Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.
A disintegrin and metalloproteinase 10 (ADAM10) is a zinc dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B-cell specific ADAM10 deficient mice (ADAM10B−/−). In the current study, we show that ADAM10 levels are significantly enhanced on GC B-cells. Moreover, ADAM10B−/− mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10B−/− displayed impaired germinal center formation, had fewer follicular helper T-cells, decreased follicular dendritic cell networks and altered chemokine expression in draining lymph nodes. Interestingly, when spleen and lymph node structures from immunized mice were analyzed for B- and T-cell localization, tissues structure was aberrant in ADAM10B−/−mice. Importantly, when ADAM10-deficient B-cells were stimulated in vitro, they produced comparable Ab as wild type B-cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B-cell activation, likely due to the decrease in follicular helper T-cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure following antigen challenge.
Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (Ͼ10 M) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK (75)) isolated for its ϳ20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1-3 M) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2-4 Gy) of 60 Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches. Cancer Gene Therapy (2000) 7, 879 -884
Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2Ј-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2Ј-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4 -2.3) compared with Adgal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose-(10 -100 M) and time-dependent (24 -48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5-to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100 -150 L) of virus delivered at rates of Յ1 L/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 Ͻ P Ͻ .009) tumor regression 6 days after radiation (30 Gy delivered as 2 ϫ 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas. Cancer Gene Therapy (2000) 7, 778 -788 Key words: Cancer gene therapy; drug delivery; halogenated pyrimidines; osmotic pump; radiation.T he halogenated pyrimidines (HPs) 5-bromo-2Ј-deoxyuridine (BrdU) and 5-iodo-2Ј-deoxyuridine (IdU) have been studied extensively as radiosensitizers of various mammalian cells in culture. [1][2][3] Preclinical and clinical studies have revealed that both BrdU and IdU are relatively efficient radiosensitizers if incorporated at sufficient levels into DNA. 4,5 However, although some clinical success has been achieved with head and neck tumors, attempts to radiosensitize gliomas with HPs have been disappointing, perhaps due to poor incorporation of the halogenated nucleosides into the DNA of these types of tumor cells. 6 Therefore, improved or alternative approaches are needed to treat this disease. The thymidine kinase (tk) gene of herpes simplex virus (HSV) has been used extensively to sensitize cancer cells to the HSV-TK-specific drug ganciclovir (GCV), and quite promising results with glioma have been obtained using various animal tumor mode...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.