Methods to Study Z-DNA-Induced Genetic Instability.

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic and eukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems.

Diagnostic delay of gynaecological cancer in women with postmenopausal bleeding.

INTRODUCTION: Postmenopausal bleeding is the primary reason for referral to the gynaecological fast-track suspected cancer programme due to an elevated risk of endometrial cancer. The aim of this study was to examine the diagnostic flow among women with postmenopausal bleeding. METHODS: Data were collected and analysed from 362 women at Sygehus Sønderjylland referred with the International Classification of Diseases, tenth edition, diagnosis "DN950 postmenopausal bleeding" from 2015 to 2019. RESULTS: We found a mean 83-day period between the initial consultation and the final cancer diagnosis. Combined, the 362 women underwent 354 diagnostic procedures of which 204 were endometrial sampling with aspiration (vabrasio). In 44% of the cases, sampling by vacuum aspirator was either unsuccessful due to pain or cervical stenosis or was deemed insufficient for pathological assessment. Gynaecological cancer was diagnosed in 16 (4%) of the women, hereof 13 (3.6%) had endometrial cancer. CONCLUSIONS: We found a remarkable delay not complying with the intentions of national guidelines with respect to final diagnostics of endometrial cancer. Vacuum aspirator is a frequently used diagnostic tool, but has shortcomings in relation to the success rate of the procedure and insufficient sampling. Gynaecological cancer was found at a rate of 3-5% as reported by other Danish studies. Because of the limitations associated with a one-step diagnostic procedure with vabrasio, attention to follow-up may reduce diagnostic delay. FUNDING: none. TRIAL REGISTRATION: This study was registered with the Region of South Denmark (no. 21/18387) and Sygehus Sønderjylland (no. 1298-001) as a quality improvement project.

Distinct DNA repair pathways cause genomic instability at alternative DNA structures.

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF-dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the sub-molecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.

Lexical access in semantic variant PPA: Evidence for a post-semantic contribution to naming deficits.

The most salient clinical symptom of semantic variant primary progressive aphasia (PPA) is a profound and pervasive anomia. These patients' naming impairments have been shown to reflect in large part a domain-general deterioration of conceptual knowledge that impacts both linguistic and non-linguistic processing. However, it is possible that post-semantic stages of lexical access may also contribute to naming deficits. To clarify the stages at which lexical access breaks down in semantic variant PPA, eleven French-speaking patients were asked to name objects, and were then queried for semantic, lexical-syntactic, and word form information pertaining to the items they could not name. Specifically, our goal was to determine whether patients can access intermediate representations known as lemmas, which mediate the arbitrary mapping between semantic representations and word forms (phonological and orthographic forms). The French language was chosen for this study because nouns in French are marked for grammatical gender, a prototypical type of lexical-syntactic information, represented at the level of the lemma. Access to word form information is also dependent on lemma access under some theoretical views. We found that six of the eleven patients showed partial access to either lexical-syntactic properties of unnamed items (grammatical gender), word form information (initial letter), or both. Access to these types of information suggests that a lemma has been retrieved, implying a breakdown at the post-semantic stage of word form retrieval. Our results suggest that although degraded conceptual knowledge is the main cause of naming deficits in semantic variant PPA, in some patients, a post-semantic component also contributes to the impairment.

Dissecting Cellulitis of the Scalp with Excellent Response to Isotretinoin.

An 18-year-old man presented for evaluation of a 1-year history of painful nodules on the scalp with associated hair loss. Physical examination revealed multiple confluent, fluctuant, boggy nodules on the scalp with overlying alopecic patches. Based on these findings, a diagnosis of dissecting cellulitis of the scalp was made and the patient was successfully treated with oral isotretinoin therapy.

Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer.

Antimetabolite chemotherapy remains an essential cancer treatment modality, but often produces only marginal benefit due to the lack of tumor specificity, the development of drug resistance, and the refractoriness of slowly proliferating cells in solid tumors. Here, we report a novel strategy to circumvent the proliferation-dependence of traditional antimetabolite-based therapies. Triplex-forming oligonucleotides (TFOs) were used to target site-specific DNA damage to the human c-MYC oncogene, thereby inducing replication-independent, unscheduled DNA repair synthesis (UDS) preferentially in the TFO-targeted region. The TFO-directed UDS facilitated incorporation of the antimetabolite, gemcitabine (GEM), into the damaged oncogene, thereby potentiating the anti-tumor activity of GEM. Mice bearing COLO 320DM human colon cancer xenografts (containing amplified c-MYC) were treated with a TFO targeted to c-MYC in combination with GEM. Tumor growth inhibition produced by the combination was significantly greater than with either TFO or GEM alone. Specific TFO binding to the genomic c-MYC gene was demonstrated, and TFO-induced DNA damage was confirmed by NBS1 accumulation, supporting a mechanism of enhanced efficacy of GEM via TFO-targeted DNA damage-induced UDS. Thus, coupling antimetabolite chemotherapeutics with a strategy that facilitates selective targeting of cells containing amplification of cancer-relevant genes can improve their activity against solid tumors, while possibly minimizing host toxicity.

