Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.

Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.

Using Budding Yeast to Identify Molecules That Block Cancer Cell 'Mitotic Slippage' Only in the Presence of Mitotic Poisons.

Research on the budding yeast Saccharomyces cerevisiae has yielded fundamental discoveries on highly conserved biological pathways and yeast remains the best-studied eukaryotic cell in the world. Studies on the mitotic cell cycle and the discovery of cell cycle checkpoints in budding yeast has led to a detailed, although incomplete, understanding of eukaryotic cell cycle progression. In multicellular eukaryotic organisms, uncontrolled aberrant cell division is the defining feature of cancer. Some of the most successful classes of anti-cancer chemotherapeutic agents are mitotic poisons. Mitotic poisons are thought to function by inducing a mitotic spindle checkpoint-dependent cell cycle arrest, via the assembly of the highly conserved mitotic checkpoint complex (MCC), leading to apoptosis. Even in the presence of mitotic poisons, some cancer cells continue cell division via 'mitotic slippage', which may correlate with a cancer becoming refractory to mitotic poison chemotherapeutic treatments. In this review, knowledge about budding yeast cell cycle control is explored to suggest novel potential drug targets, namely, specific regions in the highly conserved anaphase-promoting complex/cyclosome (APC/C) subunits Apc1 and/or Apc5, and in a specific N-terminal region in the APC/C co-factor cell division cycle 20 (Cdc20), which may yield molecules which block 'mitotic slippage' only in the presence of mitotic poisons.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase.

In Saccharomyces cerevisiae, the S-phase cyclin Clb6 is expressed shortly before the G1/S transition. It has been shown that in S phase the SCF(Cdc4) ubiquitin ligase controls Clb6 proteolysis, which requires cyclin-dependent kinases activity. A Clb6-3A mutant, bearing non-phosphorylatable mutations at S6A, T39A, and S147A, was observed to be hyperstabilized in S-phase but was unstable in mitosis. In this study, we found that the APC(Cdh1) form of the Anaphase-Promoting Complex (APC) was required for Clb6 proteolysis in both early and late G1. An in vitro ubiquitination assay confirmed that Clb6 is a substrate for APC(Cdh1). A KEN box and a destruction box in the Clb6N-terminus were identified. Mutations in the KEN box (mkb) and/or the destruction box (mdb) enhanced Clb6 stability in G1. Expression of Clb6mkd, bearing both mutations in the mkb and mdb, allowed cells to bypass the late G1 arrest caused by cdc4-1. This bypass phenotype was observed to depend upon CDK phosphorylation at residues S6, T39 and S147. Compared to Clb6, overexpression of Clb6ST, bearing all five mutations of S6A, T39A, S147A, mkb and mdb in combination, had a greater effect on promoting expression of Clb2 and S-phase entry, caused a greater G2 delay and a greater defect in cell division. Swe1 was also required for bud emergence when Clb6ST was overexpressed. Our observations suggest that both APC(Cdh1) and SCF(Cdc4)-dependent proteolysis of Clb6 at the G1/S border are crucial for multiple cell cycle regulated events including proper expression of Clb2, the G1/S and G2/M cell cycle transitions and for proper completion of cell division at mitotic exit.

The Mad1-Mad2 balancing act--a damaged spindle checkpoint in chromosome instability and cancer.

Cancer cells are commonly aneuploid. The spindle checkpoint ensures accurate chromosome segregation by controlling cell cycle progression in response to aberrant microtubule-kinetochore attachment. Damage to the checkpoint, which is a partial loss or gain of checkpoint function, leads to aneuploidy during tumorigenesis. One form of damage is a change in levels of the checkpoint proteins mitotic arrest deficient 1 and 2 (Mad1 and Mad2), or in the Mad1:Mad2 ratio. Changes in Mad1 and Mad2 levels occur in human cancers, where their expression is regulated by the tumor suppressors p53 and retinoblastoma 1 (RB1). By employing a standard assay, namely the addition of a mitotic poison at mitotic entry, it has been shown that checkpoint function is normal in many cancer cells. However, in several experimental systems, it has been observed that this standard assay does not always reveal checkpoint aberrations induced by changes in Mad1 or Mad2, where excess Mad1 relative to Mad2 can lead to premature anaphase entry, and excess Mad2 can lead to a delay in entering anaphase. This Commentary highlights how changes in the levels of Mad1 and Mad2 result in a damaged spindle checkpoint, and explores how these changes cause chromosome instability that can lead to aneuploidy during tumorigenesis.

Sonic hedgehog antagonists induce cell death in acute myeloid leukemia cells with the presence of lipopolysaccharides, tumor necrosis factor-α, or interferons.

Due to the development of drug resistance, the outcome for the majority of patients with acute myeloid leukemia (acute myelogenous leukemia; AML) remains poor. To prevent drug resistance and increase the therapeutic efficacy of treating AML, the development of new combinatory drug therapies is necessary. Sonic hedgehog (Shh) is expressed in AML biopsies and is essential for the drug resistance of cancer stem cells of AML. AML patients are frequently infected by bacteria and exposed to lipopolysaccharide (LPS). LPS itself, its derivatives, and its downstream effectors, such as tumor necrosis factor-α (TNF-α) and interferons (IFNs), have been shown to provoke anti-tumor effects. The application of a Shh inhibitor against AML cells in the presence of LPS/TNF-α/IFNs has not been investigated. We found that the Shh inhibitor cyclopamine in combination with LPS treatment synergistically induced massive cell apoptosis in THP-1 and U937 cells. The cytotoxic effects of this combined drug treatment were confirmed in 5 additional AML cell lines, in primary AML cells, and in an AML mouse model. Replacing cyclopamine with another Shh inhibitor, Sant-1, had the same effect. LPS could be substituted by TNF-α or IFNs to induce AML cell death in combination with cyclopamine. Our results suggest a potential strategy for the development of new therapies employing Shh antagonists in the presence of LPS/TNF-α/IFNs for the treatment of AML patients.

Development of a Chemical Cocktail That Rescues Mouse Brain Demyelination in a Cuprizone-Induced Model.

Oligodendrocytes are glial cells located in the central nervous system (CNS) that play essential roles in the transmission of nerve signals and in the neuroprotection of myelinated neurons. The dysfunction or loss of oligodendrocytes leads to demyelinating diseases such as multiple sclerosis (MS). To treat demyelinating diseases, the development of a therapy that promotes remyelination is required. In the present study, we established an in vitro method to convert human fibroblasts into induced oligodendrocyte-like cells (iOLCs) in 3 days. The induced cells displayed morphologies and molecular signatures similar to oligodendrocytes after treatment with valproic acid and exposure to the small molecules Y27632, SU9516, and forskolin (FSK). To pursue the development of a cell-free remyelination therapy in vivo, we used a cuprizone-induced demyelinated mouse model. The small molecules (Y27632, SU9516, and FSK) were directly injected into the demyelinated corpus callosum of the mouse brain. This combination of small molecules rescued the demyelination phenotype within two weeks as observed by light and electron microscopy. These results provide a foundation for exploring the development of a treatment for demyelinating diseases via regenerative medicine.

Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis Pathway.

Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, podocalyxin-like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self-renewal, protein expression of c-MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown-mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single-cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human-mouse chimeras, laminin and collagen signaling-related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.

Functions of the mitotic B-type cyclins CLB1, CLB2, and CLB3 at mitotic exit antagonized by the CDC14 phosphatase.

In the budding yeast Saccharomyces cerevisiae, cell cycle progression and cytokinesis at mitotic exit are proposed to be linked by CDC14 phosphatase antagonizing the function of mitotic B-type cyclin (CLBs). We have isolated a temperature-sensitive mutant, cdc14(A280V), with a mutation in the conserved phosphatase domain. Prolonged arrest in the cdc14(A280V) mutant partially uncoupled cell cycle progression from the completion of cytokinesis as measured by bud re-emergence, in the form of elongated apical projections, and DNA re-replication. In contrast to previous mitotic exit mutants, cdc14(A280V) mutants displayed a strong bias for the first apical projection to form in the mother cell body. Using cdc14(A280V) mutant phenotypes, the functions of the B-type cyclins at mitotic exit were investigated. The preference in mother-daughter apical projection formation was observed to be independent of any individual CLB function. However, cdc14(A280V)clb1Δ cells displayed a pronounced increase in apical projections, while cdc14(A280V)clb3Δ cells were observed to form round cellular chains. While cdc14(A280V) cells arrested at mitotic exit, both cdc14(A280V)clb1Δ and cdc14(A280V)clb3Δ cells completed cytokinesis, but failed cell separation. cdc14(A280V)clb2Δ cells displayed a defect in actin ring assembly. These observations differentiate the functions of CLB1, CLB2, and CLB3 at mitotic exit, and are consistent with the hypothesis that CLB activities are antagonized by the CDC14 phosphatase in order to couple cell cycle progression with cytokinesis at mitotic exit.

Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity.

The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.

Protein Complexes Form a Basis for Complex Hybrid Incompatibility.

Proteins are the workhorses of the cell and execute many of their functions by interacting with other proteins forming protein complexes. Multi-protein complexes are an admixture of subunits, change their interaction partners, and modulate their functions and cellular physiology in response to environmental changes. When two species mate, the hybrid offspring are usually inviable or sterile because of large-scale differences in the genetic makeup between the two parents causing incompatible genetic interactions. Such reciprocal-sign epistasis between inter-specific alleles is not limited to incompatible interactions between just one gene pair; and, usually involves multiple genes. Many of these multi-locus incompatibilities show visible defects, only in the presence of all the interactions, making it hard to characterize. Understanding the dynamics of protein-protein interactions (PPIs) leading to multi-protein complexes is better suited to characterize multi-locus incompatibilities, compared to studying them with traditional approaches of genetics and molecular biology. The advances in omics technologies, which includes genomics, transcriptomics, and proteomics can help achieve this end. This is especially relevant when studying non-model organisms. Here, we discuss the recent progress in the understanding of hybrid genetic incompatibility; omics technologies, and how together they have helped in characterizing protein complexes and in turn multi-locus incompatibilities. We also review advances in bioinformatic techniques suitable for this purpose and propose directions for leveraging the knowledge gained from model-organisms to identify genetic incompatibilities in non-model organisms.

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells.

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are 'true' totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely 'cluster 3', as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Combination Vaccination With Tetanus Toxoid and Enhanced Tumor-Cell Based Vaccine Against Cervical Cancer in a Mouse Model.

Cervical cancer is the fourth most common cancer in women with an estimated 570,000 new cases in 2018 which constitute about 6. 6% of all cancers in women according to WHO report 2018. Approximately 90% of the 270,000 deaths from cervical cancer in 2015 occurred in low- and middle-income countries. In cervical cancers, which is caused by human papillomavirus (HPV) infection, the expression of HPV 16 E6 and E7 proteins are essential for tumor cell transformation and maintenance of malignancy. Prophylactic vaccines against cervical cancer caused by human papillomavirus have not proven successful. Although virus-like particle-based (VLPs) vaccines have been developed with prophylactic activities to prevent most HPV infections, the therapeutic effect of VLP vaccines has yet to be demonstrated for those who were already infected. A recent study showed that pre-conditioning mice with a potent antigen such as tetanus toxoid significantly improves lymph node homing and efficacy of dendritic cells. Tetanus toxoid has also been used in combination with DNA vaccines designed from tumor based antigens. In the present study, we pre-conditioned mice with tetanus toxoid followed by vaccination with a Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) overexpressing tumor-cell based vaccine (GVAX). We observed that pre-conditioning with tetanus toxoid followed by vaccination with GVAX regressed tumor growth and enhanced the overall survival of the mice. Pre-conditioning with tetanus toxoid enhanced the immune response which was observed by enlarged spleen size, higher proliferation rate of lymphocytes, a higher level of IFN-γ, TNF-α, and IL-4 antigen-specific secretions by the splenocytes. Pre-conditioning with tetanus toxoid increased memory T cell migration into the tumor site and spleen. The antigen-specific cytotoxic T cell lysis percentage was also found to be higher in the group of mice vaccinated with the combination of tetanus toxoid and GVAX. Hence, pre-conditioning with tetanus toxoid prior to vaccination with a tumor-cell based vaccine overexpressing GM-CSF might be an effective strategy for targeting E7-specific HPV-associated cervical malignancy.

Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates.

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins.

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

An in vitro assay for Cdc20-dependent mitotic anaphase-promoting complex activity from budding yeast.

