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.

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.

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.

Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.

Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

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.

Treatment of anthracycline extravasation with Savene (dexrazoxane): results from two prospective clinical multicentre studies.

BACKGROUND: The purpose of this study was to assess the efficacy and tolerability of i.v. dexrazoxane [Savene (EU), Totect (US)] as acute antidote in biopsy-verified anthracycline extravasation. PATIENTS AND METHODS: Two prospective, open-label, single-arm, multicentre studies in patients with anthracycline extravasation were carried out. Patients with fluorescence-positive tissue biopsies were treated with a 3-day schedule of i.v. dexrazoxane (1000, 1000, and 500 mg/m(2)) starting no later than 6 h after the incident. Patients were assessed for efficacy (the possible need for surgical resection) and toxicity during the treatment period and regularly for the next 3 months. RESULTS: In 53 of 54 (98.2%) patients assessable for efficacy, the treatment prevented surgery-requiring necrosis. One patient (1.8%) required surgical debridement. Thirty-eight patients (71%) were able to continue their scheduled chemotherapy without postponement. Twenty-two patients (41%) experienced hospitalisation due to the extravasation. Mild pain (10 patients; 19%) and mild sensory disturbances (nine patients; 17%) were the most frequent sequelae. Haematologic toxicity was common as expected from the fact that the extravasation occurred during a chemotherapy course. Other toxic effects were transient elevation of alanine aminotransferases, nausea, and local pain at the dexrazoxane injection site. CONCLUSION: Dexrazoxane proved to be an effective and well-tolerated acute treatment with only one out of 54 assessable patients requiring surgical resection (1.8%).

Dexrazoxane is a potent and specific inhibitor of anthracycline induced subcutaneous lesions in mice.

BACKGROUND: Recently, we have shown that dexrazoxane (ICRF-187) is an effective antidote against accidental extravasation of anthracyclines. Thus, it inhibits the lesions induced by subcutaneous (s.c.) daunorubicin, idarubicin, and doxorubicin in mice and has proven to be successful clinically as well. Dexrazoxane is a potent metal ion chelator as well as being a catalytic inhibitor of DNA topoisomerase II. However, the mechanism behind the protection against anthracycline extravasation is not known. MATERIALS AND METHODS: Mice were injected s.c. with daunorubicin or doxorubicin. Systemic N-acetylcysteine, alfa-tocoferol, amifostine, merbarone, aclarubicin, ADR-925, and EDTA were administered i.p. immediately hereafter or as a triple-treatment over six hours. Intralesional (i.l.) adjuvants were injected immediately after and into the same area as the anthracycline. The frequency, duration, and sizes of wounds were observed until complete healing of all wounds. RESULTS: Triple-treatment with systemic dexrazoxane was superior to single dosage and completely prevented lesions after s.c. daunorubicin and doxorubicin. Low-dose i.l. dexrazoxane was effective in protecting as well. In contrast, none of the other seven adjuvants was effective. Protection was not achieved with local cooling, however, topical ice did not impair the efficacy of dexrazoxane. CONCLUSIONS: Dexrazoxane is extremely effective and apparently quite specific in protecting against lesions after s.c. doxorubicin and daunorubicin.

Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines.

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.

Linker length in podophyllotoxin-acridine conjugates determines potency in vivo and in vitro as well as specificity against MDR cell lines.

We have synthesized two podophyllotoxin-acridine conjugates-pACR6 and pACR8. In these compounds an 9-acridinyl moiety is beta linked to the C4 carbon of the four ring system in 4'-demethylepipodophyllotoxin (epiDPT) via eighter an N-6-aminohexanylamide linker (pACR6) or via an N-8-aminooctanylamide linker containing two more carbon atoms (pACR8). The acridine-linker moiety occupies the position where different glucoside moieties, dispensable for activity, are normally linked to epiDPT in the well known epipodophyllotoxins VP-16 and VM-26. As with VP-16 and VM-26, pACR6 and pACR8 show evidence of being topoisomerase II poisons as they stimulate topoisomerase II mediated DNA cleavage in vitro and induce DNA damage in vivo. This in vivo DNA damage, as well as pACR6/pACR8 mediated cytotoxicity, is antagonized by the catalytic topoisomerase II inhibitors ICRF-187 and aclarubicin, demonstrating that topoisomerase II is a functional biological target for these drugs. Despite their structural similarities, pACR6 was more potent than pACR8 in stimulating topoisomerase II mediated DNA cleavage in vitro as well as DNA damage in vivo and pACR6 was accordingly more cytotoxic towards various human and murine cell lines than pACR8. Further, marked cross-resistance to pACR6 was seen among a panel of multidrug-resistant (MDR) cell lines over-expressing the MDR1 (multidrug resistance protein 1) ABC drug transporter, while these cell lines remained sensitive towards pACR8. pACR8 was also capable of circumventing drug resistance among at-MDR (altered topoisomerase II MDR) cell lines not over-expressing drug transporters, while pACR6 was not. Two resistant cell lines, OC-NYH/pACR6 and OC-NYH/pACR8, were developed by exposure of small cell lung cancer (SCLC) OC-NYH cells to gradually increasing concentrations of pACR6 and pACR8, respectively. Here, OC-NYH/pACR6 cells were found to over-express MDR1 and, accordingly, displayed active transport of 3H-labeled vincristine, while OC-NYH/pACR8 cells did not, further suggesting that pACR6, but not pACR8, is a substrate for MDR1. Our results show that the spatial orientation of podophyllotoxin and acridine moieties in hybrid molecules determine target interaction as well as substrate specificity in active drug transport.

