Deficiency in myosin light-chain phosphorylation causes cytokinesis failure and multipolarity in cancer cells.

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which are an important part of malignant transformation in the most recent model of tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. In this study, we show that cancer cells often failed at cytokinesis because of decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light-chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light-chain phosphorylation was restored to normal levels by phosphatase knockdown, multinucleation and multipolar mitosis were markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light-chain kinase in nonmalignant cells could recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating that these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.

Inverted duplication pattern in anaphase bridges confirms the breakage-fusion-bridge (BFB) cycle model for 11q13 amplification.

The homogeneously staining region (hsr) involving chromosome band 11q13 includes amplified genes from this chromosome segment and carries a relatively poor prognosis in oral squamous cell carcinomas (OSCC), with shorter time to recurrence and reduced overall survival. We previously identified an inverted duplication pattern of genes within the 11q13 hsr in OSCC cells, supporting a breakage-fusion-bridge (BFB) cycle model for gene amplification. To validate our hypothesis that 11q13 gene amplification in OSCC occurs via BFB cycles, we carried out fluorescence in situ hybridization (FISH) using probes for band 11q13 on 29 OSCC cell lines. We demonstrate that all OSCC cell lines with 11q13 amplification express a significantly higher frequency of anaphase bridges containing 11q13 sequences compared to cell lines without amplification, providing further experimental evidence that 11q13 gene amplification in OSCC cells occurs via BFB cycles. Elucidation of mechanisms responsible for initiating and promoting gene amplification provides opportunities to identify new biomarkers to aid in the diagnosis and prognosis of oral cancer, and may be useful for developing novel therapeutic strategies for patients with OSCC.

RIM4 encodes a meiotic activator required for early events of meiosis in Saccharomyces cerevisiae.

RIM4 was previously found to be required for both the IME1- and IME2-dependent pathways of meiotic gene expression in Saccharomyces cerevisiae. We now demonstrate that RIM4 is also required for meiotic division and recombination. Furthermore, rim4Delta mutants show defects in premeiotic DNA synthesis, which can be suppressed by deletion of the SIC1 gene, which encodes a Cdk inhibitor. Expression of RIM4 is induced early in meiosis, and is dependent on IME1 but not IME2. Indeed, RIM4 itself is essential for the meiotic expression of IME2. These results suggest that RIM4 is epistatic to IME2, and is required for multiple steps during sporulation. In agreement with this interpretation, overexpression of RIM4 induces low levels of sporulation in rich medium.

ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae.

ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.

The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis.

Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division. Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.

Chromosomal instability and cytoskeletal defects in oral cancer cells.

Oral squamous cell carcinomas are characterized by complex, often near-triploid karyotypes with structural and numerical variations superimposed on the initial clonal chromosomal alterations. We used immunohistochemistry combined with classical cytogenetic analysis and spectral karyotyping to investigate the chromosomal segregation defects in cultured oral squamous cell carcinoma cells. During division, these cells frequently exhibit lagging chromosomes at both metaphase and anaphase, suggesting defects in the mitotic apparatus or kinetochore. Dicentric anaphase chromatin bridges and structurally altered chromosomes with consistent long arms and variable short arms, as well as the presence of gene amplification, suggested the occurrence of breakage-fusion-bridge cycles. Some anaphase bridges were observed to persist into telophase, resulting in chromosomal exclusion from the reforming nucleus and micronucleus formation. Multipolar spindles were found to various degrees in the oral squamous cell carcinoma lines. In the multipolar spindles, the poles demonstrated different levels of chromosomal capture and alignment, indicating functional differences between the poles. Some spindle poles showed premature splitting of centrosomal material, a precursor to full separation of the microtubule organizing centers. These results indicate that some of the chromosomal instability observed within these cancer cells might be the result of cytoskeletal defects and breakage-fusion-bridge cycles.

Slk19p is a centromere protein that functions to stabilize mitotic spindles.

We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.

Action at the ends of microtubules.

