A synthetic lethal screen identifies the Vitamin D receptor as a novel gemcitabine sensitizer in pancreatic cancer cells.

Overcoming chemoresistance of pancreatic cancer (PCa) cells should significantly extend patient survival. The current treatment modalities rely on a variety of DNA damaging agents including gemcitabine, FOLFIRINOX, and Abraxane that activate cell cycle checkpoints, which allows cells to survive these drug treaments. Indeed, these treatment regimens have only extended patient survival by a few months. The complex microenvironment of PCa tumors has been shown to complicate drug delivery thus decreasing the sensitivity of PCa tumors to chemotherapy. In this study, a genome-wide siRNA library was used to conduct a synthetic lethal screen of Panc1 cells that was treated with gemcitabine. A sublethal dose (50 nM) of the drug was used to model situations of limiting drug availability to PCa tumors in vivo. Twenty-seven validated sensitizer genes were identified from the screen including the Vitamin D receptor (VDR). Gemcitabine sensitivity was shown to be VDR dependent in multiple PCa cell lines in clonogenic survival assays. Sensitization was not achieved through checkpoint override but rather through disrupting DNA repair. VDR knockdown disrupted the cells' ability to form phospho-γH2AX and Rad51 foci in response to gemcitabine treatment. Disruption of Rad51 foci formation, which compromises homologous recombination, was consistent with increased sensitivity of PCa cells to the PARP inhibitor Rucaparib. Thus inhibition of VDR in PCa cells provides a new way to enhance the efficacy of genotoxic drugs.

Comparative study between endoscopic ultrasonography and positron emission tomography-computed tomography in staging patients with esophageal squamous cell carcinoma.

Treatment strategy of esophageal cancer mainly depends on accurate staging. At present, no single ideal staging modality is superior to another in preoperative tumor-node-metastasis (TNM) staging of patients with esophageal cancer. We aimed to investigate the efficacy of endoscopic ultrasonography (EUS) and positron emission tomography-computed tomography (PET-CT) for staging of esophageal cancer. We retrospectively studied 118 consecutive patients with esophageal squamous cell carcinoma who underwent esophagectomy with or without neoadjuvant chemoradiotherapy (CRT) over a near 3-year period between January 2005 and November 2008 at a tertiary hospital in Taiwan. Patients were separated into two groups: without neoadjuvant CRT (group 1, n= 28) and with CRT (group 2, n= 90). Medical records of demographic data and reports of EUS and PET-CT of patients before surgery were reviewed. A database of clinical staging by EUS and PET-CT was compared with one of pathological staging. The accuracies of T staging by EUS in groups 1 and 2 were 85.2% and 34.9%. The accuracies of N staging by EUS in groups 1 and 2 were 55.6% and 39.8%. The accuracies of T and N staging by means of PET-CT scan were 100% and 54.5% in group 1, and were 69.4% and 86.1% in group 2, respectively. In group 2, 38 of 90 patients (42.2%) achieved pathologic complete remission. Among them, two of 34 (5.9%) and 12 of 17 (70.6%) patients were identified as tumor-free by post-CRT EUS and PET-CT, respectively. EUS is useful for initial staging of esophageal cancer. PET-CT is a more reliable modality for monitoring treatment response and restaging. Furthermore, the accuracy of PET-CT with regard to N staging is higher in patients who have undergone CRT than those who have not.

Dual inhibition of SRC and Aurora kinases induces postmitotic attachment defects and cell death.

Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These functions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being used in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a postmitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y(416)-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.

CENP-F is a novel microtubule-binding protein that is essential for kinetochore attachments and affects the duration of the mitotic checkpoint delay.

Centromeric protein F (CENP-F) is a 367-kDa human kinetochore protein that was identified a decade ago, but its function was only recently revealed by studies that used small interfering RNA to deplete the protein from cells. All studies showed that CENP-F is important for chromosome alignment, but these studies differed as to whether CENP-F is important to the mitotic checkpoint. We report here that CENP-F is essential for cells to sustain a prolonged mitotic delay in response to unattached kinetochores. Cells depleted of CENP-F exit mitosis in the presence of defective kinetochore attachments resulting from treatment with nocodazole, or the depletion of kinetochore proteins CENP-E and hSgo1. Kinetochores depleted of CENP-F exhibited a reduction in the amounts of the mitotic checkpoint proteins Mad1, Mad2, hBUBR1, hBUB1, and hMps1. We postulate that CENP-F is not an essential component of the mitotic checkpoint but facilitates the duration of the mitotic delay. Separately, we show that CENP-F is a novel microtubule-binding protein that possesses two microtubule-binding domains at opposite ends of the molecule. The C-terminal microtubule-binding domain was found to stimulate microtubule polymerization in vitro. These activities provide a biochemical explanation for how CENP-F contributes to kinetochore attachments in vivo.

Terahertz magnetic response from artificial materials.

We show that magnetic response at terahertz frequencies can be achieved in a planar structure composed of nonmagnetic conductive resonant elements. The effect is realized over a large bandwidth and can be tuned throughout the terahertz frequency regime by scaling the dimensions of the structure. We suggest that artificial magnetic structures, or hybrid structures that combine natural and artificial magnetic materials, can play a key role in terahertz devices.

Targeting the kinetochore for mitosis-specific inhibitors.

Microtubule poisons such as taxol and vinblastine are widely used to treat a variety of cancers. These drugs are believed to kill cells by blocking mitosis. However, there is a critical need to identify new drugs because tumors can often become refractory to treatment with existing drugs. Studies over the past decade on chromosome segregation have uncovered a plethora of novel proteins that function specifically in mitosis. Centrosomes and kinetochores are two organelles that specify formation of the spindle and the attachment of chromosomes to the spindle, respectively. The focus of this review is to highlight the kinetochore as a rich source of targets for the development of mitosis-specific drugs.

Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells.

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.

Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.

Detection of repair activity during the DNA damage-induced G2 delay in human cancer cells.

All eukaryotic cells manifest cell cycle delay after exposure to DNA damaging agents. It has been proposed that such cell cycle checkpoints may allow DNA repair but direct evidence of such activity during the radiation-induced G2 delay has been lacking. We report here that cells arrested in G2 by radiation (2-3 Gy) and etoposide incorporate bromodeoxyuridine (BrdU) at discrete foci in the nucleus. We detected G2 cells with CENP-F, a nuclear protein maximally expressed in G2. Caffeine and okadaic acid, both established radiosensitizers, inhibit the incorporation of BrdU in G2 cells. Radioresistant HT29 and OVCAR cells demonstrate BrdU foci formation more frequently during the G2 delay when compared to the more radiosensitive A2780 cell line. The repair foci formed during G2 may be followed through mitosis and observed in daughter cells in G1. Taken together, these observations are consistent with the detection of DNA repair activity during the radiation-induced G2 delay after relatively low doses of radiation.

The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins.

The mRNA export factor RAE1 (also called GLE2) and the mitotic checkpoint protein BUB3 share extensive sequence homology in yeast as well as higher eukaryotes, although the biological relevance of their similarity is unclear. Previous work in HeLa cells has shown that human (h)RAE1 binds the nuclear pore complex protein hNUP98 via a short NUP98 motif called GLEBS (for GLE2p-binding sequence). Here we report that the two known binding partners of hBUB3, the mitotic checkpoint proteins hBUB1 and hBUBR1, both carry a region with remarkable similarity to the GLEBS motif of hNUP98. We show that the GLEBS-like motifs of mouse (m)BUB1 and mBUBR1 are sufficient for mBUB3 binding. mBUB3 lacks affinity for the hNUP98 GLEBS, demonstrating its binding specificity for GLEBS motifs of mitotic checkpoint proteins. Interestingly, mRAE1 does not exclusively bind to the GLEBS motif of hNUP98 and can cross-interact with the mBUB1 GLEBS. We show that full-length RAE1 and BUB1 proteins interact in mammalian cells and accumulate both at the kinetochores of prometaphase chromosomes. Our findings demonstrate that GLEBS motifs reside in mammalian nucleoporins and mitotic checkpoint proteins and apparently serve as specific binding sites for either BUB3, RAE1, or both.

A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression.

Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BRCA2-Associated Factor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression.

Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses.

Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated killing [corrected] and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.

The farnesyltransferase inhibitor, FTI-2153, blocks bipolar spindle formation and chromosome alignment and causes prometaphase accumulation during mitosis of human lung cancer cells.

Even though farnesyltransferase inhibitors (FTIs), a novel class of therapeutic agents presently in clinical trials, have preclinically outstanding anticancer activity and impressive lack of toxicity, their mechanism of action is not well understood. To enhance our understanding of how FTIs inhibit the growth of tumors, we have investigated their effects on cell cycle progression of two human lung cancer cell lines, A-549 and Calu-1. In this report, we show in synchronized A-549 and Calu-1 cells that FTI-2153 treatment resulted in a large accumulation of cells in the mitosis phase of the cell division cycle, with some cells in the G(0)/G(1) phase. Furthermore, microtubule immunostaining and 4,6-diamidino-2-phenylindole DNA staining demonstrated that the FTI-2153-induced accumulation in mitosis is due to the inability of these cells to progress from prophase to metaphase. FTI-2153 inhibited the ability of A-549 and Calu-1 cells to form bipolar spindles and caused formation of monoasteral spindles. Furthermore, FTI-2153 induced a ring-shaped chromosome morphology and inhibited chromosome alignment. Time-lapse videomicroscopy confirmed this result by showing that FTI-2153-treated cells are unable to align their chromosomes at the metaphase plate. FTI-2153 did not affect the localization to the kinetochores of two farnesylated centromeric proteins, CENP-E and CENP-F. Thus, a mechanism by which FTIs inhibit progression through mitosis and tumor growth is by blocking bipolar spindle formation and chromosome alignment.

Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere--kinetochore complexes.

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.

HZwint-1, a novel human kinetochore component that interacts with HZW10.

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes.

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.