The psychosocial experience and needs of children newly diagnosed with type 1 diabetes from their own perspective: a systematic and narrative review.

AIM: To understand the psychosocial experience of children and identify their primary support needs following a type 1 diabetes diagnosis. METHODS: A systematic review and narrative synthesis of the literature in this area was conducted. RESULTS: A total of 32 studies were included in the review. At diagnosis, the majority of children experienced high distress, including grief, anxiety, anger, irritation and injection anxiety. The intensity of this reaction decreased rapidly over the following weeks. At diagnosis, rates of depressive symptoms, anxiety, stress disorders and suicidal ideation were elevated. The initial reaction tended to peak shortly after diagnosis and declined over the following year. Thereafter, symptoms of depression and anxiety appeared to increase once again, corresponding with the children's experience of diabetes management and implications as being more difficult and upsetting. Injection anxiety, distress and depressive symptoms persisted for a smaller group of children. CONCLUSION: The initial high prevalence of depressive symptoms following diagnosis is transitional and should be regarded as a normal adaptive response. To facilitate this adaptive process, specific child-centred support should be prioritized as an integrated part of early diabetes care. Our findings point to five inter-related support needs following a type 1 diabetes diagnosis: (1) children need time to adjust to the diagnosis; (2) children need supportive relationships; (3) children need an opportunity for meaningful participation and appropriate protection; (4) children need to engage and explore; and (5) children need to feel supported, but not different.

Families' Adherence to a Family-Based Childhood Obesity Intervention: A Qualitative Study on Perceptions of Communicative Authenticity.

Childhood obesity is associated with severe physical and psychological health problems. Interventions are often directed at the whole family, but the literature provides no clear indication of the characteristics of an effective family-based intervention. The objective of the present paper is to study whether and how an analytical framework focusing on communicative authenticity can be used to observe and elaborate upon aspects of adherence in relation to health behavior change in a concrete family-based intervention. We do this by focusing on the families' experiences with a Shared-care health education intervention and thus explore the association between families' self-reported experience and their adherence to the intervention. The dataset consists of 21 in-depth semi-structured family interviews. The study shows that the Shared-care model has potential, but that this potential is rarely fulfilled in the intervention form under study. The sharing of care adds potential for several kinds of communicative authenticity because families are met by both the medical knowledge authority at the hospital and the local nurses in their municipality. It is, however, a significant finding that the families rarely benefit from this potential authenticity. Using theories of authenticity in this context adds theoretical and analytical potential and manages to incorporate elements of participation in tasks and practices of value, a sense of who we are and what we know, negotiation of meaning, emphatic caring, consistency between values and actions, and horizons of significance. The article brings new perspectives on how family-based interventions could be tailored to communicatively suit individual families.

Adolescents' health identities: a qualitative and theoretical study of health education courses.

In this paper we highlight the role of health identity in health education for adolescents. In school-based approaches to health education, it is often difficult to present health information and health communication in ways that make sense and appeal to adolescents. The concept of health identity has the potential of providing an analytical framework as well as practical recommendations for these issues and problem areas. The paper reports on an empirical study of elements of health identity in the context of health courses for adolescents--using interview data, observation studies and a theoretical construction focussing on self-observation, horizons of significance, expectational structures and social imaginaries. We present our findings in four main themes: 1) Adolescents' health identities are observed and developed when things matter, 2) Adolescents' health identities are observed and developed in relational contexts, 3) Adolescents' health identities are developed on the basis of observations of past, present and future health and 4) Adolescents' health identities are clearly defined. The paper provides health practitioners with important knowledge about why and how health-educational approaches should focus on health identity in order to provide conditions that create a significant health educating effect for all adolescents--not just for those who are already healthy.

DNA topoisomerase II enzymes as molecular targets for cancer chemotherapy.

