Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative status of normal human hematopoietic cells

Relative levels of the nuclear oncoproteins c-myb, c-myc, and c-fos were determined in selected subpopulations of normal human bone marrow (BM) cells using a flow cytometric assay which simultaneously detects a cell-surface antigen (as a marker of lineage and stage of maturation) and levels of an intracellular protein. At least two monoclonal antibodies directed against each oncoprotein and specific peptide inhibition controls were used for these determinations. Hematopoietic progenitor cells (CD34+) express the highest levels of c-myb and c-myc, whereas c-fos levels in CD34+ progenitor cells are similar to c-fos levels in mature monocytes and granulocytes. Granulocytes are the only hematopoietic cells examined which do not express detectable levels of c-myb and c-myc. The levels of these oncoproteins in these normal, unstimulated BM cell populations were more closely linked to lineage and maturation stage than to the proliferative status of the given population, as determined by either DNA staining or expression of the cell-cycle specific nuclear protein, Ki67. This flow cytometric assay helps in interpreting the significance of oncoprotein levels in leukemia cells by allowing direct comparisons of a leukemia with the phenotypically similar "normal counterpart control" cell population in normal BM.     

5′–3′-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage

Optimal induction of p53 protein after DNA damage requires RPL26-mediated increases in p53 mRNA translation. We report here the existence of a dsRNA region containing complementary sequences of the 5′- and 3′-untranslated regions (UTRs) of human p53 mRNA that is critical for its translational regulation by RPL26. Mutating as few as 3 bases in either of the two complementary UTR sequences abrogates the ability of RPL26 to bind to p53 mRNA and stimulate p53 translation, while compensatory mutations restore this binding and regulation. Short, single-strand oligonucleotides that target this 5′–3′-UTR base-pairing region blunt the binding of RPL26 to p53 mRNA in cells and reduce p53 induction and p53-mediated cell death after several different types of DNA damage and cellular stress. The ability to reduce stress induction of p53 with oligonucleotides or other small molecules has numerous potential therapeutic uses.     

The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11(ATLD1) and Nbs1(Delta)(B) alleles conferred similar rescue of Rad50(S/S)-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM-Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex-ATM pathway. Conversely, the Rad50(S) mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50(S) allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50(S/S) Atm(-/-) mice.     

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.     

Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells

To compare the differentiation of early B- and T-lymphoid precursors, we have used immune adherence combined with analytical flow cytometric techniques to enrich and characterize subsets of the small population of bone marrow mononuclear cells that express the enzyme terminal deoxynucleotidyl transferase (TdT) but lack the CD19 B-lymphoid marker. Two percent to five percent of bone marrow TdT + mononuclear cells belong to the T-lymphoid lineage by virtue of expression of CD7 or CD5. Three-color immunofluorescence studies showed that, like early B-lymphoid precursors, most bone marrow TdT + T cells express HLA-DR and the progenitor cell antigen CD34, and about half express CD10. All CD5 + TdT + cells express surface CD3 and T-cell receptor alpha, beta, while a subset of CD7 + TdT + cells lack these "mature" T cell features. CD2 is low or absent on CD5 + TdT + cells. Examination of isolated CD34 + cells showed that approximately 70% of CD34 + TdT + cells expressed neither CD19, CD22, CD7, nor CD5, and 15% to 50% also lacked CD10. Thus, a major subset of CD34 + TdT + cells lack lineage-specific surface antigens. TdT expression may be the earliest available marker of lymphoid differentiation, and CD34 + TdT + cells are likely to include progenitor cells for both the B and T lineages.