A new monoclonal antibody against DNA ligase I is a suitable marker of cell proliferation in cultured cell and tissue section samples

Abstract

The extensive characterization of the replicative human DNA ligase I (LigI) undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation. We have produced a new monoclonal antibody (5H5) against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded) demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry.

Cellular DNA Ligase I Is Recruited to Cytoplasmic Vaccinia Virus Factories and Masks the Role of the Vaccinia Ligase in Viral DNA Replication

“…Our data can explain the previously described decreased pathogenicity of a VACV ligase deletion mutant (Kerr et al, 1991), since the majority of cells in an animal are in the quiescent state and consequently have low levels of Lig1 (Vitolo et al, 2005). The early expression of the viral ligase presumably provides VACV with a head start in DNA replication.…”

RETRACTED: Sealing of Chromosomal DNA Nicks during Nucleotide Excision Repair Requires XRCC1 and DNA Ligase IIIα in a Cell-Cycle-Specific Manner

“…The results were identical with various fixation procedures and two different Lig1-specific antibodies, 2B1 and 5H5, recognizing both the unmodified and phosphorylated forms of Lig1 and only the phosphorylated form of Lig1, respectively (Rossi et al, 1999;Vitolo et al, 2005). Lig1 is downregulated in serum-starved (Ki67-negative) human fibroblasts (quiescent cells, G0) when compared to growing human fibroblasts that expressed Ki67 (Vitolo et al, 2005). Local UV irradiation of quiescent cells failed to induce accumulation of Lig1 at sites of UV damage ( Figures 1G and 1H).…”

“…We found recruitment of Lig1 to UV spots but surprisingly not in all cells harboring damage: only about 30%-50% of the damaged cells showed Lig1 accumulation ( Figure 1D), coinciding with cells that expressed the Ki67 antigen, a marker for proliferation ( Figure 1E). The results were identical with various fixation procedures and two different Lig1-specific antibodies, 2B1 and 5H5, recognizing both the unmodified and phosphorylated forms of Lig1 and only the phosphorylated form of Lig1, respectively (Rossi et al, 1999;Vitolo et al, 2005). Lig1 is downregulated in serum-starved (Ki67-negative) human fibroblasts (quiescent cells, G0) when compared to growing human fibroblasts that expressed Ki67 (Vitolo et al, 2005).…”

DNA Replication Is Intrinsically Hindered in Terminally Differentiated Myotubes

“…Bonapace), and Ligase 1 [42]; pAbs to mouse Apaf-1 (Millipore), caspase-3 (Cell Signaling), cdk4 (Ab-5) from NeoMarkers, Thermo, Waltham, MA, USA; cdc7, p21, cyclin D3, cdk2, cdk6, cyclin A, cyclin E, and MCM-2 (sc-9839) from Santa Cruz Biotechnology. Peroxidase-conjugated antisera to mouse and rabbit IgG were from Bio-Rad Laboratories.…”