DNA topoisomerase II enzymes regulate essential cellular processes by altering the topology of chromosomal DNA. These enzymes function by creating transient double-stranded breaks in the DNA molecule that allow the DNA strands to pass through each other and unwind or unknot tangled DNA. Because of the integral role of topoisomerases in regulating DNA metabolism, these enzymes are vital for cell survival. Several clinically active antitumor agents target these enzymes. Mammalian cells contain two topoisomerase II isozymes that are encoded by different genes: topoisomerase IIα and IIß. Although, both isozymes are homologous and exhibit similar catalytic activity, they are differentially regulated and are involved in distinct biological functions. The topoisomerase IIα and topoisomerase IIß enzymes are regulated by post-translational modifications, including sumoylation, ubiquitination and phosphorylation. These post-translational modifications influence the biologic and catalytic activity of the enzyme and affect sensitivity of cells to topoisomerase II-targeted drugs. In this review, we describe how the catalytic and biologic activity of the topoisomerase II enzyme is regulated and discuss the mechanisms by which chemotherapeutic agents that target these enzymes function. Given the potential importance of site-specific modifications, in particular phosphorylation, in regulating sensitivity to topoisomerase II-targeted drugs, we discuss the potential role of altered topoisomerase II phosphorylation in development of drug resistance, which is often a limiting factor in the treatment of cancer.

Downregulation of topoisomerase IIbeta in myeloid leukemia cell lines leads to activation of apoptosis following all-trans retinoic acid-induced differentiation/growth arrest.

Among the topoisomerase (topo) II isozymes (alpha and beta), topo IIbeta has been suggested to regulate differentiation. In this study, we examined the role of topo IIbeta in all-trans retinoic acid (ATRA)-induced differentiation of myeloid leukemia cell lines. Inhibition of topo IIbeta activity or downregulation of protein expression enhanced ATRA-induced differentiation/growth arrest and apoptosis. ATRA-induced apoptosis in topo IIbeta-deficient cells involved activation of the caspase cascade and was rescued by ectopic expression of topo IIbeta. Gene expression profiling led to the identification of peroxiredoxin 2 (PRDX2) as a candidate gene that was downregulated in topo IIbeta-deficient cells. Reduced expression of PRDX2 validated at the mRNA and protein level, in topo IIbeta-deficient cells correlated with increased accumulation of reactive oxygen species (ROS) following ATRA-induced differentiation. Overexpression of PRDX2 in topo IIbeta-deficient cells led to reduced accumulation of ROS and partially reversed ATRA-induced apoptosis. These results support a role for topo IIbeta in survival of ATRA-differentiated myeloid leukemia cells. Reduced expression of topo IIbeta induces apoptosis in part by impairing the anti-oxidant capacity of the cell owing to downregulation of PRDX2. Thus, suppression of topo IIbeta and/or PRDX2 levels in myeloid leukemia cells provides a novel approach for improving ATRA-based differentiation therapy.

Topoisomerase II poisoning by ICRF-193.

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.

High body mass index does not predict mortality in older people: analysis of the Longitudinal Study of Aging.

OBJECTIVE: To determine the excess mortality associated with obesity (defined by body mass index (BMI)) in older people, with and without adjustment for other risk factors associated with mortality and for demographic factors. DESIGN: Retrospective cohort analysis of the Longitudinal Study of Aging (LSOA). SETTING: Nationally representative sample of community-dwelling older people. PARTICIPANTS: Seven thousand five hundred and twenty-seven participants age 70 and older in 1984. MEASUREMENTS: We used Cox regression to calculate proportional hazards ratios for mortality over 96 months. We tested the hypothesis that increased BMI (top 15%) increased mortality rates in older people. RESULTS: Death occurred in 38% of the cohort: 54% of the thin (lowest 10% of the population, BMI <19.4 kg/m(2)), 33% of the obese (highest 15%, BMI> 28.5 kg/m(2)), and 37% of the remaining participants (normal) died. Adjustment for demographic factors, health services utilization, and functional status still demonstrated reduced mortality in obese older people (hazard ratio 0.86, 95% confidence interval (CI) = 0.77-0.97) compared with normal. After adjustment, thin older people remained more likely to die (hazard ratio 1.46, 95% CI = 1.30-1.64) than normal older people. Sensitivity analyses for income, mortality during the first two years of follow-up, and medical comorbidities did not substantively alter the conclusions. CONCLUSION: Obesity may be protective compared with thinness or normal weight in older community-dwelling Americans.

