Paclitaxel-induced aberrant mitosis and mitotic slippage efficiently lead to proliferative death irrespective of canonical apoptosis and p53.

Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (<5 h) often followed by aberrant mitosis, and about 90% of the cells of each line had lost their clonogenicity after 48 h of the treatment. A combination of the same concentration of paclitaxel with the kinesin-5 inhibitor, S-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (∼20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios — acute MOMP, mitotic slippage or aberrant mitosis ― has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment

Characterization of PAX9 variant P20L identified in a Japanese family with tooth agenesis.

Transcription factors PAX9 and MSX1 play crucial roles in the development of permanent teeth at the bud stage, and their loss-of-function variants have been associated with congenital tooth agenesis. We sequenced the coding regions of the PAX9 and MSX1 genes from nine patients with non-syndromic tooth agenesis, and identified a missense mutation, P20L, of PAX9 in a single familial case involving three patients in two generations. Identical mutation was previously reported by other authors, but has not been characterized in detail. The mutation was located in a highly conserved N-terminal subdomain of the paired domain and co-segregated as a heterozygote with tooth agenesis. The patients showed defects primarily in the first and second molars, which is typical for cases attributable to PAX9 mutation. Luciferase reporter assay using the 2.3-kb promoter region of BMP4 and electrophoretic mobility shift assay using the CD19-2(A-ins) sequence revealed that P20L substitution eliminated most of the transactivation activity and specific DNA binding activity of PAX9 under the experimental conditions we employed, while some residual activity of the mutant was evident in the former assay. The hypomorphic nature of the variant may explain the relatively mild phenotype in this case, as compared with other PAX9 pathogenic variants such as R26W

Paclitaxel-induced aberrant mitosis and mitotic slippage efficiently lead to proliferative death irrespective of canonical apoptosis and p53

<p>Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (<5 h) often followed by aberrant mitosis, and about 90% of the cells of each line had lost their clonogenicity after 48 h of the treatment. A combination of the same concentration of paclitaxel with the kinesin-5 inhibitor, <i>S</i>-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (∼20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios — acute MOMP, mitotic slippage or aberrant mitosis ― has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment.</p

Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells

Distinct Profiles of CD163-Positive Macrophages in Idiopathic Interstitial Pneumonias

Background. The types of cells most significantly linked to individual subtypes of idiopathic interstitial pneumonias (IIPs) remain unclear. Few studies have examined CD163+ macrophages in IIPs. Objective. We retrospectively aimed to immunohistochemically characterize the CD163+ macrophages in IIPs. Methods. Paraffin-embedded lung tissue samples were obtained from 47 patients with IIPs, including idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (NSIP), and cryptogenic organizing pneumonia (COP), and 12 normal controls were immunohistochemically analyzed, using primary antibodies against CD68 and CD163 as indicators of pan and M2 macrophages, respectively. Results. CD68+ macrophage density was significantly increased in the 3 subtypes of IIPs relative to that in the control group, although no difference was detected within the different IIPs. CD163+ macrophage density was significantly increased in NSIP and COP samples relative to that in IPF samples. The density ratio of CD163+ macrophages to CD68+ macrophages was significantly decreased in IPF/UIP samples relative to that in the others, while the densities in NSIP and COP were significantly higher than those in control cases. Conclusion. CD163+ macrophages show distinct profiles among IIPs, and the standardized numerical density is decreased in IPF cases that have poor prognoses

Electrophoretic mobility shift assay of PAX9^WT and PAX9^P20L.

<p>Nuclear extract was prepared at 48 h after transfection with PAX9-Myc-expressing (WT or P20L) or 1st ATG-deleted plasmids (Null), and analyzed using the CD19-2(A-ins) probe. Ectopic expression of PAX9-Myc and integrity of nuclear extract were confirmed with immunoblotting (bottom). Excessive amount of the non-biotinylated DNA eliminated the mobility shift signals in PAX9^WT, whereas addition of anti-Myc antibody resulted in a super-shift, confirming the specificity of the bipartite interaction. No detectable degree of signal shift was observed in PAX9^P20L. No NE, no nuclear extract added; L probe, biotin-labelled probe; NL probe, non-labelled probe.</p

Position of the identified PAX9 mutation.

<p>(A) Locations of P20L and previously reported missense variants are shown with arrows above and below the PAX9 diagram, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref031" target="_blank">31</a>]. Reported pathogenic variants with nonsense mutations or frameshifts are omitted. NSD, N-terminal subdomain of the paired domain; CSD, C-terminal subdomain of the paired domain. (B) Modeled pair-domain structure of the wild-type (blue and red with green backbone) and P20L (yellow) PAX9. P20L mutation results in Van del Waals clashes with the carbonyl oxygen of Leu²¹ (red disc) and the side chain of Pro⁶⁸ (brown disc). This results in a slight displacement of Pro⁶⁸. DNA is shown in pink with a gray surface.</p

Pedigree with tooth genesis involving three patients.

<p>(A) Proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>), her father (ID 7) and brother (ID 10) are affected (indicated with filled symbols), while her mother is unaffected (indicated with an open symbol). The inheritance pattern is consistent with autosomal dominance. The three affected members invariably show C/T heterozygosity at the second position of the Pro²⁰ codon, while the unaffected member shows C/C homozygosity. (B) Radiogram of the proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>). Missing teeth other than 3rd molars are indicated with asterisks.</p