Viral HSV1-TK gene, radiolabeled FIAU, and ganciclovir: combined gene targeted radiotherapy and suicide gene therapy for prostate cancer

The strategy of suicide gene therapy in cancer is based on the idea of enabling tumour cells, by gene transfer, to convert a non-toxic pro-drug into a toxic product. Previous work has shown that the combination of herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transfer with the pro-drug ganciclovir (GCV) to be a promising suicide gene therapy in cancer. Unlike several other gene therapy systems, early-phase clinical trials of this strategy have shown encouraging results. Therefore, methods to improve its therapeutic efficacy are urgently sought. The thymidine analogue 5-iodo-2’-fluoro-2’-deoxy-1-ß-D-arabino-furonosyluracil (FIAU) is an alternative substrate of the HSV1-TK enzyme. The iodine atom of FIAU can be substituted with radioactive iodine, for example; [123I]-iodine, and thereby utilised for the delivery of ionising radiation into tumour cells expressing the viral tk gene. The aim of this study was primarily to investigate the therapeutic potential of combining HSV1-tk gene transfer and [123I]FIAU for the targeted radiation cytotherapy of prostate cancer cells alone or in combination with GCV. The HSV1-tk gene was cloned into the plasmid vector pcDNA3.1. This plasmid, driven by the ubiquitous promoter of CMV, was then used to transfect the prostate cancer cell line DU145 and the glioma cell line UVW. A viral TK positive, commercially available cell line derived from osteosarcoma (143B-TK) along with its TK-negative clone were also used for comparison. The viral tk gene transfection efficiency was assessed by three independent methods. Firstly, the uptake of [123I]FIAU normalised to the uptake of tritiated thymidine ([methyl-3H]TdR); secondly, GCV sensitivity, assessed by the MTT assay; and thirdly, by the detection of HSV1-tk gene by RT-PCR. The highest specific activity of [123I]FIAU was obtained by the use of a no-carrier added method of synthesis. The cytotoxicity of [123I]FIAU was assessed by clonogenic assay after incubating monolayers of parental and TK-positive clones of the cell lines with a range of doses of [123I]FIAU for the periods of 4 h, 8 h and a period equal to their doubling times. The effect of this treatment on cell cycle progression was assessed by FACS analysis after staining the cellular DNA with propodium iodide. Combination therapy using GCV and [123I]FIAU for the treatment of TK-positive clones of the prostate cancer cell line DU-145 and the osteosarcoma cell line 143B was assessed by the method of median effect analysis and combination index. Monolayers were treated with a constant ratio of various doses of [123I]FIAU for 4h or GCV for 72h. The combination therapy followed three different timing schedules of GCV-before-[123I]FIAU, [123I]FIAU-before-GCV, or simultaneous therapy. The expression of HSV1-tk gene by the three cell lines was confirmed by the three methods described above. For instance, the TK positive clone of the cell line DU145 exhibited 4.25 ± 0.15 times higher [123I]FIAU/ [methyl-3H]TdR uptake ratio and 43 times higher sensitivity to GCV compared with the parental cell line. The three cell lines demonstrated sensitivity to radiolabelled FIAU, which was significantly enhanced by HSV1-tk gene expression. This sensitivity was time-, dose-, and proliferation-dependent. Maximum cell kill was achieved when the monolayers were exposed to [123I]FIAU for a period equavelant to the cellular doubling time. For example, the sensitivity enhancement factor by tk gene expression of the cell line DU145 increased from 5.2 to 7.6 when the treatment period was prolonged from 4 h to 26 h (doubling time of DU145). Following the treatment with [123I]FIAU for a period equal to the doubling time, cells were arrested at G2/M phase of the cell cycle. For instance, 49% of DU145-TK cells treated with 1 MBq/ml for 26 h were at G2/M phase compared with 21.9% of the untreated cells. In contrast, incubation of DU145-TK or 143B-TK cell lines with lethal doses of [123I]FIAU for 4 h and GCV for 72 h had no significant effect on cell cycle progression. Comparison of the effectiveness of [123I]FIAU in the monolayer and spheroid cultures indicated that clonogenic cell kill resulting from Auger electron bombardment was restricted to targeted rather than bystander cells. The combination therapy of [123I]FIAU and GCV of the cell line DU145-TK resulted in antagonistic effect throughout the examined dose range of the schedules of FIAU-before-GCV and simultaneous therapy and the low toxicity concentration range (lower surviving fractions) of the GCV-before-FIAU schedule. The high toxicity concentration range of the latter schedule has shown evidence of additive effect. For the osteosarcoma cell line 143B-TK, synergistic effect was observed at the high toxicity concentration range of the three combination schedules and antagonism at the low toxicity concentration range of the combinations. We concluded from this in vitro study that the combination of HSV1-tk gene transfer and the delivery of radiolabelled FIAU is a promising strategy for targeted radiation cytotherapy of prostate cancer. This proliferation-dependent therapy has caused significant cell cycle arrest that warrants further investigation. Furthermore, the combination of GCV and radiolabelled FIAU for the treatment of tumour cells expressing the gene of viral TK resulted in a dose- and schedule-dependent synergism. We believe that these encouraging results should be substantiated by in vivo experiments in the near future

