Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n .[Cancer Biology & ABSTRACTIn this study, we investigated the influence of bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/bcl-2 cells (SF 2 =0.84; D 0 =437cGy), the PC-3/vector cells (SF 2 =0.4; D 0 =235cGy) were significantly sensitive to ionizing radiation (p<0.001). Exposure of DX at 5µM concentration prior or post to radiation in both PC-3/vector and PC-3/bcl-2 transfectants caused an increase in radiation enhancement ratios. A significant reduction in G 2 M phase was observed in cells exposed to DX post IR when compared to cells exposed to IR alone. Exposure to DX after radiation in PC-3/vector significantly abrogated radiation-induced bcl-2 upregulation, with a concomitant induction of bax protein. In PC-3/bcl-2 transfectants, DX exposure after IR caused an induction of bax protein. Gel shift assays indicated that in PC-3/vector cells when exposed to IR caused an induction of NFκ-B activity however, DX down regulated the NFκ-B activity. Radiation-induced NFκ-B activity was abrogated in pre and post DX exposure in combination with IR. These findings indicate that DX mediates a potent radiosensitizing effect in p53 null prostate cancer cells by overcoming radiation induced NFκ-B activity and bcl-2 expression.

BackgroundGraft-versus-host disease is the single most important obstacle facing successful allogeneic stem cell transplantation (SCT). Even with current immunosuppressive therapies, morbidity and mortality rates are high. Current therapies including cyclosporine A (CyA) and related compounds target IL-2 signaling. However, although these compounds offer great benefit, they are also associated with multiple toxicities. Therefore, new compounds with a greater efficacy and reduced toxicity are needed to enable us to overcome this hurdle.MethodsThe allogeneic mixed lymphocyte reaction (MLR) is a unique ex vivo method to study a drug's action on the initial events resulting in T-cell activation and proliferation, synonymous to the initial stages of tissue and organ destruction by T-cell responses in organ rejection and Graft-versus-host disease. Using this approach, we examined the effectiveness of two ribonucleotide reductase inhibitors (RRI), Didox and Trimidox, to inhibit T-cell activation and proliferation.ResultsThe compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-γ, TNF-α, IL-2, IL-13, IL-10 and IL-4.ConclusionsIn conclusion, these data provide critical information to justify further investigation into the potential use of these compounds post allogeneic bone marrow transplantation to alleviate graft-versus-host disease thereby achieving better outcomes.

No abstract

No abstract

No abstract

No abstract

Despite promising MCMV inhibition in vitro, DX had no beneficial effect on MCMV disease in our model and paradoxically had adverse effects when administered prophylactically. The lack of correlation between in vitro activity and in vivo efficacy emphasizes the importance of selecting appropriate antiviral targets and of using animal models when testing new drugs.