High-dose chemotherapy augments the efficacy of recombinant adenovirus vaccines and improves the therapeutic outcome.

We have investigated the therapeutic potential of a prototypic melanoma vaccine based on recombinant adenovirus expressing human dopachrome tautomerase in the B16F10 murine melanoma model. We found that in the presence of a tumor, the magnitude of T-cell immunity evoked by the vaccine was significantly reduced. This impairment was compounded by defects in cytokine production and degranulation within the tumor-infiltrating lymphocytes (TILs). We showed that the combination of vaccination with high-dose cyclophosphamide was able to skew the response toward the target antigen and enhanced both the quantity and quality of antigen-specific CD8+ and CD4+ T-cell responses in tumor-bearing mice, which resulted in the inhibition of tumor growth. Furthermore, when tumor-specific antigens were targeted by the vaccine, the combination therapy could actually produce tumor regression, which appeared to result from the high frequency of antigen-specific T cells. These data show that recombinant adenovirus vaccines are compatible with conventional high-dose chemotherapy and that the combined treatment results in improved therapeutic outcomes relative to either agent individually.

A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast.

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).

Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway.

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

spo12 is a multicopy suppressor of mcs3 that is periodically expressed in fission yeast mitosis.

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.

Control of the eukaryotic cell cycle by MAP kinase signaling pathways.

In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane-bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross-regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen-activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.

A role for the START gene-specific transcription factor complex in the inactivation of cyclin B and Cut2 destruction.

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature. Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1. The mcs1-77 mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation. We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene-specific transcription factor complex MBF (also known as DSC-1). The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue. Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phase mcs1-77 cells. Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature. Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay. We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex. These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators.

The COP9/signalosome complex is conserved in fission yeast and has a role in S phase.

The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.

Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway.

The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stress-activated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

Stress-activated MAP kinase (mitogen-activated protein kinase) pathways of budding and fission yeasts.

All eukaryotic cells share the ability to sense and rapidly respond to environmental stress by initiating cyto-protective programmes of gene expression, protein translation and protein degradation. The molecular basis underlying these processes is, however, not well understood. Recently, attention has become focused on an evolutionarily conserved family of protein kinases called the stress-activated mitogen-activated protein kinases (SAPKs) that are activated when cells are challenged with a variety of environmental stresses or cytotoxic agents. Two members of the SAPK family, HOG1 and Sty1/Spc1, have been identified in the distantly related budding and fission yeasts, respectively. This has allowed researchers to genetically and biochemically dissect the structure of these pathways to begin to understand how they are activated and the role of the SAPKs in the cyto-protective response. In this chapter, I compare the structure of the SAPK pathways in the two yeasts and illustrate how this knowledge may benefit our understanding of stress sensing in mammalian cells.

Evidence for a novel MAPKKK-independent pathway controlling the stress activated Sty1/Spc1 MAP kinase in fission yeast.

The fission yeast Sty1/Spc1 MAP kinase, like the mammalian JNK/SAPK and p38/CSBP1 kinases, is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, heat shock, UV light and the protein synthesis inhibitor anisomycin. Sty1 is activated by a single MAPKK, Wis1. We demonstrate that the conserved MAPKKK phosphorylation sites Ser 469 and Thr 473 in the catalytic domain of Wis1 are normally essential for Sty1 activation. However, when mildly overexpressed, a mutant Wis1 kinase lacking these conserved phosphorylation sites is able to support stress inducible gene expression and activation of the Sty1 MAP kinase in response to an oxidative or osmotic stress or to a mild heat shock. We show that phosphorylation and activation of Sty1 under these conditions is not due to inactivation of the Pyp1 MAP kinase phosphatase. These results reveal a novel MAPKKK-independent pathway by which the Wis1 MAPKK can activate the Sty1 MAPK in response to stress in fission yeast.

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase.

The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sicl, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase.

The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway.

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

Regulated vacuole fusion and fission in Schizosaccharomyces pombe: an osmotic response dependent on MAP kinases.

