In an attempt to overcome the drug resistance phenotype, a NK-based molecular approach taking advantage of a chimeric antigen receptor (CAR) system targeting B7-H6 was established and tested in cells with acquired resistance to fulvestrant. Here we show that fulvestrant resistance was accompanied by increased expression of a number of innate immune response genes including the natural killer (NK) cell ligand B7-H6 on the cell surface. However, resistance to fulvestrant still occurs and unfortunately it leaves few choices other than chemotherapy as the later-line treatments to fulvestrant-resistant tumors. The anti-estrogen fulvestrant is currently the only clinically approved pure anti-estrogen which causes ER degradation. ER inhibitors through modulating the transcriptional function of ER have been the frontline anti-estrogens to which refractory phenotype often developed in advanced cancer. (B,D,F,G) *p<0.05, **p<0.01 and ***p<0.001 Student’s two-tailed t-tests.Īnti-estrogens as hormone therapy are the mainstay treatment for estrogen receptor (ER)-positive breast cancer. mRNA levels were set relative to control sister cultures infected with a construct carrying scrambled1-3 amiRNAs (dashed line). (G) Quantification of nlgn1, 2 and 3 mRNA levels by qRT-PCR from 14DIV cultured hippocampal neurons infected at saturating levels with lentivirus carrying a pEpic_Lite mCMV:eSIBR no-pA vector with amiRNAs against nlgn1, 2 and 3. Number of biological replicates (n) is noted on or above bars. (E) Representative quantitative western blots for antibodies against Cadm1, Cadm3, or Cadm1-3 and (F) quantification of protein knockdown from 14DIV cultured hippocampal neurons infected with lentivirus carrying amiRNAs against cadm1-3 compared to corresponding scrambled1-3 amiRNA infected control neurons. For scrambled1-3 and cadm1-3 groups, GFP intensity was set relative to UbiC-promoted GFP levels at an arbitrary value of 1 (dashed line). (D) Mean GFP intensity as measured by flow cytometry of HEK293T cells infected at single-copy levels with UbiC or CMVmin-promoted eSIBR vectors carrying scrambled1-3 or cadm1-3 amiRNAs. pME-nlsGFP contains a stop codon so the HA epitope in p3E-HA no-pA is not expressed. (C) Schematic of LR recombination reaction used to create pEpic_Lite UbiC:eSIBR-nlsGFP no-pA vectors. Number of biological replicates (n) are shown on or above the bars. (B) Lentiviral titers obtained from mCMV:eSIBR-nlsGFP vectors with or without a pA signal sequence. (A) Schematic of LR recombination reactions used to create pEpic_Lite mCMV:eSIBR-nlsGFP pA and no-pA vectors. Inset shows neuronal cell bodies in the spinal cord.Ĭreation and optimization of potent multi-target lentiviral knockdown constructs using eSIBR-based artificial miRNA vectors. (F) Representative fluorescent confocal microscope image for GFP, tdTomato, and E2Crimson in 48 hpf zebrafish embryo showing neuronal cell bodies in the spinal cord and motor axons within several adjacent somites. Embryos were heat-shocked at 7 hours post fertilization (hpf) to induce Cre expression for lox recombination of genomic UAS:BrainbowTEC copies, then imaged at 48 hpf. Fish carrying multiple copies of UAS:BrainbowTEC were crossed with a dual inducible-Cre and motoneuron-specific GAL4 driver line (mnx1:GAL4 hsp70l:Cre). (E) Experimental design for UAS:BrainbowTEC labeling of motoneurons in developing zebrafish spinal cords. (D) Possible fluorophore combinations and resulting observed color from three copy expression of UAS:BrainbowTEC. (C) Schematic of LR recombination reaction used to create UAS:BrainbowTEC. (B) No recombination, loxP recombination or lox2272 recombination lead to distinct fluorophore expression. Overview of pME-BrainbowTEC and application in developing zebrafish spinal cord. coli ccdB toxin CmR = chloramphenicol resistance mPGK = mouse phosphoglycerate kinase promoter WPRE = woodchuck hepatitis virus posttranslational regulatory element. LTR = long terminal repeat RRE = Rev response element cPPT = central polypurine tract ccdB = E. pEpic_Lite lacks puromycin resistance (PuroR). attR3 and 4 sites flanking the ccdB/CmR selection cassette are positioned in an anti-sense orientation to viral RNA expression driven by a Rous sarcoma virus (RSV) promoter. (B) Schematic of lentiviral destination vectors pEpic and pEpic_Lite. Site-specific recombination events (red lines) between attR and attL sites from a 5’, middle, and 3’ entry vector with a destination vector replaces the ccdB/CmR selection cassette of the destination vector with the mobile DNA elements from the entry vectors, leaving destination vector-specific 5’ and 3’ sequences intact. (A) Schematic of an LR recombination reaction and the resulting vector. Overview of three-fragment MultiSite Gateway cloning and novel lentiviral destination vectors.
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