Biomedical subjects

G Capranico

Publications and source records attributed to G Capranico.

80 records · Page 5Linked to original sources

PubMed1989

Formation, resealing and persistence of DNA breaks produced by 4-demethoxydaunorubicin in P388 leukemia cells.

The formation and disappearance of DNA single-strand breaks (SSB) produced by 4-demethoxydaunorubicin (4-dmDR) in P388 murine leukemia cells and in a resistant subline were examined by alkaline elution methods in relation to cellular pharmacokinetics. DNA strand breaks produced by this intercalating agent were essentially DNA lesions mediated by topoisomerase II, even at very high drug concentrations, since they were detected as protein-associated breaks by filter elution. Similarly, the appearance of delayed DNA breaks in cells exposed to high concentrations, following drug removal, showed predominance of protein-associated breaks, thus supporting a similar mechanism of breakage induction. This finding indirectly suggests that, in this experimental model, free radical production makes little (if any) contribution to DNA damage, and also that DNA effects are not the consequence of early cell death. In contrast to a rapid disappearance of protein-associated strand breaks produced by intercalating agents and topoisomerase II inhibitors of different classes, DNA breaks induced by low concentrations of the anthracycline derivative are only partially reversible following drug removal, but they persisted and even increased with high concentrations. Thus, not only the extent of DNA breaks but also their persistence may contribute to the cytotoxic potency of anthracyclines. The importance of DNA lesions to cytotoxic action of the anthracycline is also emphasized by drug effect on the resistant line. A negligible effect on DNA of resistant cells was detected at drug concentrations lethal to sensitive cells. However, exposure to equitoxic drug concentrations resulted in a comparable amount of DNA breaks in sensitive and resistant cells. Although faster DNA rejoining in resistant cells may be in part attributable to increased efflux of drug, no correlation exists between cell drug accumulation and extent of DNA lesions. With equitoxic drug concentrations cellular drug content was higher in resistant cells, suggesting an intrinsic insensitivity of this variant to DNA cleavage effects of the anthracycline.

Animals↗

PubMed1989

P-glycoprotein gene amplification and expression in multidrug-resistant murine P388 and B16 cell lines.

P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs. The multidrug-resistant phenotype was obtained in different conditions of selection (in vivo and in vitro for P388/DX and B16VDXR, respectively). In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance. In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells. Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number. These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1988

Doxorubicin cellular pharmacokinetics and DNA breakage in a multi-drug resistant B16 melanoma cell line.

Mechanisms of anthracycline resistance have been investigated in a B16 murine melanoma cell subline selected by continuous in vitro exposure to increasing concentrations of doxorubicin (DX). Altered drug pharmacokinetics were observed in resistant B16 cells as compared to the sensitive counterpart. In fact, cellular DX uptake - as determined by a fluorescence method - was lower in resistant than in sensitive cells. Furthermore, drug efflux rate was shown to be higher in resistant than in sensitive cells; treatment of cells with the metabolic inhibitor sodium azide decreased drug efflux rate in resistant but not in sensitive cells, suggesting the presence of an energy-dependent drug extrusion mechanism in the resistant B16 cells. However, since drug-induced cell killing did not correlate with cellular DX contents in sensitive and resistant cells, drug resistance of B16 subline could not be completely explained by the observed differences in drug pharmacokinetics. Since drug-induced DNA breaks have been related to drug cytotoxicity, DNA cleavage was also measured by alkaline elution methods. The number of DNA breaks produced by DX was decreased in resistant cells as compared to sensitive cells at the same cellular drug accumulation. The results are consistent with the view that anthracycline resistance may be multifactorial and probably arises following multiple biochemical changes.

Animals↗

PubMed1987

Markedly reduced levels of anthracycline-induced DNA strand breaks in resistant P388 leukemia cells and isolated nuclei.

DNA single-strand and double-strand breaks produced by doxorubicin and two anthracycline derivatives (4-demethoxy-daunorubicin and 4'-deoxy-4'-iododoxorubicin) were measured in doxorubicin-sensitive and -resistant P388 leukemia cell lines, using filter elution methods, and compared with cellular drug accumulation to account for major differences in their cytotoxic activities and cross-resistance. The increased cytotoxic potency of the two derivatives reflects at least in part the enhanced drug accumulation by cells that results from their increased lipophilicity. However, the level of protein-linked DNA breaks was not directly related to cellular accumulation of drug analogues. It is possible that enhanced cytotoxicity may also be the consequence of the greatly enhanced ability of analogues to cause DNA strand breaks. The resistant line showed only a modest degree of resistance to both anthracycline derivatives compared with the high degree of resistance to doxorubicin. Although for all the anthracyclines tested drug accumulation was reduced in the resistant line, this did not correlate with the degree of resistance. A differential sensitivity of resistant and parental cell lines to DNA cleavage activity was consistently found for all three drugs tested. However, in contrast to a lack of effect of doxorubicin, the derivatives caused appreciable DNA strand breakage in resistant cells. The enhanced ability of these analogues to break DNA in resistant cells is consistent with the slight cross-resistance with doxorubicin. DNA double-strand breaks produced in isolated nuclei from these cells paralleled the pattern found in whole cells, thus indicating that a nuclear alteration, presumably involving DNA topoisomerases, is associated with anthracycline resistance. Our findings strongly support the hypothesis that anthracycline resistance in these cell variants may be mediated by multiple mechanisms, involving alterations of plasma membrane and changes of nuclear enzymatic activities responsible for DNA strand breaks.

Animals↗

PubMed1986

Comparison of doxorubicin-induced DNA damage in doxorubicin-sensitive and -resistant P388 murine leukemia cells.

Doxorubicin-induced DNA damage was studied in the P388 leukemia cell line and in a doxorubicin-resistant subline by alkaline elution techniques. DNA single-strand breaks and DNA-protein cross-links were determined. Whereas, in the sensitive line, 1 hr exposure to drug induced DNA damage in a concentration-dependent manner, in the resistant line only a marginal effect was observed at high drug concentrations. In contrast, elution kinetics of DNAs from cells irradiated with X-rays were similar in both lines. Although a reduced intracellular drug accumulation was found in resistant cells, this difference could not account for the marked reduction in doxorubicin-induced DNA damage. The degree of resistance of the P388 subline was reduced about 7-fold by verapamil, whereas the extent of DNA damage was unaffected. These results suggest the presence of alternative modes of resistance, independent of membrane changes, in highly resistant cells.

Animals↗

PubMed1986

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells.

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.

Animals↗

PubMed1986

Single-strand DNA breaks induced by chromophore-modified anthracyclines in P388 leukemia cells.

Single-strand DNA breaks induced by chromophore-modified anthracyclines related to doxorubicin (including 11-deoxydaunorubicin, 4-demethoxydaunorubicin, 4-demethoxy-11-deoxy-4'-epi-daunorubicin, 4-demethyl-6-O-methyldoxorubicin) in cultured P388 leukemia cells were determined by the filter alkaline elution method. The tested analogues differed markedly in their cytotoxic potency. In the range of cytotoxic concentrations, 11-deoxydaunorubicin produced single-strand DNA break frequency of the same order of magnitude as that produced by doxorubicin, while other derivatives caused much more marked damage on DNA than doxorubicin. Since DNA breaks were found to be protein associated, the type of DNA damage produced by all tested derivatives presumably resulted by action of DNA topoisomerases II, as proposed for doxorubicin and other intercalating agents. Although the "potent" (with respect to DNA damage) derivatives, except 4-demethyl-6-O-methyldoxorubicin, showed an increased cellular drug accumulation as compared to doxorubicin, this did not account for the marked differences in ability to damage DNA. 4-Demethyl-6-O-methyldoxorubicin was the most efficient derivative, producing DNA breaks in a lower range of cellular drug content. A striking biphasic dose-response curve was observed for the 4-demethoxy derivatives, suggesting a complex mechanism of interaction among drug, DNA, and enzyme. A lack of correlation was noted among DNA binding affinity, induction of strand breaks, and cytotoxic activity of these chromophore-modified derivatives. From these observations, it is suggested that multiple actions of anthracyclines at the DNA level are responsible for their cytotoxic activity, which is not simply related to inhibition of a specific DNA-dependent enzyme and/or function.

Animals↗

PubMedDate not supplied

New developments in antitumor anthracyclines.

Doxorubicin is a major anticancer agent introduced to extended clinical use in the early 1970s. The fulfillment of a wide program of analogue synthesis led to the development of the better tolerated epirubicin and of a highly potent antileukemic drug, idarubicin. In recent years, on the basis of the available information on the molecular requirements for action, a new synthetic program, coupled with target-oriented pharmacological experiments, was carried out. Various interesting derivatives, namely, the 8- and 10-fluoro compounds and the disaccharides, were obtained. The latter compounds exhibited a strong dependence of biological activity on the orientation (axial vs. equatorial) of the second sugar moiety, daunosamine. A member of this group, namely, 7-O-(4'-O-alpha-L-daunosaminyl-2'-deoxy-alpha-L-fucosyl)-4-demetho xy-adriamycinone, is presently undergoing clinical trials as a third generation antitumor anthracycline.

Animals↗