BACKGROUND: The budding yeast Saccharomyces cerevisiae uses two mitogenactivated protein (MAP) kinase cascades, the Hog1p and the Mpk1p pathways, to signal responses to hypertonic and hypotonic stress, respectively. Mammalian cells and the fission yeast Schizosaccharomyces pombe have functional homologues of Hog1p - p38/RK/CSBP and Sty1 - which, unlike Hog1p, also mediate other responses. We have investigated the involvement of S. pombe MAP kinase pathways in signalling a newly described response to osmotic stress - that of vacuole fusion and fission. RESULTS: When S. pombe is placed into water, its vacuoles rapidly fuse into larger structures enclosing a greater proportion of the cell's volume. Under some conditions, its vacuoles can slowly fragment in response to salt. Fission requires the Sty1 pathway and also Pmk1, the homologue of S. cerevisiae Mpk1p. Fusion requires Pmk1, Ypt7 - the homologue of a protein involved in S. cerevisiae vacuole fusion - and part of the Sty1 pathway, although Sty1 phosphorylation is unaffected by hypotonic conditions. CONCLUSIONS: Vacuole fusion and fission appear to be homeostatic mechanisms that restore the concentration of the cytosol. Vacuole fusion, like stimulated secretion in higher eukaryotes, is a rapid and specific process of membrane fusion in response to an external stimulus. The Sty1 pathway, in addition to its role in responding to hypertonic stress, is required at a basal level for the expression of factors required to respond to hypotonic stress - a mechanism that may allow the cell to use a common pathway for different responses.

The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle.

The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK. We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.

The Atf1 transcription factor is a target for the Sty1 stress-activated MAP kinase pathway in fission yeast.

The atf1+ gene of Schizosaccharomyces pombe encodes a bZIP transcription factor with strong homology to the mammalian factor ATF-2. ATF-2 is regulated through phosphorylation in mammalian cells by the stress-activated mitogen-activated protein (MAP) kinases SAPK/JNK and p38. We show here that the fission yeast Atf1 factor is also regulated by a stress-activated kinase, Sty1. The Sty1 kinase is stimulated by a variety of different stress conditions including osmotic and oxidative stress and heat shock. Deletion of the atf1+ gene results in many, but not all, of the phenotypes associated with loss of Sty1, including sensitivity to environmental stress and inability to undergo sexual conjugation. Furthermore, we identify a number of target genes that are induced rapidly in a manner dependent upon both the Sty1 kinase and the Atf1 transcription factor. These genes include gpd1+, which is important for the response of cells to osmotic stress, the catalase gene lambda important for cells to combat oxidative stress, and pyp2+, which encodes a tyrosine-specific MAP kinase phosphatase. Induction of Pyp2 by Atf1 is direct in that it does not require de novo protein synthesis and results in a negative feedback loop that serves to control signaling through the Sty1/Wis1 pathway. We show that Atf1 associates stably and is phosphorylated by the Sty1 kinase in vitro. Taken together, these results indicate that the interaction between AM and Sty1 is direct. These findings highlight a remarkable level of conservation in transcriptional control by stress-activated MAP kinase pathways between fission yeast and mammalian cells.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Identification of a cdk-activating kinase in fission yeast.

We have identified a second cyclin-dependent kinase (cdk) in fission yeast, crk1, which encodes a 335 amino acid protein that is most closely related to the KIN28 gene product from Saccharomyces cerevisiae and to a cdk activating kinase (CAK) encoded by the MO15 gene from Xenopus laevis, crk1 is essential for viability and delta crk1 cells arrest with septa and condensed chromatin. We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. We demonstrate that crk1 is allelic to the mcs6 mitotic catastrophe suppressor and that the X.laevis MO15 gene rescues the cell-cycle arrest of an mcs6-13 cdc2-3w cdc25-22 at high temperature. Together these data suggest that the Crk1-Mcs2 complex is a CAK that interacts genetically with Cdc2 in fission yeast.

Coding of voice source information in the Nucleus cochlear implant system.

Two studies are reported in which the effectiveness of explicitly coding voicing and fundamental frequency information for the Nucleus cochlear implant was investigated. In the first study, the voicing perception of a group of three experienced Multipeak users was evaluated when they were using Multipeak and a modified Multipeak in which the explicit fundamental frequency and voicing cues were eliminated and replaced with a 250-Hz constant rate of stimulation. The results of consonant and monosyllabic word tests showed that there was no significant difference in the subjects' ability to discriminate voicing. In the second study, the ability of a group of five experienced users of the constant rate spectral maxima sound processor (SMSP) strategy to discriminate suprasegmental contrasts was evaluated when they were using the SMSP strategy and a modified SMSP strategy that included a rate-encoded representation of the fundamental frequency on the most apical stimulation channel. The results of intonation, roving stress, and question-statement tests showed that there was no significant difference between the scores recorded with these strategies. Since the temporal voicing cue is not a primary cue to voicing discrimination for Multipeak users, and the provision of an additional rate cue to the SMSP strategy does not improve SMSP users' ability to discriminate suprasegmental contrasts, the results of these studies indicate that in the cases investigated, the coding of voice source information by rate of stimulation does not significantly augment the cues present in the spatially distributed constant rate stimulation pattern.

Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast.