D'importantes recherches en biologie moléculaire ont permis de mettre en évidence l'existence de régulateurs du cycle cellulaire. Ces régulateurs peuvent jouer un rôle dans le déroulement du cycle cellulaire en effectuant une sorte de " contrôle qualité " à chaque étape du cycle. Ils peuvent bloquer sa progression lorsqu'une anomalie est détectée, par exemple lorsque l'ADN est endommagé, et permettre sa réparation. On note ainsi deux points de contrôle du cycle cellulaire, en G1/S et G2/M. Récemment, deux composés, la granulatimide et l'isogranulatimide, ont été isolés d'une ascidie, Didemnum granulatum. Ces composés ont montré une activité inhibitrice intéressante sur le point de contrôle en G2. Cet intérêt provient du fait que la plupart des cellules cancéreuses ont un point de contrôle en G1 déficient suite à une mutation du gène p53 et que seul le point de contrôle en G2, bien que partiellement défectueux, permet l'arrêt du cycle cellulaire nécessaire à la réparation de l'ADN endommagé. Ainsi, les inhibiteurs du point de contrôle en G2, couplés à l'action d'agents endommageant l'ADN, vont entraîner les cellules cancéreuses vers une mitose précoce et létale. Dans ce travail une première partie bibliographique est consacrée à la présentation du point de contrôle en G2 du cycle cellulaire, et plus particulièrement de la checkpoint 1 kinase Chk1 et de ses inhibiteurs. Dans une seconde partie la synthèse d'analogues de la granulatimide et de l'isogranulatimide pour lesquels l'hétérocycle imidazole est remplacé par un maléimide est décrite. Nous avons exploré plusieurs voies d'accès à de nouveaux analogues substitués par divers groupements sur les différentes parties de la molécule de référence. Nous avons également effectué la synthèse de dérivés pour lesquels l'indole a été remplacé par un 7- azaindole. Une troisième partie présente les résultats des tests d'activités biologiques effectués. Tous les analogues synthétisés ont été testés sur plusieurs lignées cellulaires cancéreuses par les Laboratoires SERVIER pour évaluer leur cytotoxicité. Les effets sur le cycle cellulaire ont également été étudiés sur quelques analogues.

Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells.

Vinclozolin (VCZ), an antiandrogenic fungicide, is an endocrine disrupting chemical that is known to possess high affinity for the androgen receptor (AR) and modulate expression of critical androgen-dependant genes in the prostate. In this study, viability and expression of AR, NKX3.1 and CYP3A4 genes were measured in androgen-sensitive prostate cells LNCaP after exposure to VCZ and VCZ treated with S9 microsomes in a time and dose dependent manner. NKX3.1 is an androgen regulated gene that plays a vital role in prostate development. CYP3A4 is involved in xenobiotic metabolism. VCZ decreased the viability at high doses after 48 hours which was slightly mitigated by treatment with S9 metabolites. Expression of NKX3.1 and CYP3A4 was upregulated while an initial downregulation of AR was observed. NKX3.1 upregulation corroborates with possibility of antiandrogens to act as androgens in LNCaP. The results illustrate that VCZ can interfere with the expression of critical prostate genes.

Aberrant DNA methylation in gene promoters causes gene silencing and is a common event in prostate cancer development and progression. While commonly identified methylated genes have been analyzed for their potential clinical utility in a variety of cancers, few studies have attempted a genome-wide methylation approach to discover new and possibly improved biomarkers for prostate cancer. In order to identify DNA methylation changes associated with aggressive prostate cancer, we performed a genome-wide analysis of 40 prostate cancers using Agilent human CpG island microarrays. Methylation profiles of candidate genes were validated using quantitative MethyLight technology in an independent series of 219 radical prostatectomies and compared to clinicopathological parameters. The effects of methylation on expression of HOXD3 and HOXD8 and the possible role of HOXD8 in progression of PCa were also investigated. We discovered previously unidentified methylation in the HOXD cluster of genes, namely HOXD3 and HOXD8, as well as TGFβ2 and GENE X as potential prognostic biomarkers. Furthermore, unsupervised clustering of samples by methylation signature indicated ERG oncogene expression as significantly different between clusters. Within the independent cohort, we observed strong correlations between Gleason score (GS) and HOXD3 as well as GENE X, while HOXD3 and HOXD8 methylation were associated with ERG expresson. TGFβ2 was an independent predictor of disease recurrence using Cox multivariate regression analysis. In gene expression studies, both HOXD3 and HOXD8 were elevated in cancers with poor prognosis, while DNA methylation did not correlate with expression levels. Both genes were found to contain alternative transcription start sites, explaining the poor correlation between methylation and expression. Finally, knockdown of HOXD8 expression did not have any effect on viable cells or cell motility in an in vitro model. These results indicate that a panel of novel DNA methylation markers distinguish indolent prostate cancers from aggressive ones, and that expression of HOXD3 and HOXD8 is regulated by mechanisms including, but not dependent on, DNA methylation.

Prostate cancer is prevalent among men and one of the few cancer sites where local therapies currently target the entire organ instead of tumour. MRI holds promise in accurately depicting regions of cancer burden within the prostate gland and guiding tumour-targeted diagnostics and therapeutics. The clinical performance of a novel stereotactic MRI-guided needle navigation system for prostate cancer targeting was evaluated. Mean absolute in-plane stereotactic needle-targeting error for 10 patients was 2.2 mm and mean absolute depth error was 6.5 mm, highlighting a need to improve technical accuracy of the system. Consequently, alternative stereotactic registration techniques were investigated. Metrics of performance were in-plane stereotactic needle-targeting error, depth error, and registration time. A Z-shaped fiducial motif using automated registration performed best in phantom experiments with an in-plane error of 2.0 mm and depth error of 1.0 mm. These results will guide further software and hardware development to improve clinical performance.

Interstitial laser thermal therapy is a novel local approach to treating prostate cancer. During treatment, thermal ablation may occur on the adjacent rectal wall. The aim of this thesis was therefore twofold: to study the effects of rectal cooling on lesion formation, and secondly, to engineer a computer controlled rectal cooling unit. To study the effects of the coolant temperatures and flow rate, thermal simulations were executed, followed by testing the phenomenon using agar gel phantoms which thermally mimic prostate tissue. Further simulations were run using a treatment planning software, which predicted the required coolant temperatures to protect the outer rectal wall while subsequently determining the shape and size of the resulting coagulated lesion at various laser settings. Results suggest that low coolant temperatures and low flow rates cause maximum cooling rates. Furthermore, the shape and size of the coagulated region is affected by coolant temperatures at specific laser powers and positions within the prostate.

Prostate cancer is prevalent among men and one of the few cancer sites where local therapies currently target the entire organ instead of tumour. MRI holds promise in accurately depicting regions of cancer burden within the prostate gland and guiding tumour-targeted diagnostics and therapeutics. The clinical performance of a novel stereotactic MRI-guided needle navigation system for prostate cancer targeting was evaluated. Mean absolute in-plane stereotactic needle-targeting error for 10 patients was 2.2 mm and mean absolute depth error was 6.5 mm, highlighting a need to improve technical accuracy of the system. Consequently, alternative stereotactic registration techniques were investigated. Metrics of performance were in-plane stereotactic needle-targeting error, depth error, and registration time. A Z-shaped fiducial motif using automated registration performed best in phantom experiments with an in-plane error of 2.0 mm and depth error of 1.0 mm. These results will guide further software and hardware development to improve clinical performance.

Interstitial laser thermal therapy is a novel local approach to treating prostate cancer. During treatment, thermal ablation may occur on the adjacent rectal wall. The aim of this thesis was therefore twofold: to study the effects of rectal cooling on lesion formation, and secondly, to engineer a computer controlled rectal cooling unit. To study the effects of the coolant temperatures and flow rate, thermal simulations were executed, followed by testing the phenomenon using agar gel phantoms which thermally mimic prostate tissue. Further simulations were run using a treatment planning software, which predicted the required coolant temperatures to protect the outer rectal wall while subsequently determining the shape and size of the resulting coagulated lesion at various laser settings. Results suggest that low coolant temperatures and low flow rates cause maximum cooling rates. Furthermore, the shape and size of the coagulated region is affected by coolant temperatures at specific laser powers and positions within the prostate.

Molecular Imaging provides new aspects in cancer diagnosis and treatment. With the ap-plication of imaging and biological techniques, molecular imaging can monitor molecular and cellular changes of different diseases. To interpret the mechanism of disease, more and more at-tention is focused on the development of new probes for molecular imaging. Magnetic resonance imaging (MRI) is a powerful, non-invasive clinical diagnostic tool with high spatial resolution without the limitation of the depth of tissues. Applications of MRI contrast agents can amply the MRI signal during imaging. Many studies have been devoted to developing targeted MR contrast agents. Proteins and peptides have been widely used for target-ing cancer cells in cancer diagnosis and treatments. GRP, gastrin-releasing peptide, is one of a well-characterized group of mammalian bombesin-like peptides. GRP acts through its cell surface receptors, GRP receptor (GRPR). It has been reported that there is a high density of GRP receptors in the majority of prostate carci-noma. In contrast, the GRPRs are not highly expressed in normal cells of most tissues. Thus, this tumor specific expression pattern provides an advantage for cancer targeting. A novel class of MRI contrast agent was designed by adding the Gd3+ binding sites into a stable host protein, the domain 1 of rat CD2. Due to the unique features of the designed metal binding properties, the protein contrast agent (ProCA1) exhibits more than 10-fold enhanced MRI relaxivity compared to that of the more commonly used Gd-DTPA. The high relaxivity of the designed protein contrast agent largely overcomes the major barrier of low sensitivity of MRI techniques. A peptide of ten amino acids from the C-terminal of GRP was grafted onto ProCA1. GRP-grafted protein contrast agents (ProCA1.GRPs) showed the targeting capability to the GRPRs which are over-expressed on prostate cancer cells. Cell MRI Imaging demonstrated that ProCA1.GRP(52) grafted between Lys51 and Ser52 had better targeting capability than C-terminal one. Administration of ProCA1.GRP into xenograft tumor model enhances the contrast in the GRPR+ prostate tumor under MRI and optical imaging. Study demonstrated a potential application for disease marker targeted MR imaging by using our developed protein contrast agent.

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