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Impulscytophotometric studies on the effects of daunomycin on sychronoized L cells
Abstract
Mechanically synchronized cultures of L-cells were treated in various phases of the cell cycle with daunomycin. After incubation for 4 hr at a concentration of 1 μg/ml the percentage of cells in the various phases was determined using an impulse-cytophotometer†. Applying daunomycin in G1 phase produced an arrest of cells in G1 that had not been observed so far or had been considered negligible. When daunomycin was applied in S phase the progression of cells throughout this phase was not influenced and the end of S phase was not delayed, indicating that DNA synthesis of cells having already started DNA synthesis, is not disturbed by daunomycin at this low concentration. Daunomycin treatment in the late S phase or in the G2 phase resulted in an accumulation of cells in G2 (G2 block).
The effect of irradiation on the permeability of cell membranes of L-929 cells was investigated. Efflux of fluorescein, accumulated by hydrolysis of fluoresceindiacetate in the cells, was measured using flow cytometry. The changes of rate constants for the permeation of fluorescein through the cell membrane at different temperatures and after various X-ray doses were studied. Decreasing temperatures yielded substantially slower efflux of fluorescein. After irradiation of cells, the rate constant of fluorescein efflux increased linearly with dose; 180 Gy are required to cause an increase by a factor of 2. The results are discussed with respect to radiation damage of active and passive transport mechanisms.
MELANOTROPIN (MSH) binds to specific receptors at the surface of mouse
melanoma cells, preferentially in the G-2 phase of the cell
cycle1. On prolonged incubation, a fluorescein-MSH conjugate
is internalised and appears intracellularly in small
vesicles2. The same phenomenon has been demonstrated by
electron microscopy with an MSH-ferritin-fluorescein
conjugate3. Although a role for the internalisation of MSH
receptors in the execution of the hormonal message is still open to
question, the fact that these conjugates of MSH are specifically
recognised and internalised by melanoma cells offers a possibility for
specific, site-directed chemotherapy of melanomas. We have therefore
assumed that MSH-toxin conjugates might be specifically recognised and
internalised and thereby have a selective toxic effect on cells that
display MSH receptors on their surface. Even though the principle of
site-directed chemotherapy has been known for a long time, it is only
recently that this field became active4. Conjugates of
specific antibodies and cytotoxic substances5-7, as well as a
DNA-daunomycin complex8 have been used in an attempt to
increase the selectivity of the toxic agents. In this report we describe
the synthesis of a melanotropin-daunomycin conjugate and its MSH
receptor mediated cytotoxic effect on mouse melanoma cells in vitro.
The combined action of Daunomycin and irradiation was investigated using mouse L-929 cells in culture. Survival of cells was measured with the colony assay. Sedimentation in alkaline sucrose gradients was used to study repair of DNA single-strand breaks (SSB) in the presence of various concentrations of Daunomycin. A small increase in radio-sensitivity, as measured by decreasing Do, was obtained for doses of Daunomycin that are considerably toxic to the cells (0.1 microgram/ml). However, the Dq values remained constant even at high concentrations indicating that Daunomycin does not interfere with recovery processes. The rate of rejoining of SSB remained constant up to 1.0 microgram/ml, whereas concentrations of Daunomycin as high as 10 microgram/ml reduced the velocity of repair by a factor of 13. Our data show that concentrations of Daunomycin similar to those required for other DNA-binding drugs are required to inhibit SSB repair. For clinical purposes, no increase in tumour-killing efficiency may be expected from a combined treatment with Daunomycin and radiation.
Publisher Summary Daunomycin is an antitumor antibiotic of the anthracycline group, which was isolated in the Farmitalia Research Laboratories in Milan, Italy, from cultures of Streptomyces peucetius. The major part of the drug was found in the culture media of the mycelium and was isolated by means of solvent extraction, cation resins, and paper or column chromatography. The yield was 5–15 mg of product per liter of culture media. The soil sample was from Castel del Monte, near Puglie, and hence the name of the species S. peucetius. The drug is classified as an anthracycline and has similar physicochemical characteristics to rhodomycins, cynerubines, pyrromicins, and rutilantines. Daunomycin consists of a pigmented aglycone (daunomycinone) in glycoside linkage with an amino sugar (daunosamine). Daunomycin inhibits the multiplication of bacteriophage. It strongly inhibits DNA phages by acting during the replication cycle of the phage and not on absorption, injection, or lysis. The RNA phage growth was unaffected by daunomycin. The antiphage activity was proportional to the concentration of daunomycin. The antiphage activity was partially blocked by DNA but not by nucleotides or nucleosides.
