Ataxia telangiectasia and rad3-related kinase contributes to cell cycle arrest and survival after cisplatin but not oxaliplatin.

The DNA cross-linking agents cisplatin and oxaliplatin are widely used in the treatment of human cancer. Lesions produced by these agents are widely known to activate the G1 and G2 cell cycle checkpoints. Less is known about the role of the intra-S-phase checkpoint in the response to these agents. In the present study, two different cell lines expressing a dominant-negative kinase dead (kd) version of the ataxia telangiectasia and rad3-related (ATR) kinase in an inducible fashion were examined for their responses to these two platinating agents and a variety of other DNA cross-linking drugs. The expression of the kdATR allele markedly sensitized the cells to cisplatin, but not to oxaliplatin, as assessed by inhibition of colony formation, induction of apoptosis, and cell cycle analysis. Similar differences in survival were noted for melphalan (ATR dependent) and 4-hydroperoxycyclophosphamide (ATR independent). Further experiments showed that ATR function is not necessary for removal of Pt-DNA adducts. The predominant difference between the responses to the two platinum drugs was the presence of a drug-specific ATR-dependent S-phase arrest after cisplatin but not oxaliplatin. These results indicate that involvement of ATR in the response to DNA cross-linking agents is lesion specific. This observation might need to be taken into account in the development and use of ATR or Chk1 inhibitors.

Naildex: pilot evaluation of an onychodystrophy severity instrument.

A valid and reliable measure that captures onychodystrophy disease severity is important for both clinical and research applications. Three hundred and twenty-two patients at two Veterans Affairs Medical Centers with clinical evidence of onychodystrophy suggesting onychomycosis (at least 25% in a distal subungual pattern) were examined using Naildex parameters. Naildex scores were calculated by a combination of: per cent of each nail infected, area of each nail and number of infected nails. Patients also completed a nail-specific quality of life questionnaire (NailQoL) and nail samples were collected and examined mycologically. Data was analysed for all enrolled patients (n = 322) and patients with mycologically-confirmed onychomycosis (n = 243). Inter-rater reliability was calculated from two examiners who each evaluated 17 patients with mycologically-confirmed onychomycosis. Significant correlations (P < 0.01) between Naildex and NailQoL as well as proxy measures (duration of infection) indicated construct validity of the instrument for all patients as well as mycologically-confirmed cases. Strong correlation (r = 0.754, P < 0.01, n = 17) indicated high inter-rate reliability. This pilot evaluation suggests that Naildex is a valid and reliable measure of onychomycosis severity.

Use of diagnostic tests by dermatologists, podiatrists and family practitioners in the United States: pilot data from a cross-sectional survey.

Before treating onychomycosis, it is important to exclude other conditions such as lichen planus and psoriasis. The purpose of this study was to evaluate physician preferences and uses of diagnostic tests for toenail onychomycosis (TO) by surveying dermatologists (D), podiatrists (P) and family practitioners (FP) in the United States. Surveys were mailed to approximately 1000 randomly sampled physicians from each of the three specialities. The questionnaire consisted of 15 items regarding physician and practice characteristics, number of patients with TO seen and treated, tests used to diagnose TO and reasons for using the tests. Results were analysed using several statistical methods. Response rates were low (D33.7%; P16.6%; FP28.4%). Ds and Ps (75.2%) and FPs (43.4%) reported feeling 'very confident' at diagnosing onychomycosis. KOH was the preferred diagnostic test for all three specialities. More Ds (75.4%) felt 'very confident' interpreting potassium hydroxide (KOH) exams than Ps (24.9%) and FPs (18.5%). Use of KOH exams was statistically associated with confidence interpreting exams (P P = 0.04092; D & FP P < 0.0001). Some FPs (46.6%) and Ps (21.6%) did not obtain a confirmatory diagnostic test prior to the treatment of onychomycosis while 63.6% of Ds 'almost always/always' did. While limited by low-response rate, this study provides pilot information on the diagnostic preferences for TO by American D, P and FP.

Cost-effectiveness of diagnostic tests for toenail onychomycosis: a repeated-measure, single-blinded, cross-sectional evaluation of 7 diagnostic tests.

OBJECTIVE: Our purpose was to estimate and compare the cost-effectiveness of the most commonly used diagnostic tests for onychomycosis: potassium hydroxide preparation (KOH), interpreted both by a dermatologist (KOH-CLINIC) and a laboratory technician (KOH-LAB); KOH with dimethyl sulfoxide (KOH-DMSO) and with chlorazol black E (KOH-CBE), interpreted by a dermatologist; culture using dermatophyte test medium, culture with Mycobiotic and Inhibitory Mold Agar (Cx); and histopathologic analysis using periodic acid-Schiff stain (PAS). METHODS: This was a repeated-measure, blinded, cross-sectional study conducted at the Minneapolis Veterans Affairs Medical Center. Inclusion criteria included: at least one toenail with 25% or more clinical disease, which was defined as subungual debris with onycholysis and/or onychauxis. Exclusion criteria included other nail dystrophies, use of oral antifungal medication for 2 months or longer within the past year, or topical ciclopirox lacquer within 6 weeks of enrollment. The main outcome measure was the cost-effectiveness (Medicare and non-Medicare costs) of 7 diagnostic tests. Sensitivity (at least 3 positive tests) was the unit of effectiveness. RESULTS: Two hundred four participants were enrolled; their average age was 69.5 years and 95.5% were male. PAS was the most sensitive test (98.8%); it was statistically significantly more sensitive than all other diagnostic tests except KOH-CBE (94.3%). Dermatophye test medium was the least sensitive test (57.3%). KOH-CBE was statistically significantly more cost effective than any other test, with the exception of KOH-CLINIC and KOH-LAB. PAS was the least cost effective. LIMITATIONS: Test specificities were not evaluated. CONCLUSION: KOH-CBE should be the test of choice for practitioners confident in interpreting KOH preparations because of its combination of high sensitivity and cost-effectiveness.

S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function.

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.