Metastasis‐focused cell‐based SELEX generates aptamers inhibiting cell migration and invasion

Metastasis, the capacity of tumour cells to disseminate and grow at distant sites, is the main factor in cancer mortality. Compounds inhibiting migration and invasion of cancer cells are promising candidates for anticancer therapy strategies. We have generated nuclease‐resistant RNA ligands (aptamers) recognizing highly metastatic cells with high affinity and specificity, and inhibiting their migratory and invasive potentials. Aptamers were generated by a cell‐based subtractive SELEX technology using isogenic cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness. Two aptamers, E37 and E10, bound specifically to the metastatically aggressive cell line and altered the phosphorylation of several tyrosine kinases. Fluorescent microscopy showed intracellular uptake of E37, in contrast to membrane binding of E10. Both aptamers inhibited migration of tumour cells in culture (50 and 85% inhibition with respect to control pool for E10 and E37, respectively) while only E10 inhibited cell invasion (?75% with respect to control pool). This proof‐of‐concept study demonstrates the potential of cell‐based SELEX to yield ligands that selectively recognize aggressive metastatic cells and inhibit phenotypes linked to metastatic potential.     

miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD)

MicroRNA contribute to tumor radiation resistance, which is an important clinical problem, and thus we are interested in identifying and characterizing their function. We demonstrate that miR-620 contributes to radiation resistance in cancer cells by increasing proliferation, and decreasing the G2/M block. We identify the hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (HPGD/15-PGDH) tumor suppressor gene as a direct miR-620 target, which results in increased prostaglandin E2 (PGE2) levels. Furthermore, we show that siRNA targeting of HPGD or administration of exogenous PGE2 recapitulates radioresistance. Targeting of the EP2 receptor that responds to PGE2 using pharmacological or genetic approaches, abrogates radioresistance. Tumor xenograft experiments confirm that miR-620 increases proliferation and tumor radioresistance in vivo. Regulation of PGE2 levels via targeting of HPGD by miR-620 is an innovative manner by which a microRNA can induce radiation resistance.     

The epidemiology of Graves' disease: Evidence of a genetic and an environmental contribution

Previous family and twin studies have indicated that Graves' disease has a heritable component. Family studies have also shown that some autoimmune disease cluster in families and genetic studies have been able to show shared susceptibility genes. In the present nation-wide study we describe familial risk for Graves' disease among parents and offspring, singleton siblings, twins and spouses with regard to age of onset, gender and number and type of affected family members. Additionally familial association of Graves' disease with any of 33 other autoimmune and related conditions was analyzed. The Swedish Multigeneration Register on 0–75-year-old subjects was linked to the Hospital Discharge Register from years 1987–2007. Standardized incidence ratios (SIRs) were calculated for individuals whose relatives were hospitalized for Graves' disease compared to those whose relatives were unaffected. The total number of hospitalized Graves' patients was 15,743. Offspring with an affected family member constituted 3.6% of all patients among offspring. The familial SIR was 5.04 for individuals whose sibling was affected but it increased to 310 when two or more siblings were affected; the SIR in twins was 16.45. Familial risks were higher for males than for females. The SIR was increased to 6.22 or 30.20 when parental age was limited to 50 or 20 years, respectively. Graves' disease associated with 19 other autoimmune and related conditions, including Addison's disease, type 1 diabetes mellitus, Hashimoto/hypothyroidism, pernicious anemia, polymyositis/dermatomyositis, myasthenia gravis, discoid lupus erythematosus and localized scleroderma. Remarkably, there was a high disease concordance of 2.75 between spouses. The clustering between spouses suggests environmental effects on Graves' disease which may contribute to the observed gender effects. The demonstrated high risks should be considered in clinical counseling and in prevention plans.     

Repair of UV dimers in skin DNA of patients with basal cell carcinoma

Epidemiologic studies suggest that exposure to sunlight is the primary etiologic agent for basal cell carcinoma. Formation of UV-induced DNA damage is believed to be a crucial event in the process leading to skin cancer. In this study, repair of photoproducts in DNA was followed in the skin of patients with basal cell carcinoma and control subjects. The subjects were exposed to 800 J/m(2) Commission Internationale de 1'Eclairag of solar-simulating radiation on buttock skin. Biopsies were taken at 0 hour, 24 hours, and 3 weeks after the exposure. Two cyclobutane pyrimidine dimers, TT=C and TT=T, were measured using a sensitive (32)P-postlabeling assay. Initial levels of both TT=C and TT=T differed between individuals in both groups. The levels of TT=T in patients with basal cell carcinoma and controls were similar (9.9 +/- 4.0 and 9.2 +/- 2.9 products per 10(6) normal nucleotides), whereas the level of TT=C was significantly lower in controls than in patients with basal cell carcinoma (6.2 +/- 3.1 versus 10.9 +/- 4.5 products per 10(6) normal nucleotides). The fractions of TT=T remaining after 24 hours and 3 weeks were significantly higher in patients with basal cell carcinoma (72% and 11%) compared with controls (48% and 5%). A slower removal in patients with basal cell carcinoma than in controls was indicated also for TT=C (52% versus 42% remaining at 24 hours); however, the difference between groups was not significant. When including data from our previously reported small-scale study, the fraction of dimers remaining at 24 hours was significantly higher in patients with basal cell carcinoma for both TT=C and TT=T. The data suggest that patients with basal cell carcinoma have a reduced capacity to repair UV-induced DNA lesions.     

Protective effects of Hibiscus tiliaceus L. methanolic extract to V79 cells against cytotoxicity and genotoxicity induced by hydrogen peroxide and tert-butyl-hydroperoxide

Plants of the genus Hibiscus thrives produce a diversity of molecules with bioactive properties. In a previous study of Hibiscus tiliaceus L. methanolic extract (HME) using bacteria and yeast, as test media, it has been shown that HME strongly inhibited the mutagenic action of H₂O₂ or tert-butyl-hydroperoxide (t-BHP). Here, our interest is to evaluate the genotoxicity and the antigenotoxic/antimutagenic properties of HME using oxidative challenge with H₂O₂ and t-BHP in V79 cells. We determined cytotoxicity using clonal survival assay; evaluated DNA damage using the comet assay and the micronucleus test in binucleated cells besides of the lipid peroxidation degree and the reduced glutathione content. We examined the ability of HME in quenching hydroxyl radical by means of a HPLC-based method utilizing the hypoxanthine/xanthine oxidase assay. At concentrations ranging from 0.001 to 0.1 mg/mL, HME was not cytotoxic, genotoxic or mutagenic. Treatment with non-cytotoxic concentrations of HME increased cell survival after H₂O₂ and t-BHP exposure and prevented DNA damage. The pre-treatment with HME also was able to decrease the mutagenic effect of these genotoxins, evaluated using the micronucleus test. HME prevented the increase in lipid peroxidation and decrease in GSH content in response to the oxidative challenge. Therefore, the ability in preventing against H₂O₂- and t-BHP-induced GSH depletion and lipid peroxidation was probably a major contribution to the cytoprotective effects. Moreover, HME acts as a hydroxyl radical scavenger. In summary, HME did not have a harmful or inhibitory effect on the growth of V79 cells and presented antioxidant activity, consequently, both antigenotoxic and antimutagenic effects against oxidative DNA damage.     

p53 mutations in larynx cancer

The objective of this work was an analysis of mutations in thep53 gene detected from fresh tumor samples of larynx cancerpatients using single-strand conformation polymorphism (SSCP)and direct DNA sequencing of exons 5–8. From 40 patientsamples, 15 showed an extra band in SSCP. In 13 samples mutationswere detected in exons 5–8. They constituted six transitionsand seven transversions, four of them being T to A transversions.Mutations in codons 205 and 248 occurred in two and in codon246 in three samples. Larynx cancer is strongly associated withtobacco smoking and alcohol consumption. The typical p53 mutationsin lung cancer, G to T transversions and G to A and C to T transitions,associated with smoking, accounted for 46% of the mutationsdetected. Fifty-four per cent of the mutations were detectedin a reported hotspot region covering codons 238–248.     

Familial relationships in squamous cell carcinoma of the skin

The Swedish Family-Cancer Database, which was updated in 1999 to cover individuals born after 1934 with their biological parents, totals 9.6 million persons. We used this resource to study invasive and in situ skin cancers. We identified 198 families in which a parent and an offspring had skin cancer. The familial standardized incidence ratios (SIRs) were 2.4 for invasive and 2.8 for in situ skin cancers in offspring. The SIRs for offspring depended only weakly on the age at diagnosis, as evaluated in two age groups. Compared with offspring whose parents had a single skin cancer, offspring whose parents had multiple skin cancers had a 70% increase in SIR. The discordant parental cancer sites that showed associations with skin cancer in offspring were melanoma, ocular melanoma, and a group of cancers observed in immunosuppressed patients.     

32P-Postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA

A ³²P-postlabelling method is reported for the detection of7-alkylguanines, the major adducts formed by the reaction of1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitrowith different epoxides (ethylene oxide through octylene oxide)and digested with micrococcal nuclease and spleen phosphodiesteraseto 3'-nucleotides. The adduct enrichment was carried out byan ion-exchange method and adducts were labelled with [     

Ambulation versus oxytocin in protracted labour: a pilot study

We compared ambulation with oxytocin in the treatment of protracted labour with a randomized, controlled trial of 57 patients. Sixty percent of the women in the ambulant group delivered their babies without oxytocin. In the ambulant group, the mean length of the second stage of labour was shorter and the women themselves held relatively positive views on their experiences. In the oxytocin group, on the other hand, the women experienced stronger contractions before pushing and also suffered from more excessively strong contractions. Our trial included too few women to judge which treatment is better for the infant's health. Nevertheless, the women's opinions and the quality of their contractions demonstrate that more attention should be paid to ambulation as a treatment for protracted labour.