Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Capacitance cytometry: Measuring biological cells one by one

Measuring the DNA content of eukaryotic cells is a fundamental task in biology and medicine. We have observed a linear relationship between the DNA content of eukaryotic cells and the change in capacitance that is evoked by the passage of individual cells across a 1-kHz electric field. This relationship is species-independent; consequently, we have developed a microfluidic technique—“capacitance cytometry”—that can be used to quantify the DNA content of single eukaryotic cells and to analyze the cell-cycle kinetics of populations of cells. Comparisons with standard flow cytometry demonstrate the sensitivity of this new technique.

The Sda1 Protein Is Required for Passage through Start

We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.

Maize as a model for the evolution of plant nuclear genomes

The maize genome is replete with chromosomal duplications and repetitive DNA. The duplications resulted from an ancient polyploid event that occurred over 11 million years ago. Based on DNA sequence data, the polyploid event occurred after the divergence between sorghum and maize, and hence the polyploid event explains some of the difference in DNA content between these two species. Genomic rearrangement and diploidization followed the polyploid event. Most of the repetitive DNA in the maize genome is retrotransposable elements, and they comprise 50% of the genome. Retrotransposon multiplication has been relatively recent—within the last 5–6 million years—suggesting that the proliferation of retrotransposons has also contributed to differences in DNA content between sorghum and maize. There are still unanswered questions about repetitive DNA, including the distribution of repetitive DNA throughout the genome, the relative impacts of retrotransposons and chromosomal duplication in plant genome evolution, and the hypothesized correlation of duplication events with transposition. Population genetic processes also affect the evolution of genomes. We discuss how centromeric genes should, in theory, contain less genetic diversity than noncentromeric genes. In addition, studies of diversity in the wild relatives of maize indicate that different genes have different histories and also show that domestication and intensive breeding have had heterogeneous effects on genetic diversity across genes.

Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.

To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

The genome of Arabidopsis thaliana.

Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae. It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project. The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented. Examples are given of genes isolated by using map-based cloning. The importance of the Arabidopsis project for plant biology in general is discussed.

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Changes in nucleic acid and protein levels in atrophying skeletal muscle in cancer cachexia

Background: To investigate factors responsible for muscle loss in cachexia changes in nucleic acid and protein levels have been determined and compared with those induced by a tumour-produced cachectic factor, proteolysis-inducing factor (PIF). Materials and Methods: Mice were transplanted with the MAC16 tumour, while non-tumour bearing mice received PIF (1.5 mg/kg; i.v.) over a 24 h period. Results: There was an exponential decrease in RNA and protein in gastrocnemius muscle with weight loss without an effect on the DNA content. Levels of myosin followed the decrease in total protein, while actin levels remained constant. There was also a significant loss of protein from soleus muscle and spleen, but not from heart, liver and kidney. PIF also produced a significant loss of RNA and protein in spleen and reduced the protein content of soleus muscle. Conclusion: This suggests that PIF may be responsible for changes in protein and RNA content of tissues with the development of cachexia.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

Isolation and characterization of the U1 and U2 snRNAs in the silk gland of the 5th instar larval stage silk moth Bombyx mori

In isolation and characterization studies, expression level U1 and U2 snRNA isoforms were obtained from the 5th instar larval stage silk gland (SG). The DNA content of the SG cells is approximately 200,000-fold higher compared to the usual (2N) somatic cells of B. mori due to endoreduplication. In this study, the existence of U1 and U2 snRNA isoforms in the SG of the organism is investigated. Bombyx mori U1 and U2-specific RT-PCR libraries from the silk gland were generated. Five U1 and eight U2 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were analyzed in these variants. For the U1 snRNA variants, they were compared to the previously reported BmN isoforms. In all these U-snRNA variants, polymorphic sites do not predominate at the core of known functional sequences, which were interspecifically conserved. Variant sites and inter-species differences are located in moderately conserved regions. Free energy (ΔG) values for the entire U1 and U2 snRNA secondary structures and for the individual stem/loops domains of the isoforms were generated and compared to determine their structural stability. This will be the first time that U1 and U2 variants are shown specific for a development stage (larval) other than embryonic or adult. ^ Using phylogenetic analysis, evolutionary trees were generated for the U1 and U2 snRNAs using animal, plant, protista and fungal species. The resulting trees were boostrapped for robustness and rooted with the self-splicing RNA group II intron sequence from the cyanobacterium Calothrix. Using phylogenetic analyses, possible structural and functional evolutionary interdependence between the U1 and U2 snRNAs was investigated. ^

Defining the Role of the Histone Methyltransferase, PR-Set7, in Maintaining the Genome Integrity of Drosophila Melanogaster

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

Establishing an EGFR mutation screening service for non-small cell lung cancer - sample quality criteria and candidate histological predictors.

INTRODUCTION: EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. METHODS: Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. RESULTS: In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (2.2 ng/μL), the mutation rate was 9.2%. CONCLUSION: Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation.

Effects of sodium hyaluronate and triamcinolone acetonide on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1

The objective of this study is to determine if the effects of a high molecular weight sodium hyaluronate (HA) alone or in combination with triamcinolone acetate (TA) can mitigate chondrocyte proteoglycan catabolism caused by interleukin-1 (IL-1) administration. Chondrocytes were collected from fetlock joints of ten horses euthanized for reasons unrelated to joint disease. Chondrocyte pellets were treated with media (negative control); media containing IL-1 only (positive control); or media containing IL-1 with HA only (0.5 or 2.0 mg/mL), TA only (0.06 or 0.6 mg/mL), or HA (0.5 or 2.0 mg/mL) and TA (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA levels of collagen type II, aggrecan, and COX-2. The high concentration of HA (2.0 mg/mL) increased GAG synthesis while the high concentration of TA (0.6 mg/mL) decreased loss of GAG into the media. Both the high concentration of HA and TA increased the total GAG content within the pellet. There was no change in pellet DNA content with either treatment. TA reduced COX-2 mRNA levels as well as aggrecan and collagen type II expression. Treatment with HA had no effect on mRNA levels of COX-2, aggrecan or collagen type II. These results indicate that the high concentration of HA or TA alone or in combination will mitigate effects of IL-1 administration on proteoglycan catabolism of equine articular chondrocytes.