Trends in antidepressant use among children and adolescents: a Scandinavian drug utilization study.

OBJECTIVE: To compare antidepressant utilization in individuals aged 5-19 years from the Scandinavian countries. METHODS: A population-based drug utilization study using publicly available data of antidepressant use from Denmark, Norway, and Sweden. RESULTS: In the study period from 2007 to 2017, the proportion of antidepressant users increased markedly in Sweden (9.3-18.0/1000) compared to Norway (5.1-7.6/1000) and Denmark (9.3-7.5/1000). In 2017, the cumulated defined daily doses (DDD) of selective serotonin reuptake inhibitors were 5611/1000 inhabitants in Sweden, 2709/1000 in Denmark, and 1848/1000 in Norway. The use of 'other antidepressants' (ATC code N06AX) also increased in Sweden with a higher DDD in 2017 (497/1000) compared to Denmark (225/1000) and Norway (170/1000). The use of tricyclic antidepressants was generally low in 2017 with DDDs ranging between 30-42 per 1000. The proportion of antidepressant users was highest among 15- to 19-year-old individuals. Girls were more likely to receive treatment than boys, and the treated female/male ratios per 1000 were similar in Sweden (2.39), Denmark (2.44), and Norway (2.63). CONCLUSION: Even in highly comparable healthcare systems like the Scandinavian countries', variation in antidepressant use is considerable. Swedish children and adolescents have a markedly higher and still increasing use of antidepressants compared to Danish and Norwegian peers.

Societal costs of diabetes mellitus in Denmark.

AIM: To provide comprehensive real-world evidence on societal diabetes-attributable costs in Denmark. METHODS: National register data are linked on an individual level through unique central personal registration numbers in Denmark. All patients in the Danish National Diabetes Register in 2011 (N = 318 729) were included in this study. Complication status was defined according to data from the Danish National Hospital Register. Diabetes-attributable costs were calculated as the difference between costs of patients with diabetes and the expected costs given the annual resource consumption of the diabetes-free population. RESULTS: Societal costs attributable to diabetes were estimated to be at least 4.27 billion EUR in 2011, corresponding to 14,349 EUR per patient-year. A twofold higher healthcare resource usage was found for patients with diabetes as compared with the diabetes-free population. Attributable costs, grouped according to different components, were 732 million EUR for primary and secondary care services, 153 million EUR for pharmaceutical drugs, 851 million EUR for nursing services, 1.77 billion EUR in lost productivity and 761 million EUR for additional costs. A steep increase in diabetes-attributable costs was found for patients with major complications compared with patients without complications across all cost components. For attributable healthcare costs this increase was estimated to be 6,992 EUR per person-year after controlling for potential confounders. CONCLUSIONS: Nearly half of the total costs of patients with diabetes can be attributed directly to their diabetes. The majority of costs are incurred among patients with major complications pointing to the importance of secondary preventive efforts among patients with diabetes.

Foetal proglucagon processing in relation to adult appetite control: lessons from a transplantable rat glucagonoma with severe anorexia.

We have previously reported severe anorexia abruptly induced in rats 2-3 weeks after they have been transplanted subcutaneously with the glucagonoma MSL-G-AN. Vagotomy did not affect the time of onset and severity of anorexia, and the anorectic state resembles hunger with strongly elevated neuropeptide Y (NPY) mRNA levels in the nucleus arcuatus. We now show that circulating levels of bioactive glucagon-like peptide-1 (GLP-1) (7-36amide) start to increase above control levels exactly at the time of onset of anorexia. At this time-point, bioactive glucagon as well as total glucagon precursors and GLP-1 metabolites are already vastly elevated compared to controls. We further show that intravenous administration of very high concentrations of GLP-1 to hungry schedule-fed rats causes anorexia in a dose-dependent manner, which is blocked by the GLP-1 receptor antagonist exendin (9-39). GLP-1 (7-36amide) has a well-characterized anorectic effect but also causes taste aversion when administered centrally. The anorectic effect is blocked in rats treated neonatally by monosodium glutamate (MSG). We show that MSG treatment does not prevent the MSL-G-AN-induced anorexia, thereby suggesting a different type of anorectic function. We show a very strong component of taste aversion as anorectic rats, when presented to novel or known alternative food items, will resume normal feeding for 1 day, and then redevelop anorexia. We hypothetize that the anorexia in MSL-G-AN tumour-bearing rats correlates with a foetal processing pattern of proglucagon to both glucagon and GLP-1 (7-36amide), and is due to taste aversion. The sudden onset is characterized by a dramatic increase in circulating levels of biologically active GLP-1 (7-36amide), suggesting eventual saturation of proteolytic inactivation of its N-terminus.

Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma.

AIMS: Aberrant histone acetylation has been associated with malignancy and histone deacetylase (HDAC) inhibitors are currently being investigated in numerous clinical trials. So far, the malignancy most sensitive to HDAC inhibitors has been cutaneous T-cell lymphoma (CTCL). The reason for this sensitivity is unclear and studies on HDAC expression and histone acetylation in CTCL are lacking. The aim of this study was to address this issue. METHODS AND RESULTS: The immunohistochemical expression of HDAC1, HDAC2, HDAC6, and acetylated H4 was examined in 73 CTCLs and the results related to histological subtypes and overall survival. HDAC1 was most abundantly expressed (P < 0.0001), followed by HDAC2; HDAC6 and H4 acetylation were equally expressed. HDAC2 (P = 0.001) and H4 acetylation (P = 0.03) were significantly more common in aggressive than indolent CTCL subtypes. In contrast, no differences were observed for HDAC1 and HDAC6. In a Cox analysis, elevated HDAC6 was the only parameter showing significant influence on survival (P = 0.04). CONCLUSIONS: High expression of HDAC2 and acetylated H4 is more common in aggressive than indolent CTCL. HDAC6 expression is associated with a favorable outcome independent of the subtype.

Drug response prediction in high-risk multiple myeloma.

A Drug Response Prediction (DRP) score was developed based on gene expression profiling (GEP) from cell lines and tumor samples. Twenty percent of high-risk patients by GEP70 treated in Total Therapy 2 and 3A have a progression-free survival (PFS) of more than 10years. We used available GEP data from high-risk patients by GEP70 at diagnosis from Total Therapy 2 and 3A to predict the response by the DRP score of drugs used in the treatment of myeloma patients. The DRP score stratified patients further. High-risk myeloma with a predicted sensitivity to melphalan by the DRP score had a prolonged PFS, HR=2.4 (1.2-4.9, P=0.014) and those with predicted sensitivity to bortezomib had a HR 5.7 (1.2-27, P=0.027). In case of predicted sensitivity to bortezomib, a better response to treatment was found (P=0.022). This method may provide us with a tool for identifying candidates for effective personalized medicine and spare potential non-responders from suffering toxicity.

Transplantable rat glucagonomas cause acute onset of severe anorexia and adipsia despite highly elevated NPY mRNA levels in the hypothalamic arcuate nucleus.

We have isolated a stable, transplantable, and small glucagonoma (MSL-G-AN) associated with abrupt onset of severe anorexia occurring 2-3 wk after subcutaneous transplantation. Before onset of anorexia, food consumption is comparable to untreated controls. Anorexia is followed by adipsia and weight loss, and progresses rapidly in severity, eventually resulting in reduction of food and water intake of 100 and 80%, respectively. During the anorectic phase, the rats eventually become hypoglycemic and hypothermic. The tumor-associated anorexia shows no sex difference, and is not affected by bilateral abdominal vagotomy, indicating a direct central effect. The adipose satiety factor leptin, known to suppress food intake by reducing hypothalamic neuropeptide Y (NPY) levels, was not found to be expressed by the tumor, and circulating leptin levels were reduced twofold in the anorectic phase. A highly significant increase in hypothalamic (arcuate nucleus) NPY mRNA levels was found in anorectic rats compared with control animals. Since elevated hypothalamic NPY is among the most potent stimulators of feeding and a characteristic of most animal models of hyperphagia, we conclude that the MSL-G-AN glucagonoma releases circulating factor(s) that overrides the hypothalamic NPY-ergic system, thereby eliminating the orexigenic effect of NPY. We hypothesize a possible central role of proglucagon-derived peptides in the observed anorexia.

Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen.

Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.

Relationship of VP-16 to the classical multidrug resistance phenotype.

The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism. The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16. The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines. Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells. The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines. In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells. VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45%. However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells. Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells. Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil. These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives.

Targeting the cytotoxicity of topoisomerase II-directed epipodophyllotoxins to tumor cells in acidic environments.

The epipodophyllotoxins etoposide and teniposide are probably the most important drugs in the treatment of small cell lung cancer. The drugs are used in maximally tolerated doses, and the toxicity of the drugs precludes significant dose increments. The cellular target is the nuclear enzyme topoisomerase II which, in the presence of these drugs, causes an extensive fragmentation of DNA. The cell kill can be antagonized by distinct drug types. We have demonstrated previously that the intercalating drug aclarubicin and the cardioprotecting agent ICRF-187 antagonize the cytotoxicity of etoposide in vitro. We have studied possible ways of using this antagonism as a means of differentially protecting normal tissue. Here we demonstrate that the intercalating agent chloroquine prevents the introduction of topoisomerase II-mediated DNA breaks and thereby antagonizes the cytotoxicity of etoposide. This interaction depends on the extracellular pH. Chloroquine, in contrast to etoposide, is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. We propose that such a pH-dependent drug interaction may be useful in directing topoisomerase II drug effects toward solid tumors. Thus, by lowering the extracellular pH (pHe) from neutral (pHe = 7.4) to acidic (pHe = 6.0), [3H]chloroquine accumulation was decreased 5-fold in a human small cell lung cancer cell line, OC-NYH, and in murine leukemia L1210 cells. In parallel, the antagonism exhibited by chloroquine on etoposide cytotoxicity was pHe dependent. Thus, no protection by chloroquine was observed at pHe = 6.5, whereas at pHe = 7.4, etoposide cytotoxicity was almost completely antagonized with a 460-fold protection or more than eight doublings of the cell population. This exploitation of antagonist extracellular trapping by acidic pH is a novel model for regulation of anticancer drug effects.

Loss of amino acids 1490Lys-Ser-Lys1492 in the COOH-terminal region of topoisomerase IIalpha in human small cell lung cancer cells selected for resistance to etoposide results in an extranuclear enzyme localization.

The human small cell lung cancer NCI-H69 cell line selected for resistance to etoposide (H69/VP) has been reported previously to sequentially overexpress both the MRP and MDR1 multidrug resistance-conferring genes. In addition, immunocytochemistry of H69/VP cells demonstrated a distinct extranuclear localization of the nuclear enzyme topoisomerase IIalpha, the target of etoposide. Immunoblots showed a decrease in Mr 170,000 topoisomerase IIalpha in nuclear extracts in H69/VP but equal amounts of the enzyme in whole-cell extracts. Topoisomerase II catalytic activities in H69 and H69/VP whole-cell extracts were equal, as were their inhibition by etoposide. Sequencing of the entire H69/VP topoisomerase IIalpha cDNA showed a homozygous 9-nucleotide deletion encompassing nucleotides 4468-76, coding for Lys-Ser-Lys, overlapping two potential bipartite nuclear localization signals. The deletion occurred at the initial nine nucleotides of an exon, suggesting alternative splicing of topoisomerase IIalpha mRNA. Subsequent sequencing of H69/VP genomic DNA revealed a G-->T point mutation in the 3' acceptor splice site consensus sequence, resulting in the use of an alternate splice site. Comparison with previous reports on three drug-resistant cell lines with large truncations/deletions in the COOH-terminal region of topoisomerase IIalpha and extranuclear localization point to a pivotal role for the basic cluster 1490Lys-Ser-Lys1492 in the nuclear import of this enzyme.

Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small cell lung cancer cells: relationship to DNA integrity and topoisomerase II.

The effect of combinations of the anthracyclines aclarubicin and daunorubicin was investigated in a clonogenic assay using the human small cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations of aclarubicin. Coordinately, aclarubicin inhibited the formation of daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein cross-links in OC-NYH cells when assayed by the alkaline elution technique. Aclarubicin had no influence on the accumulation of daunorubicin in these cells. In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by more than 300% when the cells were simultaneously incubated with the MDR modulator verapamil, aclarubicin, or the two agents combined. Yet the cytotoxicity of daunorubicin was potentiated significantly only by verapamil. The increased cytotoxicity of daunorubicin in the presence of verapamil was completely antagonized when aclarubicin was used together with the MDR modulator. Finally, the effect of daunorubicin on the DNA cleavage activity of purified topoisomerase II in the presence and absence of aclarubicin was examined. It was found that daunorubicin stimulated DNA cleavage by topoisomerase II at specific DNA sites. The addition of aclarubicin completely inhibited the daunorubicin-induced stimulation of DNA cleavage. Taken together, these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced cytotoxicity is due mainly to a drug interaction with the nuclear enzyme topoisomerase II. This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even though aclarubicin is able to increase the intracellular accumulation of daunorubicin in a MDR cell line.

Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform.

Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.

Sequential coexpression of the multidrug resistance genes MRP and mdr1 and their products in VP-16 (etoposide)-selected H69 small cell lung cancer cells.

Resistance to drugs included in the multidrug-resistance phenotype has been attributed to overexpression of either mdr1 or MRP genes and their products in numerous cell lines, while coexpression, to our knowledge, has not previously been reported in the same cells. Human small cell lung cancer H69/VP cells were developed by continuous incubation in increasing doses of VP-16. In reverse transcription-PCR assays we found over-expression of both mdr1 and multidrug-resistance protein (MRP) genes, and immunoblots showed both elevated P-glycoprotein and MRP in H69/VP cells. Double immunocytochemical staining demonstrated the expression of both MRP and P-glycoprotein in the same cells, indicating that the observations do not result from the selection of two independent clones. Examination of early passages of H69/VP cells showed that overexpression of MRP mRNA occurred prior to mdr1. Thus, cell lines and clinical samples in the future should be tested for both mdr1/P-glycoprotein and MRP since a positive result for one of the phenotypes does not preclude the existence of the other.

Chinese hamster ovary cells resistant to the topoisomerase II catalytic inhibitor ICRF-159: a Tyr49Phe mutation confers high-level resistance to bisdioxopiperazines.

