Synchronization of murine erythroleukemic cells. Nuclear volume measurements for monitoring cell cycle traverse.
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Animals↗
Biomedical subjects
A Krishan
Publications and source records attributed to A Krishan.
Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry.
The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracycline group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference of these compounds with the binding of propidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence of these antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use of these two methods for intracellular detection and quantitation of a number of important anthracyclines.
Animals↗
Cytokinetic comparison of thymidine and leucovorin rescue of marrow in humans after exposure to high-dose methotrexate.
The cytokinetics of marrow recovery were compared in patients receiving a standard exposure to high-dose methotrexate followed by either thymidine rescue, leucovorin rescue at the doses used in most clinical protocols (10 mg/sq m every 6 hr), or leucovorin rescue at a 5-fold higher dose rate (50 mg/sq m every 6 hr). Thymidine rescue initiated a prompt recovery of DNA synthesis, as detected by [3H]deoxycytidine incorporation, and progression of cells through the cell cycle monitored by flow cytometry, even in the presence of methotrexate levels that prevented initiation of rescue by the lower doses of leucovorin. Dose dependency for leucovorin in vivo in humans was suggested by the observation that the higher leucovorin dose rate was successful in initiating recue within the first 24 hr, whereas the lower dose was not. Recovery of DNA synthesis is more rapid and/or complete with thymidine rescue than with either dose of leucovorin. Thymidine rescue was accomplished without requirement for purines over and above those present in plasma. These results suggest that the kinetics of marrow recovery is quite different for thymidine and leucovorin rescue.
Bone Marrow↗
Characterization of a unique cell line (LAZ 221) from human acute lymphocytic ("null" cell) leukemia.
A unique human cell line designated LAZ 221 has been established from the peripheral blood of a patient with acute lymphocytic leukemia of the "null" cell type. The cell line does not possess the Epstein-Barr virus nuclear antigen and has a karyotype of 45,XX,-9,-12,+(9q12q). Both the established cell line and the patient's uncultured blast cells share the same phenotypic markers. They both lack T-cell markers. They fail to form sheep erythrocyte rosettes and do not react with T-cell-specific antisera (TH1-, HTL-), nor do they possess B-cell markers. They do not form rosettes with erythrocytes sensitized with complement, and they are surface immunoglobulin negative. However, they do possess an HLA-D-related glycoprotein complex of 23,000 to 30,000 daltons, an la-like antigen. Thus, LAZ 221 shares the phenotype of the patient's uncultured blasts and is a cell line representative of about 75% of all human acute lymphocytic leukemias. In this respect it differs from previously described human hematopoietic cell lines.
Adult↗
Actinomycin D in childhood acute lymphocytic leukemia.
Eighteen evaluable children who relapsed with acute lymphocytic leukemia (ALL) were treated with intermittent, high-dose actinomycin D. Objective responses occurred in four of 11 children who had relapsed with chemotherapy which did not contain an anthracycline. The major toxic effects included thrombocytopenia and granulocytopenia. Minor toxic effects included nausea, vomiting, skin rash, and stomatitis. The onset of the maculopapular skin rash coincided with the platelet count nadir. These data suggested that actinomycin D is active in ALL.
Child↗
Thymidine rescue of high-dose methotrexate in humans.
Thymidine rescue was administered following 63 courses of high-dose methotrexate in 20 patients. In the first part of this study, the methotrexate was given as a 24-hr infusion and the dose was escalated from 0.14 to 8.54 g/sq m; in the second part, methotrexate was infused to maintain a serum concentration of 15 micrometer for 30, 36, or 40 hr. Thymidine rescue was started immediately after the end of the methotrexate infusions, and consisted of 8 g/sq m/day for 3 days or until serum methotrexate was below a toxic level. Mucositis and myelosuppression were the major toxicities. Neither was dose related. Serum methotrexate levels were proportional to the logarithm of the methotrexate dose. There was a mean 6-fold increase in thymidine concentration during rescue. However, thymidine levels prior to and during rescue were not related to the incidence of subsequent toxicity. Recovery of DNA synthesis in bone marrow cells was evident by nucleoside precursor incorporation at 24 hr after the start of rescue. Two of 16 evaluable patients achieved partial responses. This study indicates that thymidine is an effective rescue agent for high-dose methotrexate in humans.
Adult↗
Effect of adriamycin and analogs on the nuclear fluorescence of propidium iodide-stained cells.
Adriamycin (ADR) and N-trifluoroacetyladriamycin-14-valerate, respectively, inhibit and enhance the nuclear fluorescence of cells stained with propidium iodide for DNA per cell estimation by flow cytometry. In cells incubated with ADR, the reduction in fluorescence is gradually manifested due to the slow intracellular drug transport. In contrast the effect of N-trifluoroacetyladriamycin-14-valerate on propidium iodide nuclear fluorescence is seen within 5 min of incubation. The effect of ADR on propidium iodide nuclear fluorescence could be detected in vivo even after 24 hr of ADR administration.
Animals↗
Flow cytometry: long-term storage of propidium iodide/citrate-stained material.
Single cell suspensions stained by the propidium iodide/hypotonic citrate method for DNA content analysis by flow cytometry can be mixed with an equal amount of 70% alcohol for long-term storage and shipping without introduction of artifacts or loss of fluorescence.
Cells, Cultured↗
1,10-Phenanthroline inhibition of lymphoblast cell cycle.
