Cell Multinucleation Research Articles

Pterocarpans induce tumor cell death through persistent mitotic arrest during prometaphase.

Pterocarpans, a family of isoflavonoids found in the diverse Fabaceae, display potent cytotoxic activity over a panel of tumor cell lines, and among those tested, 2,3,9- trimethoxypterocarpan displays the most potent activity. This study evaluates the effects of 2,3,9-trimethoxypterocarpan and its related derivatives on cell cycle progression and microtubule function in select breast cancer cell lines (MCF7, T47d and HS578T). The pterocarpans, with the exception of 3,4-dihydroxy-9-methoxipterocarpan, induced increased frequencies of mitotic cells by inducing arrest in prometaphase. While microtubule organization in interphase cells was not modified during treatment, mitotic cells exhibited high frequencies of monastral spindles surrounded by condensed chromosomes. Immunofluorescence staining with an anti-γ-tubulin antibody showed double-dot labeling in the spindle polar region, suggesting that pterocarpan treatment blocked centrosome segregation. We found that this mitotic arrest was reversible when the cells were treated for up to 24h followed by recovery in drug-free medium, but not after 48-h treatment followed by incubation in drug-free medium. In that case, treated cells typically underwent cell multinucleation and apoptosis.

First events in embryo development predict embryo quality on day 2 and 3

Time-lapse technology allows us to evaluate almost the whole development process of the embryo without exposing it to an undesirable atmosphere. The aim of this study was to correlate the first events of embryo development with morphological grading on day 2 and 3. A retrospective study between 2011 and 2012. A total of 540 embryos from 73 recipient patients were described on day 2 and 3 for cell number, symmetry, multinucleation and fragmentation and were classified as A, B, C or D following ASEBIR (Asociación para el estudio de la biología de la reproducción) criteria. After ICSI oocytes were placed in the Embryoscope (Unisense-Fertilitech, Denmark) in droplets of 25 μl of G1 (Vitrolife, Sweden) until day 3. The included kinetic variables are those published as being associated with implantation: time of appearance and disappearance of pronuclei (PN), time point of 2 cell stage (T2), length of second cell cycle (CC2) and synchrony from 2 to 4 cells (S2). All values are given as an average in hours ± standar deviation. Significant differences were found between each embryo classification grade and kinetic markers.Table 1Relationship between embryo quality and dinamic markersT2Appearance 2PNDisappearance 2PNS2CC2Day 2A26.27±2.53a10.05±3.15a23.62±2.63a1.16±2.01a11.10±2.03aB29.00±4.52b11.16±3.39b25.64±4.26b1.69±2.28b12.67±5.15bC28.97±4.20b11.39±3.83b25.73±3.84b7.76±7.38c11.02±8.17a,cD28.95±4.47b10.42±3.38a,b26.24±4.76b3.37±5.46a,b10.13±5.62cDay 3A26.73±2.46a10.06±3.15a24.03±2.36a0.96±1.59a11.54±1.84aB27.29±4.03a,b10.48±3.25a24.76±3.97a,b1.46±2.20b11.07±3.19bC28.22±3.39b10.89±3.75a,b25.35±3.41b,c4.15±4.84c9.79±5.53cD29.80±5.42c11.38±3.49b26.01±4.55c5.13±7.40c13.41±8.06aa≠b≠c < P<0.05. Open table in a new tab a≠b≠c < P<0.05. The dynamic markers which really differentiate good (A, B) from bad (C, D) embryos are time to 2 cell and disappearance of PN for day 2 and 3, and S2 for day 3, even though significant differences were found for each kinetic marker.

P14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell’s inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

Tumor Cell Anaplasia and Multinucleation Are Predictors of Disease Recurrence in Oropharyngeal Squamous Cell Carcinoma, Including Among Just the Human Papillomavirus-Related Cancers

Oropharyngeal squamous cell carcinoma (SCC) is frequently related to high risk human papillomavirus. This tumor expresses p16, frequently has a nonkeratinizing morphology, and has improved outcomes. Despite having a good prognosis, tumors can have focal or diffuse nuclear anaplasia or multinucleation, the significance of which is unknown. From a database of 270 oropharyngeal SCCs with known histologic typing (using our established system) and p16 immunohistochemistry, all surgically resected cases (149) were reviewed. Anaplasia was defined as any × 40 field with ≥ 3 tumor nuclei with diameters ≥ 5 lymphocyte nuclei (~25 μm), and multinucleation was defined as any × 40 field with ≥ 3 tumor cells with multiple nuclei. p16 was positive in 128 cases (85.9%), 64 cases (43.0%) showed anaplasia, and 71 (47.7%) showed multinucleation. Anaplasia and multinucleation were highly related (P<0.001), and both also correlated with histologic type (P<0.001 and P=0.01, respectively), p16 status (P=0.09 and 0.03, respectively), and partially with nodal extracapsular extension. There was no correlation with any of the other variables. In univariate analysis, cases showing anaplasia or multinucleation had worse overall, disease-specific, and disease-free survival (P<0.006 for all). Higher T-stage, keratinizing histologic type, extracapsular extension, and smoking also all correlated with worse survival. In multivariate analysis, anaplasia and multinucleation both predicted worse disease-specific survival (hazard ratio 9.9, P=0.04; and hazard ratio 11.9, P=0.02, respectively) independent of the other variables. In summary, among surgically resectable oropharyngeal SCC (including among just the p16-positive cohort), tumor cell anaplasia and multinucleation independently correlated with disease recurrence and poorer survival.