Cell cycle transitions are controlled, in part, by ubiquitin-dependent proteolysis. In mitosis, the metaphase to anaphase transition is governed by an E3 ubiquitin ligase called the cyclosome or Anaphase-Promoting Complex (APC), and a WD40-repeat protein co-factor called Cdc20. In vitro Cdc20-dependent APC (APC(Cdc20)) assays have been useful in the identification and validation of target substrates, and in the study of APC enzymology and regulation. Many aspects of the regulation of cell cycle progression have been discovered in the budding yeast Saccharomyces cerevisiae, and proteins purified from this model organism have been employed in a wide variety of in vitro assays. Here we outline a quantitative in vitro mitotic APC(Cdc20) assay that makes use of a highly active form of the APC that is purified from budding yeast cells arrested in mitosis.

Effect of Multiple Vaccinations with Tumor Cell-Based Vaccine with Codon-Modified GM-CSF on Tumor Growth in a Mouse Model.

Ectopic expression of codon-modified granulocyte-macrophage colony-stimulating factor (cGM-CSF) in TC-1 cells (TC-1/cGM-CSF), a model cell line for human papillomavirus (HPV)-infected cervical cancer cells, increased the expression level of GM-CSF and improved the efficacy of tumor cell-based vaccines in a cervical cancer mouse model. The number of vaccine doses required to induce a long-term immune response in a cervical cancer mouse model is poorly understood. Here, we investigated one, three, and five doses of the irradiated TC-1/cGM-CSF vaccine to determine which dose was effective in inducing a greater immune response and the suppression of tumors. Our findings showed that three doses of irradiated TC-1/cGM-CSF vaccine elicited slower tumor growth rates and enhanced survival rates compared with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Consistently, mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine exhibited stronger interferon gamma (IFN-γ) production in HPV E7-specific CD8⁺ T cells and CD4⁺ T cells. A higher percentage of natural killer cells and interferon-producing killer dendritic cells (IKDCs) appeared in the splenocytes of the mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with those of the mice vaccinated with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Our findings demonstrate that single or multiple vaccinations, such as five doses, with irradiated TC-1/cGM-CSF vaccine suppressed the immune response, whereas three doses of irradiated TC-1/cGM-CSF vaccine elicited a greater immune response and subsequent tumor suppression.

Peptide inhibitors of the anaphase promoting-complex that cause sensitivity to microtubule poison.

There is an interest in identifying Anaphase Promoting-Complex/Cyclosome (APC/C) inhibitors that lead to sensitivity to microtubule poisons as a strategy for targeting cancer cells. Using budding yeast Saccharomyces cerevisiae, peptides derived from the Mitotic Arrest Deficient 2 (Mad2)-binding motif of Cell Division Cycle 20 (Cdc20) were observed to inhibit both Cdc20- and CDC20 Homology 1 (Cdh1)-dependent APC/C activity. Over expression of peptides in vivo led to sensitivity to a microtubule poison and, in a recovery from a microtubule poison arrest, delayed degradation of yeast Securin protein Precocious Dissociation of Sisters 1 (Pds1). Peptides with mutations in the Cdc20 activating KILR-motif still bound APC/C, but lost the ability to inhibit APC/C in vitro and lost the ability to induce sensitivity to a microtubule poison in vivo. Thus, an APC/C binding and activation motif that promotes mitotic progression, namely the Cdc20 KILR-motif, can also function as an APC/C inhibitor when present in excess. Another activator for mitotic progression after recovery from microtubule poison is p31comet, where a yeast predicted open-reading frame YBR296C-A encoding a 39 amino acid predicted protein was identified by homology to p31comet, and named Tiny Yeast Comet 1 (TYC1). Tyc1 over expression resulted in sensitivity to microtubule poison. Tyc1 inhibited both APC/CCdc20 and APC/CCdh1 activities in vitro and bound to APC/C. A homologous peptide derived from human p31comet bound to and inhibited yeast APC/C demonstrating evolutionary retention of these biochemical activities. Cdc20 Mad2-binding motif peptides and Tyc1 disrupted the ability of the co-factors Cdc20 and Cdh1 to bind to APC/C, and co-over expression of both together in vivo resulted in an increased sensitivity to microtubule poison. We hypothesize that Cdc20 Mad2-binding motif peptides, Tyc1 and human hp31 peptide can serve as novel molecular tools for investigating APC/C inhibition that leads to sensitivity to microtubule poison in vivo.

The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit.

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.