Gamma secretase inhibitors enhance vincristine-induced apoptosis in T-ALL in a NOTCH-independent manner.

Activating mutations in the NOTCH1 gene are found in over 50 % of T-ALL cases. Since Notch signaling contributes to the leukemia cell survival and growth, targeting Notch signaling using γ-secretase inhibitors (GSI) has been proposed as a molecularly targeted therapy for the treatment of T-ALL. However, not all T-ALL with NOTCH1 activating mutations respond to GSI treatment. We examined whether GSI could enhance the cytotoxic effect of anti-leukemic agents in the GSI-resistant T-ALL cells although GSI does not have anti-tumor effect as a single agent. GSI significantly increased cell death induced by Vincristine (VCR) but not other anti-leukemic drugs (Methotrexate, Asparaginase, and Cytarabine). The GSI effect in enhancing VCR efficacy was not the result of inhibition of Notch signaling. GSI augmented VCR-induced mitotic arrest, followed by apoptosis. GSI accelerated VCR-triggered loss of mitochondrial membrane potential and caspase-mediated apoptosis. Our finding suggests that GSI has other functions besides inhibiting Notch signaling in T-ALL and incorporating GSI into the conventional regimen containing VCR may offer therapeutic advantage by potentiating VCR treatment in leukemia patients.

GSI promotes vincristine-induced apoptosis by enhancing multi-polar spindle formation.

Gamma secretase inhibitors (GSI), cell-permeable small-molecule inhibitors of gamma secretase activity, had been originally developed for the treatment of Alzheimer disease. In recent years, it has been exploited in cancer research to inhibit Notch signaling that is aberrantly activated in various cancers. We previously found that GSI could synergize with anti-microtubule agent, vincristine (VCR) in a Notch-independent manner. Here, we delineate the underlying cell cycle-related mechanism using HeLa cells, which have strong mitotic checkpoints. GSI enhanced VCR-induced cell death, although GSI alone did not affect cell viability at all. GSI augmented VCR-induced mitotic arrest in a dose-dependent manner, which was preceded by apoptotic cell death, as shown by an increase in Annexin V-positive and caspase-positive cell population. Furthermore, GSI amplified multi-polar spindle formation triggered by VCR. Altogether, we show the evidence that GSI enhances VCR-induced apoptosis in HeLa cells via multi-polar mitotic spindle formation, independent of Notch signaling. These data suggest that one or more GS substrates, yet to be identified, in a post-GS processed form, may play a role in maintaining functional centrosomes/mitotic spindles. More significantly, the synergistic effect of GSI in combination with VCR could be exploited in clinical setting to improve the efficacy of VCR.

Intracellular Enhanced Cyan Fluorescent Protein/Angiotensin II Does Not Modify Angiotensinogen Accumulation in Transgenic Mice.

BACKGROUND: Several studies suggest that extracellular angiotensin can upregulate renin and angiotensinogen (AGT). We have shown that enhanced cyan fluorescent protein/angiotensin II (ECFP/AngII) transgenic mice, in which AngII is fused downstream of ECFP and regulated by the mouse metallothionein housekeeping gene, possess elevated blood pressure and kidney thrombotic microangiopathy. The present study evaluated the effect of intracellular AngII on AGT messenger RNA (mRNA) and protein levels in ECFP/AngII transgenic mice. METHODS: The traditional guanidinium thiocyanate method was used to extract total mRNA. Proteins were extracted by homogenization in a tissue extraction reagent buffer. Northern blots for AGT mRNA and an 18S ribosomal RNA control were performed. Immunoblots for AGT protein levels with actin and tubulin controls were evaluated. RESULTS: Northern blot densitometry showed liver mRNA levels an average of 12-fold greater than levels in the brain or kidney in both Lines A and D (different copies of the transgene) with no quantifiable differences between wild-type (WT) and homozygous (HO) transgenic mice. Immunoblots showed liver AGT protein levels 3.2-fold greater than levels in the brain or kidney, with no differences observed between WT and HO transgenic mice. CONCLUSION: ECFP/AngII transgene expression does not alter AGT mRNA or protein levels in major organs (kidney, liver, and brain) of transgenic mice. The altered blood pressure and kidney thrombosis observed in these transgenic mouse lines are not the result of increased intracellular AGT synthesis and resultant increases in free extracellular AngII. This finding is consistent with our published studies that indicate no increase in circulating AngII by radioimmunoassay.

Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.