Colchicine: an ancient drug with novel applications.

Colchicine is a treatment for gout that has been used for more than a millennium. It is the treatment of choice for familial Mediterranean fever and its associated complication, amyloidosis. The 2009 U.S. Food and Drug Administration approval of colchicine as a new drug had research consequences. Recent investigations with large cohorts of patients with gout who have been taking colchicine for years have demonstrated novel applications within oncology, immunology, cardiology and dermatology. Some emerging dermatological uses include the treatment of epidermolysis bullosa acquisita, leucocytoclastic vasculitis, aphthous stomatitis and others. In this work we relate the history and the new horizon of this ancient medicine.

Isolated penile lymphedema in an adolescent male: a case of metastatic Crohn's disease.

OBJECTIVE: Metastatic Crohn's disease is a rare and complex inflammatory condition distinguished by cutaneous granulomatous lesions outside the gastrointestinal tract. Genital involvement is rare; with less than 10 cases reported involving isolated penile lymphedema. Here, we present a case of isolated lymphedema of the penis as a consequence of extra-intestinal Crohn's disease. CASE REPORT: The patient is an 18-year-old African American male with a complex history of inflammatory bowel disease, who initially presented with a chief complaint of 6 weeks of swelling of his penis at age 13. A modified circumcision and lymphangectomy of the penis were performed; histopatholgy demonstrated a granulomatous infiltrate and interstitial edema of the distal penis. CONCLUSION: Surgical intervention is an applicable therapeutic alternative and last-line therapy for treatment of isolated penile lymphedema in select patients with appropriate pathology, and can be a suitable adjunct to medical management in these cases.

Impaired p53 binding to importin: a novel mechanism of cytoplasmic sequestration identified in oxaliplatin-resistant cells.

Previous studies have described one nuclear localization signal (NLSI) in p53 and speculated on two additional sites termed NLSII and NLSIII. Drug-resistant KB cells selected with cisplatin or oxaliplatin were found to have increased p53 levels and in oxaliplatin-selected cells, a larger p53 predominantly in the cytoplasm. In oxaliplatin-selected cells a single nucleotide deletion in the sequence-encoding amino acid 382, part of NLSIII, resulted in a frame shift and a 420 amino acid protein (p53(420)). We investigated explanations for the cytoplasmic sequestration of p53(420) while assessing the role, if any, of NLSII and NLSIII in p53 nuclear import. We found that neither NLSII nor NLSIII are essential for p53 nuclear localization. Furthermore, we confirmed p53(420) is able to tetramerize, transactivate a p21 promoter, bind dynein and that the reduced nuclear accumulation is not a consequence of increased p53 nuclear export. However, the association of p53(420) with importin-beta, essential for nuclear import, was significantly impaired. We conclude that despite sequence similarity to consensus NLSs neither NLSII nor NLSIII have roles in p53 nuclear transport. We also identified impaired association with importin as a novel mechanism of p53 cytoplasmic sequestration that impairs nuclear transport rendering cells functionally deficient in p53.

Quantitative method to study the network formation of endothelial cells in response to tumor angiogenic factors.