Impaired Manganese Metabolism Causes Mitotic Misregulation

Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn(2+) acts as a cofactor for many enzymes and excess of Mn(2+) is toxic. Alterations in Mn(2+) homeostasis affect metabolic functions and mutations in the human Mn(2+)/Ca(2+) transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn(2+) on cell cycle progression and show that an excess of cytosolic Mn(2+) alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn(2+) pool requires a functional morphology checkpoint to avoid the formation of polyploid cells.

SCFFbxo45 controls cytokinesis through ubiquitin‐mediated proteolysis of GEF‐H1

The rho guanine nucleotide exchange factor H1 (GEF‐H1) is a critical regulator of cytokinesis, but the requirement and mechanism for its timely destruction for proper execution of cytokinesis are unknown. Here we show that GEF‐H1 is regulated by the evolutionarily conserved E3 ligase SCFFbxo45. Fbxo45 promotes GEF‐H1 ubiquitylation upon PLK1‐mediated phosphorylation of GEF‐H1 on serine 644, and this event is necessary for GEF‐H1 degradation during mitosis. Fbxo45 silencing causes stabilization and abnormal cellular distribution of GEF‐H1 with diffuse RhoA hyperactivation. A GEF‐H1 mutant that is unable to bind Fbxo45 is stabilized in mitosis, and results in an uncoordinated hyperactive membrane phenotype, with abnormal aberrant cytokinesis leading to cell death or multinucleation. Our studies provide evidence that limiting activation of the RhoAGTPase via Fbxo45‐mediated degradation of the GEF‐H1 activator is essential for proper execution of cytokinesis. This work was supported by NIH grants R01 DE119249 and R01 CA136905 to KSJ E‐J, R01 CA140806 to MSL.

Phosphorylation at serine 331 is required for Aurora B activation

Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.

In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.

The Enzymatic Motor Activity of Nonmuscle Myosin II-B is not Critical for Cardiac Myocyte Cytokinesis

Nonmuscle myosin (NM) II is believed to drive contractile ring constriction during cytokinesis and NM II-B is the major nonmuscle myosin II isoform expressed in cardiac myocytes. Ablation of NM II-B in mice resulted in premature binucleation in cardiac myocytes consistent with a role for NM II-B in cytokinesis. Surprisingly binucleation can be rescued by expressing a motor-impaired mutant form of NM II-B (R709C) in the mouse heart, suggesting that NM II-B enzymatic motor activity is not essential for cytokinesis. We tested this hypothesis directly by using a COS-7 cell line which resembles cardiac myocytes in containing only NM II-B and small quantities of NM II-C (<7%) but no NM II-A. Previous work had shown that using siRNA to deplete NMHC II-B in this cell line resulted in multinucleation due to a failure in cytokinesis. We now show that COS-7 cell multinucleation can be rescued by transfecting with mutant forms of NM II-B (R709C) or NM II-A (N93K). These two mutant NM IIs have previously been shown to have marked reductions in their actin-activated MgATPase activity (70% in the case of NM II-B and 96% in the case of NM II-A) and neither could propel actin filaments in an in vitro motility assay. Importantly, cytokinesis is inhibited by the myosin II inhibitor blebbistatin in COS-7 cells expressing either wild-type NM II-B or the mutant form of NM II-A (or II-B). Whereas blebbistatin blocks myosin in an actin-detached state, the mutant NM IIs (R709C II-B and N93K II-A) have no defect in their actin-binding activity. Our results therefore argue that the role of NM II in cytokinesis depends more on its actin binding (cross-linking) property than its enzymatic Mg2+-ATPase activity.

Kif18A is involved in human breast carcinogenesis

Microtubule (MT) kinesin motor proteins orchestrate various cellular processes (e.g. mitosis, motility and organelle transportation) and have been implicated in human carcinogenesis. Kif18A, a plus-end directed MT depolymerase kinesin, regulates MT dynamics, chromosome congression and cell division. In this study, we report that Kif18A is overexpressed in human breast cancers and Kif18A overexpression is associated with tumor grade, metastasis and poor survival. Functional analyses reveal that ectopic overexpression of Kif18A results in cell multinucleation, whereas ablation of Kif18A expression significantly inhibits the proliferative capability of breast cancer cells in vitro and in vivo. Inhibition of Kif18A not only affects the critical mitotic function of Kif18A but also decreases cancer cell migration by stabilizing MTs at leading edges and ultimately induces anoikis of cells with inactivation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Together, our results indicate that Kif18A is involved in human breast carcinogenesis and may serve as a potential therapeutic target for human breast cancer.

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.

Role of Prosurvival Molecules in the Action of Lidamycin toward Human Tumor Cells

Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses. Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin. Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.

HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells

Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

MLN3897, a novel CCR1 inhibitor, impairs osteoclastogenesis and inhibits the interaction of multiple myeloma cells and osteoclasts

AbstractThe interaction between osteoclasts (OCs) and multiple myeloma (MM) cells plays a key role in the pathogenesis of MM-related osteolytic bone disease (OBD). MM cells promote OC formation and, in turn, OCs enhance MM cell proliferation. Chemokines are mediators of MM effects on bone and vice versa; in particular, CCL3 enhances OC formation and promotes MM cell migration and survival. Here, we characterize the effects of MLN3897, a novel specific antagonist of the chemokine receptor CCR1, on both OC formation and OC-MM cell interactions. MLN3897 demonstrates significant impairment of OC formation (by 40%) and function (by 70%), associated with decreased precursor cell multinucleation and down-regulation of c-fos signaling. OCs secrete high levels of CCL3, which triggers MM cell migration; conversely, MLN3897 abrogates its effects by inhibiting Akt signaling. Moreover, MM cell-to-OC adhesion was abrogated by MLN3897, thereby inhibiting MM cell survival and proliferation. Our results therefore show novel biologic sequelae of CCL3 and its inhibition in both osteoclastogenesis and MM cell growth, providing the preclinical rationale for clinical trials of MLN3897 to treat OBD in MM.

Low-Grade Squamous Intraepithelial Lesions of the Cervix With Marked Cytological Atypia-Clinical Follow-Up and Human Papillomavirus Genotyping

The majority of low-grade squamous intraepithelial lesions (LSILs) of the cervix regress spontaneously; however, a small proportion of LSILs progress to high-grade squamous intraepithelial lesion (HSIL) if the lesion is not excised. The guidelines for which patients with LSIL should be treated and which may be followed safely are not well established. The goal of this study was to identify a subgroup of patients with LSIL who may require surgical treatment. We hypothesized that patients with LSILs with marked cytological atypia (LSIL-MA) may be at higher risk for subsequent HSIL. In addition, we were interested in whether LSIL-MA was associated with specific human papillomavirus genotypes. Consecutive patients with biopsy diagnosis of LSIL (n = 30) and LSIL-MA (n = 30) were identified. Marked atypia was defined as 5 or more cells with nuclear enlargement of at least 5 times the size of an intermediate cell nucleus or multinucleation with 5 or more nuclei. Patient follow-up was recorded for up to 24 months. Human papillomavirus genotyping was performed using SPF10 PCR and line probe assay. High-grade squamous intraepithelial lesion on follow-up was significantly more common in patients with LSIL-MA (36%) than in patients with LSIL (7%), and negative follow-up was significantly more common in patients with LSIL (50%) than LSIL-MA (23%). Cases of LSIL and LSIL-MA showed similar spectrum of human papillomavirus genotypes. Marked cytological atypia in LSILs identifies a subset of patients with a high rate of HSIL on follow-up. In such patients, an excisional cone biopsy should be strongly considered.

Expression profiling reveals alternative macrophage activation and impaired osteogenesis in periprosthetic osteolysis

Interactions between periprosthetic cells and prosthetic wear debris have been recognized as an important event in the development of osteolysis and aseptic loosening. Although the ability of wear debris to activate pro-inflammatory macrophage signaling has been documented, the full repertoire of macrophage responses to wear particles has not been established. Here, we examined the involvement of alternative macrophage activation and defective osteogenic signaling in osteolysis. Using real-time RT-PCR analysis of periprosthetic soft tissue from osteolysis patients, we detected elevated levels of expression of alternative macrophage activation markers (CHIT1, CCL18), chemokines (IL8, MIP1 alpha) and markers of osteoclast precursor cell differentiation and multinucleation (Cathepsin K, TRAP, DC-STAMP) relative to osteoarthritis controls. The presence of cathepsin K positive multinuclear cells was confirmed by immunohistochemistry. Reduced expression levels of the osteogenic signaling components BMP4 and FGF18 were detected. Expression levels of TNF-alpha, IL-6, and RANKL were unchanged, while the anti-osteoclastogenic cytokine OPG was reduced in osteolysis patients, resulting in elevated RANKL:OPG ratios. In vitro studies confirmed the role of particulate debris in alternative macrophage activation and inhibition of osteogenic signaling. Taken together, these results suggest involvement in osteolysis of alternative macrophage activation, accompanied by elevated levels of various chemokines. Increased recruitment and maturation of osteoclast precursors is also observed, as is reduced osteogenesis. These findings provide new insights into the molecular pathogenesis of osteolysis, and identify new potential candidate markers for disease progression and therapeutic targeting.

