Danielle Londos-Gagliardi

Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review

Daunorubicin and doxorubicin, two antibiotics belonging to the anthracycline group, are widely used in human cancer chemotherapy. Their activity has been attributed mainly to their intercalation between the base pairs of native DNA. Complex formation between daunorubicin or doxorubicin with polydeoxyribonucleotides and DNAs of various base composition or chromatins has been investigated by numerous techniques. Many authors have tried to correlate biological and therapeutic activities with the affinity of the drugs for DNA or some specific sequences of DNA. In vivo these anthracycline drugs cause DNA damage such as fragmentation and single-strand breaks. The mechanism of action of anthracyclines involves the inhibition of RNA and DNA syntheses. There exists two limiting factors in the use of anthracyclines as antitumoral agents: a chronic or acute cardiotoxicity and a spontaneous or acquired resistance. In both cases, there is probably an action at the membrane level. It has to be noted that daunorubicin and doxorubicin have a particular affinity for phospholipids and that the development of resistance is linked to some membrane alterations.

Subcellular-localization of Daunorubicin in Sensitive and Resistant Ehrlich Ascites Tumor-cells

Abstract Cellular uptake and subcellular localization of daunorubicin were studied in Ehrlich ascites tumor cells, either in vivo after inoculation of the drug to mice or in vitro after incubation of cultured Ehrlich ascites tumor cells with the drug. These cells were either sensitive (wild-type) or resistant to daunorubicin. The uptake of daunorubicin was 2–4 times less in resistant cells. About 90% of the drug which had penetrated in the cells was recovered in the nuclei. By cell fractionation, it has been established that the extranuclear daunorubicin was localized in the lysosomes. The lower uptake of the drug in resistant cells and the similar subcellular localization in both types of cells were in favour of an increased active outward transport of the drug in resistant cells.

Metabolism of daunorubicin in sensitive and resistant Ehrlich ascites tumor cells

SummaryThe intracellular metabolism of daunorubicin (DNR) has been studied in sensitive and resistant Ehrlich ascites tumor (EAT) cells. The subcellular localization of metabolites has been followed by normal-phase and reverse-phase high-pressure liquid chromatography (HPLC). The metabolism of DNR by either sensitive or resistant EAT cells is not significant; unmetabolized DNR is always the main intracellular compound. Daunorubicinol (DOL) accounts for less than 5% after 24h and an unidentified product is also observed. This highly apolar compound, having an intrinsic fluorescence one order of magnitude greater than that of DNR is formed in acellular conditions and could be a chemical artifact. DNR and DOL are mainly associated with DNA-containing fractions. No significant differences can be observed in the metabolism of DNR in sensitive and resistant EAT cells.

Subcellular localization of some anthracycline derivatives in Ehrlich ascites tumor cells

Abstract With the whole set of the tested anthracyclines, a bimodal localization, nuclear and lysosomal, is observed. But the percentage of the drug which is stored in the nuclei is different according to the drug. With some derivatives which have a high therapeutic efficiency, 82–87 % of the drug is recovered in the nuclei. In a second group whatever the biological activity of the drug only 49–52 % of the drug accumulates in the nuclei.

In vivo effects of several anthracyclines on DNA integrity

Anthracycline antibiotics are widely used in human cancer chemotherapy. Their activity has been attributed mainly to their intercalation between the base pairs of native DNA. In the last few years, however, a number of authors have attributed their tyrotoxic action to DNA damage, such as single strand breaks or alkaline labile regions. Using neutral and alkaline sucrose gradient ceatrifugation [ 1 ] or hydroxyapatite column chromatography at 60°C [2], it was shown that doxorubicin needs the cellular environmeat to induce DNA single-strand breaks. By use of alkaline sucrose-gradient centrifugation [3], doxorubicin was shown to induce both singleand doublestrand breaks. Doxorubicin and daunorubicin induce the formation of regions in nuclear DNA which can be susceptible to hydrolysis by Neurospora crassa endonuclease and which can be demonstrated by change in the sedimentation properties of nuclear DNA in neutral sucrose gradients [4]. Doxorubicin [5], daunorubicin [5,6] and aclacinomycin A [7] induce single strand scissions in the presence of reducing agents. Both free radical of anthracycline quinones and hydroxyl radical may react with DNA strands. The in vivo effects of anthracycline upon the integrity of DNA has been studied using cell lysates or nuclear structures of various kinds of cells: human lymphoblastic cells [1 ], leukemia cells [2], HeLa, L~21o leukemia and Me-180 cells and a rat excision repair-deficient line [3] and mouse fibroblasts [4]. in [5-7] DNA cleavage was followed with a superhellcal DNA. In [8] no degradation of Ehrlich ascites tumor cell DNA was shown by treatment of the animals with daunorubicin or doxorubicin, contrary to the damage observed with L12~o tumor cells. Results obtained with one population of tumor cells, in our case Ehrllch ascites tumor cells, cannot be applied to another kind of cells. For example, using leukemia cells from several patients, DNA damages were different depending on the population of cells [9] when uptake and retention of the drug are characteristic of each drug and uniform for the different cell population. Here, we report the results obtained with daunorubicin and some of its derivatives after administration of the drugs to mice bearing an Ehrlich ascites tumor. The use of a technique based on the isopycnic ceatrifugation of nuclei extracted from Ehrlich ascites tumor cells followed by a CsCI gradient allows the separation of undegraded DNA [10]. The sedimentation constant of this DNA has been determined in an analytical ultracentrifuge, before and after alkaline degradation and subsequently the number of single strand breaks has been calculated.

Template activity of brominated DNA for DNA polymerase.

Bromination of DNA affects only guanine and cytosine which are converted into 8bromoguanine (8-BrC) and S-bromocytosine (5.BrC) respectively [I] . If the Br:nucleotide ratio is below 1, the percentage of brominated bases is proportional to the amount of bromine added. The brominated DNA molecules are more flexible than native DNA [2] and electronmicroscope photographs of material brominated to a low degree reveal that they are composed of two species: clusters resembling the denatured DNA and extended chains simulating native molecules [3]. The presence of two distinct species is also revealed by the melting curve profile of partially brominated DNA and these two species can be separated by fractionation on a hydroxylapatite column. DNA isolated from antibiotic-resistant strains of Haemophilus influenzae looses some of its transforming activity after bromination. A similar inactivation is observed in brominated Bacillus subtilis DNA; however, in both cases, a significantly different degree of inactivation is found for various markers [4] . In the course of this study, the template activity of calf thymus DNA, brominated to different extents, was tested with two DNA polymerases: E. coli DNA polymerase, insensitive to the secondary structure of the primer; and regenerating rat liver replicative DNA polymerase requiring preferentially denatured DNA [51. The action of both polymerases is inhibited by the presence of 8BrC and S-BrC in the template DNA and Ear the mammalian enzyme, modifications of the secondary structure of DNA are not responsible for the inhibition. North-Holland Publishing Company Amsterdam 2. Methods

Deoxyribonucleic acid of Cancer pagurus: IV. Elution behaviour on hydroxyapatite chromatographic column

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.