Characterizing the Cellular Response to Nitrogen-Doped Carbon Nanocups.

Carbon nanomaterials, specifically, carbon nanotubes (CNTs) have many potential applications in biology and medicine. Currently, this material has not reached its full potential for application due to the potential toxicity to mammalian cells, and the incomplete understanding of how CNTs interface with cells. The chemical composition and structural features of CNTs have been shown to directly affect their biological compatibility. The incorporation of nitrogen dopants to the graphitic lattice of CNTs results in a unique cup shaped morphology and minimal cytotoxicity in comparison to its undoped counterpart. In this study, we investigate how uniquely shaped nitrogen-doped carbon nanocups (NCNCs) interface with HeLa cells, a cervical cancer epithelial cultured cell line, and RPE-1 cells, an immortalized cultured epithelial cell line. We determined that NCNCs do not elicit a cytotoxic response in cells, and that they are uptaken via endocytosis. We have conjugated fluorescently tagged antibodies to NCNCs and shown that the protein-conjugated material is also capable of entering cells. This primes NCNCs to be a good candidate for subsequent protein modifications and applications in biological systems.

Cell division patterns and chromosomal segregation defects in oral cancer stem cells.

Oral squamous cell carcinoma (OSCC) is a serious public health problem caused primarily by smoking and alcohol consumption or human papillomavirus. The cancer stem cell (CSC) theory posits that CSCs show unique characteristics, including self-renewal and therapeutic resistance. Examining biomarkers and other features of CSCs is critical to better understanding their biology. To this end, the results show that cellular SOX2 immunostaining correlates with other CSC biomarkers in OSCC cell lines and marks the rare CSC population. To assess whether CSC division patterns are symmetrical, resulting in two CSC, or asymmetrical, leading to one CSC and one cancer cell, cell size and fluorescence intensity of mitotic cells stained with SOX2 were analyzed. Asymmetrical SOX2 distribution in ≈25% of the mitoses analyzed was detected. Chromosomal instability, some of which is caused by chromosome segregation defects (CSDs), is a feature of cancer cells that leads to altered gene copy numbers. We compare chromosomal instability (as measured by CSDs) between CSCs (SOX2+) and non-CSCs (SOX2-) from the same OSCC cell lines. CSDs were more common in non-CSCs (SOX2-) than CSCs (SOX2+) and in symmetrical CSC (SOX2+) mitotic pairs than asymmetrical CSC (SOX2+/SOX2-) mitotic pairs. CSCs showed fewer and different types of CSDs after ionizing radiation treatment than non-CSCs. Overall, these data are the first to demonstrate both symmetrical and asymmetrical cell divisions with CSDs in OSCC CSC. Further, the results suggest that CSCs may undergo altered behavior, including therapeutic resistance as a result of chromosomal instability due to chromosome segregation defects. © 2016 Wiley Periodicals, Inc.

Targeted inhibition of ATR or CHEK1 reverses radioresistance in oral squamous cell carcinoma cells with distal chromosome arm 11q loss.

Oral squamous cell carcinoma (OSCC), a subset of head and neck squamous cell carcinoma (HNSCC), is the eighth most common cancer in the U.S.. Amplification of chromosomal band 11q13 and its association with poor prognosis has been well established in OSCC. The first step in the breakage-fusion-bridge (BFB) cycle leading to 11q13 amplification involves breakage and loss of distal 11q. Distal 11q loss marked by copy number loss of the ATM gene is observed in 25% of all Cancer Genome Atlas (TCGA) tumors, including 48% of HNSCC. We showed previously that copy number loss of distal 11q is associated with decreased sensitivity (increased resistance) to ionizing radiation (IR) in OSCC cell lines. We hypothesized that this radioresistance phenotype associated with ATM copy number loss results from upregulation of the compensatory ATR-CHEK1 pathway, and that knocking down the ATR-CHEK1 pathway increases the sensitivity to IR of OSCC cells with distal 11q loss. Clonogenic survival assays confirmed the association between reduced sensitivity to IR in OSCC cell lines and distal 11q loss. Gene and protein expression studies revealed upregulation of the ATR-CHEK1 pathway and flow cytometry showed G2 M checkpoint arrest after IR treatment of cell lines with distal 11q loss. Targeted knockdown of the ATR-CHEK1 pathway using CHEK1 or ATR siRNA or a CHEK1 small molecule inhibitor (SMI, PF-00477736) resulted in increased sensitivity of the tumor cells to IR. Our results suggest that distal 11q loss is a useful biomarker in OSCC for radioresistance that can be reversed by ATR-CHEK1 pathway inhibition.

Kinesin molecular motor Eg5 functions during polypeptide synthesis.

The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.

The role of molecular microtubule motors and the microtubule cytoskeleton in stress granule dynamics.

Stress granules (SGs) are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly.

The c-Myc target glycoprotein1balpha links cytokinesis failure to oncogenic signal transduction pathways in cultured human cells.

An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbalpha), a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbalpha in a p53-deficient background. GpIbalpha was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbalpha.

Widespread genomic instability mediated by a pathway involving glycoprotein Ib alpha and Aurora B kinase.

c-Myc (Myc) oncoprotein induction of genomic instability (GI) contributes to its initial transforming function and subsequent tumor cell evolution. We describe here a pathway by which Myc, via its target protein glycoprotein Ibalpha (GpIb alpha), mediates GI. Proteomic profiling revealed that the serine/threonine kinase Aurora B is down-regulated by GpIb alpha in p53-deficient primary human fibroblasts. The phenotypes of Aurora B deficiency are strikingly reminiscent of Myc or GpIb alpha overexpression and include double-stranded DNA breaks, altered nuclear size and morphology, chromatin bridges, cleavage furrow regression, and tetraploidy. During mitosis, GpIb alpha and Aurora B redistribute to the cleavage furrow along with other cleavage furrow proteins. GpIb alpha overexpression at levels comparable with those seen in some tumor cells causes the dispersal of these proteins but not Aurora B, resulting in furrow regression and cytokinesis failure. Aurora B normalization redirects the mislocalized furrow proteins to their proper location, corrects the cleavage furrow abnormalities, and restores genomic stability. Aurora B thus appears necessary for a previously unrecognized function in guiding and positioning a number of key proteins, including GpIb alpha to the cleavage furrow. These findings underscore the importance of maintaining a delicate balance among cleavage furrow-associated proteins during mitosis. Suppression of Aurora B via GpIb alpha provides a unifying and mechanistic explanation for several types of Myc-mediated GI.

Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1.

BACKGROUND: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol. RESULTS: In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1. CONCLUSION: Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.

Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A(1), was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Actin and microtubules: working together to control spindle polarity.

Centrosomes are frequently amplified in cancer cells, but centrosome clustering pathways act to minimize their detrimental impact on mitosis. Recent data published online in Genes & Developments by Kwon et al. (2008) suggest that these pathways involve microtubule motors, actin, and focal adhesions. Since centrosomal amplification is rarely seen in normal cells, could blocking clustering lead to the selective killing of tumor cells?

A tale of too many centrosomes.

Having the correct number of centrosomes is crucial for proper chromosome segregation during cell division and for the prevention of aneuploidy, a hallmark of many cancer cells. Several recent studies (Basto et al., 2008; Kwon et al., 2008; Yang et al., 2008) reveal the importance of mechanisms that protect against the consequences of harboring too many centrosomes.

Loss of distal 11q is associated with DNA repair deficiency and reduced sensitivity to ionizing radiation in head and neck squamous cell carcinoma.

About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage-fusion-bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of gamma-H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor.

DNA repair pathways involved in anaphase bridge formation.

Cancer cells frequently exhibit gross chromosomal alterations such as translocations, deletions, or gene amplifications an important source of chromosomal instability in malignant cells. One of the better-documented examples is the formation of anaphase bridges-chromosomes pulled in opposite directions by the spindle apparatus. Anaphase bridges are associated with DNA double strand breaks (DSBs). While the majority of DSBs are repaired correctly, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging. To identify the cellular repair pathways used to form these aberrant structures, we tested a requirement for either of the two major DSB repair pathways in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). Our observations show that neither pathway is essential, but NHEJ helps prevent bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to have a positive trend with induction of bridges from DNA damage.

Overexpression of Cdc20 leads to impairment of the spindle assembly checkpoint and aneuploidization in oral cancer.

Defects in the spindle assembly checkpoint are thought to be responsible for an increased rate of aneuploidization during tumorigenesis. Despite a plethora of information on the correlation between BUB-MAD gene expression levels and defects in the spindle checkpoint, very little is known about alteration of another important spindle checkpoint protein, Cdc20, in human cancer and its role in tumor aneuploidy. We observed overexpression of CDC20 in several oral squamous cell carcinoma (OSCC) cell lines and primary head and neck tumors and provide evidence that such overexpression of CDC20 is associated with premature anaphase promotion, resulting in mitotic abnormalities in OSCC cell lines. We also reconstituted the chromosomal instability phenotype in a chromosomally stable OSCC cell line by overexpressing CDC20. Thus, abnormalities in the cellular level of Cdc20 may deregulate the timing of anaphase promoting complex (APC/C) in promoting premature anaphase, which often results in aneuploidy in the tumor cells.

Relationship between FRA11F and 11q13 gene amplification in oral cancer.

Common fragile sites (CFS) are nonstaining gaps or breaks in chromosomes that are expressed under conditions inducing replicative stress. CFS have been suggested to play a role in epithelial cancers by their association with loss of heterozygosity, loss of gene expression, and/or gene amplification in the form of homogeneously staining regions (hsrs). In oral squamous-cell carcinomas (OSCC), amplification of chromosomal band 11q13 occurs in the form of an hsr. We suggested previously that CFS flanking 11q13 may be susceptible to breakage induced by tobacco or other carcinogens and/or human papillomavirus, promoting formation of the 11q13 amplicon. Examination of OSCC cell lines with 11q13 amplification using fluorescence in situ hybridization showed loss of FRA11F sequences, whereas cell lines without 11q13 amplification displayed an intact FRA11F site. Cell lines with more complex 11q rearrangements expressed FRA11F in the form of an inverted duplication, characteristic of breakage-fusion-bridge cycles. Our findings suggest that gene amplification involving chromosomal band 11q13 in OSCC may be initiated by breakage at FRA11F.

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends.

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

Spindle multipolarity is prevented by centrosomal clustering.

Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. Here, we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization, promoting multipolarity.

Cigarette smoke induces anaphase bridges and genomic imbalances in normal cells.

Exposure to cigarette smoke has long been linked to carcinogenesis, but the emphasis has been placed on mutational changes in the DNA sequence caused by the carcinogens in smoke. Here, we report an additional role for cigarette smoke exposure in contributing to chromosomal aberrations in cells. We have found that cigarette smoke condensate (CSC) induces anaphase bridges in cultured human cells, which in a short time lead to genomic imbalances. The frequency of the induced bridges within the entire population decreases with time, and this decrease is not dependent upon the p53-mediated apoptotic pathway. Additionally, we show that CSC induces DNA double stranded breaks (DSBs) in cultured cells and purified DNA. The reactive oxygen species (ROS) scavenger, 2' deoxyguanosine 5'-monophosphate (dGMP) prevents CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Furthermore, since the amount of CSC added to the cultures was substantially less than that extracted from a single cigarette, our results show that even low levels of cigarette smoke can cause irreversible changes in the chromosomal constitution of cultured cells.

Chromosomal instability and marker chromosome evolution in oral squamous cell carcinoma.

Squamous cell carcinoma of the head and neck and its subset, oral squamous cell carcinoma (OSCC), arise through a multistep process of genetic alterations as a result of exposure to environmental agents, such as tobacco smoke, alcoholic beverages, and viruses, including human papillomavirus. We and others have shown that the karyotypes of OSCC are near-triploid and contain multiple structural and numerical abnormalities. However, despite a background of clonal chromosomal aberrations, individual cells within a culture express many nonclonal numerical and structural abnormalities, termed chromosomal instability (CIN). To evaluate CIN in oral cancer cells, we isolated clones from two OSCC cell lines and carried out classical cytogenetic analysis, fluorescence in situ hybridization using centromere-specific probes, and spectral karyotyping. We observed variation in chromosome number within clones and between clones of the same cell line. Although similar numbers of centromeric signals for a particular chromosome were present, "homologs" of a chromosome varied structurally from cell to cell (marker chromosome evolution) as documented by classical and spectral karyotyping. In addition to the numerical chromosome variations within a clone, we observed marker chromosome evolution by structural chromosome alterations. It appears that both intrinsic structural alterations and extrinsic cytoskeletal factors influence chromosome segregation, resulting in individual tumor cells that express unique karyotypes. We show that CIN and marker chromosome evolution are essential acquired features of neoplastic cells. Proliferation of this heterogeneous cell population may provide some cells with the ability to evade standard therapies.

Resolution of anaphase bridges in cancer cells.

Chromosomal instability is a key step in the generation of the cancer cell karyotype. An indicator of unstable chromosomes is the presence of chromatin bridges during anaphase. We examined in detail the fate of anaphase bridges in cultured oral squamous cell carcinoma cells in real-time. Surprisingly, chromosomes in bridges typically resolve by breaking into multiple fragments. Often these fragments give rise to micronuclei (MN) at the end of mitosis. The formation of MN is shown to have important consequences for the cell. We found that MN have incomplete nuclear pore complex (NPC) formation and nuclear import defects and the chromatin within has greatly reduced transcriptional activity. Thus, a major consequence of the presence of anaphase bridges is the regular sequestration of chromatin into genetically inert MN. This represents another source of ongoing genetic instability in cancer cells.