E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins.

A class of helix-loop-helix (HLH) proteins, including E2A (E12 and E47), E2-2, and HEB, that bind in vitro to DNA sequences present in the immunoglobulin (Ig) enhancers has recently been identified. E12, E47, E2-2, and HEB are each present in B cells. The presence of many different HLH proteins raises the question of which of the HLH proteins actually binds the Ig enhancer elements in B cells. Using monoclonal antibodies specific for both E2A and E2-2, we show that both E2-2 and E2A polypeptides are present in B-cell-specific Ig enhancer-binding complexes. E2-box-binding complexes in pre-B cells contain both E2-2 and E2A HLH subunits, whereas in mature B cells only E2A gene products are present. We show that the difference in E2-box-binding complexes in pre-B and mature B cells may be caused by differential expression of E2A and E2-2.

Novel green fluorescent protein (GFP) baculovirus expression vectors.

Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the gfp open reading frame allows visualization of the produced GFP-fusion protein using UV light. The presence of an in-frame 6 x His tag between the gfp cDNA and the multiple cloning site allows purification of the fusion protein on a Ni-NTA (nickel-nitrilo-triacetic acid) agarose matrix.

Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation.

Phosphorylation of the protein encoded by retinoblastoma susceptibility gene (pRb) is the key event of the cell cycle committing the cell to enter S phase and also required for progression through S and G2. We describe a new methodology to monitor pRb phosphorylation in individual cells and correlate it with the cell cycle position. Specifically, pRb phosphorylation in human lymphocytes was assayed immunocytochemically using mAb which recognizes underphosphorylated pRb (pRbP-) conjugated with a fluorochrome of one color combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT) tagged with another color fluorochrome. DNA was stained with still another color fluorochrome and cell fluorescence was measured by multiparameter flow cytometry. Specificity of anti-pRbP- mAb was confirmed by preincubation of the permeabilized cells with phosphatase. Analysis of pRbP- or a ratio of pRbP-/pRbT revealed that pRb was underphosphorylated in over 98% of the nonstimulated lymphocytes. The proportion of cells with underphosphorylated pRb dropped to 20% between 3 and 8 h after addition of the mitogen phytohemagglutinin (PHA). Phosphorylation of pRb within a cell was rapid and complete since reactivity of individual lymphocytes with anti-pRbP-mAb was lost abruptly rather than step-wise during stimulation. Phosphorylation of pRb coincided with the appearance of cyclin D3, which was induced 3 h and peaked 12 h after addition of PHA. The nonspecific protein kinase inhibitor staurosporine at a concentration known to arrest lymphocytes in G1 but not to interfere with the induction of cyclin D3 (20 nM) prevented pRb phosphorylation. The present assay can be applied for screening antitumor drugs targeting CDKs and be useful for monitoring pRb phosphorylation in human tumors, the feature of a possible prognostic value in oncology.

Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase.

The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein.

Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: characterization and diagnostic utility.

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1;19) (q23;p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1;19)-positive from t(1;19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1;19)-positive childhood ALL.