The response of cultured Chinese hamster ovary (CHO) fibroblast cells to hyperthermia at 43° and 45°C and its modification by additional hyperthermic treatments at temperatures from 38° to 45°C were determined. Treatment of CHO cells at temperatures below 43°C prior to exposure at 43° or 45°C induced pronounced thermal resistance, with an increase in surviving fraction by factors up to 20. In contrast, hyperthermia at lower temperatures enhanced the effectiveness of hyperthermia at 43° or 45°C if the order of application was reversed. Pre- or post-incubation at 44° or 45°C enhanced the effectiveness of hyperthermia at 43°C, irrespective of the order of application.
The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine liver cells. At low daunomycin concentrations (1 and 2 μM) the rate of [ ³ H]thymidine incorporation decreased progressively with the duration of exposure to the inhibitor. This was accompanied by a shift of nascent DNA intermediates of replicon size to higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells.
At high daunomycin concentrations (12 μM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [ ³ H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre‐fork mode of DNA synthesis.
Mouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age reponse functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D
0 and extrapolation numbern were found to vary during the cell cycle. TheD
0 has a maximum value of 176 ± 1 rad in the middle ofG
1 phase and a minimum of 71 ± 1 rad at theS/G
2 transition, while the extrapolation number is rather constant from the beginning ofG
1 phase (1.9 ± 0.1) to the middle ofS phase (2.3 ± 0.1) and reaches a steep maximum of 9.3 ± 1.1 atS/G
2 transition. The values ofn in the various phases of cell cycle are compared with the respective values of the recovery factorγ determined after fractionated irradiation. - Cell survival after a single dose of 616 rad has minima for irradiation atG
1/S transition and in earlyG
2 phase; the survival in earlyG
2 being about 40 times smaller than in earlyG
1 phase. Implications for a cell cycle specific therapy are discussed.
Changes in the membrane potential of cultured L-929lcells were investigated after irradiation with doses ranging from 5-200 Gy. Immediately after irradiation a depolarisation is observed that is followed by a damped oscillation of the membrane potential and finally by a rapprochement to the control value. Whereas the magnitude of depolarisation does not show any dependence on irradiation dose, the time required to reach the control value again increases with increasing dose. Up to 10 Gy, the period of the first half-oscillation rises rapidly to about 12 min, at higher doses a slow linear increase follows reaching a value of 23 min after 200 Gy.
Cell doubling time, DNA distribution pattern, frequency distribution of chromosome numbers per cell and degree of synchrony obtained after mitotic selection were studied in 4 different sublines of mouse fibroblast cells, NCTC L-929, in culture. The 4 sublines did not differ with respect to their cell kinetic data but showed changes of the chromosome pattern compared to the stemline and compared to each other suggesting that chromosome analysis provides a very sensitive means to detect evolutianary changes of a permanent cell line.
This chapter discusses the cytotoxic and antitumor antibiotics produced by microorganisms. Microorganisms are among the most prolific producers of new biologically active compounds. Many antibiotics are able to suppress or retard the growth of tumors; an extensive effort has been made in many laboratories to find new antibiotics with antineoplastic properties, either by screening new soil isolates or by chemical modification of the existing antibiotics. This chapter provides information on 373 antibiotics possessing antineoplastic properties for which reasonably complete data is available. It discusses the names and synonyms of 373 antibiotics with reported antineoplastic activities and is listed in alphabetical order, and presents a list of the most common experimental tumors.
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.
