Proliferating HeLa Cells Research Articles

Biocatalytic Nucleobase Diversification of 4'-Thionucleosides and Application of Derived 5-Ethynyl-4'-thiouridine for RNA Synthesis Detection.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Biocatalytic Nucleobase Diversification of 4′‐Thionucleosides and Application of Derived 5‐Ethynyl‐4′‐thiouridine for RNA Synthesis Detection

AbstractNucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4′‐thiouridine in a one‐pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4′‐thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5‐iodo‐4′‐thiouridine on multi‐milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5‐ethynyl‐4′‐thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Exhaustive analysis of human endogenous retrovirus expression in cytotrophoblast differentiation into syncytiotrophobloast

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and historic 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in ahCG, pregnancy-specific β1-glycoprotein and 3β-hydroxysteroid dehydrogenase (3βOHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of αhCG or 3βOHSD mRNA, but did prevent extensive syncytialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes.

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Serum-stimulated cell cycle entry promotes ncOGT synthesis required for cyclin D expression

Nuclear and cytoplasmic O-GlcNAc transferase (OGT) is a unique and universally expressed enzyme catalyzing O-GlcNAcylation of thousands of proteins. Although OGT interferes with many crucial intracellular processes, including cell cycle, only few studies have focused on elucidating the precise role of the glycosyltransferase during cell cycle entry. We first demonstrated that starved MCF7 cells reincubated with serum quickly induced a significant OGT increase concomitantly to activation of PI3K and MAPK pathways. Co-immunoprecipitation experiments performed upon serum stimulation showed a progressive interaction between OGT and β-catenin, a major factor in the regulation of cell cycle. OGT expression was also observed in starved HeLa cells reincubated with serum. In these cells, the O-GlcNAcylation status of the β-catenin-2XFLAG was increased following stimulation. Moreover, β-catenin-2XFLAG was heavily O-GlcNAcylated in exponentially proliferating HeLa cells when compared to confluent cells. Furthermore, blocking OGT activity using the potent inhibitor Ac-5SGlcNAc prevented serum-stimulated cyclin D1 synthesis and slightly delayed cell proliferation. At last, interfering with OGT expression (siOGT) blocked cyclin D1 expression and decreased PI3K and MAPK activation. Together, our data indicate that expression and catalytic activity of OGT are necessary and essential for G0/G1 transition.

Interaction of Peptidomimetics with Bilayer Membranes: Biophysical Characterization and Cellular Uptake

Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (ΔG) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized ΔG being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, ΔG extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.

Study on the Role of PML on Cell Proliferation and Differentiation In Human Mesenchymal Stem Cells

AbstractAbstract 4711Promyelocytic leukemia protein (PML) was found in discrete, punctuate subnuclear structures known as PML nuclear bodies (PML NBs) in normal somatic cells and is an important factor acting downstream of wnt/β-catenin in the regulation of cell proliferation and differentiation. It is well-known that PML can bind cbp/p300 to promote TCF/LEF dependent transcription of downstream genes in 293T cells. However, its function in human mesenchymal stem cells (hMSCs) remains unclear. In our former studies, we found PML NBs in mitosis metaphase of hMSCs were more abundant than in Hela cells and 293T cells, which indicates PML may have special functions in hMSCs compared with tumor cells and normal somatic cells. The aim of our study is to investigate PML's function during proliferation and differentiation in hMSCs in contrast to Hela cells and 293T cells. hMSCs were isolated from bone marrow of healthy volunteers by density gradient centrifugation using Ficoll-paque and cultured (LG-DMEM, 10%FBS) with their characteristic adherence and morphology. hMSC immunophenotype was analyzed by flow cytometry and demonstrated uniform positivity for CD29, CD44, CD166 and CD105, and negativity for CD34, CD45 and HLA-DR. MTT assay and flow cytometry were used to evaluate proliferation of hMSCs. Osteogenic differentiation of hMSCs was perfoprmed and differentiated cells were identified with biochemical and morphological approaches. The levels of PML mRNA and protein expression during proliferation and differentiation were detected by RT-PCR, western blot and immunofluorescence while Hela cells and 293T cells were used as controls. Results showed the mean levels of mRNA and protein expression significantly increased during proliferation of hMSCs and 293T cells and positively correlated with the proliferation status of hMSCs. However, the expression of PML decreased in proliferating Hela cells (Fig. 1A, B). Intranuclear NBs of MSCs immunostained with PML monoclonal antibody and showed increased levels during proliferation, but remained very low in Hela cells throughout their proliferation. (Fig2). The above results indicate PML may take part in regulating the proliferation of hMSCs, and the mechanism involved is different from that in tumor cells. In tumor cells, the main function of PML maybe a suppressor of malignant cell growth. And PML protein is reduced or almost completely lost by post-transcriptional mechanisms. This loss is associated with tumor cell proliferation while regulation of cell proliferation and differentiation is the more important function of PML in hMSCs. During osteogenic differentiation of MSCs, ALP which is an early marker of osteogenic differentiation increased while PML's expression accordingly increased as detected by RT-PCR. (Fig 1C). Intranuclear NBs during differentiation are bigger but unequal in size compared with controls. (Fig3) These changes indicated that PML may interact with other molecules in NBs and act as a complex to regulate the process of osteogneic differentiation, but the exact mechanism involved is unclear and needs further study. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin

BackgroundThe process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells.ResultsWe have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin.ConclusionTopoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

DEK Represses hTERT Expression during Leukemogenesis

hTERT encodes telomerase reverse transcriptase which is the rate-limiting factor for telomerase activity (TA). Its expression is primarily regulated at the transcriptional level. High TA participates to the immortalization of malignant cells but the positive correlation between hTERT overexpression, increased TA and leukemogenesis does not seem to be a rule as hTERT underexpression has been evidenced in chronic myeloid leukemia, adult T-cell leukemia/lymphoma (ATLL), chronic lymphocytic leukemia, and in acute myeloid leukemia (AML). Certain oncogenes such as BCR-ABL or the HTLV-1-encoded oncoprotein Tax, which are respectively involved in the development of chronic myeloid leukemia and ATLL, negatively interfere with hTERT, resulting in its transcriptional repression. To investigate the molecular control of hTERT transcriptional repression during leukemogenesis, we carried out a proteomic screening of hTERT promoter occupancy in Tax+ and Tax− cells. Nuclear extracts (NEs) were prepared from HeLa cells transfected with a Tax or a control plasmid. NEs were incubated with the hTERT core promoter (hCP) and with an irrelevant control DNA fragment (CDF). For Tax+ and Tax− cells, protein interacting with the hCP or the CDF were purified and then analyzed by mass spectrometry. 270 proteins were identified from NEs incubated with hCP. Of those, 230 were present in both Tax+ and Tax− NEs whereas 22 and 18 were identified in NEs derived from Tax− and Tax+ cells. Values for CDF were 11, 1, and 0. The Tax+-hCP peptidome included DEK, a chromatin protein first identified as a fusion protein with CAN in AML with t(6;9). DEK is overexpressed in the majority of AML and displays multifunctional properties. Cotransfections and western blotting with proteins purified from NEs showed that DEK and Tax are present along the hCP in Tax+ cells but not in cells transfected with the control vector. DEK recruitment to the endogenous hTERT promoter was also evidenced by ChIP assays in Tax+- but not in Tax–cells. Cotransfection assays with a hTERT promoter-luciferase reporter plasmid and various concentrations of DEK and Tax expressors revealed that DEK inhibits hTERT transcription in a dose-dependent manner and that Tax and DEK synergize for repressing hTERT. Physical interaction between Tax and DEK was further demonstrated in vivo by co-immunoprecipitation assays. Cotransfections with modified reporter plasmids demonstrated that DEK represses hTERT transcription through the same E-box as Tax. We showed that a phosphorylation-dependent increased DNA binding activity of DEK stimulates its negative effect on hTERT transcription. SiRNA-mediated knockdown of DEK expression abolished the negative effect of Tax on hTERT, indicating that DEK is necessary for Tax-mediated hTERT repression. We quantified by qRTPCR the expression of 3 additional E-box containing genes known to be repressed by Tax, in cells stably expressing tax before and after siRNA-mediated knockdown of DEK: DEK knockdown resulted in a 3.5-, 2-, 1.5-, 14-fold increased expression of hTERT, lck, DNA polymerase beta and p18INK4C genes, respectively. Whether DEK could be involved in hTERT repression in other hematological malignancies than ATLL was finally demonstrated by identifying DEK as a member of the hCP peptidome in bone marrow blasts derived from patients with de novo AML with hTERT transcriptional repression but not in normal bone marrow mononuclear cells derived from donors. In conclusion our proteomic analysis of hTERT transcriptional regulation allowed the detection of specific preleukemic factors and provided evidence that in proliferating HeLa cells 15% of the hTERT promoter peptidome depends on the absence (9%) or the presence (6%) of Tax. The Tax-specific factor DEK physically interacts with Tax, is recruited to the hTERT promoter in Tax+ cells in which it represses hTERT transcription synergistically with Tax, through the same downstream E-box involved in Tax-dependent hTERT repression, and in a phosphorylation-dependent manner. The interplay between Tax and DEK in repressing the transcription of additional genes suggests that previous scenarios of Tax-mediated transcriptional repression in HTLV-1 should be revisited and updated to a new model in which DEK connects tax expression and gene repression. Finally, DEK and hTERT interactions appear to pertain to other malignancies than ATLL, such as AML.

