The role of MYH and microsatellite instability in the development of sporadic colorectal cancer

Biallelic germline mutations in MYH are associated with colorectal neoplasms, which develop through a pathway involving somatic inactivation of APC. In this study, we investigated the incidence of the common MYH mutations in an Australian cohort of sporadic colorectal cancers, the clinicopathological features of MYH cancers, and determined whether inactivation of mismatch repair and base excision repair (BER) were mutually exclusive. The MYH gene was sequenced from lymphocyte DNA of 872 colorectal cancer patients and 478 controls. Two compound heterozygotes were identified in the cancer population and all three cancers from these individuals displayed a prominent infiltration of intraepithelial lymphocytes. In total, 11 heterozygotes were found in the cancer group and five in the control group. One tumour from an individual with biallelic germline mutation of MYH also demonstrated microsatellite instability (MSI) as a result of biallelic hypermethylation of the MLH1 promoter. Although MYH-associated cancers are rare in a sporadic colorectal population, this study shows that these tumours can develop through either a chromosomal or MSI pathway. Tumours arising in the setting of BER or mismatch repair deficiency may share a biological characteristic, which promotes lymphocytic infiltration

Localization and Characterization of STRO-1+ Cells in the Deer Pedicle and Regenerating Antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process

Amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status

<p>Abstract</p> <p>Background</p> <p>Amifostine is an efficient cytoprotector against toxicity caused by some chemotherapeutic drugs. Doxorubicin, a potent anticancer anthracycline, is known to produce spermatogenic damage even in low doses. Although some studies have suggested that amifostine does not confer protection to doxorubicin-induced testicular damage, schedules and age of treatment have different approach depending on the protocol. Thus, we proposed to investigate the potential cytoprotective action of amifostine against the damage provoked by doxorubicin to prepubertal rat testes (30-day-old) by assessing some macro and microscopic morphometric parameters 15, 30 and 60 days after the treatment; for fertility evaluation, quantitative analyses of sperm parameters and reproductive competence in the adult phase were also carried out.</p> <p>Methods</p> <p>Thirty-day-old male rats were distributed into four groups: Doxorubicin (5 mg/kg), Amifostine (400 mg/kg), Amifostine/Doxorubicin (amifostine 15 minutes before doxorubicin) and Sham Control (0.9% saline solution). "Standard One Way Anova" parametric and "Anova on Ranks" non-parametric tests were applied according to the behavior of the obtained data; significant differences were considered when p < 0.05.</p> <p>Results</p> <p>The rats killed 30 and 60 days after doxorubicin treatment showed diminution of seminiferous epithelium height and reduction on the frequency of tubular sections containing at least one type of differentiated spermatogonia; reduction of sperm concentration and motility and an increase of sperm anomalous forms where observed in doxorubicin-treated animals. All these parameters were improved in the Amifostine/Doxorubicin group only when compared to Doxorubicin group. Such reduction, however, still remained below the values obtained from the Sham Control group. Nevertheless, the reproductive competence of doxorubicin-treated rats was not improved by amifostine pre-administration.</p> <p>Conclusions</p> <p>These results suggest that amifostine promotes a significant reduction of the doxorubicin long-term side effects on the seminiferous epithelium of prepubertal rats, which is reflected in the epidydimal fluid parameters in the adult phase. However, fertility status results suggest that such protection may not be effective against sperm DNA content damage. Further investigation of sperm DNA integrity must be carried out using amifostine and doxorubicin-treated experimental models.</p

The effect of early pregnancy following chemotherapy on disease relapse and foetal outcome in women treated for gestational trophoblastic tumours

Little literature exists on the safety of early pregnancy following chemotherapy. Here we assess the rate of relapse and foetal outcome in women who have completed single and multi-agent chemotherapy for gestational trophoblastic tumours. The records of 1532 patients treated for persistent gestational trophoblastic tumours at Charing Cross Hospital between 1969 and 1998 were reviewed. Patients were defined as receiving single agent or multi-agent treatment. Relapse rates and foetal outcome were reviewed in the 230 patients who became pregnant within 12 months of completing chemotherapy. In the single agent group 153 (22%) of 691 patients conceived early. Three subsequently relapsed. In the multi-agent group, 77 (10%) of 779 patients conceived early, two then relapsed. Relapse rates were 2% (3 out of 153) and 2.5% (2 out of 77) for each group compared to 5% and 5.6% in the comparative non-pregnant groups. Outcomes of 230 early pregnancies: 164 (71%) delivered at full term, 35 (15%) terminations, 26 (11%) spontaneous abortions, three (1.3%) new hydatidiform moles and two (1%) stillbirths. Early pregnancies were more common in the single agent group (P<0.001), but spontaneous miscarriages and terminations were more likely to occur in the multi-agent group (P=0.04 and 0.03, respectively). Of the full-term pregnancies, three (1.8%) babies were born with congenital abnormalities. Patients in either group who conceive within 12 months of completing chemotherapy are not at increased risk of relapse. Though, we still advise avoiding pregnancy within 12 months of completing chemotherapy, those that do conceive can be reassured of a likely favourable outcome

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants

Background: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. Methods: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. Results: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). Conclusions: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers

AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11

Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy

Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a−/− mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance

Hoxb1 Controls Anteroposterior Identity of Vestibular Projection Neurons

The vestibular nuclear complex (VNC) consists of a collection of sensory relay nuclei that integrates and relays information essential for coordination of eye movements, balance, and posture. Spanning the majority of the hindbrain alar plate, the rhombomere (r) origin and projection pattern of the VNC have been characterized in descriptive works using neuroanatomical tracing. However, neither the molecular identity nor developmental regulation of individual nucleus of the VNC has been determined. To begin to address this issue, we found that Hoxb1 is required for the anterior-posterior (AP) identity of precursors that contribute to the lateral vestibular nucleus (LVN). Using a gene-targeted Hoxb1-GFP reporter in the mouse, we show that the LVN precursors originate exclusively from r4 and project to the spinal cord in the stereotypic pattern of the lateral vestibulospinal tract that provides input into spinal motoneurons driving extensor muscles of the limb. The r4-derived LVN precursors express the transcription factors Phox2a and Lbx1, and the glutamatergic marker Vglut2, which together defines them as dB2 neurons. Loss of Hoxb1 function does not alter the glutamatergic phenotype of dB2 neurons, but alters their stereotyped spinal cord projection. Moreover, at the expense of Phox2a, the glutamatergic determinants Lmx1b and Tlx3 were ectopically expressed by dB2 neurons. Our study suggests that the Hox genes determine the AP identity and diversity of vestibular precursors, including their output target, by coordinating the expression of neurotransmitter determinant and target selection properties along the AP axis

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses