New-onset Diabetes Mellitus After Kidney Transplantation-A Paired Kidney Analysis.

BACKGROUND: New-onset diabetes mellitus (NODAT) is a severe complication after kidney transplantation. It is associated with increased risk of graft failure, cardiovascular disease, mortality and infections. METHODS: We retrospectively (partially using the registry database) analyzed risk factors and clinical consequences of NODAT in patients after kidney transplantation performed at the University Transplant Centre between 2001 and 2016. To minimize the donor variability and bias, a paired kidney analysis was applied. Diabetes was defined as the need for insulin therapy for a minimum 30 days after transplantation. RESULTS: The incidence of NODAT was 7.6% (109 of 1424), but only 74 patients with NODAT had their pairs of patients without NODAT, who received kidneys from the same donor and were included to the analysis. The NODAT group was older, and with a significantly higher Charlson Comorbidity Index (2.97 vs 3.39; P = .02). The groups did not differ significantly with respect to immunosuppressive protocols and number of mismatches (2.65 vs 2.78). The incidence of acute rejection (AR; not biopsy proven) was significantly higher in the NODAT group (30% vs 13%), but the incidence of delayed graft function (DGF) was similar (40%). Creatinine concentration and estimated glomerular filration rate (using the Modified Diet in End-stage Renal Disease equation) 1-month after kidney transplantation did not differ: 1.5 vs 1.54 mg/dL and 49.3 vs 50.2 mL/min, respectively. Body mass index (BMI) was higher in the NODAT group. On multivariate analysis, factors significantly associated with NODAT were: age; AR; Charlson Comorbidity Index; and pretransplant dialysis time. BMI was higher in the NODAT group. NODAT was not a predictor of early graft loss and patient survival in the short-term analysis. CONCLUSION: AR, older age, higher comorbidity index, and BMI were risk factors for NODAT. We did not identify an influence of NODAT on early graft function and loss, but further analysis with a longer follow-up is necessary.

Clinical Consequences of Diabetes Mellitus in Patients After Kidney Transplantation: A Paired Kidney Analysis.

BACKGROUND: Diabetes mellitus (DM) has been acknowledged as the most common disorder leading to end-stage renal failure in adults. Diabetic patients show higher survival rates after kidney transplantation (KTx) compared with dialysis therapy. The aim of the study was to evaluate follow-up after KTx in patients with DM as a reason of end-stage renal disease (ESRD), or with long-lasting diabetes before transplantation, compared with patients without DM. METHODS: We retrospectively analyzed the clinical consequences of DM in patients after KTx performed at the Gdansk Transplantation Centre between 2000 and 2016. To minimize donor bias, a paired kidney analysis was applied. RESULTS: The incidence of DM (types 1 and 2) was 13%; 145 patients with DM had pairs of nondiabetic patients, who received kidneys from the same donor and were included to the analysis. The DM group was older. The incidence of AR was similar among the 2 groups, DGF was observed more often in patients with diabetes. Kidney graft function 1 month after transplantation was equal in both groups (mean serum creatinine concentration 1.4 mg/dL). Five-year patient survival was better in the non-DM group (96.7% vs 81.5%). Kaplan-Meier survival curves did not differ significantly between the DM and non-DM groups. DM was not associated graft loss. In the univariate analysis age was the only factor associated with death. CONCLUSION: Diabetic patient survival after KTx seems to be worse than in patients without diabetes, but generally the follow-up among diabetics is good, with graft survival similar to that observed in patients without DM.

Rapamycin as a therapy of choice after renal transplantation in a patient with tuberous sclerosis complex.

