Philip A. Burns

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

Dietary, lifestyle and clinicopathological factors associated with APC mutations and promoter methylation in colorectal cancers from the EPIC-Norfolk study†

The tumour suppressor APC is the most commonly altered gene in colorectal cancer (CRC). Genetic and epigenetic alterations of APC may therefore be associated with dietary and lifestyle risk factors for CRC. Analysis of APC mutations in the extended mutation cluster region (codons 1276‐1556) and APC promoter 1A methylation was performed on 185 archival CRC samples collected from participants of the European Prospective Investigation into Cancer (EPIC)‐Norfolk study, with the aim of relating these to high‐quality seven‐day dietary and lifestyle data collected prospectively. Truncating APC mutations (APC+) and promoter 1A methylation (PM+) were identified in 43% and 23% of CRCs analysed, respectively. Distal CRCs were more likely than proximal CRCs to be APC+ or PM+ (p = 0.04). APC+ CRCs were more likely to be moderately/well differentiated and microsatellite stable than APC− CRCs (p = 0.05 and 0.03). APC+ CRC cases consumed more alcohol than their counterparts (p = 0.01) and PM+ CRC cases consumed lower levels of folate and fibre (p = 0.01 and 0.004). APC+ or PM+ CRC cases consumed higher levels of processed meat and iron from red meat and red meat products (p = 0.007 and 0.006). Specifically, CRC cases harbouring GC‐to‐AT transition mutations consumed higher levels of processed meat (35 versus 24 g/day, p = 0.04) and iron from red meat and red meat products (0.8 versus 0.6 mg/day, p = 0.05). In a logistic regression model adjusted for age, sex and cigarette‐smoking status, each 19 g/day (1SD) increment increase in processed meat consumption was associated with cases with GC‐to‐AT mutations (OR 1.68, 95% CI 1.03–2.75). In conclusion, APC+ and PM+ CRCs may be influenced by diet and GC‐to‐AT mutations in APC are associated with processed meat consumption, suggesting a mechanistic link with dietary alkylating agents, such as N‐nitroso compounds. Copyright

ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival

Background:The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.Methods:Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.Results:A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.Conclusion:Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

Synthesis and Biological Evaluation of Colchicine B‑Ring Analogues Tethered with Halogenated Benzyl Moieties

Deacetylcolchicine was reacted with substituted benzyl halides to provide a library of compounds for biological analysis. Compound 7 (3,4-difluorobenzyl-N-aminocolchicine) was shown to possess cytotoxicity in cancer cell lines in the low nanomolar range. Significantly, it showed no loss of activity in the resistant A2780AD ovarian carcinoma cell line known to overexpress the ABCB1 drug transporter and was also unaffected by overexpression of class III β-tubulin in HeLa transfected cells.

NuMA Overexpression in Epithelial Ovarian Cancer

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon. NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis. Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (pu200a=u200a0.009), lymph node involvement (pu200a=u200a0.03) and patient age (pu200a=u200a0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (pu200a=u200a0.02) but increased with disease stage (pu200a=u200a0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (pu200a=u200a0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (pu200a=u200a0.05), increased binucleation (pu200a=u200a0.021) and multinucleation (pu200a=u200a0.007), and aneuploidy (pu200a=u200a0.008). NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.

Kinetic analysis of intracellular Hoechst 33342—DNA interactions by flow cytometry: Misinterpretation of side population status?

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342‐loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351–364 nm) flow cytometry was used to confirm ABCG2‐specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT‐PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342–DNA fluorescence in live cells. A live cell “kinetic SP gate” tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye–DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic—critical for functional assay design and the interpretation of heterogeneity.

Helping students to improve their academic performance: A pilot study of a workbook with self-monitoring exercises

Background: There is increasing interest in developing student self-regulated learning skills, especially self-monitoring, to improve academic performance. Aims: A pilot study to investigate the impact of self-monitoring exercises on calibration accuracy and academic performance in undergraduate medical students on a Biomedical Science (BMS) module. Method: A randomised trial of 51u2009second-year students comparing a structured workbook with and without self-monitoring exercises. Results: Participants significantly improved calibration accuracy after completing the intervention, as well as increased self-efficacy and greater satisfaction with performance. The intervention group significantly improved their BMS exam score compared with the control group. Conclusion: A relatively simple intervention seems to have the potential to improve self-monitoring skills and academic performance. Further research is recommended to identify if the development of self-monitoring skills by a similar intervention leads to long-term improvement in academic performance, if low-performing students can significantly benefit from a similar intervention and if there is transfer of improved self-monitoring skills from one context to another.

Comparison of induced and cancer-associated mutational spectra using multivariate data analysis

One of the most useful tools for investigating the aetiopathology of cancer is the mutation spectrum, which comprises the type and distribution of mutations within a gene sequence. Many studies have generated mutagen-induced spectra using in vitro or in vivo model systems in an attempt to find correlations with those observed in cancer-associated genes such as the TP53 tumour suppressor gene. Consequently, meaningful similarities in the types of mutation found in induced and human spectra have been demonstrated. However, it is more difficult to draw such conclusions about the distribution or sequence context of mutations when they arise in different target sequences. We have developed an analytical approach for base substitution spectra that capture information for both sequence context and mutation type simultaneously. The resulting mutation signature is a fixed set of data points that allows comparison of multiple mutation spectra regardless of sequence. We have applied this method to a mixed set of mutation spectra observed in exons 5, 7 and 8 of TP53 from cancers of brain, breast, skin, colon, oesophagus, liver, head and neck, stomach and lung (smokers and non-smokers) and spectra induced by benzo[a]pyrene diol epoxide, ultraviolet (UV) B, UVC, simulated sunlight and hydroxyl radicals in the cII, supF and yeast p53 model systems. We demonstrate that this approach allows human cancer and mutagen-induced signatures to be grouped together according to similarity. Specifically, the analysis reveals key differences between smoking- and non-smoking-related lung cancer for TP53 mutations and the mutability of CpG sites between exons in skin cancer.