Tumor Necrosis Factor Receptor-Associated Factor 4 Is a Dynamic Tight Junction-Related Shuttle Protein Involved in Epithelium Homeostasis

BackgroundDespite numerous in vivo evidences that Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) plays a key biological function, how it works at the cellular and molecular level remains elusive.Methodology/Principal FindingsIn the present study, we show using immunofluorescence and immuohistochemistry that TRAF4 is a novel player at the tight junctions (TJs). TRAF4 is connected to assembled TJs in confluent epithelial cells, but accumulates in the cytoplasm and/or nucleus when TJs are open in isolated cells or EGTA-treated confluent cells. In vivo, TRAF4 is consistently found at TJs in normal human mammary epithelia as well as in well-differentiated in situ carcinomas. In contrast, TRAF4 is never localized at the plasma membrane of poorly-differentiated invasive carcinomas devoid of correct TJs, but is observed in the cytoplasm and/or nucleus of the cancer cells. Moreover, TRAF4 TJ subcellular localization is remarkably dynamic. Fluorescence recovery after photobleaching (FRAP) experiments show that TRAF4 is highly mobile and shuttles between TJs and the cytoplasm. Finally, we show that intracellular TRAF4 potentiates ERK1/2 phosphorylation in proliferating HeLa cells, an epithelial cell line known to be devoid of TJs.Conclusions/SignificanceCollectively, our data strongly support the new concept of TJs as a dynamic structure. Moreover, our results implicate TRAF4 in one of the emerging TJ-dependent signaling pathways that responds to cell polarity by regulating the cell proliferation/differentiation balance, and subsequently epithelium homeostasis. Drastic phenotypes or lethality in TRAF4-deficient mice and drosophila strongly argue in favor of such a function.

Sororin Is Required for Stable Binding of Cohesin to Chromatin and for Sister Chromatid Cohesion in Interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.

Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication

Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G(1)-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G(1)-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

The Role of Mitochondrial dNTP Levels in Cells with Reduced TK2 Activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.

Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: Principal component and artificial neural network analysis

We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.

Peutz–Jeghers LKB1 mutants fail to activate GSK-3β, preventing it from inhibiting Wnt signaling

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition-and in contrast to the wild type protein-these LKB1 mutants fail to activate the GSK-3beta kinase, which otherwise phosphorylates beta-catenin. The increase in beta-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/beta-catenin pathway could contribute to the cancer predisposition of PJS patients.

The Replicative Regulator Protein Geminin on Chromatin in the HeLa Cell Cycle

Geminin is believed to have a major function in the regulation of genome replication and cell proliferation. Published evidence shows that geminin specifically interacts with Cdt1 to block its function in the assembly of prereplication complexes. However, in proliferating HeLa cells geminin and Cdt1 are co-expressed during a relatively short time at the G(1)-to-S phase transition. Under these conditions, nearly all Cdt1 and a major part of geminin are bound to chromatin and reside at the same or closely adjacent sites as shown here by chromatin immunoprecipitation. Cdt1 is rapidly degraded early in S phase, but geminin remains bound to the chromatin sites. One function that chromatin-bound geminin could perform is to prevent access to Cdt1 that may escape S phase-dependent degradation or is synthesized in excess. Indeed, Cdt1 continues to be synthesized in HeLa cells in S phase but never accumulates because of the efficient degradation. Therefore, geminin can be eliminated by RNA interference without detectable effects on cell cycle parameters.

14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.

Chromatin Association of Replication Protein A

Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.

In vitro cultured human term cytotrophoblast: A model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [ 3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and historic 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in ahCG, pregnancy-specific β 1-glycoprotein and 3β-hydroxysteroid dehydrogenase (3βOHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of αhCG or 3βOHSD mRNA, but did prevent extensive syncytialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.