The H-35 rat hepatoma, a cell line which is relatively resistant to the classical anthracycline antibiotics such as Adriamycin [the concentration of drug which inhibits cell proliferation by 5090 (IC50) = 2.5 microM] and daunorubicin (IC50 of 0.5 microM), is markedly more sensitive to the 4-demethoxydaunorubicin derivative, idarubicin (IC50 of 0.025 microM). In contrast to daunorubicin, which has previously been shown to inhibit hepatoma cell proliferation in the absence of perceptible DNA cleavage, idarubicin induces concentration-dependent DNA damage which may account for its enhanced capacity to inhibit proliferation of the rat hepatoma. Free radical scavengers fail to interfere with inhibition of cell proliferation induced by idarubicin. Damage to the cell membrane or alterations in mitochondrial integrity do not appear to represent components of idarubicin toxicity in this tumor cell line. Inhibition of DNA synthesis by idarubicin parallels inhibition of cell growth; however, sensitivity of DNA synthesis to idarubicin is significantly less than that for cell proliferation (IC50 values of 0.5 microM and 0.025 microM, respectively). It is postulated that the antiproliferative effects of idarubicin in the H-35 rat hepatoma model may be a consequence of alterations in DNA integrity which ultimately result in the inhibition of cellular biosynthetic processes.
We have utilized digitized video microscopy to investigate the influence of verapamil, a calcium channel blocker, on the in vitro effects of daunomycin in P388 sensitive and resistant sublines. In this study, verapamil enhanced the uptake of daunomycin and its cytotoxicity in both sublines, but the effect was more pronounced in the resistant cells. Flow cytometry showed a pronounced accumulation of resistant cells at G2-M when treated with daunomycin in the presence of verapamil, with no effects observed in the absence of verapamil. Analysis of individual resistant cells using the digitized video fluorescence microscopy technique demonstrated that the influence of verapamil on daunomycin uptake affected all cells and was not restricted to certain subpopulations of cells.
We have investigated the in vitro drug-cell interaction and therapeutic effects of a low-density lipoprotein (LDL)-daunomycin complex as compared to free daunomycin. Uptake and retention of daunomycin were significantly enhanced in sensitive and resistant P388 leukemia cells when they were perfused with LDL-daunomycin complex relative to free drug. The interaction of the LDL-daunomycin complex with P388 cells appeared to be through a LDL-specific pathway since competition with free LDL but not high-density lipoprotein significantly reduced daunomycin uptake. The LDL-daunomycin complex was cytotoxic to both daunomycin-sensitive and daunomycin-resistant P388 sublines. Colony growth studies showed the LDL-daunomycin complex to be more cytotoxic at shorter exposure times relative to free drug. Resistant cells showed 55 and 12% colony growth with 10-min and 2-hr exposures, respectively, to 0.4 microgram daunomycin/ml of the LDL-daunomycin complex. Free drug at 0.4 microgram/ml drug concentration and similar exposure times resulted in no loss in colony growth. The resistant cells were subjected to cell cycle analysis based on DNA content and showed an accumulation of cells at the G2-M phase of the cell cycle when treated with the LDL-daunomycin complex, with no effects being observed with free daunomycin.
Kinetic and cytotoxic effects of cytosine arabinoside (Ara-C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony-forming assay, respectively. With Ara-C alone, increasing drug concentrations between 10(-7) M and 10(-4) M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10(-4) M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 X 10(-7) M. It decreased the rate of progression through S phase and caused cells to accumulate in G2, except at the highest concentration (2 X 10(-5) M, at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara-C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non-lethal dose of Ara-C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug-free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by drug-induced redistribution of cells within the cell cycle, but may have been related to the effects of the non-lethal drug on DNA synthesis rate.
The influence of an in vivo treatment with optimally tumor-inhibiting doses of cis-diamminedichloroplatinum(II) (DDP), titanocene dichloride (TDC), and vanadocene dichloride (VDC) on the DNA distribution pattern, determined by pulse-cytophotometry, and on the, mitotic activity of EAT cells is investigated.
Whereas DDP causes an immediate and, long-lasting decrease of mitoses but no disturbances of the EAT cell kinetics, a marked G2 block with a maximum at 10–12 h a. t. and an irreversible and extensive mitotic depression is evoked by TDC. In the case of DDP and TDC, the tumor cells are removed several days a. t. by cells belonging to the defensive system of the host animals.
In contrast, VDC induces partially synchronized waves after a transient suppression of mitoses and reversible cell accumulation in the late S and in the G2 phases. Additionally, 5–16% of the cells become polyploid after VDC treatment.
The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G1-phase, at the G1/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by 14C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G1, a fraction of cells was prevented from entering S-phase, after irradiation at G1/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G2 blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G1 are partly released from the block after 10 h. Irradiation at G1/S caused a persisting accumulation of 50% of the cells in G2, and for irradiation in S more than 80% of the cells were arrested in G2.