Androgen receptor expression in human prostate cancer cell lines

Androgens are essential for the growth and differentiation of the prostate. Androgen signalling is mediated by the androgen receptor (AR), a ligand-dependent transcription factor. Androgen ablation is the standard treatment for prostate cancer but nearly all patients relapse with androgen-independent disease. Progression of prostate cancer is often associated with changes in the AR-signalling pathway. The project aimed to investigate molecular mechanisms underlying the regulation of AR gene expression in hormone-relapsed prostate cancer. An inducible and a constitutive gene expression system were used to overexpress AR in human prostate cancer cell lines. The TetOff inducible gene expression system, which offers the advantage of quantitatively regulating AR gene expression in response to varying concentrations of tetracycline, was used. A highly tTA-expressing stable ceil line (DUTetOff) was established in the AR- negative DU145 cell line, and a functional AR expression vector (pTRE-AR) was constructed. Transient assays with pTRE-AR in DUTetOff, DU145, PC-3, DUSF and COS-1 cells indicated that, while AR mRNA was expressed in all cells tested, the AR transcript was not translated in DUTetOff and DU145 cells. To develop a constitutive AR gene expression system, the full-length human AR cDNA was introduced into a DU145-derived serum-free subline (termed DUSF). Stable clones were screened for AR expression by immunocytochemistry, Western analysis and RT-PCR. Up- regulation of AR mRNA and protein was detected in DUSF transfectants following androgen treatment. Endogenous PSA mRNA expression was observed in untransfected DUSF cells, while androgen treatment of the transfectants implied an AR- and androgen-independent mechanism for PSA regulation. The work described in this thesis indicates that overexpression of AR in AR-negative cells permits androgen-mediated AR gene expression, and implies an alternative mechanism for PSA activation. The stable AR-expressing DUSF cells provide a useful model system for investigating androgen-independent regulatory elements involved in PSA gene regulation and, elucidating the mechanisms involved in prostate cancer progression

Targeting the splice factor kinase CLK1 in prostate cancer cells

Prostate cancer is the most rampant diagnosed cancer and the second leading cause of cancer related death in men between middle age and old age. There is a need to identify the molecular genetic processes that underpin prostate cancer and to search for new treatments. Alternative splicing affects over 94% of human genes and aberrant splicing is implicated in prostate cancer. Splicing is regulated by splice factors and protein kinases (the latter including SRPK1 and CLK1). The CLK1 protein kinase specifically regulates alternative splicing by phosphorylation of SR proteins within the nucleus, particularly in nuclear speckles (splice factor storage sites). CLK1 is also found to be overexpressed during malignant prostate cell transformation-; therefore targeting the splice factor kinase CLK1 by its specific chemical inhibitor (TG003) could be therapeutically useful. We confirm that CLK1 expression is itself regulated through alternative splicing: skipping of exon 4 or retention of intron 4 results in truncated, inactive CLK1. The aims of this project are to study CLK1 alternative splicing and to explore the effects of targeting the splice factor kinase CLK1 in prostate cancer.Prostate cancer cell lines (androgen independent PC3 and DU145 cells) were treated independently for 24, 48 and 72hrs with varying concentrations (10nM -100μM) of thebenzothiazole compound (TG003) a specific inhibitor of CLK1. Results suggest that chemical inhibition of CLK1 with TG003 treatment suppressed growth and induced apoptosis in prostate cancer cell lines, as well as causing decreased cell migration and invasion. Similar effects were observed when CLK1 expression was reduced with an siRNA. There was also increased E-cadherin expression with TG003 treatment; incontrast, vimentin expression was reduced suggesting reversal of endothelialmesenchymal transition following TG003 treatment. RT-PCR analysis revealed that TG003 treatment altered CLK1’s own splicing by altering exon 4 skipping rates in a iv dose dependent manner, suggesting that a feedback loop mechanism contributes to the regulation of CLK1 expression. In vivo mouse work with PC3 cell line xenograftsshowed a highly significant reduction in tumour growth and volume following intraperitoneal TG003 administration (10 and 50μM). In conclusion, the findings presented in this thesis suggest that targeting CLK1 may bring considerable anticancer benefits

Υπολογισμός του PSA density με διακοιλιακό υπερηχογράφημα, διορθικό υπερηχογράφημα και αξονική τομογραφία και συσχέτισή του με το PSA density του παρασκευάσματος ριζικής προστατεκτομής σε ασθενείς με καρκίνο του προστάτη

Σκοπός: Η εξέταση της ακρίβειας της PSA density υπολογιζόμενης με τρεις διαφορετικές απεικονιστικές μεθόδους και συγκρινόμενης με την PSA density του παρασκευάσματος της ριζικής προστατεκτομής. Ασθενείς και μέθοδοι: Η PSA density 60 ασθενών με κλινικά εντοπισμένο καρκίνο του προστάτη υπολογίστηκε με διακοιλιακό υπερηχογράφημα (TAUS), διορθικό υπερηχογράφημα (TRUS) και αξονική τομογραφία (CT) πριν από τη διενέργεια ριζικής προστατεκτομής και έγινε σύγκρισή της με την PSA density του χειρουργικού παρασκευάσματος, χρησιμοποιώντας την κατά ζεύγη t-test δοκιμασία. Η σχέση του πραγματικού όγκου του προστάτη και της διαφοράς μεταξύ της PSA density της υπολογιζόμενης με τις τρεις απεικονιστικές μεθόδους και αυτής του παρασκευάσματος αναλύθηκε χρησιμοποιώντας το συντελεστή συσχέτισης του Pearson ( r ). Αποτελέσματα: Η μέση διαφορά μεταξύ της PSA density της υπολογιζόμενης με κάθεμια από τις τρεις απεικονιστικές μεθόδους και της PSA density του παρασκευάσματος ήταν -0,01 ng/ml/cm3 (p=0,28) για το TAUS, 0,01 ng/ml/cm3 (p=0,37) για το TRUS και -0,03 ng/ml/cm3 (p=0,001) για την CT. Αυτή η διαφορά δεν φάνηκε να εξαρτάται από το πραγματικό μέγεθος του προστάτη (r=0,056, p=0,673 για το TAUS, r=-0,014, p=0,917 για το TRUS και r=0,184, p=0,159 για την CT). Η ευαισθησία της PSA density της υπολογιζόμενης με τα TAUS, TRUS και την CT ήταν 58,3%, 65% και 45% αντίστοιχα, ενώ αυτή του παρασκευάσματος ήταν 70%. Συμπεράσματα: Αν και συνολικά η ευαισθησία της PSA density ήταν μέτρια, το TAUS και το TRUS είναι οι απεικονιστικές μέθοδοι που την υπολογίζουν πλησιέστερα στην πραγματική PSA density του χειρουργικού παρασκευάσματος.Objective: To examine the accuracy of PSA density, calculated with three different imaging modalities and compared to the PSA density of the radical prostatectomy specimen. Patients and methods: The PSA density of 60 men with clinically localized prostate cancer was calculated with transabdominal ultrasound (TAUS), transrectal ultrasound (TRUS) and computed tomography (CT) before radical prostatectomy and was compared with the PSA density of the surgical specimen using the paired t-test. The relationship of the real prostate volume and the difference between the PSA density calculated with the three imaging modalities and that of the PSA density of the specimen was analysed using the Pearson’s correlation coefficient. Results: The mean difference between the PSA density calculated by each one of the three imaging modalities and the PSA density of the specimen was -0,01 ng/ml/cm3 (p=0,28) for TAUS, 0,01 ng/ml/cm3 (p=0,37) for TRUS and -0,03 ng/ml/cm3 (p=0,001) for CT. This difference has not been shown to depend on the real prostate volume (r=0,056, p=0,673 for TAUS, r=-0,014, p=0,917 for TRUS and r=0,184, p=0,159 for CT). The sensitivity of PSA density calculated with TAUS, TRUS and CT was 58,3%, 65% and 45% respectively, while that of the specimen was 70%. Conclusions: Although overall PSA density showed a moderate sensitivity, TRUS and TAUS are the imaging modalities that calculate it closer to the real PSA density of the surgical specimen

Rôle de l'endogline et de la protéine kinase c-tak1 dans le cancer de la prostate

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal