Mutational analysis of Cak1p, an essential protein kinase that regulates cell cycle progression

In Saccharomyces cerevisiae, entry into S phase requires the activation of the protein kinase Cdc28p through binding with cyclin Clb5p or Clb6p, as well as the destruction of the cyclin-dependent kinase inhibitor Sic1p. Mutants that are defective in this activation event arrest after START, with unreplicated DNA and multiple, elongated buds. These mutants include cells defective in CDC4, CDC34 or CDC53, as well as cells that have lost all CLB function. Here we describe mutations in another gene, CAK1, that lead to a similar arrest. Cells that are defective in CAK1 are inviable and arrest with a single nucleus and multiple, elongated buds. CAK1 encodes a protein kinase most closely related to the Cdc2p family of protein kinases. Mutations that lead to the production of an inactive kinase that can neither autophosphorylate, nor phosphorylate Cdc28p in vitro are also incapable of rescuing a cell with a deletion of CAK1. These results underscore the importance of the Cak1p protein kinase activity in cell cycle progression. Received: 2 January 1997 / Accepted: 20 June 1997     

A model for random genetic damage directing selection of diploid or aneuploid tumours

Objectives: To test whether genetic instability may determine whether tumours become aneuploid or diploid. Materials and methods: We have identified genes needed for cell survival or replication by combining Affymetrix gene expression array data from 12 experimental cell lines with in silico GEO+GNF and expO databases. Specific loss of heterozygosis (LOHs), chromosomal abnormalities (called derivative chromosomes) and numbers of normal homologues were identified by SNP and SKY analyses. Random gene losses were calculated under the assumption that bi‐allelic MMR gene inactivation causes a 20‐fold increase in rate of gene loss. Results: There were ～1.23 × 10⁴ genes widely dispersed throughout the genome and possibly expressed by all cells for survival or proliferation, many of these genes performed housekeeping functions. Conservation of the genes may explain the complete haploid genomes found for 15 different cell types and derivative chromosomes selectively retained in aneuploid cancer cell lines after LOH formations, and normal homologue losses. Loss of cell survival/replication genes was calculated to be higher in colon stem cells of carriers of MMR gene mutations than carriers of APC gene mutations. Conclusion: Random loss of cell survival/replication genes was calculated to be low enough for colon stem cells with APC gene mutations to ‘select’ LOH and derivative chromosome combinations favouring tumour cell proliferation. However, cell survival/replication gene loss was calculated to be too high for colonic stem cells lacking MMR genes to survive chromosomal instability, explaining why MMR mutations only produce tumours with diploid chromosome cells.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.     

Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast

The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins. Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events. Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5. Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START. Downregulation of Far1 protein at START is normal in cln−GAL1::CLB5 cells. Even though the Clb5-Cdc28 kinase activity in cln−GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells. However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln−GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA. Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle. Received: 25 September 1997 / Accepted: 9 November 1997     

Yeast as a model system for understanding the control of DNA replication in eukaryotes

In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the initiation of DNA replication is controlled at a point called START. At this point, the cellular environment is assessed; only if conditions are appropriate do cells traverse START, thus becoming committed to initiate DNA replication and complete the remainder of the cell cycle. The cdc2⁺ / CDC28⁺ gene, encoding the protein kinase p34, is a key element in this complex control. The identification of structural and functional homologues of p34 suggests that it has a role in the control of DNA replication in all eukaryotes. The WHI1⁺, CLN1⁺ and CLN2⁺ gene products, identified in S. cerevisiae, are positive regulators that function at START and may interact with p34. Determining how passing the START control point leads to the initiation of DNA replication is a major outstanding challenge in cell cycle studies.     

Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development

 The tactile bristles of the fly comprise four cells that originate from a single precursor cell through a fixed lineage. The gene tramtrack (ttk) plays a crucial role in defining the fates of these cells. Here we analyse the normal pattern of expression of ttk, as well as the effect of ttk overexpression at different steps of the lineage. We show that ttk is never expressed in cells having a neural potential, and that in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. Furthermore, this expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. Our results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice. Received: 10 October 1996 / Accepted: 11 February 1997     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function

Mutation of the essential Schizosaccharomyces pombe rad4/cut5 gene causes sensitivity to UV and ionising radiation at the permissive temperature whilst at the restrictive temperature cells fail to undergo DNA replication but still attempt mitosis owing to a defective S-phase checkpoint response. Many mutations in genes encoding DNA replication proteins also abolish checkpoint responses, possibly because the replication machinery is a pre-requisite for the generation of the signal. We demonstrate here that rad4/cut5 cells fail to arrest cell division when treated with the replication inhibitor hydroxyurea at the semi-permissive temperature 32° C, but retain essentially normal replicative capacity. This demonstrates that the replication and checkpoint function of the rad4/cut5 gene product can be separated and that the Rad4 protein differs from other replication proteins in being directly involved in generating the S-phase checkpoint signal. Furthermore, we have investigated the checkpoint response or rad4/cut5-deficient cells to γ-irradiation and UV-mimetic drugs. We find that, at the restrictive temperature, the rad4 ⁻ /cut5 ⁻ cells fail to delay mitosis in response to γ-irradiation whilst retaining a normal checkpoint response to the UV-mimetic drug 4-nitroquinoline-1-oxide. The lack of the γ-irradiation checkpoint is reminiscent of the deficiency associated with mutation of the human ATM locus, the causative deficiency of the heritable disorder ataxia telangiectasia. The implications of our results for the organisation of distinct checkpoint-response pathways in both fission yeast and mammalian cells are discussed. Moreover the data are consistent with a model in which the generation of the S-Phase checkpoint signal is DNA polymerase ɛ dependent. Received: 29 October 1996 / Accepted: 14 January 1997     

Life without geminin

The interplay of proliferation and differentiation is essential for normal development and organogenesis. Geminin is a cell cycle regulator which controls licensing of origins for DNA replication, safeguarding genomic stability. Geminin has also been shown to regulate cellular decisions of self-renewal versus commitment of neuronal progenitor cells. We discuss here our recent analysis of mice with conditional inactivation of the Geminin gene in the immune system. Our data indicate that Geminin is not indispensable for every cell division: in the absence of Geminin, development of progenitor T cells appears largely unaffected. In contrast, rapid cell divisions, taking place in vitro upon TCR receptor activation or in vivo during homeostatic proliferation, are defective.     

