Expression of CREB in leukemia cell lines and bone marrow samples from patients with acute leukemia.

Shown are results of Western blot analyses of cell lines and of bone marrow specimens from patients with and without leukemia. (A) Leukemia and nonleukemia cell lines were assessed for CREB expression by Western blot analysis. Cells were grown to a density of 1 × 10⁶ cells/mL, and lysates were prepared from 1 × 10⁶ cells. Twenty micrograms of protein from cell lysates was loaded on a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel. Blots were probed with anti-CREB antiserum (0.5 μg/mL), stripped, and reprobed with actin (60 μg/mL) as the loading control. The arrows represent CREB (43 kd) or the actin control (43 kd). (B) Mononuclear cells from bone marrow obtained from patients with leukemia or without active disease were processed with the Ficoll-Hypaque method. Lysates were prepared from 1 × 10⁶ mononuclear cells. Twenty micrograms of protein was loaded on a 10% SDS-polyacrylamide gel. Western blot analysis was done with anti-CREB antiserum. Shown are results with positive controls (32Dcl3 cell lysates; lane 1), results with the AML (lanes 2-5) and ALL (lanes 6-7) samples, and results with samples from patients without active leukemia or with normal bone marrow (lanes 11-13). (C) Western blot analysis of lysates from bone marrow from patients with ALL and AML at diagnosis (lanes 2, 5, and 8), remission (lanes 3 and 7), and relapse (lane 4); lanes 2-4 are results from the same patient with ALL at diagnosis, remission, and relapse, respectively. Lanes 6 and 7 are results from the same patient at diagnosis and remission, respectively. (D) Bone marrow from 9 patients without leukemia was obtained and isolated as described above. Lane 1 shows results with the positive (plus sign) control (32Dcl3 cell lysates). All lanes had equal protein loading on Ponceau S staining (data not shown). Densitometry measurements showed that the CREB-to-actin ratio ranged from 0.1 to 3.0 and the fold difference between leukemia and nonleukemia samples ranged from 3.5 fold to 10 fold. (E) Immunohistochemical analysis with CREB antiserum (10 μg/mL) was done on bone marrow biopsy specimens from (E) a representative patient with ALL and (F) a normal lymph node (× 40 magnification). Nuclear staining of CREB was observed in leukemia blast cells but not in normal lymphocytes. Staining was not detected with rabbit IgG (data not shown).