Human leucocytes were cultured for 3 days at 37°C, and during this time treated with rubidomycin (also known as daunomycin) for periods up to 48 hr. The effects of this treatment were studied by examining mitotic indices, uptake of 3H-thymidine, and patterns of DNA content (measured by microdensitometry on Feulgen-stained cells). A low concentration of rubidomycin (0.1 μg/ml) caused accumulation of cells in the G2 period, which in turn resulted in a decrease in the mitotic index. A secondary effect was a slight drop in 3H-thymidine uptake after 12 hr. Higher doses (up to 10 μg rubidomycin per ml) caused an inhibition of DNA synthesis, with accumulation of unlabelled cells between G2 and G2. The probable mode of action of rubidomycin, as presented by earlier authors, is the intrusion of the drug molecule between DNA strands, forming a complex with DNA, and hindering its normal folding. This is discussed with respect to the present findings.
It is reported about the clinico-pharmacologic evaluation (Phase I) of the combination of cytostatic drugs Daunorubicine — Vincristine — Prednisone (ViDaP) in all acute leukemias treated at the Medizinische Universitätsklinik Freiburg between 3-1-1967 and 12-31-1968. The observations were continued till to 30-4-70. In the treatment two phases were distinguished: in the first phase the goal was to obtain a remission (=induction phase). If the bone marrow examination had shown a remission (M1 or M2), the maintenance treatment was instituted (reinduction-pulses). This consisted of 6-Mercaptopurine with ViDaP at 6 weeks intervals.
Classification of acute leukemias was made according to morphologic features of the Pappenheim stain, and cytochemical criteria in 3 types: AML=acute myelocytic leukemia; ALL=acute lymphoblastic leukemia; AUL=acute undifferentiated leukemia. Leukemias of the peroxydase, mixed peroxydase-esterase and esterase types were called acute myelogenous leukemias in contrast to acute lymphatic leukemias. In addition quantitative cytophotometric DNS measurements were carried out.
The therapeutic effects of ViDaP-therapy were evaluated by the rate and duration of remissions in relation to the different types of leukemias.
22 Adults were treated with ViDaP. The remission rate (complete and partial) was 32%. The highest rate (3/7) was seen in ALL. Among the 12 patients with AML 2 complete and 1 partial remission were achieved. Only one of 3 patients with AUL went into complete remission.
By April 30-1970 the remissions had lasted 227, 446+, and 667+ days for ALL, 210, 213, 646+ days for AML and 666+ for AUL respectively.
There was a correlation between effect of therapy and degree of ploidie of leukemic cells. Cells with diploid stem lines responded less favorable to therapy than polyploid ones.
Side effects of ViDaP-therapy were moderate. The therapy was well tolerated. The side-effects were mainly neurological caused by Vincristine. The toxic effects of Daunorubicine on the heart are discussed. The hematologic toxicity was tolerable. Fatal bone marrow aplasias were not observed.
Infolge der geringen Haftbarkeit von Zellen whrend der Zellteilung knnen 95–98% Mitosezellen aus Monolayer-Kulturen gewonnen werden. Die Zusammensetzung der Glasoberflche, Ca++-Mg++-Gehalt des Mediums, Temperatur, Dichte des Zellrasens und Strke der Scherkrfte bestimmen die Reinheit des Prparates. Die Zellen bestehen aus etwa 50% Telophasen und 20% Metaphasen, 10% Anaphasen, 10% Prophasen, 2% Interphasen und 3% Pyknosen. Nach Wiedereinpflanzung durchlaufen 95% dieser Zellen einen ganzen Zellzyklus. Mikrokinematographisch wurde eine mittlere Generationszeit von 22 Std17,5% ermittelt. Der Unterschied in der Generationszeit von Zelle zu Zelle und die damit verbundene progrediente Desynchronisierung ist der grte Nachteil bei der Verwendung dieses Zellsystems.Preferential detachment of dividing cells from a monolayer of confluent L-cells is used as a method to synchronize cells. 95% of the selected cells are in mitosis. Purity of the cell preparation depends on the surface qualities of the glass, Ca++-Mg++ concentration in the medium, temperature, density of the monolayer and intensity of sheering force used. The cell preparation consists of 50% telophases, 20% metaphases, 10% anaphases, 10% prophases, 2% interphases and 3% pyknotic cells. Upon reimplantation 95% of these cells reach the 2. mitosis in a synchronous wave. Microcinematographic registration showed an average generation time of 22 hours17,5%. Progressive desynchronization within one cycle is due to differences in generation time.