Herein we have reported the use of rapamycin in immunosuppressive treatment after renal transplantation as a therapy of choice in a patient with diagnosis of tuberous sclerosis complex (TSC). TSC is a genetic disorder, caused by mutations of TSC1 or TSC2 genes. Products of these genes, hamartin and tuberin, create a complex that inhibits mammalian target of rapamycin (mTOR), a key protein engaged in regulation of the cell cycle. Mutations of TSC genes lead to constitutive activation of mTOR resulting in uncontrolled proliferation, differentiation, and migration of cells. As a consequence malformations of many organs arise. We have presented a case of a 47-year-old female TSC patient with multisystem involvement (skin, brain, lungs, and kidneys), who developed end-stage renal disease ESRD due to angiomyolipomas with subsequent bilateral nephrectomy. At the age of 44 years, she started hemodialysis treatments and 10 months later underwent kidney transplantation. Immunosuppressive treatment included the mTOR inhibitor rapamycin. Since the patient was discharged from hospital, she has remained in good clinical condition with stable graft function. Clinical evaluation after 2 years treatment with rapamycin revealed significant regression of skin lesions. Brain, chest, and abdominal cavity computed tomography images remained stable. No complications of immunosuppressive treatment or TSC were observed. Experimental and clinical studies have confirmed that rapamycin exerts beneficial effects in TSC, providing a new therapeutic option. Therefore an immunosuppressive regimen with rapamycin should be considered as the treatment of choice after kidney transplantation among patients with TSC seeking to avoid development or progression of disease complications.

Pre-clinical toxicology and pathology of 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), a novel anti-cancer agent in male Beagle dogs.

We have developed a group of 4-substituted-1-nitroacridines with potent anti-tumor activity against prostate cancer and less toxic than parent 1-nitroacridines. The most active 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748) was selected for pre-clinical studies. The current study was undertaken to evaluate clinical and/or morphological adverse effects of C-1748 as a single intravenous dose at concentrations ranging from 0.16 to 4.6 mg/kg administered to male Beagle dogs. The maximum tolerated dose was 1.5 mg/kg. Emesis was observed in all groups lasting an average of 30 min to 12 h post-dosing. At high dose, extreme aggression was observed in one dog followed by disorientation and depression lasting for 48 h a frequent observation with chemotherapy. Reductions in platelets and white blood cells were observed which was similar to that seen with other chemotherapeutic agents. A compensatory hyperplasia of lymph nodes and a transient and limited extravasation in the intestinal mucosa were also observed. Increases in aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase were transient with normal levels restored by day 9. These enzyme increases were accompanied by epithelial hypertrophy of larger bile ductules in the periportal triads of the liver. The low toxicity profile and high tumor target activity make this novel class of drug a promising chemotherapeutic agent.

Expression of TNFalpha by CD3+ and F4/80+ cells following irradiation preconditioning and allogeneic spleen cell transplantation.

The pathogenesis of acute graft-versus-host disease (aGVHD) includes tumor necrosis factor-alpha (TNFalpha) expression by macrophages and T cells. However, the temporal comparison of donor vs host cells to TNFalpha expression in response to irradiation conditioning and alloreactivity has not been reported. This study compared intracellular TNFalpha expression in donor vs host spleen T cells and macrophages using a murine model of aGVHD. Total body irradiation conditioning alone resulted in increased frequency of F4/80+/TNFalpha+ cells, but no increase in CD3+/TNFalpha+ cells. Syngeneic transplantation resulted in an increased frequency of F4/80+/TNFalpha+ cells, while CD3+/TNFalpha+ cells increased on days 1 and 3 but declined on day 5. Allogeneic transplantation resulted in an increased frequency of donor CD3+/TNFalpha+ cells, while the frequency of host CD3+/TNFalpha+ cells declined. Similarly, donor F4/80+/TNFalpha+ cells also increased in frequency after allotransplantation, while the frequency of host F4/80+/TNFalpha+ cells was increased on day 1 and declined through days 3 and 5. In absolute cell numbers, CD3+/TNFalpha+ cells were greater than F4/80+/TNFalpha+ cells post allotransplantation. We conclude that (1) both donor and host CD3+ and F4/80+cells are present in the post transplant period and contribute to TNFalpha production and (2) in terms of frequency, the majority of TNFalpha producing cells in the spleen after allogeneic BMT are CD3+.

Enzymatic activation of a new antitumour drug, 5-diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311, observed after its intercalation into DNA.