To study the network formation of endothelial cells (ECs) in an extracellular matrix (ECM) environment, we have devised an EC aggregation-type model based on a diffusion limited cluster aggregation model (DLCA), where clusters of particles diffuse and stick together upon contact. We use this model to quantify EC differentiation into cord-like structures by comparing experimental and simulation data. Approximations made with the DLCA model, when combined with experimental kinetics and cell concentration results, not only allow us to quantify cell differentiation by a pseudo diffusion coefficient, but also measure the effects of tumor angiogenic factors (TAFs) on the formation of cord-like structures by ECs. We have tested our model by using an in vitro assay, where we record EC aggregation by analysing time-lapse images that provide us with the evolution of the fractal dimension measure through time. We performed these experiments for various cell concentrations and TAFs (e.g. EVG, FGF-b, and VEGF). During the first six hours of an experiment, ECs aggregate quickly. The value of the measured fractal dimension decreases with time until reaching an asymptotic value that depends solely on the EC concentration. In contrast, the kinetics depend on the nature of TAFs. The experimental and simulation results correlate with each other in regards to the fractal dimension and kinetics, allowing us to quantify the influence of each TAF by a pseudo diffusion coefficient. We have shown that the shape, kinetic aggregation, and fractal dimension of the EC aggregates fit into an in vitro model capable of reproducing the first stage of angiogenesis. We conclude that the DLCA model, combined with experimental results, is a highly effective assay for the quantification of the kinetics and network characteristics of ECs embedded in ECM proteins. Finally, we present a new method that can be used for studying the effect of angiogenic drugs in in vitro assays.

Active-control equivalence trials and antihypertensive agents.

PURPOSE: To identify methodological features that affect the validity of conclusions drawn from active-control equivalence trials and to apply these criteria to recently published trials comparing antihypertensive agents from different classes. METHODS: Standard methodological criteria for randomized clinical trials and six additional methodological features that affect the validity of active-control equivalence trials were applied to four recently published large trials that compared different antihypertensive classes and that concluded that their results showed equivalence. RESULTS: All four of these trials fulfilled standard criteria for randomized trials. However, none fulfilled all of the six additional methodological criteria that affect the validity of active-control equivalence trials, one fulfilled five criteria, two fulfilled two criteria, and one failed to fulfill any of the criteria. CONCLUSION: Standard methodological criteria for evaluating superiority trials are inadequate for the interpretation of active-control equivalence trials. The methodological criteria outlined in this article for judging the validity of active-control equivalence trials are not specific to antihypertensive trials and may be applied to trials that test a wide variety of interventions.

Criteria for evaluating evidence that laboratory abnormalities are associated with the development of venous thromboembolism.

The identification of conditions associated with an increased risk of venous thromboembolism may indicate the need for aggressive prophylaxis during periods of high risk, prolonged anticoagulant therapy after an initial venous thromboembolic episode, the investigation of asymptomatic family members and the avoidance of oral contraceptives. Advances in laboratory medicine have led to the identification and assessment of many proteins responsible for normal hemostasis, and associations between abnormalities in a number of these proteins and venous thromboembolism have been reported. Without the ability to appraise this information critically, physicians may be unable to determine whether or how they should modify their clinical practice. Criteria for determining whether specific laboratory abnormalities have a relationship with venous thromboembolism are proposed here, and one example of the application of these guidelines is provided.

p53 is associated with cellular microtubules and is transported to the nucleus by dynein.

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.

FR901228 causes mitotic arrest but does not alter microtubule polymerization.

FR901228, a natural cyclic depsipeptide, shows high cytotoxicity against human cancer cell lines (low nM IC50 values). Cells exposed to FR901228 arrest with G1 or G2/M DNA content; S phase is depleted. G2/M cells include cells arrested in mitosis. We wished to understand the mitotic arrest by this compound. Mitotic arrest is often due to interference with microtubules and COMPARE testing in the NCI drug screen indicated a possible taxane-like mechanism. Testing of FR901228 for tubulin binding or alteration of in vitro MT assembly failed to reveal any effect. Likewise, examination of cellular microtubules following exposure to FR901228 did not reveal any change. Similar G2/M accumulation was observed in MCF7, MCF10 and PC3 cells. About 50% of G2/M cells were mitotic and contained microtubule spindles. Mitotic cells peaked at about 14-16 h drug exposure and declined to near 0% by 24-30 h. The block was at prometaphase, with numerous chromosomes unattached to the spindle. We conclude that FR901228 induces formation of aberrant spindles probably by interfering with chromosome attachment, causing mitotic accumulation without affecting mitotic microtubules.

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228.

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21(WAF1/CIP1)in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity.