A Re-Emerging Marker for Prognosis in Hepatocellular Carcinoma: The Add-Value of FISHing c-myc Gene for Early Relapse

Hepatocellular carcinoma is one leading cause of cancer-related death and surgical resection is still one of the major curative therapies. Recently, there has been a major effort to find mechanisms involved in carcinogenesis and early relapse. c-myc gene abnormality is found in hepatocarcinogenesis. Our aim was to analyze the role of c-myc as prognostic factor in terms of overall survival and disease-free survival and to investigate if c-myc may be an important target for therapy. We studied sixty-five hepatocellular carcinomas submitted to surgical resection with curative intent. Size, macro-microvascular invasion, necrosis, number of nodules, grading and serum alfa-fetoprotein level were registered for all cases. We evaluated the c-myc aberrations by using break-apart FISH probes. Probes specific for the centromeric part of chromosome 8 and for the locus specific c-myc gene (8q24) were used to assess disomy, gains of chromosomes (polysomy due to polyploidy) and amplification. c-myc gene amplification was scored as 8q24/CEP8 > 2. Statistical analysis for disease-free survival and overall survival were performed. At molecular level, c-myc was amplified in 19% of hepatocellular carcinoma, whereas showed gains in 55% and set wild in 26% of cases. The 1- and 3-year disease-free survival and overall survival for disomic, polysomic and amplified groups were significantly different (p=0.020 and p=.018 respectively). Multivariate analysis verified that the AFP and c-myc status (amplified vs. not amplified) were significant prognostic factors for overall patients survival. c-myc gene amplification is significantly correlated with disease-free survival and overall survival in patients with hepatocellular carcinoma after surgical resection and this model identifies patients with risk of early relapse (≤12 months). We suggest that c-myc assessment may be introduced in the clinical practice for improving prognostication (high and low risk of relapse) routinely and may have be proposed as biomarker of efficacy to anti-c-myc targeted drugs in clinical trials.     

Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating

Hyaluronan (hyaluronic acid, HA) was depolymerised by ultrasonication (US), microwave irradiation (MW) and conventional heating (CH), and the effect of pH and oxidants was investigated. The degradation was followed by viscometry and size exclusion chromatography coupled with low-angle light scattering. The results demonstrated that depolymerisation of HA by US leveled off to a limiting molecular mass, and the degradation was significantly enhanced by acidic and alkaline pH only in the presence of oxidants. In contrast to US, the course of depolymerisation by MW was strongly pH-dependent, and the degradation rate increased with decreasing pH. The expected enhancement of depolymerisation by MW in comparison to CH was marked only at very short heating time at pH <4. The NMR and FTIR spectral analyses indicated that HA in the whole M_w-range studied retained almost the backbone of the parent polysaccharide independently on the degradation method used. At harsh degradation conditions (long-term treatments, particularly at acidic pH or alkaline pH and in presence of oxidants) the depolymerisation was accompanied by destruction of both constituent sugar residues and formation of unsaturated structures detectable by UV-absorption at 230–240 and 260–270 nm. US-assisted oxidative degradation under mild reaction conditions was shown to be the most appropriate procedure to reduce the molecular mass of HA to 100 kDa without significant chemical modification of the polysaccharide.     

Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex

Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.     

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, -methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated -methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.     

Contrasting growth strategies of pond versus marine populations of nine-spined stickleback (Pungitius pungitius): a combined effect of predation and competition?

Gigantism in isolated ponds in the absence of sympatric fish species has previously been observed in nine-spined sticklebacks (Pungitius pungitius). Patterns in sexual size dimorphism suggested that fecundity selection acting on females might be responsible for the phenomenon. However, the growth strategy behind gigantism in pond sticklebacks has not been studied yet. Here, we compared von Bertalanffy growth parameters of four independent nine-spined stickleback populations reared in a common laboratory environment: two coastal marine (typical size) and two pond (giant size) populations. We found that both pond populations had larger estimated final size than marine populations, which in turn exhibited higher intrinsic growth rates than the pond populations. Female growth strategies were more divergent among marine and pond populations than those of males. Asymptotic body size and intrinsic growth rate were strongly negatively correlated. Hence, pond versus marine populations exhibited different growth strategies along a continuum. Our data suggest that quick maturation—even with the cost of being small (low fecundity)—is favoured in marine environments. On the contrary, growth to a giant final size (high fecundity)—even if it entails extended growth period—is favoured in ponds. We suggest that the absence (ponds) versus presence (marine environment) of sympatric predatory fish species, and the consequent change in the importance of intraspecific competition are responsible for the divergence in growth strategies. The sex-dependence of the patterns further emphasizes the role of females in the body size divergence in the species. Possible alternative hypotheses are also discussed.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.