Analysis of CBS transcripts by RT-PCR, protein levels by Western blot, transient transfections of CBS −1b promoter and ara-C sensitivities/metabolism in clinically relevant AML cell lines.
(A) Results are shown for an RT-PCR analysis of CBStranscripts in the CMK (DS) and CMS (non-DS) AMkL cell lines normalized to 18S RNA transcript levels, demonstrating CBS transcripts in the DS CMK cell line and a lack of transcripts in the non-DS CMS cell line. (B) A Western blot analysis of CBS protein levels in CMS and CMK cells, as described in “Materials and methods.” (C) Data are shown for transient transfections of the CMS and CMK cell lines with the full-length CBS luciferase reporter gene construct (positions −4046 to −3565) demonstrating approximately 40-fold greater promoter activity in the CMK compared with CMS cell line. The data are presented as means ± SE from 3 independent experiments performed in duplicate. (D) In vitro ara-C sensitivities of the CMK and CMS cell lines measured by the percentage growth inhibition of the cell lines incubated for 4 days with ara-C. (Left) Following in vitro incubation of the cell lines with 5 μM 3H-ara-C for 3 hours, the 3H-ara-CTP generation was assessed by high-performance liquid chromatography (HPLC). The metabolites are the mean of 3 independent experiments ± SE (right).