Characterization and drug resistance patterns of Ewing's sarcoma family tumor cell lines.

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.

FOXM1 is an oncogenic mediator in Ewing Sarcoma.

Ewing Family Tumors (Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor) are common bone and soft tissue malignancies of childhood, adolescence and young adulthood. Chromosomal translocation in these tumors produces fusion oncogenes of the EWS/ETS class, with EWS/FLI1 being by far the most common. EWS/ETS chimera are the only well established driver mutations in these tumors and they function as aberrant transcription factors. Understanding the downstream genes whose expression is modified has been a central approach to the study of these tumors. FOXM1 is a proliferation associated transcription factor which has increasingly been found to play a role in the pathogenesis of a wide range of human cancers. Here we demonstrate that FOXM1 is expressed in Ewing primary tumors and cell lines. Reduction in FOXM1 expression in Ewing cell lines results in diminished potential for anchorage independent growth. FOXM1 expression is enhanced by EWS/FLI1, though, unlike other tumor systems, it is not driven by expression of the EWS/FLI1 target GLI1. Thiostrepton is a compound known to inhibit FOXM1 by direct binding. We show that Thiostrepton diminishes FOXM1 expression in Ewing cell lines and this reduction reduces cell viability through an apoptotic mechanism. FOXM1 is involved in Ewing tumor pathogenesis and may prove to be a useful therapeutic target in Ewing tumors.

Treatment outcomes with methylphenidate formulations among patients with ADHD: retrospective claims analysis of a managed care population.

OBJECTIVE: Describe treatment patterns, resource use, and predictors of methylphenidate (MPH) switch among children (6-12 years), adolescents (13-17 years), and adults (≥ 18 years) with attention-deficit/hyperactivity disorder (ADHD). METHODS: This retrospective U.S. managed care database study used medical, pharmacy, and enrollment data to examine treatment patterns among patients with ≥ 1 ADHD diagnosis code (ICD-9 314.00-314.9), MPH pharmacy claims during 01/01/2004-09/30/2006, and no ADHD pharmacy claims in prior 6 months. Patients were followed for 1 year for dosage change, switch (change to non-MPH treatment), augmentation, persistence (number days on index medication) and adherence (days supplied/days persistent). End points were assessed by age group and MPH formulation. Cox proportional hazards modeling was conducted to determine predictors of MPH switch. RESULTS: Among 23,860 MPH users, 51.4% had a dosing change, 14% switched to a non-MPH agent, and 4% augmented MPH therapy. Among those prescribed long-acting (LA) MPH (N = 14,681), switching rates were 14% for children, 13% for adolescents, and 16% for adults. Augmentation rates for LA MPH were <5%. Overall, 53% of patients were adherent with mean persistence of 219 days. For the subgroup of patients prescribed LA MPH (n = 14,681), adherence ranged from 49% (adolescents) to 59% (children); persistence varied between 183 days (adults) to 256 days (children). During the 1-year follow-up, office/clinic visits were the major driver of health care resource use in MPH patients (mean 9.7 visits/patient). Patients with psychiatric comorbidity utilized significantly greater services. Predictors of MPH switch included psychiatric comorbidity (hazards ratio [HR] 1.37; 95% confidence interval [CI] = 1.26-1.48; p < 0.0001) and specialty prescribers (HR 1.19, 95% CI = 1.04-1.35; p = 0.011). Potential limitations of this study include use of claims data for definition of drug usage; inclusion of medications approved for use in ADHD; assessment of switching that may not have captured short-term augmentation; absence of economic, clinical and other variables from the claims dataset that may have influenced treatment selection, and outcomes. The 6-month baseline period to determine newly treated patients may not guarantee exclusion of all previously treated patients who restart therapy after an extended period. CONCLUSIONS: Children exhibited the highest persistence of MPH users. ADHD patients on MPH therapy with a psychiatric comorbidity may require additional follow-up to help improve adherence and reduce health care resource use.

Direct costs of chronic obstructive pulmonary disease among managed care patients.

PURPOSE: To estimate patient- and episode-level direct costs of chronic obstructive pulmonary disease (COPD) among commercially insured patients in the US. METHODS: In this retrospective claims-based analysis, commercial enrollees with evidence of COPD were grouped into five mutually exclusive cohorts based on the most intensive level of COPD-related care they received in 2006, ie, outpatient, urgent outpatient (outpatient care in addition to a claim for an oral corticosteroid or antibiotic within seven days), emergency department (ED), standard inpatient admission, and intensive care unit (ICU) cohorts. Patient- level COPD-related annual health care costs, including patient- and payer-paid costs, were compared among the cohorts. Adjusted episode-level costs were calculated. RESULTS: Of the 37,089 COPD patients included in the study, 53% were in the outpatient cohort, 37% were in the urgent outpatient cohort, 3% were in the ED cohort, and the standard admission and ICU cohorts together comprised 6%. Mean (standard deviation, SD) annual COPD-related health care costs (2008 US$) increased across the cohorts (P < 0.001), ranging from $2003 ($3238) to $43,461 ($76,159) per patient. Medical costs comprised 96% of health care costs for the ICU cohort. Adjusted mean (SD) episode-level costs were $305 ($310) for an outpatient visit, $274 ($336) for an urgent outpatient visit, $327 ($65) for an ED visit, $9745 ($2968) for a standard admission, and $33,440 for an ICU stay. CONCLUSION: Direct costs of COPD-related care for commercially insured patients are driven by hospital stays with or without ICU care. Exacerbation prevention resulting in reduced need for inpatient care could lower costs.

Pharmacological treatment patterns among patients with attention-deficit/hyperactivity disorder: retrospective claims-based analysis of a managed care population.

OBJECTIVE: To develop a descriptive profile of attention-deficit/hyperactivity disorder (ADHD) pharmacological treatment patterns in terms of persistence, adherence, augmentation, switching, and dosing changes; and to assess differences in treatment patterns with regard to ADHD medication type, class, and duration of action. METHODS: This retrospective claims database analysis used medical data, pharmacy data, and enrollment information to examine treatment patterns among patients with at least one claim with a diagnosis code for ADHD and a filled prescription for ADHD medication (index therapy) during the period 01 January 2004 through 30 September 2006. Treatment persistence and adherence (days supplied/days persistent) were calculated. Dose changes, medication switching, and augmentation were analyzed at three levels of comparison: class (stimulant vs nonstimulant [atomoxetine]), drug type (amphetamine vs methylphenidate), and duration of action (short, intermediate, long). Statistical comparisons were made using the chi-square test for proportions and Student's t-test or the F-test from one-way ANOVA for means. RESULTS: Of 60,010 patients meeting eligibility criteria, 58.4% were younger than age 18. Most (78.4%) were prescribed a stimulant as their index therapy. Persistence and adherence were greater for patients on stimulants (vs the nonstimulant), for patients on amphetamines (vs methylphenidates), and for patients on long-acting medications (vs short- and intermediate-acting medications; all p < 0.0001). Index drug dose changes were least likely among individuals taking the nonstimulant (vs stimulants), methylphenidates (vs amphetamines), or intermediate-acting drugs (vs short- and long-acting drugs; all p < 0.0001), and medication switches were more frequent among those on nonstimulants, methylphenidates, or short-acting drugs (all p < 0.0001). Subjects taking long-acting medication were less likely to augment with a drug with a different duration of action than those taking intermediate- or short-acting medication (p < 0.0001). This claims-based study is limited by possible discrepancies between claims and patient behaviors (i.e., a claim for a prescription does not necessarily indicate that the medication was taken as prescribed). CONCLUSIONS: Patients were more stable on treatment compared with their respective comparator groups if their index therapy was a stimulant, long-acting drug, or amphetamine.

GLI1 is a central mediator of EWS/FLI1 signaling in Ewing tumors.

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

Pre-transplant risk factors for chronic renal dysfunction after pediatric heart transplantation: a 10-year national cohort study.