Microtubule-based motility in the cell is directly associated with changes in microtubule numbers through nucleation and growth and shrinkage of the polymer from the ends. Recent analysis of spindle pole bodies and kinetochores in yeast reveal how the cell builds specialized structures for association with the ends of microtubules.

The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers.

Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.

Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation.

The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle. The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle. In this study, the roles of these motor proteins in anaphase chromosome segregation were examined. While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation. As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force. Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double-deletion mutants are inviable. Our findings indicate that the bulk of anaphase chromosome segregation in S. cerevisiae is accomplished by the combined actions of these three motors.

Mitotic spindle pole separation.

Eukaryotic cells utilize a microtubular spindle to segregate chromosomes during mitosis. Chromosome segregation requires the timely separation of the mitotic spindle poles to which the chromosomes are attached. Recent studies at the molecular and cellular levels have provided new insights into the mechanism and regulation of this process. On the one hand, the process now seems more complex, as redundant mechanisms apparently overlap in function during cell division. On the other hand, some of these processes may be acting continuously during the various stages of spindle pole separation, suggesting an underlying simplicity.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anti-centromere autoantibodies with anti-chromo specificity.

We have identified a novel autoantibody specificity in scleroderma that we term anti-chromo. These antibodies recognize several chromosomal antigens with apparent molecular mass of between 23 and 25 kDa, as determined by immunoblots. Anti-chromo autoantibodies occur in 10-15% of sera from patients with anti-centromere antibodies (ACA). We used anti-chromo antibodies to screen a human expression library and obtained cDNA clones encoding a 25 kDa chromosomal autoantigen. DNA sequence analysis reveals this protein to be a human homologue of HP1, a heterochromatin protein of Drosophila melanogaster. We designate our cloned protein HP1Hs alpha. Epitope mapping experiments using both human and Drosophila HP1 reveal that anti-chromo antibodies target a region at the amino terminus of the protein. This region contains a conserved motif, the chromo domain (or HP1/Pc box), first recognized by comparison of Drosophila HP1 with the Polycomb gene product. Both proteins are thought to play a role in creating chromatin structures in which gene expression is suppressed. Anti-chromo thus defines a novel type of autoantibody that recognizes a conserved structural motif found on a number of chromosomal proteins.

Kinesin-related proteins required for structural integrity of the mitotic spindle.

For S. cerevisiae cells, the assembly of a bipolar mitotic spindle requires the action of either Cin8p or Kip1p, gene products related to the mechanochemical enzyme kinesin. In this paper we demonstrate that the activity of either one of these proteins is also required following spindle assembly. When their function was eliminated, preanaphase bipolar spindles rapidly collapsed, with previously separated poles being drawn together. In contrast, anaphase spindles were apparently resistant to collapse. Deletion of kinesin-related KAR3 partially suppressed the phenotypes associated with loss of Cin8p/Kip1p function. Our findings suggest that the structure of the preanaphase bipolar spindle is maintained by counteracting forces produced by kinesin-related proteins.

Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.

Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a. CIN9), were identified by their requirement for normal chromosome segregation. Both genes encode polypeptides related to the heavy chain of the microtubule-based force-generating enzyme kinesin. Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute. At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function. Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles. Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.

Compartmentalization within the nucleus: discovery of a novel subnuclear region.

Antibodies to a set of structurally related autoantigens (p23-25) bind to a previously uncharacterized, large structural domain in the nucleus of a variety of human cell types. This subnuclear domain is visible by phase contrast alone as a region of decreased density after several different fixation protocols. The morphology of this region changes dramatically during the cell cycle and we have given it the name PIKA (for polymorphic interphase karyosomal association) based on preliminary evidence that the PIKA proteins may be associated with chromatin. The function of the PIKA is not yet known, but our immunolocalization data indicate that it is unlikely to be associated with regions of ongoing DNA replication, heterogeneous nuclear RNA storage, or mRNA processing. The discovery of the PIKA provides evidence supporting an emerging model of nuclear structure. It now appears that the nucleus is organized into distinct domains which include not only the nucleolus, but also previously unidentified regions such as the PIKAs. Furthermore, structural rearrangements undergone by the nucleolus and the PIKAs may be indicative of a broad tendency for nuclear organization to change in a cell cycle-specific fashion.

Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.

Autoantibodies present in the sera of patients with bullous pemphigoid (BP) bind to the basement membrane zone of normal human skin and commonly recognize two epidermal proteins, the BP240 and BP180 antigens. Two BP antigen cDNA clones from a lambda gt11 human keratinocyte library have been identified on the basis of reactivity with a BP serum. The fusion protein (FP) produced by one clone immunoadsorbed autoantibodies, which specifically recognized the BP180 by antigen, showing no cross-reactivity with BP240 by immunoblot analysis. The FP produced by the second clone immunoadsorbed autoantibodies which specifically reacted with the BP240 epidermal antigen. Northern blot analysis demonstrated that the BP180 and BP240 antigens are encoded by distinct RNA transcripts with lengths of 6.0 and 8.5 kb, respectively. Immunoblot analysis of the BP180 lysogen extract identified a 135-kD FP which was recognized by 7 of 16 BP sera and 7 of 8 herpes gestationis sera. A rabbit antiserum prepared against the lysogenic BP180 FP specifically recognized the BP180 antigen from human epidermal extracts by immunoblotting, labeled the BMZ by indirect immunofluorescence, and bound to human epidermal hemidesmosomes by immuno-electron microscopy. These results indicate that the BP180 antigen recognized by BP and herpes gestationis autoantibodies is a unique hemidesmosomal polypeptide, distinguishable from the BP240 antigen.

Opiate and alpha receptor antagonists block the pressor responses of conscious rats given intravenous dynorphin.

Conscious, unrestrained rats were used to determine the hemodynamic (blood pressure and heart rate) responses following intravenous (IV) injection of dynorphin A(1-13) and the possible receptor mechanisms mediating those changes. Male Sprague-Dawley rats (300 g) were given IV bolus injections (via femoral venous catheter) of 6.0 to 600 nmoles/kg of dynorphin A(1-13), 8.0 nmoles/kg of norepinephrine HCl (NE), 14.3 pmoles/kg of angiotensin II or a vehicle control solution. Blood pressure (BP) and heart rate (HR) were monitored via femoral arterial catheter (into abdominal aorta) over 90 sec postpeptide or -amine administration before and 10 min after IV injection of 4.2 mumoles/kg of naloxone HCl (opiate antagonist), yohimbine HCl (alpha 2 receptor antagonist) or prazosin HCl (alpha 1 receptor antagonist). Dynorphin A(1-13) caused a transient but dose-related rise in mean arterial pressure (MAP) whereas mean pulse pressures (MPP) and mean heart rates (MHR) concomitantly fell, from preinjection control values in a dose-dependent fashion. Pretreatment with naloxone blocked the pressor response of only a subsequent injection with 20 nmoles/kg but not 60 nmoles/kg of dynorphin A or NE (8.0 nmoles/kg). Pretreatment with yohimbine suppressed the marked pressor responses of subsequent NE or Dyn A (60 nmoles/kg) administration whereas prazosin antagonized the rise in MAP of only the lower doses of dynorphin as well as NE. The suppression of the pressor responses of dynorphin by opiate or alpha receptor antagonists were not caused by tachyphylaxis for repeated injections of 6.0 or 60 nmoles/kg of dynorphin caused the same rise in MAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressor, tachycardic and feeding responses in conscious rats following i.c.v. administration of dynorphin. Central blockade by opiate and alpha 1-receptor antagonists.

