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The complications of neurofibromatosis type 1 (NF1) are widespread, unpredictable and variable and each person’s experience of this disorder is unique. However, few studies have addressed the impact of NF1 from an individual’s perspective. This qualitative study aims to identify the ways in which NF1 impacts upon affected Australian adults. Sixty adults with NF1, with a range of disease severity and visibility participated in a semi-structured interview about the ways in which NF1 impacted upon their life and health. Data were analyzed using grounded theory methodology. Results indicated that NF1 impacts upon affected adults in five major ways: 1) cosmetic burden of disease 2) learning difficulties 3) concerns about the risk of passing NF1 to offspring 4) uncertain disease progression, and 5) pain. Participants identified the aspects of NF1 that bothered them the most, creating a hierarchy of NF1 concerns within the cohort. Importantly, mildly affected adults shared many of the same concerns as those more severely affected. This study enhances our current understanding of the impact of NF1 in adulthood, and augments existing recommendations for the care of these patients.

The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.

Histone acetylation is a fundamental mechanism in the regulation of local chromatin conformation and gene expression. Research has focused on the impact of altered epigenetic environments on the expression of specific genes and their pathways. However, changes in histone acetylation also have a global impact on the cell. In this study we used digital texture analysis to assess global chromatin patterns following treatment with trichostatin A (TSA) and have observed significant alterations in the condensation and distribution of higher‐order chromatin, which were associated with altered gene expression profiles in both immortalised normal PNT1A prostate cell line and androgen‐dependent prostate cancer cell line LNCaP. Furthermore, the extent of TSA‐induced disruption was both cell cycle and cell line dependent. This was illustrated by the identification of sub‐populations of prostate cancer cells expressing high levels of H3K9 acetylation in the G2/M phase of the cell cycle that were absent in normal cell populations. In addition, the analysis of enriched populations of G1 cells showed a global decondensation of chromatin exclusively in normal cells.

McKenna, D. J., Rajab, N. F., McKeown, S. R., McKerr, G. and McKelvey-Martin, V. J. Use of the Comet-FISH Assay to Demonstrate Repair of the TP53 Gene Region in Two Human Bladder Carcinoma Cell Lines. Radiat. Res. 159, 49–56 (2003). The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy γ irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after γ irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.

Doc number: 295 Abstract Background: Selective Alzheimer Disease Indicator-1 (or Seladin-1) is a multifunctional protein first discovered by downregulation of its expression in Alzheimer's disease. Interestingly, the expression of this protein is upregulated in several cancers, including primary bladder cancer. However, its role in cancer formation has yet to be discovered. Goniothalamin is a natural product that has been demonstrated to induce apoptosis in various cancer cell lines. In this study, we have elucidated the role of Seladin-1 in goniothalamin-induced cytotoxicity towards human urinary bladder cancer cell line RT4. Methods: The cytotoxicity of goniothalamin in human urinary bladder cancer cell line RT4 was assessed using MTT assay and the mode of cell death was determined by Annexin V-FITC/PI labeling assay. Finally, the expression of Seladin-1 protein in goniothalamin-treated RT4 cells was determined by Western blot. Results: MTT assay showed that the cytotoxicity of goniothalamin on RT4 cells was concentration and time dependent with IC50 values of 61 μM (24 hr), 38 μM (48 hr) and 31 μM for 72 hr, respectively. Cell death induced was confirmed through apoptosis; as assessed using the Annexin V-FITC/PI labeling assay. Furthermore, the involvement of Seladin-1 in goniothalamin-induced apoptosis was evidenced through the cleavage of 60 kDa protein to 40 kDa and 20 kDa. This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells. Conclusion: This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells. The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.