28 research outputs found
Maximizing the Efficacy of CRISPR/Cas Homology-Directed Repair Gene Targeting
Clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) is a powerful gene editing tool that can introduce double-strand breaks (DSBs) at precise target sites in genomic DNA. In mammalian cells, the CRISPR/Cas-generated DSBs can be repaired by either template-free error-prone end joining (e.g., non-homologous end joining/microhomology-mediated end joining [NHEJ]/[MMEJ]) or templated error-free homology-directed repair (HDR) pathways. CRISPR/Cas with NHEJ/MMEJ DNA repair results in various length insertions/deletion mutations (indels), which can cause frameshift mutations leading to a stop codon and subsequent gene-specific knockout (i.e., loss of function). In contrast, CRISPR/Cas with HDR DNA repair, utilizing an exogenous repair template harboring specific nucleotide (nt) changes, can be employed to intentionally edit out or introduce mutations or insertions at specific genomic sites (i.e., targeted gene knock-in). This review provides an overview of HDR-based gene-targeting strategies to facilitate the knock-in process, including improving gRNA cleavage efficiency, optimizing HDR efficacy, decreasing off-target effects, suppressing NHEJ/MMEJ activity, and thus expediting the screening of CRISPR/Cas-edited clonal cells
Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo
Intracellular activity of thymidylate synthetase and its inhibition in l1210 leukemia in vitro. Abstr.
Kinetic studies of the effects of antifolates on intracellular thymidylate synthetase activity. Abstr.
Analysis of the inhibitory effects of vp-16-213 (etoposide) and podophyllotoxin on thymidine transport and metabolism in ehrlich ascites tumor cells in vitro.
Augmentation of the intracellular levels of polyglutamyl derivatives of methotrexate by vincristine and probenecid in ehrlich ascites tumor cell.
Teniposide (vm-26)- and etoposide (vp-16-213)-induced augmenta- tion of methotrexate transport and polyglutamylation in ehrlich ascites tumor cells in vitro.
A phase II study of combined methotrexate and teniposide infusions prior to reinduction therapy in relapsed childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study.
Induction of Human Breast Cancer Cell Apoptosis from G2/M Preceded by Stimulation into the Cell Cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane
We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, AII) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of AII. MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 μM AII when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1–2 Mb fragments appeared after 6 hr, while 30–50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30–50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that AII blocks MDA-MB-231 cells in G2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. AII demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, “normal” human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that AII caused stimulation into S phase, and that G2/M was the phase of the cell cycle from which cells apoptosed. AII caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited AII-induced cell death, indicating that its toxicity requires de novo protein synthesis
Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents
Photograph of the restored Guard House at Fort Supply