Suppression of tumorigenicity in T-cell lymphoma hybrids is correlated with changes in myc expression and DNA replication of the myc chromosomal domain

Intraspecific somatic cell hybrids between T-lymphoma cells and lymphocytes are highly tumorigenic whereas fusion of T-lymphoma cells with normal fibroblasts leads to reduced or even completely suppressed tumorigenicity of the hybrid cells. A particular cytogenetic phenomenon defines these two classes of hybrids. DNA replication analysis via bromodeoxyuridine pulse labelling reveals an aberrant banding pattern in the c-myc chromosomal domain in tumour cells and highly tumorigenic hybrids. In hybrids with suppressed tumorigenicity the tumour parent derived chromosomes have reverted to normal DNA replication banding. Aberrant DNA replication in tumour cells and highly tumorigenic hybrids coincides with enhanced c-myc expression. In hybrids with suppressed tumorigenicity and with normal DNA replication banding c-myc expression is also reduced. Thus, a correlation between aberrant DNA replication and enhanced expression of a gene located in the same chromosomal domain is observed. Reversion of aberrant DNA replication and reduction of c-myc expression to normal in hybrid cells may be due to a site-specific trans effect which overrides the control brought about in cis by retroviral insertion near the c-myc gene.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a -transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Cell-cycle-specific initiation of replication

The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle: the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle. Several fundamentally different methods have been used to study these processes: Meselson—Stahl density-shift experiments, experiments with the so-called‘baby machine', sorting of cells according to size, and flow cytometry. The evidence for precise timing and co-ordination of chromosome replication in Escherichia coli is overwhelming. Similarly, the high-copy-number plasmid ColE1 and the low-copy-number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle. Data about the low-copy-number plasmids F and P1 are conflicting. This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36° C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25° C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25° C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function. Received: 10 March 1997 / Accepted: 1 September 1997     

Identifying UBA2 as a proliferation and cell cycle regulator in lung cancer A549 cells

Ubiquitin activating enzyme 2 (UBA2) is a basic component of E1-activating enzyme in the SUMOylation system. Expression and function of UBA2 in human cancers are largely unknown. In this study we investigate UBA2 expression the function in human non–small-cell lung cancer. Immunochemistry study showed that UBA2 was overexpressed in cancer tissues (53.3%, 40 of 75) compared with normal lung tissues (14.3%, 4 of 28) (P < 0.05). Immunostaining of UBA2 was mainly detected in nucleus. Overexpression of UBA2 in cancer tissues was significantly associated with poor differentiation, large tumor size ( > 5.0 cm), higher T stages (T3 + 4), lymph node metastasis and advanced TNM stages (III + IV). In vitro study showed that UBA2 was expressed in A549, 95D, H1975, and H1299 cells. Knockdown of UBA2 in A549 cells significantly inhibited cancer cell proliferation and upregulated cancer cell apoptosis (P < 0.05). Cell cycle analysis showed that knockdown of UBA2 in A549 cell significantly increased the G1 and G2/M phase cells and reduced the S phase cells (P < 0.05). Gene expression profile after knockdown of UBA2 in A549 cells showed that the most related function was cell cycle, cell death and survival, and cellular growth and proliferation. Western blot analysis study showed that knockdown of UBA2 significantly inhibited expression of poly(ADP-ribose) polymerase 1, mini-chromosome maintenance 7 (MCM7), MCM2, MCM3 and MCM7. These results indicated that UBA2 was a critical cell cycle and proliferation regulator and may be a novel cancer marker in this malignant tumor.     

Ovarian hormone concentrations and genital swelling patterns in female chimpanzees with Norplant implants

The purpose of this study was to assess the effects of Norplant implants on ovarian hormone secretion and genital swelling patterns in female chimpanzees. Urine samples were collected during one natural cycle before (n = 5 females) and one cycle ∼6 months following (n = 7 females) insertion of Norplant implants. Genital swelling data were recorded daily for all seven females. During Norplant treatment, two females exhibited minimal ovarian activity consistent with anovulation. Hormone data from five subjects indicated both follicular and luteal activity, suggesting that ovulation may have occurred during Norplant treatment. Among cycles presumed to be ovulatory, the concentration of estrone glucuronide did not differ from concentrations during the control phase. However, mean (F = 12.253, P = 0.001) and peak (F = 4.127, P = 0.003) concentrations of pregnanediol glucuronide were significantly lower during Norplant treatment. Cycle duration did not differ during Norplant treatment (F = 1.415, P = 0.200); however, duration of the total swelling phase (F = 3.311, P = 0.0011) and full swelling phase was shorter (F = 5.906, P = 0.011), whereas number of days with partial swelling increased (F = 2.635, P = 0.011). The results of this study indicate that Norplant provided effective contraception while not inhibiting ovarian hormone secretion or the expression of cyclic genital swelling in the majority of the chimpanzee subjects. Zoo Biol 16:209–223, 1997. © 1997 Wiley-Liss, Inc.     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a λgt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif ``IXCXE'. The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2–q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication. Received: 24 June 1998 / Accepted: 12 August 1998