Medicaid reimbursement and the quality of nursing home care.

An influential series of papers have found that an increase in Medicaid reimbursement decreases the level of nursing home quality in the presence of certificate-of-need (CON) and construction moratorium regulations. Using more recent national data, an outcome-oriented measure of quality, and an alternative methodology, this study finds a positive, albeit small, effect of reimbursement on quality. Although this paper does find some evidence of excess demand within the market for nursing home care, this new finding is attributed to a decline in nursing home utilization over the last two decades.

Does an increase in the Medicaid reimbursement rate improve nursing home quality?

OBJECTIVES: Numerous studies have documented poor nursing home quality over the last 3 decades. Previous research has questioned the effectiveness of Medicaid reimbursement policy in improving quality in the presence of certificate-of-need (CON) and construction moratoria regulation. This study evaluated how the Medicaid reimbursement rate may influence a home's decision to provide quality under CON and moratoria. METHODS: Linking national data from the On-Line Survey, Certification, and Reporting system, the Area Resource File, and aggregate reimbursement information, the author examined the effect of Medicaid reimbursement on a range of quality measures in the context of CON and moratoria. RESULTS: An increase in Medicaid reimbursement improved quality as measured by professional staffing, but there was not a statistically significant effect when quality was measured by nonprofessional staffing, various procedural measures, or regulatory deficiencies. However, this study did not support previous research showing a negative effect of Medicaid reimbursement on nursing home quality in the context of CON laws. DISCUSSION: This study supports recent trends suggesting that nursing home CON laws may be lessening in importance for the nursing home market. Nevertheless, further work is necessary to determine the quality returns to increased Medicaid reimbursement.

Cytotoxic mechanism of XK469: resistance of topoisomerase IIbeta knockout cells and inhibition of topoisomerase I.

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.

Cell cycle phase specificity in the potentiation of etoposide-induced DNA damage and apoptosis by KN-62, an inhibitor of calcium-calmodulin-dependent enzymes.

The cell cycle phase-dependent induction of DNA damage and apoptosis by etoposide (VP-16) and its modulation by 1-[N,O-bis(1, 5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-piperazine (KN-62), an inhibitor of calcium-calmodulin-dependent enzymes, were examined in sensitive (HL-60/S) and VP-16-resistant (HL-60/DOX0.05) HL-60 cells. Cells from exponential-phase cultures were enriched by centrifugal elutriation into G(1), S, and G(2)+M fractions. Modulation of VP-16-induced apoptosis by KN-62 in HL-60/S cells was apparent only in the S phase at the IC(50) concentration. However, in the HL-60/DOX0.05 cells, significant (P < 0.001) potentiation of VP-16-induced apoptosis by a non-cytotoxic concentration of 2 microM KN-62 was apparent in cells in the G(1), S, and G(2)+M phases, as well as over the entire concentration range tested. VP-16-induced apoptosis and its potentiation by a non-cytotoxic concentration of 2 microM KN-62 were correlative with drug-stabilized DNA cleavable complex formation based on a band depletion assay. In agreement with the results on apoptosis in the resistant HL-60/DOX0.05 cells, the enhanced depletion of the alpha and beta isoforms of topoisomerase II by VP-16 + KN-62 was observed in G(1), S, and G(2)+M cells. Results suggest that the effects of KN-62 in reversing resistance are based on its role as a potent sensitizer of VP-16-induced DNA damage and apoptosis in a cell cycle phase-independent manner.

Roles of NF-kappaB and 26 S proteasome in apoptotic cell death induced by topoisomerase I and II poisons in human nonsmall cell lung carcinoma.

Activation of signaling pathways after DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or NSCLC-5) cells with the topo I poison SN-38 or the topo II poison etoposide (VP-16) leads to activation of NF-kappaB before induction of apoptosis. Inhibiting the degradation of IkappaBalpha by pretreatment with the proteasome inhibitor MG-132 significantly inhibited NF-kappaB activation and apoptosis but not DNA damage induced by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with dominant negative mutant IkappaBalpha (mIkappaBalpha) inhibited SN-38 or VP-16 induced transcription and DNA binding activity of NF-kappaB without altering drug-induced apoptosis. Regulation of apoptosis by mitochondrial release of cytochrome c and activation of pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by effector caspases 3 and 7 was similar in neo and mIkappaBalpha cells treated with SN-38 or VP-16. In contrast to pretreatment with MG-132, exposure to MG-132 after SN-38 or VP-16 treatment of neo or mIkappaBalpha cells decreased cell cycle arrest in the S/G2 + M fraction and enhanced apoptosis compared with drug alone. In summary, apoptosis induced by topoisomerase poisons in NSCLC cells is not mediated by NF-kappaB but can be manipulated by proteasome inhibitors.

The effect of state regulations on motor vehicle fatalities for younger and older drivers: a review and analysis.

Policymakers have had a long-standing interest in improving the motor vehicle safety of both younger and older drivers. Although younger and older drivers share the distinction of having more crashes and fatalities per mile driven than other age groups, the problems posed by these two groups stem from different origins and manifest in different ways. A number of state-level policies and regulations may affect the number of motor vehicle crashes and fatalities in these two high-risk groups. A critical review of the existing literature in regard to the risk factors and the effects of various policy measures on motor vehicle crashes in these two high-risk populations provides direction for policymakers and high-priority areas of interest for the research community.

Using MT(-/-) mice to study metallothionein and oxidative stress.

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.

Altered drug interaction and regulation of topoisomerase IIbeta: potential mechanisms governing sensitivity of HL-60 cells to amsacrine and etoposide.

Topoisomerase II (topo II), an enzyme essential for cell viability, is present in mammalian cells as the alpha- and beta-isoforms. In human leukemia HL-60/S or HL-60/doxorubicin (DOX)0.05 cells, the levels of topo IIalpha- or beta-protein were similar in either asynchronous exponential or synchronized cultures. Although topo IIalpha was hypophosphorylated in HL-60/DOX0.05 compared with HL-60/S cells, both overall and site-specific hyperphosphorylation of topo IIbeta was apparent in HL-60/DOX0.05 compared with HL-60/S cells. The phosphorylation of topo IIalpha and not beta was enhanced in the S and G(2) + M phases of HL-60/S cells. In contrast, an increase in the phosphorylation of topo IIbeta compared with alpha was apparent in the G(1) and S phases of HL-60/DOX0.05 cells. The cytotoxicity and depletion of topo IIalpha or beta in cells treated with drug for 1 h revealed that mole-for-mole, amsacrine was 2-fold more effective than etoposide in killing HL-60/S or HL-60/DOX0.05 cells and in depleting the beta versus alpha topo II protein. Present results demonstrate that: 1) hyperphosphorylation of topo IIbeta in HL-60/DOX0.05 cells may be a compensatory consequence of the hypophosphorylation of topo IIalpha to maintain normal topo II function during proliferation, and 2) enhanced sensitivity of HL-60/S or HL-60/DOX0.05 cells to amsacrine may be due to the preferential interaction and depletion of topo IIbeta.

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Altered expression and activity of topoisomerases during all-trans retinoic acid-induced differentiation of HL-60 cells.

Regulation of topoisomerase II (TOPO II) isozymes alpha and beta is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIalpha and TOPO IIbeta proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (alpha and beta) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIbeta protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIbeta protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 micromol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 micromol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 micromol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIbeta protein levels are posttranscriptionally regulated and that degradation of TOPO IIbeta is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (alpha + beta) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIbeta may have a specific role in transcription of genes involved in differentiation with RA treatment.

Tumor cell resistance to topoisomerase II poisons: role for intracellular free calcium in the sensitization by inhibitors or calcium-calmodulin-dependent enzymes.

Tumor cell resistance to inhibitors of topoisomerase II (topo II) is associated frequently with the overexpression of P-glycoprotein (PGP), and strategies to overcome resistance are focused on restoring defects in drug accumulation. Inhibitors of calcium-calmodulin-dependent enzymes sensitize resistant tumor cells to the topo II poison etoposide (VP-16) by enhancing DNA damage and an apoptotic response. In the present study, we have investigated the consequences of buffering intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra(acetoxy-methyl) ester (BAPTA-AM) on the sensitizing effects of the calmodulin-dependent protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-piperazine (KN-62) in etoposide-resistant human leukemia HL-60 (HL-60/ADR0.05) cells. In cells pretreated with 20 microM BAPTA-AM for 2 hr, extracellular ATP failed to trigger intracellular calcium transients, and no effects on the accumulation of VP-16 were apparent. Also, the effect of KN-62 in significantly (P=0.002 to 0.042) enhancing the accumulation of VP-16 in HL-60/ADR0.05 cells was unaffected due to pretreatment with BAPTA-AM. In contrast, pretreatment with BAPTA-AM reduced the DNA damage induced by VP-16, and significantly (P=0.038) reversed the enhancement by KN-62 of VP-16-stabilized topo II-mediated DNA cleavable complex formation. The pretreatment of HL-60/ADR0.05 cells with BAPTA-AM was also associated with the hypophosphorylation of topo IIalpha. Consistent with the ability of BAPTA-AM to circumvent the potentiation by KN-62 of VP-16-induced DNA damage, survival of cells treated with 40 microM VP-16 in the absence of KN-62 and 10 microM VP-16 in the presence of KN-62 was significantly (P=0.026 to 0.031) higher due to BAPTA-AM pretreatment. Results demonstrate that intracellular calcium transients could play a key role in the sensitization of etoposide-resistant tumor cells by inhibitors of calcium-calmodulin-dependent enzymes.

Specific interaction of wild-type and truncated mouse N-methylpurine-DNA glycosylase with ethenoadenine-containing DNA.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, is responsible for the removal of a wide variety of alkylated base lesions in DNA, e.g., N-alkylpurines and cyclic ethenoadducts of adenine, guanine, and cytosine. These lesions, some of which are mutagenic and toxic, are generated endogenously or by genotoxic agents such as N-alkylnitrosamines and vinyl chloride. Wild-type mouse MPG, expressed from recombinant baculovirus, was purified to near homogeneity for studying its specific interaction with substrate, 1,N6-ethenoadenine- (epsilonA-) containing DNA. Electrophoretic mobility shift assays (EMSA) indicated that MPG formed a specific complex with a 50-mer epsilonA-containing duplex oligonucleotide. This complex was shown to be a transient reaction intermediate, because it could be formed only with the unreacted substrate and contained active enzyme molecules. DNA footprinting studies confirmed the specific binding of the protein to the epsilonA-containing duplex oligonucleotide; eight nucleotides on the epsilonA-containing strand and 16-17 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion. A systematic deletion analysis of MPG was carried out in order to determine the minimally sized polypeptide capable of forming a stable substrate complex that is also suitable for characterization by NMR spectroscopy and X-ray crystallography. A truncated polypeptide (NDelta100CDelta18) lacking 100 and 18 amino acid residues from the amino and carboxyl termini, respectively, was found to be the minimal size that retained activity. The truncated and wild-type enzymes have similar kinetic properties. Moreover, both EMSA and DNase I footprinting studies indicated identical pattern of specific binding by the truncated and full-length polypeptides. Removal of five and nine additional residues from the amino- and carboxyl-termini of this polypeptide, respectively, resulted in a complete loss of activity. These results suggest that minimal structural change occured as a result of truncation in the NDelta100CDelta18 mutant, which may thus be suitable for elucidating the structure and mechanism of MPG.