Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines

The CDKN2 gene, encoding the cyclin dependent kinase inhibitor p16, is a tumour suppressor gene involved in melanoma and maps to chromosome band 9p22. Mutations or interstitial deletions of this gene have been found both in the germline of familial melanoma cases and somatically in melanoma cell lines. Previous mutation analyses of melanoma cell lines have indicated a high frequency of C:G to T:A transitions, with all of these mutations occurring at dipyrimidine sites. Including three melanoma cell lines carrying tandem CC to TT mutations, the spectrum of mutations so far reported indicates a possible role for u.v. radiation in the mutagenesis of this gene in some tumours. To further examine this hypothesis we have characterised mutations of the CDKN2 gene in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous deletions of the gene. Of the remaining cell lines, eight were shown by direct sequencing of PCR products from exon 1 and exon 2 to carry a total of nine different mutations of CDKN2. Two cell lines carried tandem CC to TT mutations and a high rate of C:G to T:A transitions was observed. This study provides further evidence for the role of u.v. light in the genesis of melanoma, with one target being the CDKN2 tumour suppressor gene.

CDKN2A/p16 is inactivated in most melanoma cell lines

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-1/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines.

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Aerial image analysis using spiking neural networks with application to power line corridor monitoring

Trees, shrubs and other vegetation are of continued importance to the environment and our daily life. They provide shade around our roads and houses, offer a habitat for birds and wildlife, and absorb air pollutants. However, vegetation touching power lines is a risk to public safety and the environment, and one of the main causes of power supply problems. Vegetation management, which includes tree trimming and vegetation control, is a significant cost component of the maintenance of electrical infrastructure. For example, Ergon Energy, the Australia’s largest geographic footprint energy distributor, currently spends over $80 million a year inspecting and managing vegetation that encroach on power line assets. Currently, most vegetation management programs for distribution systems are calendar-based ground patrol. However, calendar-based inspection by linesman is labour-intensive, time consuming and expensive. It also results in some zones being trimmed more frequently than needed and others not cut often enough. Moreover, it’s seldom practicable to measure all the plants around power line corridors by field methods. Remote sensing data captured from airborne sensors has great potential in assisting vegetation management in power line corridors. This thesis presented a comprehensive study on using spiking neural networks in a specific image analysis application: power line corridor monitoring. Theoretically, the thesis focuses on a biologically inspired spiking cortical model: pulse coupled neural network (PCNN). The original PCNN model was simplified in order to better analyze the pulse dynamics and control the performance. Some new and effective algorithms were developed based on the proposed spiking cortical model for object detection, image segmentation and invariant feature extraction. The developed algorithms were evaluated in a number of experiments using real image data collected from our flight trails. The experimental results demonstrated the effectiveness and advantages of spiking neural networks in image processing tasks. Operationally, the knowledge gained from this research project offers a good reference to our industry partner (i.e. Ergon Energy) and other energy utilities who wants to improve their vegetation management activities. The novel approaches described in this thesis showed the potential of using the cutting edge sensor technologies and intelligent computing techniques in improve power line corridor monitoring. The lessons learnt from this project are also expected to increase the confidence of energy companies to move from traditional vegetation management strategy to a more automated, accurate and cost-effective solution using aerial remote sensing techniques.