The effects of the metal-chelating agent, 1,10-phenanthroline (OP), on the cell cycle progression of CCRF-CEM lymphoblasts has been studied by flow microfluorometry. Lymphoblasts were incubated with 2,3-dihydro-1H-imidazo[1,2-b]pyrazole in order to block them in G1-early S. The block was then reversed by incubating the cells in fresh media. Within 2 to 4 hr, approximately 90% of the cells were in S phase and, by 6 hr, approximately 80% were in late S. Aliquots of these latter cells then were incubated with podophyllotoxin to block them in G2-M. These cells divided within 4 hr of reversal of the podophyllotoxin block. Lymphoblast populations in G1-early S, mid-S, or G2-M were then incubated with 4 micronM OP. OP blocked entry of G1 cells into S as well as progression through S but had no effect on progression from G2-M to G1. The OP effects were reversed by addition of Zn2+, Cu2+, or Fe2+ or by dilution of the chelating agent in the growth media. Incubation of CCRF-CEM lymphoblasts with 1,7-phenanthroline, a nonchelating analog of OP, did not affect the cell cycle. The data indicate that OP reversibly blocks progression of cells from G1 to S and through S by chelation of metals involved in processes essential for those cell cycle events.
Cell Division↗
Cell cycle synchronization of human lymphoid cells in vitro by 2,3-dihydro-1h-imidazo[1,2-b]pyrazole.
2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a DNA synthesis-inhibitory drug, reversibly arrests growth of human lymphoblasts in vitro. DNA distribution histograms of cultures exposed to 0.5 to 2.0 mM IMPY show accumulation of cells with G1-early S DNA content. On reincubation in fresh medium, cell cycle traverse is resumed by the blocked cells in a synchronized manner. Maximum incorporation of [H3]thymidine into DNA (174 to 220% of control) and labeling indexes (72 to 86%) are seen after 4 hr of incubation, and a major increase in cell number is seen between the 9th and 13th hr. DNA distribution histograms of cells reincubated in fresh medium (after double block), show an initial increase in the number od cells with S-G2-M DNA content and a corresponding decrease in the number of G1-early S cells. After 4 hr of reincubation, a gradual increase in the number of G1-early S cells was seen as the earlier blocked cells completed cell cycle traverse and mitosis. Cells exposed to 2.0 mM IMPY took approximately 2 hr longer to traverse than did cells exposed to 0.5 mM IMPY.
Cell Division↗
Effect of adriamycin on the cell cycle traverse and kinetics of cultured human lymphoblasts.
Exposure of cultured human lymphoblasts to adriamycin (ADM) (0.1 mug/ml for 24 hr or 0.5 mug/ml for 1 hr) leads to an accumulation of cells with the DNA content of late S and G2. Higher concentrations of ADM (0.5 to 10 mug/ml) inhibit cell cycle traverse. Effect of ADM on cell cycle traverse, cell growth, and incorporation of labeled precursors into DNA is dependent on drug concentration and length of exposure to ADM. Synchronized cells in G1 or G2 part of the cell cycle are less sensitive to ADM than cells in S phase. Similarly, plateau-phase cells are less sensitive to ADM than cells from log-phase cultures.
Cell Division↗
Differences in cellular uptake and cytofluorescence of adriamycin and N-trifluoroacetyladriamycin-14-valerate.
Adriamycin-specific fluorescence appears slowly in living cells and is localized in nuclei and chromosomes. N-Trifluoroacetyladriamycin-14-valerate, a recently synthesized adriamycin analog, differs from the parent anthracycline in the rapid appearance of its fluorescence in the cytoplasm of living cells and the lack of any fluorescent binding to nuclei and chromosomes.
Cell Line↗
Flow microfluorometric patterns of human bone marrow and tumor cells in response to cancer chemotherapy.
A rapid propidium iodide staining method was used for analysis of single-cell suspensions of bone marrow and tumor biopsies by flow microfluorometry. With this technique, information on the proliferative status of target tissues can be obtained within 10 min of sample removal. DNA histograms and labeling index of sequential bone marrow biopsies from a patient with Stage IV diffuse lymphocytic leukemia and treated with 1-beta-D-arabinofuranosylcytosine infusion showed pronounced reduction in the percentage of cycling cells. In contrast, sequential tumor biopsies from a melanoma patient on methotrexate-citrovorum factor rescue therapy showed no changes. In sequential bone marrow biopsies of 3 patients on high-dose methotrexate-citrovorum factor rescue, initial accumulation of cells in G1-S (Day 1) was followed by a significant proliferative response (Days 4 to 7) and return to pretherapy values. In contrast, no recovery similar to that of the bone marrow was seen in tumor cells.
Antineoplastic Agents↗
Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining.
A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.
Bone Marrow↗
Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts.
Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.
Cell Count↗
Morphological basis for the cytolytic effect of vinblastine and vincristine on cultured human leukemic lymphoblasts.
Exposure of human leukemic lymphoblasts in suspension cultures to low concentrations of vinblastine and vincristine results in alterations in cell shape and leads to the formation and release of a large number of membrane-lined vesicles from the cytoplasm. Separation of these vesicles from peripheral cytoplasm is effected through alignment and fusion of small vacoules. Similar vesicle formation is seen neither in fibroblasts exposed to vinblastine nor in lymphoblasts exposed to bleomycin or adriamycin. Possible relation of this phenomenon to vinblastine- and vincristine-induced cytotoxicity, spherocytosis, and thrombocytosis is discussed.
Bleomycin↗