Experiments were designed to determine the hemodynamic responses of conscious, unrestrained rats given intracerebroventricular (i.c.v.) injections of dynorphin A-(1-13) and the possible central receptor mechanisms mediating those changes. Male Sprague-Dawley rats (300 gb. wt.) received i.c.v. injections (by gravity flow in a total volume of 3 or 5 microliter) of control solutions of sterile saline (SS) or dimethylsulfoxide (DMSO) or 1.5, 3.0 or 6.1 nmol of dynorphin A-(1-13). Blood pressure and heart rate changes were monitored over 2 h after administration; as well, feeding activity was visually assessed and scored over this period. Other groups of conscious rats were pretreated i.c.v. with equimolar doses (3.0-24.4 nmol) of specific receptor antagonists (naloxone HCl, phentolamine HCl, propranolol HCl, yohimbine HCl or prazosin HCl) 10 min before subsequent i.c.v. administration of SS or DMSO/SS or 6.1 nmol of dynorphin A-(1-13). I.c.v. injection of dynorphin A-(1-13) caused a dose-related pressor response, associated temporally with tachycardia. As well, dynorphin evoked feeding activity and some grooming, which occurred when the rats were hypertensive and tachycardic and decreased as heart rate and blood pressure returned to control levels. I.c.v. pretreatment studies indicated that naloxone HCl (12.2 nmol), phentolamine HCl (12.2 nmol) and prazosin HCl (6.1 nmol) blocked the pressor response, tachycardia as well as feeding activity of rats subsequently given dynorphin. The results suggest the pressor and tachycardic effects of conscious rats following i.c.v. dynorphin administration may, in part, be due to behavioral activation (feeding). As well, these data indicate that both opioid as well as alpha 1-adrenergic receptors within the CNS are involved in mediating the pressor, tachycardic and feeding responses of conscious rats given i.c.v. injections of dynorphin A.

Skeletal muscle thermogenesis: its role in the hyperthermia of conscious rats given morphine or beta-endorphin.

The role of skeletal muscle thermogenesis (increases in skeletal muscle tone) in the hyperthermic responses of conscious, unrestrained rats given acute or repeated i.p. or i.c.v. injections of morphine sulfate (MS) or beta-endorphin was investigated. Initial blood gas experiments showed that rats given acute i.p. injections of MS caused PO2 and pH to decrease by 60 min postadministration in a dose-related fashion whereas PCO2 increased; with repeated MS administration the respiratory acidosis seen with acute injections was reduced. Acute i.p. injections of MS (1, 10 or 20 mg/kg) caused catalepsy scores, plasma lactate levels and electromyographic (EMG) amplitude to be elevated in a dose-related fashion along with a rise in rectal temperatures (TRS). Surface (tail) temperatures also rose after the acute MS injections but only after the increase in TR. Significant increases in EMG amplitude after acute injections of MS occurred even before TRS increased and, with subsequent naloxone HCl administration (10 mg/kg i.p.), a rapid and marked fall in EMG amplitude occurred before TRS fell back to saline control levels. Acute i.c.v. injections of 1.1 nmol of either MS or beta-endorphin also caused EMG amplitudes to rise significantly before TRS began to increase. Tail temperatures again increased passively after i.c.v. injection of either drug. Subsequent naloxone injections (10 mg/kg i.p.) to these groups also caused EMG amplitudes to decrease before TRS decreased back to control TRS. Repeated i.p. injections of MS (10 mg/kg i.p. daily for 5 days) caused TRS to be higher than those seen after the initial injection but catalepsy scores, plasma lactates and EMG amplitudes were below those respective levels seen upon acute MS administration. Similar chronic findings occurred in other groups of rats given 1.1 nmol of either MS or beta-endorphin i.c.v., when they had been previously given repeated i.p. injections of MS (5 mg/kg i.p. twice daily for 2 days). The results indicate that acute peripheral or central injections of MS or beta-endorphin to conscious rats cause skeletal muscle to be activated, resulting in nonlocomotor, catatonic behavior. This skeletal muscle activation occurs before the rise in TR and is thought to be an important and possibly the primary cause of the resultant hyperthermia seen in rats after acute central or peripheral administration of MS or beta-endorphin. Repeated injections of morphine cause TRS to escalate higher, compared to that seen with acute MS administration, yet catalepsy scores, plasma lactate levels and EMG amplitude changes did not increase likewise.(ABSTRACT TRUNCATED AT 400 WORDS)