Abstract
INMAP is a spindle protein that plays essential role for mitosis, by ensuring spindle and centromere integrality. The aim of this study was to investigate the relevant functions of INMAP for genomic stability and its functional pathway. We overexpressed INMAP in HeLa cells, resulting in growth inhibition in monolayer cell cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice. In this system caused micronuclei (MNi) formation, chromosome distortion and γH2AX expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a tumour biochemical marker, lactate dehydrogenase (LDH) isoenzymes were detected to evaluate cell metabolic activity, the results confirming that total activity of LDH, as well as that of its LDH5 isoform, is significantly decreased in INMAP-overexpressing HeLa cells. The levels of p53 and p21 were upregulated, and however, that of PCNA and Bcl-2, downregulated. Indirect immunofluorescence (IIF) and coimmunoprecipitation (CoIP) analyses revealed the interaction between INMAP and p21. These results suggest that INMAP might function through p53/p21 pathways.
Highlights
INMAP is a 38.2-kDa conserved protein and plays vital roles in spindle assembly and cell proliferation
Quantitative analyses of INMAP expression was detected using an INMAP polyclonal antibody in HeLa, Flag-HeLa, Flag-INMAP cells, the result was shown in S1 Fig. INMAP expression was shown to be 2.82 Æ 0.85 fold and 1.03 Æ 0.64 (P = 0.954) fold higher in Flag-INMAP and Flag-HeLa cells than that of in HeLa cells, indicating INMAP was stable expressed in Flag-INMAP cell line
HeLa cells grew well in soft agar, as shown Fig. 1C and D, the frequency of colonies in soft agar was reduced in INMAP overexpressing cells (5.87% Æ 0.92%) compared with Flag-HeLa (8.91% Æ 1.17%, P = 0.025) and HeLa cells (8.54% Æ 0.87%, P = 0.044). These results suggested that overexpression of INMAP inhibits the proliferation of HeLa cells in vitro
Summary
INMAP (interphase nucleus/mitotic apparatus protein) is a 38.2-kDa conserved protein and plays vital roles in spindle assembly and cell proliferation. Abnormal expression of INMAP leads to mitotic aberration, malignant cell proliferation or apoptosis. Our previous studies revealed that stable overexpression of INMAP in HeLa cells causes the formation of abnormal mitotic spindles, decreased DNA content and split intracellular vesicles, thereby brings out cell-cycle arrest and apoptosis[1]. INMAP Overexpression Induces Genomic Instability through p53 Pathways and analysis, decision to publish, or preparation of the manuscript. AbMax Biotechnology Co provided support in the form of a salary for LS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of these authors are articulated in the ‘author contributions’ section
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