Budding Daughter Cells Research Articles

The subcellular location of cyclin B1 and CDC25 associated with the formation of polyploid giant cancer cells and their clinicopathological significance

Polyploid giant cancer cells (PGCCs) are key contributors to cancer heterogeneity, and the formation of PGCCs is associated with changes in the expression of cell-cycle-related proteins. This study investigated the intracellular localization and expression level of multiple cell-cycle-related proteins in PGCCs derived from BT-549 and HEY cells. In addition, the formation of PGCCs and the clinicopathological significance of cell-cycle-related proteins in human breast and ovarian cancer were examined. The expression levels of cell-cycle-related proteins, including cyclin B1, CDC25B, CDC25C, and other cell cycle phosphoproteins, including Chk2, and Aurora-A kinase, were determined using immunostaining and western blotting both in vitro and in vivo. Migration, invasion, and proliferation in control cells, cyclin B1 knockdown cells and their PGCCs following CoCl2 treatment were compared. In addition, human breast and ovarian cancer samples were collected to determine the correlation of number of PGCCs, expression of cell-cycle-related proteins, and tumor pathologic grade and metastasis. Our results confirm that cyclin B1 was localized in the cytoplasm of PGCCs and in the nuclei of their budding daughter cells. The phosphorylated proteins Chk2 and Aurora-A kinase regulated the expression and subcellular localization of cyclin B1, CDC25B, and CDC25C. The rate of positive cytoplasmic staining of cyclin B1 and positive nuclear staining of both CDC25B and CDC25C increased with increase in tumor grade and lymph node metastasis. Cell-cycle-related proteins, including cyclin B1, CDC25B, and CDC25C play an important role in regulating the formation of PGCCs. The inhibition of cyclinB1 and CoCl2 treatment significantly promoted cell proliferation, invasion, and migration abilities. The subcellular localization of these cell-cycle-related proteins was regulated by other cell cycle phosphoproteins, and was associated with pathologic grade and metastasis of tumors in cases of human breast and ovarian cancer.

Prognostic Implications of Neoadjuvant Chemoradiation in Locally Advanced Rectal Cancer and Potential Mechanisms

BACKGOUND: To evaluate the role of neoadjuvant chemoradiation therapy (nCRT) in locally advanced rectal cancer, we retrospectively analyzed the prognosis and survival in patients treated with and without nCRT, and explored the possible mechanisms of chemoradiation resistance. METHODS: Clinicopathologic data of 304 nCRT cases and 301 paired non-nCRT cases were collected. LoVo and HCT116 cell lines were treated with irradiation and chemotherapeutic drugs, and subsequently the expression levels of metastasis-related proteins before and after treatments were compared. FINDINGS: After nCRT, pathologic complete remission (pCR), partial remission (pPR), stable disease (pSD), and progressive disease (pPD) were observed in 26(8.55%), 127(41.78%), 137(45.07%), and 12(3.95%) patients, respectively. Mucinous adenocarcinomas (MC) occurred more frequently in nCRT than in non-nCRT patients (χ2=29.352, P=0.000) and the prognosis in MC patients was worse than that in non-MC patients (χ2=24.617, P=0.000). The difference of survival rates from three to eight years between the nCRT and non-nCRT groups increased gradually. The difference in survival time had statistical significance for 60 days (χ2=5.357, P=0.021) and 70 days (χ2=18.830, P=0.001) rest interval time. T stage, lymph node metastasis, recurrence and/or distant metastasis, and Duke's stage were associated with poor survival. On multivariable analysis, 60 days rest interval, Duke's stage, and recurrence and/or distant metastasis remained significant predictors of survival. Radiation and chemotherapeutic treatment induced polyploid giant cancer cells (PGCCs) formation, and the PGCCs and their daughter cells exhibited strong migratory, invasive, and proliferation abilities. INTERPRETIATION. Radiation and chemotherapy can induce the formation of PGCCs with cancer stem cell properties. PGCCs and their budding daughter cells display strong migration and invasion abilities, which may be related to the poor prognosis in patients with nCRT. Funding: This work was supported in part by grants from the National Natural Science Foundation of China (81472729 and 81672426), and the foundation of committee on science and technology of Tianjin (17ZXMFSY00120 and 17YFZCSY00700). Declaration of Interest: All authors declare no competing interests. Ethical Approval: Our hospital review board approved the use of data from the colorectal cancer database.

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Binding of DEAD-box helicase Dhh1 to the 5′-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells

Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA.

Regulation of Cell Polarity by Exocyst-Mediated Trafficking.

One requirement for establishing polarity within a cell is the asymmetric trafficking of intracellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated trafficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establishment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front-back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.

Daughter Cells and Erythroid Cells Budding from PGCCs and Their Clinicopathological Significances in Colorectal Cancer.