Lake Superior Rural Cancer Care Project, part I: an interventional trial.

PURPOSE: To date, effective cancer care and control intervention studies have been carried out largely in urban and suburban populations. This study was conducted to test innovative interventions, using experimental designs, to improve the care and outcomes of patients with cancer in rural settings. DESCRIPTION OF STUDY: The Lake Superior Rural Cancer Care Project (LSRCCP) tested an innovative, multimodal, multidisciplinary intervention that involved rural healthcare providers and their healthcare system. An experimental design was used, with the rural community as the unit of randomization. Outcomes were measured at three levels: rural providers' knowledge of cancer management, providers' practice performance, and patient outcomes. This 5-year study was conducted in rural areas of northern Minnesota, Wisconsin, and the western part of the Upper Peninsula of Michigan. RESULTS: Baseline data from the study are provided, and details of the design and methods are presented. The study outcomes are reported in part in "Lake Superior Rural Cancer Care Project Part II" in this issue and will be reported further in future issues. CLINICAL IMPLICATIONS: This article describes the hypotheses, design, and methods of the LSRCCP. The design and methods as well as the results of this study may be useful to cancer researchers and clinicians in rural areas across the United States.

Lake Superior Rural Cancer Care Project, part II: provider knowledge.

PURPOSE: The purpose of this article is to report the main learning outcomes of the Lake Superior Rural Cancer Care Project. DESCRIPTION OF STUDY: The authors designed and tested a multimodal intervention directed at rural providers and their healthcare systems in a large rural area in the north central United States. An experimental design was used to randomize rural providers at the group level. The intervention consisted of providing increased education for rural providers with a number of approaches, including the use of clinical opinion leaders. The main outcome of the intervention was knowledge scoring on discipline-specific cancer management tests. RESULTS: Knowledge scores for providers in the experimental group significantly increased from pretest to post-test: 66 to 79 for physicians (and physician assistants) (P=.02); 58 to 71 for nurses (P=.01); and 54 to 64 for pharmacists (P=.01). At post-test, participating providers in the experimental group performed significantly better on the knowledge tests (P <.01) than those in the control groups. CLINICAL IMPLICATIONS: This study may be the first to test educational interventions to improve rural providers' knowledge about cancer practice using an experimental design. The intervention may possibly change provider practice behaviors and, thus, patient outcomes, data that will be reported in a future issue. Finally, this educational intervention may prove useful for providers in other rural areas.

Repeated digital nerve block for pain control after tenolysis.

We describe a way of achieving immediate painfree mobilisation after tenolysis or tenosynovectomy in Zone II. Bupivacaine is instilled along the flexor tendon sheath through a thin percutaneous catheter with an antibacterial filter.

N-terminal and core-domain random mutations in human topoisomerase II alpha conferring bisdioxopiperazine resistance.

Random mutagenesis of human topoisomerase II alpha cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer > 37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.

Treatment of anthracycline extravasation with dexrazoxane.

Accidental extravasation of anthracyclines is a feared complication. Present treatment consists of local cooling and extensive surgical debridement, which often results in severe morbidity. All clinically important anthracyclines are topoisomerase II poisons that are antagonized by topoisomerase II catalytic inhibitors such as dexrazoxane. Therefore, we investigated whether dexrazoxane protects against extravasation lesions caused by anthracyclines. B6D2F1 mice received s.c. daunorubicin, doxorubicin, or idarubicin followed by systemic treatment with dexrazoxane or saline. One single systemic dose of dexrazoxane immediately after s.c. administration of doxorubicin, daunorubicin, or idarubicin reduced the tissue lesions (expressed as area under the curve of wound size times duration) by 96% (P < 0.0001), 70% (P < 0.0001), and 87% (P = 0.0004), respectively. Moreover, the treatment resulted in a statistically significant reduction in the fraction of mice with wounds as well as the duration of wounds. The induction of wounds was dose-dependent, as was the degree of protection by dexrazoxane. Dexrazoxane could be administered up to 3 h after the anthracycline without loss of protection. Triple-dosage of dexrazoxane tended to be more effective than a single injection. Dexrazoxane had no effect on lesions induced by hydrogen peroxide. This is the first report of use of a topoisomerase II catalytic inhibitor such as dexrazoxane in the treatment of anthracycline extravasation injuries. These convincing preclinical data represent a novel nontoxic approach that can easily be implemented into the clinical handling of accidental extravasation of anthracyclines.

A dose escalating study of topotecan preceding cisplatin in previously untreated patients with small-cell lung cancer.

BACKGROUND: The aim was to define the MTD of topotecan (TPT) given before cisplatin in patients with previously untreated SCLC. PATIENTS AND METHODS: Alternating cycles A and B to a total of 6 cycles were given. Cycle A: TPT days 1-5 and cisplatin (50 mg/m2) day 5. Cycle B consisted of teniposide, carboplatin, vincristine, and cisplatin. TPT was escalated at doses 0.75, 1.0, 1.25, and 1.5 mg/m2. DLT was defined for the first cycle as grade 4 neutropenia with fever or when lasting > 7 days, or grade 4 thrombocytopenia. RESULTS: Fifteen patients with limited disease and six patients with extensive disease were included. No episodes of DLT were recorded in the first cycles A and consequently 1.5 mg/m2 was defined as MTD. At 1.5 mg/m2 (11 patients, 30 cycles), four and three episodes of grade 4 thrombocytopenia and neutropenia lasting more than seven days occurred in subsequent cycles A. Thrombocytopenia and anaemia were cumulative as more cycles were administrated. Non-hematological toxicity was mild. The response rate was 86% (95% confidence interval (95% CI): 64%-97%) with 33% (95% CI: 15%-57%) achieving CR. CONCLUSIONS: 1.5 mg/m2 TPT can be delivered safely with 50 mg/m2 cisplatin on day 5 in patients with previously untreated SCLC.

Plasma Escherichia coli beta-galactosidase as a marker of tumor burden and response to experimental anti-neoplastic therapy in nude mice xenografted with lacZ transduced human tumor cells.

Genetic labeling of tumor cells with the Escherichia coli lacZ reporter gene, encoding the enzyme beta-galactosidase, is widely used for histochemical detection of micrometastases in mice. Recently, we have developed a novel, highly sensitive and specific immunocapture chemiluminescence assay for the quantitation of E. coli beta-galactosidase. This assay achieved a detection limit of 0.01 mU of E. coli beta-galactosidase per milliliter, and 97% signal recovery of purified enzyme added to mouse plasma. LacZ transduced MDA-MB-231 BAG human breast cancer cells grown in vitro released soluble beta-galactosidase into the culture medium, and the concentration found correlated with cell density. Growth of the same cells in nude mice produced readily measurable levels of E. coli beta-galactosidase enzyme activity in host plasma and a highly significant correlation could be demonstrated between the size of primary tumor xenografts and the host plasma level of E. coli beta-galactosidase activity. When mice bearing MDA-MB-231 BAG tumor xenografts were treated intravenously with a single injection of doxorubicin (5 mg/kg), the mean tumor volume after 16 days was reduced 4-fold in the group of doxorubicin-treated mice compared with saline-treated control mice, and the mean level of plasma E. coli beta-galactosidase was correspondingly reduced 3.8-fold in the doxorubicin-treated mice compared with control mice. Sensitive and specific measurement of soluble E. coli beta-galactosidase in blood, using an immunocapture chemiluminescence assay, thus provides objective assessment of tumor burden in mice xenografted with lacZ transduced human tumors. This assay may have important applications as a tool for determining the efficacy of new experimental anti-tumor agents.

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.

Differential cytotoxic pathways of topoisomerase I and II anticancer agents after overexpression of the E2F-1/DP-1 transcription factor complex.

The transcription factor complex E2F-1/DP-1 regulates the G1-to-S-phase transition and has been associated with sensitivity to the S-phase-specific anticancer agents camptothecin and etoposide, which poison DNA topoisomerase I and II, respectively. To investigate the relationship between E2F-1 and drug sensitivity in detail, we established human osteosarcoma U-20S-TA cells expressing full-length E2F-1/ DP-1 under the control of a tetracycline-responsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and activity as well as the number of camptothecin-induced DNA single- and double-strand breaks were unchanged in UEIDP-1/tc- cells with >10-fold E2F-1/DP-1 overexpression. However, UE1DP-1/tc- cells were hypersensitive to camptothecin in both a clonogenic assay and four different apoptotic assays. This indicates that camptothecin-induced toxicity in this model is due to the activation of an E2F-1/ DP-1-induced post-DNA damage pathway rather than an increase in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase IIalpha levels (but not topoisomerase IIbeta levels), together with topoisomerase IIalpha promoter activity, increased 2--3-fold in UE1DP-1/tc-cells. Furthermore, the number of etoposide-induced DNA single- and double-strand breaks increased in UE1DP-1/tc-cells together with a rise in clonogenic sensitivity to etoposide, but an equal apoptotic sensitivity to etoposide. The increase in topoisomerase IIalpha promoter activity in UE1DP-1/tc--cells was shown to be due to S-phase initiation per se because it was blocked by ectopic expression of dominant negative cyclin-dependent kinase 2. In conclusion, overexpression of E2F-1/DP-1 in U-20S-TA cells is sufficient to increase clonogenic sensitivity to both topoisomerase I- and II-targeted anticancer drugs. However, the mechanism by which this occurs appears to be qualitatively different. The UE1DP-1 cell model may be used to elucidate post-DNA damage mechanisms of cell death induced by topoisomerase I-directed anticancer agents.