Research on aging indicates that older adults do not, as a group, report decreased health-related quality of life (HRQOL) despite age-related declines in physical health status. Several cognitive adaptation strategies have been suggested to underlie HRQOL stability in this population. Studies of older cancer patients nevertheless show substantial variance in post-treatment HRQOL outcomes, although cognitive mechanisms for individual differences have received little attention. The current study expanded on a developmental adaptation of self-regulation theory in which aging influences both self-vulnerability and perceptions of disease. A model was tested in which older age was hypothesized to predict better HRQOL via less severe illness perceptions in men treated for localized (Stage I and II) PC. Results indicated that age was not directly associated with HRQOL. However, older age was indirectly associated with better HRQOL via less severe PC perceptions. Further, this indirection association helped account for the positive association between age and HRQOL that three risk factors (income, comorbid disease burden, and sexual function) were shown to suppress. Perceptions of PC may promote HRQOL stability by mitigating age-related declines in health and income status. Disease perceptions thus represent critical components of health assessments and interventions for PC survivors of all ages, but particularly for men facing difficulties adapting to complex health profiles or normative lifespan challenges.

Most patients undergoing androgen deprivation therapy relapse eventually and progress to androgen-independent (AI) prostate cancer. Although the mechanisms underlying progression to AI prostate cancer are not well understood, studies suggest that androgen receptor (AR) is still required for AI prostate cancer. Our lab found that Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is up-regulated during the progression of androgen-dependent human prostate cancer cells to androgen-independence in vivo and in cell-based experiments. Since Vav3 significantly increases ligand-dependent AR transcriptional activity and this action requires the Vav3 pleckstrin homology (PH) domain but not Vav3 GEF activity, we explored the role of the Vav3 PH domain in ligand-dependent AR coactivation by Vav3. We found that targeting the Vav3 PH mutant into nuclei but not the plasma membrane restored Vav3 PH mutant in AR coactivation. Targeting Vav3 to the plasma membrane eliminated the capacity of Vav3 to coactivate AR. In agreement with nuclear targeting of Vav3 via its PH domain, chromatin immunoprecipitation assays showed that Vav3 enhancement of AR transcriptional activity was accompanied by Vav3 recruitment to AR transcriptional complexes at an AR target gene enhancer. Further, Vav3 increased AR occupancy at the target gene enhancer upon androgen treatment and this may underlie the capacity of Vav3 to enhance AR transcriptional activity. Because Vav3 can also be activated by growth factors (GFs) and GFs activate AR in the absence of androgen (ligand-independent), we investigated the crosstalk between Vav3 and GF activation of AR and found Vav3 strongly enhanced AR transcriptional activity induced by GFs. GEF function and the downstream Rho GTPase, Rac1 were required for constitutively active (Ca) Vav3 activation of AR, which differs from Vav3 activation of AR in the presence of androgen. We also investigated the possible signal pathways contributing to AR activation by Ca Rac1. Ca Rac1 caused ligand-independent activation of AR in part through MAPK/ERK signaling and conferred prostate cancer growth in the absence of androgen in cell culture, soft agar and mouse tumor xenografts. Thus, our findings indicate that Vav3 activates AR in the presence or absence of ligand through two distinct mechanisms, which supports a versatile regulatory effect of Vav3 in AR signaling and prostate cancer progression.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D, inhibits the proliferation of a variety of cell types including adenocarcinoma of the prostate. The primary mechanism for the antiproliferative effects of 1,25-(OH)2D3 in prostate cancer cells is inhibition of G1 to S phase cell cycle progression. While 1,25-(OH)2D3-mediated growth inhibition requires the vitamin D receptor (VDR), a ligand activated transcription factor, expression of functional VDR is not sufficient. To define target genes that might participate in the antiproliferative actions of 1,25-(OH)2D3, we developed a derivative of the human prostate cancer cell line, LNCaP, which retains transcriptionally active VDRs but unlike parental LNCaP cells, is not growth inhibited by 1,25-(OH)2D3. Gene expression profiling of these resistant cells (termed VitD.R) compared to control LNCaP cells revealed two novel 1,25-(OH)2D3-inducible genes, GADD45G and MIG6. The expression of GADD45G and MIG6 genes was induced by 1,25-(OH)2D3 in LNCaP but not in the resistant VitD.R or in ALVA31 cells, human prostate cancer cells that exhibit natural resistance to growth inhibition by 1,25-(OH)2D3 despite expression of functional VDR. Ectopic expression of GADD45G but not MIG6 in either LNCaP or ALVA31 cells resulted in accumulation of cells in G1 and inhibition of proliferation equal to or greater than that caused by 1,25-(OH)2D3 treatment. While GADD45G is induced by androgens in prostate cancer cells, up-regulation of GADD45G by 1,25-(OH)2D3 was not dependent on androgen receptor signaling further refuting a requirement for androgens/androgen receptor in vitamin D-mediated growth inhibition in prostate cancer cells. These data introduce two novel 1,25-(OH)2D3-regulated genes and establish GADD45G as a growth inhibitory protein in prostate cancer. Further, defects in vitamin D-mediated induction of GADD45G may underlie vitamin D resistance in prostate cancer cells. We previously demonstrated that the antiproliferative actions of 1,25-(OH)2D3 in prostate cancer cells are associated with decreased CDK2 activity and increased stability of the cyclin dependent kinase inhibitor (CKI) p27KIP1. We defined a novel mechanism that may underlie these antiproliferative effects, 1,25-(OH)2D3 -mediated cytoplasmic relocalization of CDK2, which would provide a unifying mechanism for the observed effects of 1,25-(OH)2D3 on CDK2 and p27. In the present study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. In contrast, while p27KIP1 is induced by 1,25-(OH)2D3, this CKI was completely dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction of CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Since cyclin E is important for nuclear translocation of CDK2, we investigated cyclin E effects on 1,25D-mediated growth inhibition. Ectopic expression of cyclin E blocked 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma (Rb), which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3 mediated growth inhibition. VitD.R cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Importantly, targeting CDK2 to the nucleus of LNCaP cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and uncoupling of VDR in the regulation of antiproliferative target genes in the mechanisms of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells. Since 1,25-(OH)2D3 continues to be evaluated for its chemotherapeutic and chemopreventative potential, elucidating the mechanism of 1,25-(OH)2D3 antiproliferative effects is critical in the determination of 1,25-(OH)2D3 responsiveness and the design of individualized treatment strategies.

Advanced prostate cancer (PrC) is the second leading cause of death from cancer in US males. Advanced PrC cells are initially androgen-sensitive and thus androgen ablation therapy causes tumors to undergo regression and fall into a remission phase where residual cells remain dormant while androgen levels remain very low. Unfortunately, this phase usually lasts 3 to 5 years prior to tumor relapse, where the tumor cells re-grow in the absence of androgens. This form of the disease is aggressive and invariably fatal. In this study, we investigated the effects of a combination of herbal extracts on various stages of PrC, androgen-dependent and castrate-resistant, using CWR22 and CWR22Rv1 cells, respectively. Zyflamend, a commercially available product consisting of 10 different herbal extracts, had been shown to reduce pre-malignant forms of PrC in clinical trials. We expanded these earlier experiments by using Zyflamend in a model of advanced PrC. Our initial results indicated that Zyflamend could repress androgen-sensitive and castrate-resistant (androgen-insensitive) prostate tumor growth. Using a cell model for castrate-resistant PrC, Zyflamend inhibited the growth of CWR22Rv1 cells by increasing the expression of the cell cycle inhibitors p21 and p27. These effects were mediated via hyperacetylation of histone 3 through the suppression of class I and II histone deacetylases (HDACs) and an induction of CBP/p300 histone acetyl transferase activity. The latter effect was mediated by the upregulation/activation of Erk-1/-2 and Elk-1. Zyflamend also inhibited androgen receptor expression, its downstream gene target, prostate specific antigen (PSA), and increased cell death by inducing apoptosis as indicated by caspase 3 activity and PARP cleavage. The reduction of androgen receptor was confirmed in CWR22Rv1 xenograft tissues. Our results suggest the extracts of this herbal combination inhibits castrate-resistant prostate cancer cell growth epigenetically and by coordinately affecting androgen receptor signaling pathways involved in cell growth/death. In an androgen-dependent PrC tumor xenograft model, Zyflamend reduced the growth of CWR22-derived tumors and enhanced the responsiveness of tumor cells to hormone ablation. Zyflamend potentiated the regression of PrC cells and their sensitivity to androgen deprivation. These results suggest that Zyflamend may be an effective adjuvant when used with hormone ablation therapy.

In the work of this doctoral dissertation a new resonator sensor technique, first presented in 1989, has been further developed and evaluated with focus on technical characteristics and applications within the medical field. In a first part a catheter-type tactile sensor using the resonator sensor technique was evaluated in a silicone model and applied to human prostate in vitro. The main finding was that different histological compositions of prostate tissue correlated with the frequency shift, .fS, of the resonator sensor and that the common property was the hardness of the tissue. The results indicated that hardness of the prostate tissue, and maybe hardness of human tissue in general, can be expressed according to a cone penetration standard (DIN ISO 2137) and that the hardness can be measured with this tactile sensor system. The tissue hardness application for the resonator sensor technique has to be further developed and evaluated in a larger study. The study also produced results that has led to the basic understanding of the resonator sensor system. One important result was that .fS of the sensor system was related to the contact area between sensor and sample. This indicated that the resonance sensor could be used for contact area measurement. In a second part, containing three studies, the area-sensing capability from the first study was utilised in the development and evaluation of the applanation resonator sensor (ARS) for measurement of intraocular pressure (IOP). For the purpose of evaluating IOP-tonometers, an in vitro pig-eye model was developed, and it was shown that a saline column connected to the vitreous chamber could be used successfully to induce variations in IOP. A ARS sensor with a flat contact surface was applied onto the cornea with constant force and .fS was measured. A mathematical model based on the Imbert-Fick law and the assumption that .fS was linearly related to contact area was proposed and verified with a convincing result. IOP measured with the ARS correlated well (r=0.92, n=360) with the IOP elicited by a saline column. The ARS in a constant-force arrangement was evaluated on healthy human subjects in vivo. The results verified the sensor principle but revealed a nonnegligible source of error in off-centre positioning between the sensor and cornea. The sensor probe was redesigned and evaluated in the in vitro model. The new probe, with a spherical contact surface against the eye reduced the sensitivity to off-centre positioning. It was also shown that a .fS normalisation procedure could reduce the between-eye differences. The ARS method for IOP measurement was further developed using combined continuous force and area measurement during the dynamic phase when the sensor initially contacts the cornea. A force sensor was included with the resonator sensor in one probe. Evaluation was performed with the in vitro pig-eye model. The hypothesis was that the IOP could be deduced from the differential change of force and area during that phase. The study showed good accuracy and good reproducibility with a correlation of r=0.994 (n=414) between measured pressure in the vitreous chamber and IOP according to the ARS. Measurement time was short, 77 ms after initial contact. Problems with inter-eye differences and low resolution at high pressures were reduced. The ARS method is the first to combine simultaneous, continuous sampling of both parameters included in the applanation principle. Consequently, there is a potential for reducing errors in the clinical IOP tonometry.

The aims of the thesis were to • explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression • find possible effect modifiers • evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer • inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated. A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer. Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.

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.