Purpose: We previously reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride (CoCl2) exhibit cancer stem cell properties. Daughter cells generated by PGCCs possess epithelial mesenchymal transition (EMT) phenotype changes and EMT plays an important role in cancer development and progression. This study investigated the characteristics of PGCCs from LoVo and HCT116 induced by CoCl2 and the clinicopathological significances of PGCCs in colorectal cancer (CRC).Materials and Methods: Western blotting and immunocytochemical staining were used to compare the expression levels of EMT-related proteins between PGCCs with budding daughter cells and the control cells. In addition, tissue samples were collected from 159 patients with CRC for analysis of PGCCs, vasculogenic mimicry (VM), and single stromal PGCCs with budding, as well as immunohistochemical staining for cathepsin B, vimentin, and hemoglobin A.Results: Single PGCCs induced by CoCl2 formed spheroids in vitro. Poorly differentiated CRCs showed the highest numbers of PGCCs and VM, and expression of cathepsin B. There was greater expression of EMT-related proteins in PGCCs with budding daughter cells than in control cells. The expression of vimentin located in PGCC nuclei. Single stomal PGCCs with budding were detected in 27.45% of well differentiated, 50% of moderately differentiated, and 90.20% of poorly differentiated CRC samples. PGCCs can generate erythroid cells that express delta-hemoglobin to form VM. Erythroid cells generated by PGCCs were positive for hemoglobin A immunocytochemical staining.Conclusion: PGCCs from LoVo and HCT116 treated by CoCl2 exhibited cancer stem cell properties. The number of PGCCs and VM were associated with CRC differentiation and daughter cells budded from PGCCs may promote the lymph node metastasis via expression of EMT-related proteins. PGCCs and their newly generated erythroid cells form VM structures.

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence.

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.

The number of polyploid giant cancer cells and epithelial-mesenchymal transition-related proteins are associated with invasion and metastasis in human breast cancer.

BackgroundPreviously, we reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride (CoCl2) could have generated daughter cells with strong invasiveness and migration capabilities via asymmetric divisions. This study compared the expression of epithelial-mesenchymal transition (EMT)-related proteins, including E-cadherin, N-cadherin, and vimentin, between PGCCs and their daughter cells, and control breast cancer cell lines MCF-7 and MDA-MB-231. The clinicopathological significance of EMT-related protein expression in human breast cancer was analyzed.MethodsWestern blot was used to compare the expression levels of E-cadherin, N-cadherin, and vimentin in breast cancer lines MCF-7 and MDA-MB-231, between PGCCs with budding daughter cells and control breast cancer cells. Furthermore, 167 paraffin-embedded breast tumor tissue samples were analyzed, including samples obtained from 52 patients with primary breast cancer with lymph node metastasis (group I) and their corresponding lymph node metastatic tumors (group II), 52 patients with primary breast cancer without metastasis (group III), and 11 patients with benign breast lesions (group IV). The number of PGCCs was compared among these four groups.ResultsThe number of PGCCs increased with the malignant grade of breast tumor. Group IIhad the highest number of PGCCs and the differences among group I, II, III and IV had statistically significance (P =0.000). In addition, the expression of E-cadherin (P = 0.000), N-cadherin (P = 0.000), and vimentin (P = 0.000) was significantly different among the four groups. Group II exhibited the highest expression levels of N-cadherin and vimentin and the lowest expression levels of E-cadherin.ConclusionsThese data suggest that the number of PGCCs and the EMT-related proteins E-cadherin, N-cadherin, and vimentin may be valuable biomarkers to assess metastasis in patients with breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0277-8) contains supplementary material, which is available to authorized users.

Polyploid giant cancer cells with budding and the expression of cyclin E, S-phase kinase-associated protein 2, stathmin associated with the grading and metastasis in serous ovarian tumor

BackgroundWe previously reported that polyploid giant cancer cells (PGCCs) exhibit cancer stem cell properties and express cell cycle-related proteins. HEY PGCCs induced by cobalt chloride generated daughter cells and the daughter cells had a strong migratory and invasive ability. This study is to compare the expression of cyclin E, S-phase kinase-associated protein 2 (SKP2), and stathmin between PGCCs with budding and control HEY cells, and determine the clinicopathological significance of cell cycle-related protein expression in ovarian tumors.MethodsWe used western blot and immunocytochemical staining to compare the expression levels of cyclin E, SKP2 and stathmin between PGCC with budding daughter cells and control HEY cells. In addition, immunohistochemical staining for cyclin E, SKP2 and stathmin was performed on a total of 80 paraffin-embedded serous ovarian tumor tissue samples. The samples included 21 cases of primary high-grade carcinoma (group I) and their metastatic tumors (group II), 26 cases of primary low-grade carcinoma without metastasis (group III), and 12 cases of serous borderline cystadenoma (group IV).ResultsSingle PGCC with budding in the stroma showed high correlation with the metastasis of ovarian carcinoma. Group I had a significantly higher number of single PGCCs with budding in the stroma than group III (85.71% [18/21] vs. 23.08% [6/26] cases; χ2 = 18.240, P = 0.000). The expression of cyclin E, SKP2, and stathmin was compared among the four groups. The expression levels of cyclin E, SKP2, and stathmin increased with the malignant grade of ovarian tumors and group II had the highest expression levels. The expression of cyclin E (χ2 = 17.985, P = 0.000), SKP2 (χ2 = 12.384, P = 0.000), and stathmin (χ2 = 20.226, P = 0.000) was significantly different among the 4 groups.ConclusionsThese data suggest that the cell cycle-related proteins cyclin E, SKP2, and stathmin may be valuable biomarkers to evaluate the metastasis in patients with ovarian serous carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-576) contains supplementary material, which is available to authorized users.

ITRAQ-Based Proteomic Analysis of Polyploid Giant Cancer Cells and Budding Progeny Cells Reveals Several Distinct Pathways for Ovarian Cancer Development

Polyploid giant cancer cells (PGCCs) are a morphologically distinct subgroup of human tumor cells with increased nuclear size or multiple nuclei, but they are generally considered unimportant because they are presumed to be nondividing and thus nonviable. We have recently shown that these large cancer cells are not only viable but also can divide asymmetrically and yield progeny cancer cells with cancer stem-like properties via budding division. To further understand the molecular events involved in the regulation of PGCCs and the generation of their progeny cancer cells, we comparatively analyzed the proteomic profiles of PGCCs, PGCCs with budding daughter cells, and regular control cancer cells from the HEY and SKOv3 human ovarian cancer cell lines with and without CoCl2. We used a high-throughput iTRAQ-based proteomic methodology coupled with liquid chromatography-electrospray ionization tandem mass spectroscopy to determine the differentiated regulated proteins. We performed Western blotting and immunohistochemical analyses to validate the differences in the expression patterns of a variety of proteins between PGCCs or budding PGCCs and regular cancer cells identified by iTRAQ approach and also a selected group of proteins from the literature. The differentially regulated proteins included proteins involved in response to hypoxia, stem cell generation, chromatin remodeling, cell-cycle regulation, and invasion and metastasis. In particular, we found that HIF-1alpha and its known target STC1 are upregulated in PGCCs. In addition, we found that a panel of stem cell-regulating factors and epithelial-to-mesenchymal transition regulatory transcription factors were upregulated in budding PGCCs, whereas expression of the histone 1 family of nucleosomal linker proteins was consistently lower in PGCCs than in control cells. Thus, proteomic expression patterns provide valuable insight into the underlying mechanisms of PGCC formation and the relationship between PGCCs and cancer stem cells in patients with ovarian cancers.

The road not taken: new destinations for yeast mRNAs on the move

The road not taken: new destinations for yeast mRNAs on the move

Capsular Localization of theCryptococcus neoformansPolysaccharide Component Galactoxylomannan

Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.

A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein.

Checkpoint controls ensure that events of the cell-division cycle are completed with fidelity and in the correct order. In budding yeast with a mutation in the motor protein dynein, the mitotic spindle is often misaligned and therefore slow to enter the neck between mother cell and budding daughter cell. When this occurs, cytokinesis (division of the cytoplasm into two) is delayed until the spindle is properly positioned. Here we describe mutations that abolish this delay, indicating the existence of a new checkpoint mechanism. One mutation lies in the gene encoding the yeast homologue of EB1, a human protein that binds the adenomatous polyposis coli (APC) protein, a tumour suppressor. EB1 is located on microtubules of the mitotic spindle and is important in spindle assembly. EB1 may therefore, by associating with microtubules, contribute to the sensor mechanism that activates the checkpoint. Another mutation affects Stt4, a phosphatidylinositol-4-OH kinase. Cold temperature is an environmental stimulus that causes misalignment of the mitotic spindle in yeast and appears to activate this checkpoint mechanism.

Disruption of mitotic spindle orientation in a yeast dynein mutant.

Dynein motor isoforms have been implicated as potential kinetochore-associated motors that power chromosome-to-pole movements during mitosis. The recent identification and sequence determination of genes encoding dynein isoforms has now permitted the in vivo analysis of dynein function in mitosis. In this report we describe the identification and mutational analysis of the gene, DHC1, encoding a dynein heavy chain isoform in Saccharomyces cerevisiae. Sequence analysis of a 9-kb genomic fragment of the DHC1 gene predicts a polypeptide highly homologous to dynein sequences characterized from sea urchin, Dictyostelium, Drosophila, and rat. Mutations in the yeast dynein gene disrupt the normal movement of the spindle into budding daughter cells but have no apparent effect on spindle assembly, spindle elongation, or chromosome segregation. Our results suggest that, in yeast, a dynein microtubule motor protein has a nonessential role in spindle assembly and chromosome movement but is involved in establishing the proper spindle orientation during cell division.