High-content analysis identified synergistic drug interactions between INK128, an mTOR inhibitor, and HDAC inhibitors in a non-small cell lung cancer cell line.

BACKGROUND: The development of drug resistance is a major cause of cancer therapy failures. To inhibit drug resistance, multiple drugs are often treated together as a combinatorial therapy. In particular, synergistic drug combinations, which kill cancer cells at a lower concentration, guarantee a better prognosis and fewer side effects in cancer patients. Many studies have sought out synergistic combinations by small-scale function-based targeted growth assays or large-scale nontargeted growth assays, but their discoveries are always challenging due to technical problems such as a large number of possible test combinations. METHODS: To address this issue, we carried out a medium-scale optical drug synergy screening in a non-small cell lung cancer cell line and further investigated individual drug interactions in combination drug responses by high-content image analysis. Optical high-content analysis of cellular responses has recently attracted much interest in the field of drug discovery, functional genomics, and toxicology. Here, we adopted a similar approach to study combinatorial drug responses. RESULTS: By examining all possible combinations of 12 drug compounds in 6 different drug classes, such as mTOR inhibitors, HDAC inhibitors, HSP90 inhibitors, MT inhibitors, DNA inhibitors, and proteasome inhibitors, we successfully identified synergism between INK128, an mTOR inhibitor, and HDAC inhibitors, which has also been reported elsewhere. Our high-content analysis further showed that HDAC inhibitors, HSP90 inhibitors, and proteasome inhibitors played a dominant role in combinatorial drug responses when they were mixed with MT inhibitors, DNA inhibitors, or mTOR inhibitors, suggesting that recessive drugs could be less prioritized as components of multidrug cocktails. CONCLUSIONS: In conclusion, our optical drug screening platform efficiently identified synergistic drug combinations in a non-small cell lung cancer cell line, and our high-content analysis further revealed how individual drugs in the drug mix interact with each other to generate combinatorial drug response.

The molecular characteristics and functional roles of microspherule protein 1 (MCRS1) in gene expression, cell proliferation, and organismic development.

Accurate spatial and temporal regulation of cell cycle progression is essential for cell proliferation and organismic development. This review demonstrates the role of microspherule protein 58kD, commonly known as MCRS1, as a key cell cycle regulator of higher eukaryotic organisms. We discuss the isoforms and functional domains of MCRS1 as well as their subcellular localization at specific stages of the cell cycle. These molecular characteristics reveal MCRS1's dynamic regulatory role in gene expression, genome stability, cell proliferation, and organismic development. Furthermore, we discuss the molecular details of its seemingly opposite, tumor-suppressive or tumor-promoting, role in different types of cancer.

The genomic stability regulator PTIP is required for proper chromosome segregation in mitosis.

BACKGROUND: The Pax transcription activation domain-interacting protein (PTIP) is a nuclear protein that is an essential component of H3K4 methylation for gene activation in vascular, kidney, B cell, and adipocyte development. Furthermore, it plays a key role in genomic stability in higher eukaryotic cells. It binds to 53BP1 and antagonizes inappropriate homologous recombination for a proper DNA damage response. Interestingly, an early study reported mitotic defects after PTIP inactivation, but it is not clear whether PTIP directly facilitates mitotic processes. RESULTS: Here, we showed that PTIP is essential for the mitotic integrity of HeLa cells. PTIP inactivation increases cell death during mitotic exit, which appears to result from direct mitotic defects. PTIP inactivation did not affect the G2M DNA damage checkpoint during interphase upon etoposide treatment. However, in mitosis, PTIP inactivation results in prolonged mitotic time, inefficient chromosome alignment, and increased cell death. Furthermore, PTIP localizes to the mitotic centrosome via BRCT domains at the C-terminus. CONCLUSION: This study reveals a novel function of PTIP in maintaining the genomic stability of higher eukaryotes during mitosis. Therefore, its deregulation, which occurs in various tumors, may destabilize the genome by introducing an abnormal DNA damage response, as well as erroneous chromosome segregation.

Regulation of Life & Death by REGγ.

REGγ, a proteasome activator belonging to the 11S (otherwise known as REG, PA28, or PSME) proteasome activator family, is widely present in many eukaryotes. By binding to the 20S catalytic core particle, REGγ acts as a molecular sieve to selectively target proteins for degradation in an ATP- and ubiquitin-independent manner. This non-canonical proteasome pathway directly regulates seemingly unrelated cellular processes including cell growth and proliferation, apoptosis, DNA damage response, immune response, and metabolism. By affecting different pathways, REGγ plays a vital role in the regulation of cellular life and death through the maintenance of protein homeostasis. As a promoter of cellular growth and a key regulator of several tumor suppressors, many recent studies have linked REGγ overexpression with tumor formation and suggested the REGγ-proteasome as a potential target of new cancer-drug development. This review will present an overview of the major functions of REGγ as it relates to the regulation of cellular life and death, along with new mechanistic insights into the regulation of REGγ.

Mps1 regulates spindle morphology through MCRS1 to promote chromosome alignment.

Accurate partitioning of chromosomes during mitosis is essential for genetic stability and requires the assembly of the dynamic mitotic spindle and proper kinetochore-microtubule attachment. The spindle assembly checkpoint (SAC) monitors the incompleteness and errors in kinetochore-microtubule attachment and delays anaphase. The SAC kinase Mps1 regulates the recruitment of downstream effectors to unattached kinetochores. Mps1 also actively promotes chromosome alignment during metaphase, but the underlying mechanism is not completely understood. Here, we show that Mps1 regulates chromosome alignment through MCRS1, a spindle assembly factor that controls the dynamics of the minus end of kinetochore microtubules. Mps1 binds and phosphorylates MCRS1. This mechanism enables KIF2A localization to the minus end of spindle microtubules. Thus, our study reveals a novel role of Mps1 in regulating the dynamics of the minus end of microtubules and expands the functions of Mps1 in genome maintenance.

Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines.

High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them--the ORACL--best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy by means of orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power.

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells.

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1-INCENP and HP1-Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres.

Kinase signaling in the spindle checkpoint.

The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.

Structure and substrate recruitment of the human spindle checkpoint kinase Bub1.

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint.

The spindle checkpoint ensures the accuracy of chromosome segregation during mitosis. The protein serine/threonine kinase, Mps1, is a critical component of the spindle checkpoint in human cells and regulates the kinetochore localization of key checkpoint proteins. The kinase activity of Mps1 is required for the spindle checkpoint, but how Mps1 is activated during mitosis is unclear. Here, we show that the endogenous Mps1 in mitotic HeLa cells is phosphorylated on T676, a residue in the activation loop. This phosphorylation event on Mps1 is required for its kinase activity in vitro and for spindle checkpoint signaling in vivo. T676 phosphorylation of Mps1 increases during mitosis and can occur through intermolecular/trans autophosphorylation. Induced dimerization of Mps1 is sufficient to activate its kinase activity in cells. We speculate that the kinetochore localization of Mps1 raises its local concentration, leading to its activation during mitosis through more efficient trans autophosphorylation.

A protein interaction map of the mitotic spindle.

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.

Phosphorylation-facilitated sumoylation of MEF2C negatively regulates its transcriptional activity.

BACKGROUND: Sumoylation has emerged as an important posttranslational regulatory mechanism for transcription factors and cofactors. Sumoylation of many transcription factors represses their transcriptional activities. The myocyte enhancer factor 2 (MEF2) family of transcription factors plays an important role in regulating gene expression during myogenesis and has been recently shown to be sumoylated. RESULTS: Consistent with earlier reports, we show that sumoylation of MEF2C at K391 inhibits its transcriptional activity. Sumoylation of MEF2C does not block its DNA-binding activity. A small C-terminal fragment of MEF2C containing K391, referred to as delta-N2-MEF2C, is efficiently sumoylated and, when targeted to DNA, represses transcription at neighbouring promoters. Because delta-N2-MEF2C lacks the binding site for class II histone deacetylases (HDACs), this result suggests that sumoylation of MEF2C may help to recruit transcriptional repressors other than these HDACs. Intriguingly, we show that phosphorylation of S396 in MEF2C, a residue in close proximity to the major sumoylation site (K391) and known to be phosphorylated in vivo, enhances sumoylation of delta- N2-MEF2C in vitro. The S396A mutation reduces sumoylation of MEF2C in vivo and enhances the transcription activity of MEF2C in reporter assays. CONCLUSION: We propose that phosphorylation of MEF2C at S396 facilitates its sumoylation at K391, which in turn recruits yet unidentified co-repressors to inhibit transcription. Our studies further suggest that sumoylation motifs containing a phosphorylated serine or an acidic residue at the +5 position might be more efficiently sumoylated.

Crm1-mediated nuclear export of Cdc14 is required for the completion of cytokinesis in budding yeast.

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.

Systematic identification and analysis of mammalian small ubiquitin-like modifier substrates.

Small ubiquitin-like modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an in vitro expression cloning approach, we have identified 40 human SUMO1 substrates. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. We have validated the sumoylation of 24 substrates in living cells. Analysis of this panel of SUMO substrates leads to the following observations. 1) Sumoylation is more efficient in vitro than in living cells. Polysumoylation occurs on several substrates in vitro. 2) SUMO isopeptidases have little substrate specificity. 3) The SUMO ligases, PIAS1 and PIASxbeta, have broader substrate specificities than does PIASy. 4) Although SUMO1 and SUMO2 are equally efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo. 5) Most SUMO substrates localize to the nucleus, and sumoylation does not generally affect their subcellular localization. Therefore, sumoylation appears to regulate the functions of its substrates through multiple, context-dependent mechanisms.