Notch and Hedgehog activate cell-cycle progression of adult and cancer stem cells. Notch is activated by DLL and Jag presents on neighboring cells. We investigated the effects of density of the Notch-activating ligand, Jag-1, and targeting Gli-1, in activation of division of paclitaxel/taxol-resistant, (PTXRes) ovarian cancer cells SKOV3 (SKOV3). We used the specific γ-presenilin inhibitor, DAPT, to identify the specificity of activating signals for Notch-1 and created ‘butterfly-duplex-3548-Gli-1-inhibitory RNA’ (i-Gli-1.RNA) to inhibit cell division. To accurately quantify kinetics of division, the expression of CD44 and CD24 was determined in each gated population of divided cells. CD44High proliferated when activated by Jag-1Low and poorly when activated by Jag-1High. DAPT inhibited proliferation of cells activated by Jag-1Low, and increased proliferation of cells activated by Jag-1High. Only 5-10% of cells activated by Jag-1High and Jag-1Low divided fast, polynomial, and symmetric. i-Gli-1.RNA eliminated more than 50% of the small CD44High/CD24Neg cells in divisions 3 and 4. This effect appeared specific compared with cells transfected with negative control siRNA. i-Gli-1.RNA had no effect on large CD44High/CD24Neg cells, but inhibited the population of CD44High/CD24Low cells. Expansion of CD44High inversely correlated with Jag-1 density on activating autologous tumor and fibrosarcoma cells. Created i-RNAs may decrease the resting CSC pool. Notch and Gli-1 signals play an important role in proliferation/division and survival of cancer stem cells. Targeting Notch-1 through its enhancer Gl-1, should be significant for novel treatments to eliminate taxol-resistant cancer stem cells (CSC). i.Gli-1 RNA should be more effective if used together with Taxol.

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