BACKGROUND: Chronic renal dysfunction may develop after pediatric heart transplantation (PHTx). We examined the incidence of end-stage renal disease (ESRD) and chronic renal insufficiency (CRI) after PHTx, the associated pre-transplant patient characteristics, and impact of renal disease on survival. METHODS: Data sources included the Scientific Registry of Transplant Recipients, Centers for Medicare and Medicaid Services and the Social Security Death Master File. All PHTx recipients (age <18 years) in the USA from 1990 to 1999 who survived >1 year were included. ESRD was defined as long-term dialysis and/or kidney transplant. CRI was defined as creatinine >2.5 mg/dl, including those with ESRD. Relationships between pre-transplant characteristics and time to ESRD and CRI were analyzed using Cox proportional hazards models. The effect of renal disease on survival was analyzed using time-dependent Cox models. RESULTS: During the mean follow-up of 7 years (range 1 to 14 years), 61 of 2,032 (3%) PHTxs developed ESRD. Ten-year actuarial risks for ESRD and CRI were 4.3% and 11.8%, respectively. In a multivariate analysis, significant risk factors for ESRD were: hypertrophic cardiomyopathy; African-American race; intensive care unit (ICU) stay or extracorporeal membrane oxygenation (ECMO) at time of transplant; and pre-transplant diabetes. Risk factors for CRI were: pre-transplant dialysis; hypertrophic cardiomyopathy; African-American race; and previous transplant. Adjusted risk of death in those who developed CRI was 9-fold higher than in those who did not (p < 0.0001). CONCLUSIONS: After PHTx there is an increasing risk for CRI and ESRD over time. Recipients with the characteristics identified in this study may be at greater risk. Development of renal disease significantly increases the risk of post-transplant mortality.

Targeting oncogenes to improve breast cancer chemotherapy.

Despite recent advances in treatment, breast cancer remains a serious health threat for women. Traditional chemotherapies are limited by a lack of specificity for tumor cells and the cell cycle dependence of many chemotherapeutic agents. Here we report a novel strategy to help overcome these limitations. Using triplex-forming oligonucleotides (TFOs) to direct DNA damage site-specifically to oncogenes overexpressed in human breast cancer cells, we show that the effectiveness of the anticancer nucleoside analogue gemcitabine can be improved significantly. TFOs targeted to the promoter region of c-myc directly inhibited gene expression by approximately 40%. When used in combination, specific TFOs increased the incorporation of gemcitabine at the targeted site approximately 4-fold, presumably due to induction of replication-independent DNA synthesis. Cells treated with TFOs and gemcitabine in combination showed a reduction in both cell survival and capacity for anchorage-independent growth (approximately 19% of untreated cells). This combination affected the tumorigenic potential of these cancer cells to a significantly greater extent than either treatment alone. This novel strategy may be used to increase the range of effectiveness of antitumor nucleosides in any tumor which overexpresses a targetable oncogene. Multifaceted chemotherapeutic approaches such as this, coupled with triplex-directed gene targeting, may lead to more than incremental improvements in nonsurgical treatment of breast tumors.

Z-DNA-forming sequences generate large-scale deletions in mammalian cells.

Spontaneous chromosomal breakages frequently occur at genomic hot spots in the absence of DNA damage and can result in translocation-related human disease. Chromosomal breakpoints are often mapped near purine-pyrimidine Z-DNA-forming sequences in human tumors. However, it is not known whether Z-DNA plays a role in the generation of these chromosomal breakages. Here, we show that Z-DNA-forming sequences induce high levels of genetic instability in both bacterial and mammalian cells. In mammalian cells, the Z-DNA-forming sequences induce double-strand breaks nearby, resulting in large-scale deletions in 95% of the mutants. These Z-DNA-induced double-strand breaks in mammalian cells are not confined to a specific sequence but rather are dispersed over a 400-bp region, consistent with chromosomal breakpoints in human diseases. This observation is in contrast to the mutations generated in Escherichia coli that are predominantly small deletions within the repeats. We found that the frequency of small deletions is increased by replication in mammalian cell extracts. Surprisingly, the large-scale deletions generated in mammalian cells are, at least in part, replication-independent and are likely initiated by repair processing cleavages surrounding the Z-DNA-forming sequence. These results reveal that mammalian cells process Z-DNA-forming sequences in a strikingly different fashion from that used by bacteria. Our data suggest that Z-DNA-forming sequences may be causative factors for gene translocations found in leukemias and lymphomas and that certain cellular conditions such as active transcription may increase the risk of Z-DNA-related genetic instability.