Six chemotherapeutic agents were tested for effects on mammalian cell cycle traverse in synchronized cultures of Chinese hamster cells. Cytosine arabinoside was found to inhibit DNA synthesis, as well as to reduce grossly the rate of progression from Gj into S. At low dosage levels, daunomycin and adriamycin had only a slight effect upon entry of cells into S, but they were almost totally effective in preventing cells from reaching mitosis, and 5-azacytidine greatly inhibited progression into S phase. However, cells already in S at the time azacytidine was added continued to synthesize DNA but were unable to progress to mitosis. Entry into S was hardly affected by mithramycin, while completion of G2 was partially inhibited, although the sensitivity of G2 processes was much less pronounced in mithramycin-treated cells than in cells treated with daunomycin or adriamycin. Camptothecin allowed initiation of DNA synthesis, but it effectively prevented cells from progressing to mitosis in the incubation period allotted in the test protocol.
The enzyme activities of DNA polymerase and thymidine kinase were determined in peripheral leukemic leukocytes of patients with acute leukemia treated with Dauno-Rubidomycin or Adriamycin. During the first hours after the injection of each drug both enzymes are significantly augmented. After a few days there is a distinct decrease of the enzyme activities preceding the fall of the leukocyte count. The initial increase in enzyme activity could be reproduced in PHA stimulated lymphocyte cultures of healthy donors by incubating the cells with the Anthracycline antibiotics. During this increase of DNA polymerase and thymidine kinase DNA and RNA synthesis are markedly inhibited. Experiments with inhibitors of protein synthesis make it likely that the remarkable rise in enzyme activity is caused by the synthesis of new enzyme molecules. The DNA polymerase reaction is inhibited by Dauno-Rubidomycin or Adriamycin. To some extent this could be reversed by the addition of new primer-DNA.
SUMMARY The effect of daunorubicin (DNR) on nucleic acid synthesis was investigated in vitro in cells from an Ehrlich ascites tumor that was sensitive in vivo to DNR and in cells from a subline of the tumor in which resistance to DNR had been developed in vivo. DNR inhibited DNA and RNA synthesis in sensitive as well as in resistant cells, as determined from the incorporation of three different radioactive precursors. In order to give the same degree of inhibition of DNA and RNA synthesis in resistant cells as in sensitive cells, the DNR concentrations required were approximately 5 times greater. When the synthesis of different RNA species in sensitive cells was analyzed, nucleoplasmic heterodisperse RNA was found to be the least DNR sensitive, while tRNA, 5 S RNA, and two small-molecular-weight RNA components had a medium sensitivity to DNR. Labeling of rRNA and of the 45 S and 32 S nucleolar RNA was found to be inhibited most and to the same extent, indicating that it is the synthesis of 45 S RNA that is inhibited and not the processing of 45 S RNA to rRNA. The preferential inhibition of rRNA was less pronounced in resistant cells than in sensitive cells, thus indicating that the sensitivity ofrRNA synthesis was changed more Muring development of resistance than was the sensitivity of synthesis of other RNA components. The cellular uptake of DNR was less in resistant than in sensitive cells, but the difference was too small to explain the differences in inhibition of the nucleic acid synthesis.
Cytogenetic and morphologic abnormalities in bone marrow and peripheral blood and biochemical changes in peripheral blood leukocytes were studied after in-vivo and in-vitro treatment with daunomycin. Seven patients with acute lymphocytic or granulocytic leukemia were studied; the total dose varied from 80 to 420 mg/m2. The highest percentage of major chromosomal aberrations (80 to 90%) appeared right after treatment in vivo. These aberrations disappeared within 1 or 2 weeks and consisted of chromatid breaks, fragments, chromatid exchanges, ring chromosomes, dicentrics or extensive fragments. Prominent morphologic abnormalities included megaloblastic transformation of both the myeloid and erythroid series and increased cytoplasmic vacuolization. Aneuploid cells lines from two patients showed less change after daunomycin treatment than did normal diploid cells. The daunomycin dose threshold in vitro was narrow: 3 μg/ml of culture caused death of the entire cell population, while 0.03 μg/ml of culture produced little chromosomal damage. The in-vitro studies on normal human lymphocytes indicate that daunomycin affects RNA and DNA synthesis and that it interferes with the cell cycle during the G2 period. This drug appears to have the unique capacity to delay the onset of mitosis in cells which have already synthesized DNA.
