Figure 3. Clofarabine and cladribine inhibit homodimer formation of CD99 and cyclophilin & PKA binding, and the membrane-impermeable analog of clofarabine shows potent cytotoxicity in ES cells.

(A) STA-ET-7.2 ES cells were incubated with either drug at 5 μmol/L concentration or DMSO for 1 h. The cells were treated with cross-linking agent BS³ as described in Materials and Methods. The lysates were resolved in 12% SDS-PAGE followed by immunoblotting with anti-actin and anti-CD99 clone 013 antibodies. M and D represent monomeric and dimeric forms of CD99, respectively. Values given below the lanes on the immunoblot represent the relative density of the bands, and were determined using ImageJ 1.48v software. (B) STA-ET-7.2, RDES and A4573 ES cells were treated with 3 μmol/L of either drug for 6 h. Endogenous CD99 was immunoprecipitated from total cell lysates using anti-CD99 clone 013 antibody. Immunoblot (IB) analysis was performed for cyclophilin A, CD99 and PKA-RIIα using total cell lysates (TCL) and immunoprecipitated (IP) samples. Values given below the lanes on each immunoblot represent the relative density of the bands, and were determined using ImageJ 1.48v software. (C) A steady-state affinity curve and sensorgram (inset) showing binding kinetics of clofarabine-5’-triphosphate to the purified recombinant human full-length CD99. Recombinant human full-length CD99 protein produced in HEK293 cells was immobilized by amine coupling on a CM4 Biacore sensor chip in a Biacore T200 instrument. Compound was injected over the chip surface at 2.5, 5, 10 and 20 μmol/L concentrations in duplicate. (D) Cell viability was evaluated by WST-1 assay following 48 h treatment of TC-71 and A4573 ES cells with clofarabine and clofarabine-5’-triphosphate. The IC₅₀ values were determined by nonlinear regression analysis using GraphPad Prism version 6.0h software.