The imidazoacridinone derivative, C-1311, is a new antitumour agent that exhibits strong antitumour activity against experimental colorectal cancer and has been selected for entry into clinical trial. The compound has previously been shown to have DNA non-covalent binding properties in vitro and to bind irreversibly to DNA of tumour cells. The latter effect has also been observed in a cell-free system, but only in the presence of activated enzymes. The present studies were aimed at finding out whether and in what way the enzymatic activation of C-1311 and its non-covalent binding to DNA influence or depend on each other. Enzymatic activation was performed with a model system containing horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and was followed by UV-VIS spectroscopy and by HPLC with UV-VIS and electrospray ionisation mass spectrometry detection. DNA non-covalent binding was studied in the cell-free system by means of an unwinding assay and UV-VIS spectroscopy. It was shown that C-1311 was oxidised by the HRP/H2O2 system in a manner dependent on the drug:H2O2 ratio. In the case of ratios of 1:3 and 1:5, the reaction gave highly reactive species that were quickly transformed into the further products p2 and p3 that were unable to intercalate into DNA. In the presence of DNA, C-1311 first intercalated into DNA and the intercalated compound was then oxidised. This oxidation was directed to only one product. Therefore, DNA seems to play the role of a "scavenger" of the reactive oxidation product(s) yielded from the intercalated drug and prevents its further deactivation. We conclude that, under the conditions studied, intercalation of C-1311 into DNA is followed by its HRP-mediated activation, giving rise to the intercalated species that might irreversibly bind to DNA. Since peroxidase-type enzymes are present in the cell nucleus, the proposed sequence of events may also be expected to take place in the cellular environment in vivo.

Products of metabolic activation of the antitumor drug ledakrin (nitracrine) in vitro.

The aim of this work was to characterize the products of metabolic activation of the antitumor drug ledakrin (Nitracrine) in model metabolic systems, where formation of drug-DNA adducts was previously discovered. The metabolic products obtained in different biological systems were compared with those obtained in experiments where chemical reducing agents were applied. Therefore, activation products were obtained in the presence of the microsomal fraction of rat liver and in the experiments with the reducing agents dithiothreitol, hydrazine hydrate, and SnCl(2). Furthermore, transformations of the drug with oxidoreductase enzymes DT-diaphorase and xanthine oxidase were observed. The ledakrin transformation products were separated and analyzed by HPLC with diode array detection. Structural studies of the products were performed by means of ESI-MS and NMR. Proton, carbon, and nitrogen assignments were made based upon DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. It was demonstrated during the reduction of ledakrin that a key metabolite, a compound with an additional five-membered ring attached to positions 1 and 9 of the acridine core and with the retained 9-aminoalkyl side chain, was formed in all the systems that were studied. It was determined that the reactive nitrogen atoms of this additional ring underwent further transformations resulting in the formation of a six-membered ring produced by the addition of a carbon atom to the dihydropyrazoloacridine ring. Furthermore, it was observed that positions 2 and 4 of ledakrin's acridine ring are susceptible to nucleophilic substitution as revealed by the studies with dithiothreitol. Additionally, although most products from the reduction of ledakrin were extremely unstable, 1-aminoacridinone, produced enzymatically and with dithiothreitol, exhibited persistent stability under the studied conditions.

Does the antitumor cyclopropylpyrroloindole antibiotic CC-1065 cross-link DNA in tumor cells?

We have found that a cyclopropylpyrroloindole antibiotic, compound CC-1065 (benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1, 6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8, 8a-tetrahydro-7-methyl-4-oxocyclopropan[c]pyrrolo[3, 2-e]indol-2(1H)-yl)carbonyl]benzo[1,2-b:4, 3-b']dipyrrol-3(2H)-yl]-carbonyl]-1,6-dihydro-4-hydroxy-5-methoxy, (7bR,8aS)), forms interstrand DNA cross-links of an apparently covalent nature in HeLa S(3) cells. This compound induced interstrand cross-links at concentrations ranging from 0.1 to 1 nM/3 hr in whole cells, but these cross-links were absent or marginally low when the drug was added to cell lysates with inactivated cellular enzymes or isolated nuclei, which suggests that metabolic activation of the drug is a necessary step for DNA cross-linking to occur. In contrast, an analog of CC-1065, Bizelesin, which forms DNA-DNA cross-links by direct alkylation, induced interstrand DNA cross-links in both whole cells and in cell lysates. Interestingly, a demethoxy analog of CC-1065, Adozelesin, did not induce DNA cross-links under the same conditions. CC-1065 was found to be extremely potent in terms of concentrations required to cross-link DNA of tumor cells, and this may be related to its remarkable cytotoxic activity.

Mitoxantrone and ametantrone induce interstrand cross-links in DNA of tumour cells.

We show here that mitoxantrone and ametantrone induce interstrand DNA cross-links in HeLa S3 cells. These cross-links were observed only in cellular system suggesting that metabolism of the drugs is a necessary step leading to DNA cross-linking. Biologically inactive analogue of mitoxantrone, compound NSC 321458, did not induce cross-links in DNA of tumour cells which suggests that DNA cross-linking is associated with the cytotoxic and anti-tumour activity of these compounds.

The relevance of enzymatic oxidation by horseradish peroxidase to antitumour potency of imidazoacridinone derivatives.

The presented study concentrated on the oxidative enzymatic transformation of six imidazoacridinone derivatives exhibiting different antitumour activity. Horseradish peroxidase was applied as an enzymatic model system. The investigations aimed to evaluate: (1) whether the studied compounds can undergo oxidative biotransformation; (2) whether the susceptibility to such biotransformation relates to the structure and antitumour activity of these compounds; and (3) which elements of imidazoacridinone structure are involved in this kind of transformation. The reaction courses were followed by three methods: UV-VIS spectroscopy, electron paramagnetic resonance and high-performance liquid chromatography. It was shown that all the imidazoacridinones studied underwent enzymatic oxidation resulting in the formation of several products, spectra of which revealed that imidazoacridinone chromophore as well as alkylamino side-chain were involved in these biotransformations. The susceptibility to enzymatic oxidation turned out to be well correlated with antitumour activity of these compounds. It was demonstrated that the highly active antitumour 8-hydroxy derivatives underwent oxidative transformation far more readily than the less active 8-methoxy derivatives and analogues without substituent in position 8. The results indicated that the oxidation pathway of 8-hydroxy compounds was different from those observed for the remaining imidazoacridinones studied. It also differed from the pathway proposed earlier for mitoxantrone. Moreover, it was find out that not only the rate but also the mechanism of horseradish oxidation of 8-hydroxy derivatives depended on the reaction conditions. In the presence of excess of hydrogen peroxide, the drugs were exceptionally reactive giving rise to the mixture of many unstable products, among which compounds with both changed and unchanged chromophore structure were formed. However, the equimolar ratio of drug and hydrogen peroxide led to stable products, which resulted from the oxidation in aminoalkyl side-chain. The possible structures of products of imidazoacridinone enzymatic oxidation are discussed. In conclusion, the results presented in this paper indicate that the oxidative metabolic activation of imidazoacridinones may represent the crucial step in their biological action.

32P-post-labelling analysis of nucleobases involved in the formation of DNA adducts by antitumor 1-nitroacridines.

Adducts generated in vitro by the reaction of 1-nitroacridines with poly(dN)s in the presence of dithiothreitol were used to identify a kind of nucleic base involved in the formation of individual adducts. The patterns of chromatographic spots corresponding to modified nucleotides obtained by 32P-post-labelling assay for synthetic homopolymers of four deoxyribonucleotides were compared with the fingerprints detected in the case of calf thymus DNA reacted with 1-nitroacridines under conditions in which the formation of identical DNA adducts as in cellular models was demonstrated in earlier investigations. Both compounds studied (Ledakrin and C-857) turned out to bind covalently only with purine nucleotides. Ledakrin formed with dG four and C-857 five different adducts. All of them were also detected in ctDNA. The incubation with poly(dA) resulted in four Ledakrin-dA species, two of which were found in ctDNA, and in two C-857-dA adducts that were not, however, observed in DNA containing samples. Modification of purines accounted for all adducts observed in ctDNA. For both compounds studied, the level of total binding to poly(dA) was about one order of magnitude lower than to poly(dG) for which it was comparable with the extent of ctDNA modification. This indicates that dG represents a preferential site of covalent binding of 1-nitroacridines to DNA.

In vitro DNA crosslinking by Ledakrin, an antitumor derivative of 1-nitro-9-aminoacridine.

Using agarose gel electrophoresis we confirmed that Ledakrin is capable of incurring covalent crosslinking in pBR322 plasmid DNA and also in poly(dGdC) in the presence of a simple activating system containing DTT. The identification of adducts resulting from DNA crosslinking was carried out by 32P-post-labelling assay. We assumed that such adduct(s) should be brought about more readily with double-stranded than with single-stranded polynucleotides or nucleotides. Since our earlier experiments had shown that guanine is a major site of covalent binding of 1-nitroacridines, we compared DNA adduct formation by Ledakrin for ctDNA, dG-containing synthetic homopolymers and 3'-pdG. 32P-Post-labelling assay revealed two adduct spots that were enhanced in samples containing double-stranded substrates in which interstrand crosslinking between guanines was possible, namely ctDNA and poly(dGdC).

Preclinical evaluation of novel imidazoacridinone derivatives with potent activity against experimental colorectal cancer.

Novel imidazoacridinone derivatives, C1310 and C1311, have been evaluated for their potential to inhibit tumour cell growth in vitro and in vivo. A cell line panel, including seven human and murine colon carcinoma cell lines and three in vivo models, was used. The compounds were found to be potent inhibitors of tumour cell growth with IC50 values ranging between 10 nM and 2 microM in human colon cancer cell lines. Statistically significant tumour growth delay (P < 0.01) was observed after a single intraperitoneal (i.p.) dose of C1311 (100 mg kg-1 body weight) in MAC15A, MAC29 murine and HT29 human adenocarcinomas of the colon. Rapid accumulation of fluorescence of both C1310 and C1311 was seen in the nuclei of HT29 human colon tumour cells in culture. C1311 was also found to bind into calf thymus DNA as shown by spectrophotometric titration and thermal denaturation and to cause early inhibition of thymidine incorporation in HT29 cells in vitro. The results of this study suggest that C1311 should be considered as a candidate for clinical development.

Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors.

Imidazoacridinones are new antitumor compounds that exhibit strong antitumor effect against solid tumors such as human colon and breast carcinomas. The mechanism of action of imidazoacridinones is unknown, although a similarity in the chemical structure between active imidazoacridinones and mitoxantrone suggests common cellular targets. We show that imidazoacridinones inhibit the catalytic activity of purified topoisomerase II as determined by both relaxation and decatenation assays. All biologically active compounds stimulated the formation of cleavable complexes in vitro, whereas inactive compounds did not. The pattern of DNA cleavage in SV40 DNA was similar to that obtained for 4'-(9-acridinylamino)methane-sulfon-m-aniside, particularly within the matrix-associated region. Significant levels of DNA complexes were observed when DC-3F fibrosarcoma cells were treated with active compounds, whereas negligible amounts of these complexes were induced by inactive analogues. DC-3F/9-OHE cells, which are resistant to other topoisomerase II inhibitors, are 30-125-fold cross-resistant to active imidazoacridinones. The resistance is associated with a reduction in the formation of DNA/protein complexes and is highest for compounds that are potent topoisomerase II inhibitors in vitro. Interestingly, the two most active derivatives, C-1310 and C-1311, were equally cytotoxic toward fast-growing monolayer cultures and cells growing in three dimensions as multicellular spheroids, which have a slower growth fraction. In contrast, 4'-(9-acridinylamino)methanesulfon-m-aniside, mitoxantrone, and doxorubicin were more cytotoxic toward monolayer cultures. Taken together, the results suggest that DNA topoisomerase II is a major cellular target of biologically active imidazoacridinones and that these drugs show both similarities and dissimilarities compared with classic topoisomerase II inhibitors.

Structure-activity relationship for antineoplastic imidazoacridinones: synthesis and antileukemic activity in vivo.

Synthesis of several new 5-amino-substituted derivatives of 5-amino-6H-imidazo[4,5,1-de]-acridin-6-one bearing in the benzene ring OH, OCH3, CH3, tert-butyl, or OCH2O groups is described. 8-OH-substituted compounds or double-substituted 7-OH-10-OCH3 compounds demonstrated potent in vivo activity against murine P388 leukemia. The highest activity was exhibited by 5-[[2-[[2-(diethylamino)ethyl]amino]ethyl]amino]-8-hydroxy-6H- imidazo[4,5,1-de]-acridin-6-one (4c).

Imidazoacridinones arrest cell-cycle progression in the G2 phase of L1210 cells.

Imidazoacridinones are a new class of highly potent antineoplastic agents synthesised at the Technical University of Gdansk. The pharmacophoric alkyldiamine group, which is also present in anthracenediones (e.g. ametantrone, mitoxantrone), has been shown to be responsible for their antineoplastic activity. In view of their chemical similarity to anthracenediones, we anticipated that the imidazoacridinones would have a mechanism of action similar to that of these agents and that this would be reflected by a similar influence on cell-cycle progression. Flow cytometry was used to monitor the effect of three derivatives of imidazoacridinone (C-1263, C-1310 and C-1311) on L1210 cell cycle traverse at concentrations ranging from 0.01 to 0.9 microgram/ml, corresponding to their 50% and 90% effective concentrations (EC50 and EC90 values), over times of drug treatment ranging from 1 to 48 h. The results demonstrate that all of the compounds produced a similar effect, inducing preferential and complete arrest (accumulation) of cells in the G2 phase of the cell cycle (i.e. G2 block). The kinetics of the induction of G2 arrest were dependent on both the dose and the duration of treatment. Cell-cycle arrest was reversible for up to about 3 h of treatment, being quite irreversible at longer incubation times. Microscopic inspection of cells performed in parallel with flow cytometry confirmed that imidazoacridinones induced a G2, not a G2/M, block.

QSAR of acridines, III. Structure-activity relationship for antitumour imidazoacridinones and intercorrelations between in vivo and in vitro tests.

A study on quantitative relationships between the biological activity and physicochemical properties of antitumour 5-alkylaminoimidazoacridinone derivatives was carried out. The activity was based on the results of several in vitro tests as well as experimental antileukaemic therapy. The capacity factor, log k', determined by the reverse-phase HPLC method, was a measure of lipophilic properties. UV and NMR spectra of the compounds were employed to describe electronic parameters. Values of steric descriptors were calculated as topological indexes. Results obtained by means of principal component analysis (PCA) allow us to group biological tests into two subsets: the lipophilicity-dependent and lipophilicity-independent test groups. The highest intercorrelation, R = 0.92, was shown between the optimal dose, pOD, determined in leukaemia P388-bearing mice and cytotoxicity expressed as pEC50 in leukaemia cells. The equation describing this relationship could be applied to predict the therapeutic doses of imidazoacridinone derivatives which would be effective in experimental antileukaemic therapy. The quantitative structure-activity relationship (QSAR) study showed that lipophilic properties significantly influence cytotoxicity, pEC50, and antileukaemic potency, pOD, only in the case of 8-hydroxy analogues of imidazoacridinones, whereas the activity of the remaining derivatives is very low and does not depend on lipophilicity. Electronic resonance properties seem to influence this specific impact of lipophilicity on the biological activity of 8-hydroxy derivatives. Hence, it may be possible to improve the antitumour activity of 8-hydroxyimidazoacridinones by obtaining more hydrophilic derivatives, up to the optimal value of the lipophilic parameter.

32P-postlabelling analysis of adducts formed by mitoxantrone and ametantrone with DNA and homopolydeoxyribonucleotides after enzymatic activation.

DNA adduct formation by enzymatically activated mitoxantrone and ametantrone has been studied by 32P-postlabelling method. Both drugs were activated by peroxidase/hydrogen peroxide system and formed several DNA adducts when reacted with calf thymus DNA. Mitoxantrone gave 3 and ametantrone 4 different DNA adducts with apparently similar chromatographic features suggesting that DNA adducts formed by both compounds do not differ significantly in their chemical structure. Despite this similarity, the level of DNA modification is 10 times higher for mitoxantrone compared to ametantrone. We did not observe DNA adducts in control samples where both drugs were incubated with DNA in the absence of the activating system. It indicates the importance of oxidative activation of mitoxantrone and ametantrone for their ability to bind covalently DNA. In order to identify nucleobases involved in the formation of DNA adducts by anthracenediones, polydeoxyadenosine, polydeoxythymidine, deoxyguanosine 3'-monophosphate and deoxycytosine 3'-monophosphate were modified by mitoxantrone and ametantrone activated in the above mentioned oxidating system. We proved that the only nucleobase modified by both drugs is guanine with no alkylation observed at other DNA bases. The pattern of adducts formed with deoxyguanosine 3'-monophosphate is reminiscent of that obtained with calf thymus DNA. In addition, mitoxantrone was found to be phosphorylated during the postlabelling procedure, most probably at the 1,4-hydroxyl groups of the chromophore. Ametantrone which does not possess hydroxyl groups attached to the chromophore core was resistant to phosphorylation by T4 polynucleotide kinase and gamma-[32P]ATP. These results for the first time provide direct evidence that mitoxantrone and ametantrone form DNA adducts when activated by oxidation in vitro.

Interstrand DNA crosslinking induced by anthracyclines in tumour cells.

Using a new mild method it is shown for two anthracyclines, Adriamycin and Daunomycin, that these compounds are able to form DNA crosslinks in HeLa S3 cells. It was also found that other anthracyclines: Epirubicin, Rubidazone, Iodorubicin, 3'-deamino-3'-hydroxy-4'-amino-Adriamycin, Aclacinomycin, Marcellomycin, and Cinerubin A, induced crosslinks in the DNA of HeLa S3 cells in a concentration-dependent manner. DNA crosslinks formed by five anthracyclines studied, excluding Iodorubicin, were both alkali and thermally unstable. No DNA crosslinking could be detected when the compounds were added to cell lysates in which cellular enzymes had been inactivated. This implies that metabolic activation is prerequisite for DNA crosslinking by anthracyclines. The kinetics of DNA crosslinks formation by Adriamycin as well as their removal from cellular DNA were also studied. The presented results indicate that all biologically active anthracyclines studied induce DNA crosslinks, and for two of them DNA crosslinking was observed at growth inhibitory concentrations.

Relevance of interstrand DNA crosslinking induced by anthracyclines for their biological activity.

The relevance of interstrand DNA crosslinking induced by anthracyclines for their cytotoxic action was studied in several biological systems with cells differing in sensitivity towards these compounds. It was done by establishing the correlation between DNA crosslinking and cytotoxic activity of anthracyclines. The study showed that there is a strong positive correlation between cytotoxic activity of anthracyclines and their DNA crosslinking potency in HeLa S3 cells for a group of six Daunomycin derivatives (r = 0.97) as well as for all the studied 13 anthracyclines of divergent chemical structure (r = 0.95). Similar relationships between cytotoxic activity and DNA crosslinking ability was found for Adriamycin and Daunomycin in three other cellular systems: (i) in LoVo and about 20-fold Adriamycin-resistant LoVo/DX human colon adenocarcinoma cells, (ii) CHO-K1 and its Adriamycin-hypersensitive mutant CHO-ADR5 Chinese hamster ovary cells and (iii) HeLa S3 cells sensitized about 3-fold to cytotoxic action of Adriamycin and Daunomycin by lowering intracellular glutathione content, to about 10% of normal level, by buthionine sulfoximine treatment. The presented results show that DNA crosslinking induced by anthracyclines may be responsible for the cytotoxic activity of these compounds.