Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage.

Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size macromolecules is layered on the surface of a gradient whose density increases linearly from top to bottom. During centrifugation, different size macromolecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the macromolecules, as well as on the density and viscosity of the gradient. In this way, macromolecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the gradient. The method has been particularly successful in the size fractionation of large DNA molecules and has been extensively used to measure induction and repair of DNA breaks after exposure to clastogenic factors. Here, we describe an adaptation of this method that can be used in the analysis of newly synthesized DNA formed during DNA replication. Through size analysis of nascent DNA in alkaline sucrose gradients, variations in replication activity can be measured after exposure of cells to DNA-damaging agents. The method is particularly useful as it allows distinction between DNA damage-mediated effects on chain elongation vs. replicon initiation, which is essential for an in-depth analysis of the intra-S-phase checkpoint. This ability makes the technique unique and justifies its somewhat labour-intensive nature.

Pegylated granulocyte colony-stimulating factor mobilizes CD34+ cells with different stem and progenitor subsets and distinct functional properties in comparison with unconjugated granulocyte colony-stimulating factor.

BACKGROUND: Pegylated granulocyte colony-stimulating factor (G-CSF) has recently been introduced as a new compound for mobilization of CD34(+) hematopoietic stem and progenitor cells. In this study, we compared the molecular and functional characteristics of CD34(+) cells mobilized by pegylated G-CSF with those mobilized by unconjugated G-CSF. DESIGN AND METHODS: Gene expression of immunomagnetically enriched CD34(+) cells from leukapheresis products of patients who were given pegylated-G-CSF or unconjugated G-CSF was analyzed using Affymetrix HG Focus microarrays and quantitative reverse transcriptase polymerase chain reaction. Flow cytometry and fluorescence activated cell sorting was conducted to assess the CD34(+) subset composition and to obtain Lin(-), CD34(+), CD38(-) hematopoietic stem cells. Cell cycle assays and clonogenic assays were performed for functional corroboration. RESULTS: Pegylated G-CSF and unconjugated G-CSF mobilized CD34(+) and hematopoietic stem cells with different molecular phenotypes and functional properties. The CD34(+) cells mobilized by pegylated G-CSF had higher expression levels of genes indicative of early hematopoiesis, including HOXA9, MEIS1 and GATA3. We found lower expression of genes characteristic of erythroid and later stages of myeloid differentiation and a lower functional burst-forming unit erythroid/colony-forming unit-granulocyte-macrophage ratio. Consistently, greater numbers of hematopoietic stem cells and common myeloid progenitors and fewer megakaryocyte-erthrocyte progenitors were found in the pegylated-G-CSF-mobilized CD34(+) cells. Additionally, sorted pegylated-G-CSF-mobilized hematopoietic stem cells displayed higher expression of HOXA9 in comparison to G-CSF-mobilized hematopoietic stem cells. In line with the gene expression data, CD34(+) cells mobilized by pegylated G-CSF, as well as sorted hematopoietic stem cells, showed a significantly greater cell cycle activity. CONCLUSIONS: Stimulation with pegylated-G-CSF or G-CSF results in different expression of key regulatory genes and different functional properties of mobilized hematopoietic stem cells as well as their progeny, a finding that might be relevant for the application of these cells in blood stem cell transplantation.

Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines.

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.