only to ara-C (controls), the number of cells with labeled nicks increased during the first 24 h and cells with damaged DNA could be detected for 48-72 h, depending on the ara-C dose in spite of the dilution at each passage. OCI/AML-2 cells exposed to hydrocortisone before drug showed fewer nick-labeled cells than controls at the first observation and damaged cells rapidly disappeared from the population with increasing time. For cells treated with retinoic acid after ara-C, the nick-labeled cell population was greater than controls and remained greater throughout subsequent observations. We propose that in the control cultures, sublethal damage either became lethal with time and was seen as increased numbers of cells with damaged DNA, or alternatively, sublethal damage was repaired. From this point of view we consider that hydrocortisone promotes repair of sublethal damage while retinoic acid inhibits repair.
Leukemia 1994 Dec
PMID:Fluorescence-labeling of nicks in DNA from leukemic blast cells as a measure of damage following cytosine arabinoside. Application to the study of regulated drug sensitivity. 780 94

The apoptosis-associated DNA strand breaks were detected in situ, in individual leukemic cells in peripheral blood and bone marrow of over 110 patients with different types of leukemia (ALL, AML, CML in blastic crisis, APL), prior to and during routine chemotherapy. The DNA strand breaks were labeled with digoxigenin- or biotin-conjugated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase, and the cells, counterstained for DNA, were analyzed by bivariate flow cytometry. The proportion of cells with DNA strand breaks prior to therapy, most likely reflecting spontaneous apoptosis, varied from 0.1 to 16%, but in the large majority of cases was below 3%. Administration of drugs of different classes, which included DNA topoisomerase I (Topotecan) and II (mitoxantrone, VP-16) inhibitors, antimetabolite (ara-C) or microtubule poison (Taxol), all triggered the appearance of cells with extensive DNA breakage, typical of apoptosis, to up to 80%. The peak of the response, measured as maximal percent of cells with DNA strand breaks, which varied between individual patients by as much as factor 10, was generally seen between 8 to 24 h after the initial administration of DNA topoisomerase inhibitors, and somewhat later (48-72 h) during the response to Taxol or ara-C. Thus, the data show that the response to treatment with a variety of drugs, in terms of induction of apoptosis, can be conveniently measured by the present method. The prognostic value of the apoptotic index, before, as well as during treatment, is being estimated for each type of leukemia, in the ongoing prospective studies.
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PMID:Apoptotic cell death during treatment of leukemias. 807 83

A new flow cytometric method is described to detect DNA strand breaks associated with apoptosis, by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase. The method has been applied in studies on leukemic HL-60 and MOLT-4 cell lines to reveal whether it is specific to apoptotic cells, and whether it can be used in the clinic to detect DNA breakage in leukemic cells during chemotherapy. There was labeling of mononuclear cells in peripheral blood of all 11 patients studied during chemotherapy for acute lymphoblastic, acute myelogenous, or chronic myelogenous leukemia (ALL, AML, or CML) in blastic crisis, indicating induced DNA damage; the number of labeled cells increased from 1-8% before treatment up to 80% during the course of treatment. The DNA topoisomerase inhibitors mitoxantrone, VP-16 (etoposide), and m-AMSA (amsacrine) were more effective in inducing DNA breaks than was hydroxyurea or cytosine arabinoside (AraC). Cells with DNA breaks were identified in peripheral blood for up to 5 days following administration of Mitoxantrone and VP-16. In the case of DNA aneuploid leukemias, the DNA breaks were predominant in the aneuploid cell subpopulations, whereas presumably non-neoplastic diploid cells were unlabeled. In one case of ALL there were two distinct subpopulations of aneuploid cells: one responded to the treatment (by DNA breakage) and the other was non-responding. Thus, cells undergoing apoptosis can be detected by this method of labeling DNA strand breaks and the technique is applicable for analysis of response of leukemic cells to chemotherapy. With this method it may be possible to identify tumor cell sensitivity or resistance to particular drugs early in the course of treatment.
Leukemia 1993 May
PMID:Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. 848 18

To clarify whether apoptosis is involved in endometriosis, we obtained eutopic endometrial tissues along with endometriotic tissues from the uterus (adenomyosis) (n = 12) and from the ovary (n = 12) from patients undergoing gynaecological surgery. Apoptosis-induced DNA fragmentation was detected by the TdT-mediated dUTP-biotin nick-end labelling method, and immunostaining with a monoclonal antibody against the Fas, Le(y) or B-cell leukaemia/lymphoma-2 (bcl-2) was also performed using the same tissue section. Analysis showed that apoptosis was occurring in all the samples of ovarian endometriotic tissue but in only two of the 12 adenomyotic and in five of the 24 eutopic endometrial tissue samples. In none of these cases was apoptosis correlated with phases of the menstrual cycle. The expression of bcl-2 in the eutopic endometrial and adenomyotic tissues was limited to the proliferative phase, and was observed in only one of the 12 cases of ovarian endometriosis. Fas and Ley were expressed randomly across a wide range in both the eutopic and ectopic endometrial tissues. These results suggest that the features of ovarian endometriosis are different from those of adenomyosis and eutopic endometrium in terms of the involvement of apoptosis. In addition, the regulatory mechanism involved in ovarian endometriosis may differ from that in other endometrial cells.
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PMID:Detection of apoptosis in human endometriotic tissues. 923 97

Frequent apoptosis in the bone marrow of patients with myelodysplastic syndromes (MDS) was demonstrated on frozen sections using the terminal deoxytransferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. The overall mean percentage of TUNEL-positive cells was about 17% in the bone marrow of MDS, while bone marrow from control cases exhibited a mean of 3.4% (P < 0.001). To elucidate the mechanism of apoptosis in bone marrow cells of MDS, the expression of Fas antigen and Fas ligand (FasL) was examined by RT-PCR and immunohistochemistry. All MDS cases showed expression of Fas mRNA (12/12) and most exhibited an expression of FasL mRNA (10/12) by RT-PCR. Basically, control cases did not show positive signals for Fas and FasL mRNA, however, a very weak band was detected in three cases (3/10) for Fas and in one case (1/10) for FasL mRNA by RT-PCR. Immunohistochemical examination revealed positive staining for Fas (11/12) and FasL (12/12) in the bone marrow of MDS, while all the bone marrow samples from control cases were negative for anti-Fas (0/15) and for anti-FasL (0/15) antibody. Double staining clarified that TUNEL-positive apoptotic cells expressed Fas antigen on the cell surface, although not all Fas-positive cells were TUNEL positive. The Fas-positive cells of MDS bone marrow included hematopoietic cells expressing CD34 antigen, neutrophil elastase, a marker for myeloid series of cells, or glycophorin A, a marker for erythroid cells. However, CD68-positive cells which were macrophage lineage cells, did not express Fas antigen strongly. In contrast, positive staining for FasL was detected in hematopoietic cells and CD68-positive cells in the bone marrow of MDS. These results suggest that the Fas-FasL system plays an important role in inducing apoptosis in the bone marrow of MDS and works in an autocrine (hematopoietic cell-hematopoietic cell interaction) and/or paracrine (hematopoietic cell-stromal cell interaction) manner.
Leukemia 1998 Apr
PMID:Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia. 955 5

Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines (Drach et al, 1996; Raghu et al, 1996). Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines (HB8065 hepatoma cells, KG1a and K562 leukaemia cells). The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3'-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug.
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PMID:Cytotoxic effect of the cyclosporin PSC 833 in multidrug-resistant leukaemia cells with increased expression of P-glycoprotein. 974 97

Selective death of magnocellular vasopressinergic neurons in the hypothalamus has been reported in cases of hereditary and idiopathic diabetes insipidus and after experimental lesions of the hypothalamo-neurohypophyseal pathway. To identify trophic factors that promote survival of these neurons, an in vitro model system was established in which organotypic cultures of the rat hypothalamic paraventricular nucleus were maintained in chemically-defined medium. We observe that the majority of magnocellular vasopressinergic neurons die in these cultures, while other cell populations such as corticotrophin-releasing factor producing parvicellular and oxytocin producing magnocellular cells retain a well preserved cytoarchitectonic organization. Degenerating vasopressinergic cells exhibit morphological signs of apoptosis and stained positively when analysed by the terminal deoxynucleotidyl transferase biotinylated dUTP nick end-labelling assay. Partial survival of vasopressinergic neurons occurred after co-culturing the paraventricular nucleus with neurohypophyseal explants, indicating that target-derived factors may be required for the survival of these neurons. Cell survival is dramatically increased by the administration of ciliary neurotrophic factor and leukemia inhibiting factor, but not by interleukin 6 or the members of the neurotrophin family. Reverse transcription-polymerase chain reaction followed by Southern analysis shows the presence of ciliary neurotrophic factor messenger RNA in the neurohypophysis. Thus, endogenous ciliary neurotrophic factor and leukemia inhibiting factor, produced by neurohypophyseal cells may function as a physiological survival factor for neurosecretory vasopressinergic neurons.
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PMID:Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor. 975 24

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.
Leukemia 1999 May
PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. 1037 89

Background: Mutations in members of the ras gene family (H-ras, K-ras, and N-ras) have been identified in various human malignancies. A variety of techniques have been used to test for ras mutations. Methods and Results: A simplified reverse dot blot (RDB) assay was used in this study. Polymerase chain reaction products were hybridized to nitrocellulose membrane-fixed synthetic probes (20 nucleotides long) specific for codons 12, 13, and 61 of H-, K-, and N-ras mutations and their wild-type sequences. No special treatment or modification of the probes was necessary to obtain adequate results in overnight film exposure when the polymerase chain reaction was carried out using (32)P-end labeled primers. It was demonstrated that this simplified RDB assay can also be used with fluorescein-11-dUTP and a chemiluminescence detection system. The RDB assay is more reliable than the single-strand conformation polymorphism (SSCP) assay. By comparison, the SSCP assay is significantly less sensitive and less specific. It was confirmed with sequencing that 11 (12%) of 93 SSCP assays were false positive and 2 (2%) were false negative, whereas no false positive or false negative RDB assay was detected. The RDB assay also provides more additional detailed information about the specific point mutation and amino acid change, which may have clinical implications in some tumors. Conclusions: The RDB assay is very sensitive and able to detect mutations when the mutant allele is in 1% of the cells and can be used to detect minimal residual disease, particularly in some cases of leukemia and myelodysplasia.
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PMID:Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations. 1046 6


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