A synchronous cell system, based on the mechanical selection of mitotic cells, is briefly characterized. The mitotic index at the first synchronous division is better than 80%. Prophases constitute 40-55%, metaphases 25-45%, anaphases 1-5%, telophases 5-12%, interphases 4-18% and pyknoses 2-4%. Second synchronous mitotis is reached within 22 hr±10·5% by 96% of the cells. The G1, S and G2 phases measured from the incorporation of radioactive-labelled precursors into DNA constitute each 1 3 of the cell cycle. Daunomycin is found to decrease the mitotic index both by action in the interphase and by direct action on mitosis. Also morphological changes are reported and discussed in relation to the phenomenon of unbalanced growth. Some obersvations on the nature of the inhibition of nucleic acid synthesis brought about by daunomycin is discussed in relation to observation time, membrane permeability and cellular fractions. Finally the results of investigations on the sequential inhibition of nucleic acid fractions are reported.
SUMMARY The influence of daunomycin on nucleic acid synthesis during different stages of the cell cycle has been studied in mammalian cells. Explants from leg muscle of newborn rats were synchronized by two 24-hr treatments with excess thymidine, 16 hr apart. The metabolic activity of DNA and RNA was determined by autoradiographic methods by measuring the incorporation of thymidine-3 H, deoxy cytidine-3H, and uridine-3H into nuclear structures. In control cultures, DNA synthesis shows two distinct waves located in the early and late S phase, the latter being the most sensitive to the inhibiting effect of daunomycin. RNA synthesis takes place in the cells during the entire cell cycle from the end of mitosis to the late prophase, but two well-defined peaks can be recognized: the first in the G2 phase just 1 or 2 hr before the mitotic peak and the second in the middle stages of @ . These RNA syntheses involving both nucleolar and extranucleolar structures are strongly inhibited by daunomycin treatment. The significance of these RNA syntheses in control cells and the relationship between their inhibition and the antimitotic effect of daunomycin are discussed.
Daunomycin is a new antibiotic with antitumor activity against a variety of solid and ascites forms of transplantable animal tumors. It is effective in acute leukemias and certain solid tumors in children. Daunomycin has been considered to inhibit preferentially RNA synthesis, with inhibition of DNA only at higher concentrations, therefore having a biologic mechanism of action similar to actinomycin. The effect of this antibiotic on the 32P incorporation into DNA and RNA of 6C3HD ascites tumor in vitro and in vivo and also on the regenerating mouse liver after partial heptectomy has been studied. These observations indicate that Daunomycin is a more effective inhibitor of the synthesis of DNA than RNA, and hence the biologic mechanism of action of this antibiotic is probably different from that of actinomycin.
Die cytostatische Wirkung yon Daunomycin im Impulscytophotometrie Test, Arzneimittel-Forsch
- W G6hde
- W Dittrich
W. G6HDE and W. DITTRICH, Die cytostatische Wirkung yon Daunomycin im Impulscytophotometrie Test, Arzneimittel-Forsch. 21, 1656 (1971).
Untersuchungen an teilungssynchronen L-Zellen. I. Optimale Wachstumsbedingungen und Charakterisierung des synchronen Zellsystems
- Mittermayer
Synchronisation des Teilungszyklus von L-Zellen in der G2-Phase durch fraktionierte Röntgenbestrahlung und Daunomycin
- Linden
Daunomycin inhibition of DNA and RNA synthesis
- Theologides
Cell cycle analysis by impulsecytophotometry
- H Baisch
- W Göhde
- W A Linden
Die Wirkung von Dauno-Rubidomycin und Adriamycin auf Enzyme der DNS-Synthese in Leukocyten in vivo und in der Kultur
- Wilms
Impulszytophotometrische Messungen an atypischen Zellabstrichen aus Scheide und Cervix uteri
- Göhde