Anticancer drugs targeted to the nuclear enzyme DNA topoisomerase II are classified as poisons that lead to DNA breaks or catalytic inhibitors that appear to completely block enzyme activity. To examine the effects of the bisdioxopiperazine class of catalytic inhibitors to topoisomerase II, we investigated a Chinese hamster ovary (CHO) subline selected for resistance to ICRF-159 (CHO/159-1). Topoisomerase IIalpha content in CHO/159-1 cells was reduced by 40-50%, compared to wild-type CHO cells, whereas the beta isoform was increased by 10-20% in CHO/159-1 cells. However, the catalytic activity of topoisomerase II in nuclear extracts from CHO/159-1 cells was unchanged, as was its inhibition by the topoisomerase II poison etoposide (VP-16). No inhibition of topoisomerase II catalytic activity by ICRF-187 was seen in CHO/159-1 cells up to 500 microM, whereas inhibition was evident at 50 microM in wild-type CHO cells. VP-16-mediated DNA single-strand breaks and cytotoxicity were similar in the two sublines. ICRF-187 could abrogate these VP-16 effects in the wild-type line but had no effect in CHO/159-1 cells. Western blots of topoisomerase IIalpha after incubation of CHO cells with ICRF-187 demonstrated a marked band depletion, whereas this effect was completely lacking in CHO/159-1 cells, and an equal effect of VP-16 was observed in both lines. These data imply that the CHO/159-1 topoisomerase IIalpha lacks sensitivity to bisdioxopiperazines and that the mechanism of resistance in this cell line does not confer cross-resistance to topoisomerase II poisons, suggesting that mutations conferring resistance to bisdioxopiperazines can occur at sites distinct from those responsible for resistance to complex stabilizing agents. Accordingly, CHO/159-1 cDNA showed two heterozygous mutations in the proximal NH2-terminal part of topoisomerase IIalpha (Tyr49Phe and delta 309Gln-Gln-Ile-Ser-Phe313), which is in contrast to those induced by topoisomerase II poisons, which cluster further downstream. Site-directed mutagenesis and transformation of the homologous Tyr50Phe coding mutation in human topoisomerase IIalpha in a temperature-conditional yeast system demonstrated a high-level resistance to ICRF-193, compared to cells expressing wild-type cDNA, but none toward the poisons VP-16 or amsacrine, thus confirming that the Tyr50Phe mutation confers specific resistance to bisdioxopiperazines. Thus, these results indicate that the region of the protein involved in ATP-binding also plays a critical role in sensitivity to bisdioxopiperazines, a result consistent with the known requirement for the formation of an ATP-bound closed clamp for bisdioxopiperazine activity. These results may enable a more precise understanding of the interaction of topoisomerase II-directed drugs with their target enzyme.

Antagonistic effect of aclarubicin on the cytotoxicity of etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide in human small cell lung cancer cell lines and on topoisomerase II-mediated DNA cleavage.

The effect of combinations of the anthracycline aclarubicin and the topoisomerase II targeting drugs 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost completely antagonized by preincubating cells with nontoxic concentrations of aclarubicin. The inhibition of cytotoxicity was not seen when the cells were exposed to aclarubicin after exposure to VP-16. The inhibition was significant over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above which the toxicity of aclarubicin became apparent. A similar effect was seen on the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase II and naked DNA, aclarubicin did not stimulate the formation of cleavable complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1 microM inhibited the baseline formation of cleavable complexes elicited with the enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at aclarubicin concentrations above 1 microM an antagonistic effect was obtained. In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks were completely inhibitable by aclarubicin preincubation with no synergic dose levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated DNA cleavage. This inhibition could represent the mechanism of action of the drug and explain the lack of cross-resistance to the classical anthracyclines. The observed antagonism could have consequences for scheduling of aclarubicin with topoisomerase II-active anticancer drugs.

Characterization of the cooperative cross-linking of doxorubicin N-hydroxysuccinimide ester derivatives to water soluble proteins.

Protein-anthracycline interactions have been examined by using reactive N-hydroxysuccinimide ester derivatives of doxorubicin. These compounds cross-link to lysine epsilon-amino groups with high efficiency and offer the possibility for structural studies of protein-anthracycline complex formation by using gel filtration, ultracentrifugation and spectrophotometric methods. The results are in accordance with association of anthracycline to the hydrophobic ligand binding cavities of serum albumin. The results for proteins not having hydrophobic domains (IgG, serum transferrin, lactotransferrin, ovotransferrin) suggest that complex formation is cooperative and involves two steps: initial self-association of anthracycline into aggregated structures and subsequent binding of protein at the aggregate surface. With serum transferrin, anthracycline self-association makes possible the assembly of stable nanometer-sized protein-anthracycline particles held together by non-covalent bonds. This reaction, which is highly reproducible and efficient, may have applications in the field of development of anthracycline carrier systems.

A model for computer simulation of P-glycoprotein and transmembrane delta pH-mediated anthracycline transport in multidrug-resistant tumor cells.

Anthracycline resistance in multidrug-resistant (MDR) tumor cells is due in part to a reduced cellular drug accumulation. Using a simple kinetic model and numerical computer simulations, we have analyzed mathematically the following possible mechanisms controlling fluxes of the membrane permeable anthracyclines in MDR cells: (1) active outward transport via a specific drug transporter (P-glycoprotein), (2) exocytotic drug export via the endosomal vesicle system, and (3) pH-gradients across the plasma membrane. Model calculations were based on morphometric and kinetic data previously presented in the literature for daunorubicin transport in wild-type Ehrlich ascites tumor cells (EHR2) and the corresponding daunorubicin (DNR)-resistant cell line EHR2/DNR+. The results confirm the possible importance of the cell-surface pH in controlling DNR accumulation in the cells. With P-glycoprotein as the main efflux pump, a catalytic constant of the protein greater than 40 mol DNR transported/mol protein per min is predicted by the model calculations. Changes in the drug binding affinity of P-glycoprotein (Km = 10(-9)-10(-6) M) is of little importance in influencing its effectiveness to reduce DNR accumulation, which could explain the broad substrate specificity of the MDR efflux pump system. The conditions to evaluate unidirectional fluxes of DNR across the plasma membrane in cells with active P-glycoprotein are defined. An efflux mechanism which relies solely on pH-dependent drug trapping in a pH 5 endosomal compartment by a simple diffusion process followed by exocytosis, appears inadequate to account for the high rate of DNR efflux in EHR2/DNR+ cells.

Covalent modification of serum transferrin with phospholipid and incorporation into liposomal membranes.

A method is described for incorporation of water-soluble proteins into liposomal membranes using covalent protein-phospholipid conjugates in detergent solution. A disulfide derivative of phosphatidylethanolamine containing a reactive N-hydroxysuccinimide ester group is synthesized, and the derivative is reacted with serum transferrin in deoxycholate-containing buffer. Disulfide-linked transferrin-phosphatidylethanolamine conjugates containing up to 6 mol phospholipid/mol protein are prepared. The amphiphilic conjugates have solubility properties very similar to integral membrane proteins. The conjugates self-associate to form protein micelles of narrow size distribution (Stokes radii 6-7 nm), and in the presence of excess phospholipid (egg phosphatidylcholine), they readily incorporate into liposomal membranes upon removal of detergent. Stable incorporation into liposomes requires the introduction of two molecules of phosphatidylethanolamine into the transferrin. Using the disulfide linker to release transferrin from the liposomes, evidence is presented for a function of the phosphatidylethanolamine as an anchor-molecule into the liposomal lipid. Optimal conditions for preparation of homogeneous liposomes with diameters in the range 30-125 nm and with a varying content of transferrin are defined. The liposomes appear well suited for studies on liposome-cell membrane interactions.

Insulin and cortisol promote leptin production in cultured human fat cells.

The aim of this study was to investigate the regulation of leptin expression and production in cultured human adipocytes using the model of in vitro differentiated human adipocytes. Freshly isolated human preadipocytes did not exhibit significant leptin mRNA and protein levels as assessed by reverse transcriptase (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). However, during differentiation induced by a defined adipogenic serum-free medium, cellular leptin mRNA and leptin protein released into the medium increased considerably in accordance with the cellular lipid accumulation. In fully differentiated human fat cells, insulin provoked a dose-dependent rise in leptin protein. Cortisol at a near physiological concentration of 10(-8) mol/l was found to potentiate this insulin effect by almost threefold. Removal of insulin and cortisol, respectively, was followed by a rapid decrease in leptin expression, which was reversible after readdition of the hormones. These results clearly indicate that both insulin and cortisol are potent and possibly physiological regulators of leptin expression in human adipose tissue.

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.