Immunohistochemical Analysis of Embryonal Sarcoma of the Liver

Embryonal sarcoma of the liver is a rare, aggressive malignant tumor that typically occurs in children and teenagers. Microscopic features include spindle, oval, or stellate cells with poorly defined cell borders, nuclear pleomorphism and multinucleation, and variable immunoreactivity to cytokeratin, vimentin, and alpha-1-antitrypsin. Intracellular and extracellular PAS-positive, diastase-resistant hyaline globules are commonly present. The authors evaluated a panel of IHC stains to better define the pattern of immunoreactivity in this tumor. Embryonal sarcomas of the liver were identified from archival files and were immunostained with antibodies: cytokeratin AE1/3, hepatocyte, SMMS, myogenin, calponin, h-caldesmon, desmin, S100, vimentin, CD34, C-kit (CD117), CD10, ALK-1, PE10, Bcl2, p53, and Ki-67. Six cases were identified. Patient age ranged from 6 to 24 years. Tumors ranged from 10 to 20 cm and contained spindled and epithelioid areas with PAS-positive, diastase-resistant globules and atypical cells with focal multinucleation. All cases showed immunoreactivity with vimentin and five showed immunoreactivity with Bcl2. Focal immunoreactivity was seen with cytokeratin AE1/3 in three cases, CD10 in four, calponin in two, desmin in one, and p53 in four. All tumors were negative with hepatocyte, myogenin, CD34, SMMS, h-caldesmon, PE10, ALK-1, and S100. No cytoplasmic staining was seen with C-kit. The proliferation index ranged from 30% to 95%. The diagnosis of embryonal sarcoma is based on typical morphologic features in a large liver tumor occurring in a young patient. The most useful IHC stains help to exclude tumors such as hepatoblastoma, hepatocellular carcinoma, embryonal rhabdomyosarcoma, and other sarcomas.

Correlations Between Morphology Grading and Aneuploidy in Patients Undergoing Preimplantation Genetic Diagnosis

In the IVF laboratory, embryos are typically selected for transfer based on morphology criteria from development at the pronuclear, day two and day three stages. Although pregnancy data suggests that these embryos are more likely to be chromosomally normal, it is impossible to determine chromosomal status with morphology. However, trends can be ascertained between morphology and aneuploidy in patients undergoing preimplantation genetic diagnosis (PGD). The goal of this study was to determine if any correlations exist between embryo morphology and aneuploidy status after PGD. Retrospective data analysis Embryo morphology on days one through three of culture was analyzed and compared to aneuploidy status. Embryos from patients undergoing PGD between 2004 through March of 2005 were assessed based on pronuclear morphology; cell number, evenness of cell size and multinucleation on day two and day three cell number. 1310 embryos were biopsied, Of these 1264 had accurate Z scoring, 1040 day 2 cell number, 997 day 2 symmetry; 942, nucleation score and 1310 accurate day 3 score. Percent euploid is calculated from the number scored in each category to estimate the rate of euploidy/ stage-score. Embryos with good morphology on days 2 through 3 were more likely to be euploid after PGD analysis. Tabled 1 Tabled 1*P<0.05 Tabled 1*P<0.05 *P<0.05 *P<0.05 This data suggests that embryos with good morphology with regards to Zscore, day two cell number, cell symmetry and lack of mulitnucleation and day three cell number are more likely to be chromosomally normal and therefore represent a higher implantation and pregnancy potential. The most significant correlations between morphology and aneuploidy appear to be reflected in the day two symmetry and multinucleation categories. Embryos with combined good Z-scores, day 2 morphology and day 3 morphology may be significantly less aneuploid than any others and this can be used in general IVF for transitioning to single embryo transfer.

Clues to CD2-associated Protein Involvement in Cytokinesis

Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis.

Mitotic cell death in BEL-7402 cells induced by enediyne antibiotic lidamycin is associated with centrosome overduplication.

Mitotic cell death has been focused on in tumor therapy. However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells. Growth curve was established by MTT assay. Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle. Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-beta-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells. Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication. Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.