The Role of Harvey-ras in Mouse Skin Tumorigenesis

This thesis outlines two alternative approaches to investigating the role of H-ras in mouse skin tumour development. The first, the in vitro approach utilises a number of unique cell lines representative of a late stage of tumorigenesis, the conversion from a well differentiated squamous carcinoma to a undifferentiated spindle form. Through manipulation of the gene dosage ratio of mutant:normal H-ras in these cell lines, by plasmid transfection and virus infection, it was hoped to provide some clues as to the function of H-ras in this late stage transition event. The second approach involves the technique of gene targeting. The aim of this approach was to create a mouse hemizygous or completely deficient in H-ras and to examine the effects in the context of both chemical carcinogenesis studies and normal mouse development. Initial observations of the squamous/spindle transition event showed that the cell lines representative of both phenotypes were characteristically very different. The squamous cell line B9 produced a well differentiated tumour when injected into nude mice whereas the spindle cell lines A5 and D3 gave rise to aggressive, disorganised tumours with a much reduced latency. Genetic analysis of the cell lines showed that the most dramatic difference between the squamous and spindle derivatives was in the copy number and expression levels of H-ras. Both spindle cell lines A5 and D3 showed an approximate 5-10 fold increase in mutant H-ras, best explained by a genetic amplification mechanism. Experimentally overexpressing mutant H-ras in the squamous cells, and vice versa the levels of normal H-ras in the spindle variants, was unable to mimic the conversion event. However it was found that several A5 and D3 transfectant clones showed a dramatic reduction in tumorigenicity. This correlated not with the levels of the introduced normal H-ras but the absolute levels of endogenous mutant H-ras. Spontaneous loss of mutant copies of H-ras resulted in clones that morphologically resembled spindle cells yet produced tumours with a vastly increased latency period. It seems therefore that in this system mutant H-ras is the critical determinant of tumorigenicity, and relates perhaps to the growth of the tumour cell in vivo, possibly providing the driving force for the conversion. However, alteration of the H-ras gene dosage ratio was not in itself sufficient for the transition of a squamous to a spindle cell carcinoma, implying that another locus as yet unknown must play a role in controlling the epithelial phenotype. Finally, the gene targeting approach, provides a means to examining the earlier stages of mouse skin tumorigenesis, in particular the proposed role of normal H-ras in suppressing the development of papillomas from an initiated cell. For instance, if the consistent duplication of the chromosome carrying the mutant H-ras gene, seen in the development of papillomas, is related to overcoming a suppressive effect of normal H-ras, then carcinogen treatment of a mouse deficient in H-ras may result in a mouse more susceptible to tumorigenesis. Experiments with the gene targeting technique are on going and as yet no targeted ES cell clone has been isolated, but hope remains that this approach will provide new insights into the functions of H-ras both in early tumorigenesis and normal development

Proceedings of the 13th annual conference of INEBRIA

CITATION: Watson, R., et al. 2016. Proceedings of the 13th annual conference of INEBRIA. Addiction Science & Clinical Practice, 11:13, doi:10.1186/s13722-016-0062-9.The original publication is available at https://ascpjournal.biomedcentral.comENGLISH SUMMARY : Meeting abstracts.https://ascpjournal.biomedcentral.com/articles/10.1186/s13722-016-0062-9Publisher's versio

Metabolic Effects of Rexinoids: Tissue-Specific Regulation of Lipoprotein Lipase Activity

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Five-year follow-up of a phase 2 study of ibrutinib plus fludarabine, cyclophosphamide, and rituximab as initial therapy in CLL

We previously reported high rates of undetectable minimal residual disease <10-4 (uMRD4) with ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) followed by 2-year ibrutinib maintenance (I-M) in treatment-naïve chronic lymphocytic leukemia (CLL). Here, we report updated data from this phase 2 study with a median follow-up of 63 months. Of 85 patients enrolled including 5 (6%) with deletion 17p or TP53 mutation, 91% completed iFCR and 2-year I-M. 5-year progression-free survival (PFS) and overall survival were 94% (95% CI 89-100%) and 99% (95% CI 96-100%), respectively. No additional deaths have occurred with this extended follow-up. No difference in PFS was observed by IGHV status or duration of I-M. High rates of peripheral blood (PB) uMRD4 were maintained (72% at the end of iFCR, 66% at the end of 2-year I-M, 44% at 4.5 years from treatment initiation). Thirteen patients developed MRD conversion without clinical progression, mostly (77%) after stopping ibrutinib. None had BTK mutations. One patient had PLCG2 mutation. Six of these patients underwent ibrutinib re-treatment per protocol. Median time on ibrutinib retreatment was 34 months. The cumulative incidence of atrial fibrillation was 8%. Second malignancy or non-malignant hematologic disease occurred in 13%, mostly non-melanoma skin cancer. Overall, iFCR with 2-year I-M achieved durably deep responses in patients with diverse CLL genetic markers. Re-emergent clones lacked BTK mutation and retained sensitivity to ibrutinib upon retreatment. NCT02251548

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are nonessential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of nondeletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells