S-phase HeLa Cells Research Articles

Silk Sericin Hydrolysate is a Potential Candidate as a Serum-Substitute in the Culture of Chinese Hamster Ovary and Henrietta Lacks Cells.

The silk sericin hydrolysate (SSH) from the waste of silk processing as a substitute of fetal bovine serum (FBS) was used for the culture of Chinese hamster ovary (CHO) cells and Henrietta Lacks (Hela) strain of human cervical cancer cells. The survival ratio of these cells cultured in SSH media were similar to or higher than those in FBS media. Especially after the serum was replaced by low concentration of SSH at 15.0 μg/ml for 5 d, the proliferation of both cells was also similar to or higher than that of FBS group; the percentages of CHO and Hela cells in S-phase were 28.9 and 28.0%, respectively. The former is nearly two times that of FBS group, the latter is also higher than the control group. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that among the differentially expressed genes, the relative expression of CXCL12 gene of CHO cells in SSH group increased, was three times that of serum group, and the relative expression of LCN2 gene of Hela cells increased 2.8 times, indicating that these related genes were activated to promote cell growth and proliferation. These results fully illustrated the hydrolysated sericin has a potential use as serum substitutes in cell culture.

A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression.

BackgroundTo facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells.ResultsTo overcome the low CRISPR-Cas9 editing efficiency at the TERT locus, we develop a two-step “pop-in/pop-out” strategy to enrich cells that underwent homologous recombination (HR). Using this technique, we fuse an N-terminal FLAG-SNAP-tag to TERT, which allows us to reliably detect TERT in western blots, immunopurify it for biochemical analysis, and determine its subcellular localization by fluorescence microscopy. TERT co-localizes detectably with only 5–7 % of the telomeres at a time in S-phase HeLa cells; no nucleolar localization is detected. Furthermore, we extend this approach to perform single base-pair modifications in the TERT promoter; reverting a recurrent cancer-associated TERT promoter mutation in a urothelial cancer cell line results in decreased telomerase activity, indicating the mutation is causal for telomerase reactivation.ConclusionsWe develop a two-step CRISPR-Cas9 genome editing strategy to introduce precise modifications at the endogenous TERT locus in human cell lines. This method provides a useful tool for studying telomerase biology, and suggests a general approach to edit loci with low targeting efficiency and to purify and visualize low abundance proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0791-1) contains supplementary material, which is available to authorized users.

MRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'→3' and 3'→5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'→3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'→5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

Relevant study on apoptosis of cervical cancer HeLa cells induced by paeoniflorin

To explore the effects of paeoniflorin on the proliferation and apoptosis of HeLa cells. HeLa cells treated with paeoniflorin at different concentrations for different hours were assessed by the method of methyl thiazolyl tetrazolium (MTT). Cell apoptosis rate and cycle change were detected by flow cytometry with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). The morphological change of HeLa cells was observed by transmission electron microscope (TEM). The expressions of Bcl-2, Bax and Caspase-3 in HeLa cells induced by paeoniflorin were detected by immunocytochemistry. After treatment with different concentrations of paeoniflorin for different hours, the proliferation of HeLa cells was inhibited in a dose and time-dependent manner with an IC50 value of 5054, 2965, 2459 µg/ml at 24, 48 and 72 hours respectively (P < 0.05). After treated by different concentrations of paeoniflorin (control group, 1000 and 2000 µg/ml), apoptosis was induced in HeLa cells at a rate of 0.94%, 10.94% and 13.95% respectively. Such effects were dose-dependent (P < 0.05) and the proportion of HeLa cells in Sphase was under a rising trend. Typical apoptotic changes of HeLa cells under the exposure to paeoniflorin were observed under TEM. There was a lowered expression of Bcl-2 and an elevated expression of Bax and Caspase-3 genes versus the control group after a 48-hour paeoniflorin treatment (P < 0.05). Paeoniflorin can significantly induce the apoptosis of HeLa cells through a down-regulation of anti-apoptotic gene Bcl-2 and an up-regulation of pro-apoptotic genes Bax and Caspase-3.

Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate (MMS). The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS+UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.

P53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.

Cdc2-Independent Induction of Premature Mitosis by Okadaic Acid in HeLa Cells

The induction of premature mitosis by okadaic acid (OA) in HeLa cells in S-phase or in G 2-phase has been studied using light microscopy, immunofluorescence, and immunochemical techniques. The observations indicate an involvement of a cdc2-independent pathway in these cells. It has been claimed that inhibition of an OA-sensitive phosphatase, possibly of PP1, induces activation of a kinase which is sensitive to staurosporine and Zn 2+. This kinase brings about mitosis-specific cytoskeletal rearrangements, chromosome condensation, and nuclear envelope breakdown, inducing a mitosis-like state. However, other mitotic events do not follow. The possibility that this kinase may be a NIMA-like Nek2 kinase is discussed.

Differential cytotoxic effects of gamma-linolenic acid on MG-63 and HeLa cells

Gamma-linolenic acid (GLA) inhibited cell proliferation in MG-63 and HeLa cells. Different morphological lesions were present in dividing cells; abnormal spindle formation in MG-63 cells and chromosome hypercondensation in HeLa cells. Different types of cell death were also present in interphase cells, no apoptosis but only 6% pycnosis in MG-63 cells and 90% apoptosis in HeLa cells. In MG-63 cells immunofluorescence showed segregation of nucleoli components, abnormal spindle formation and decreased labelling of microtubuli during interphase. In HeLa interphase cells prominently labelled abnormally arranged microtubuli were observed. The effects of GLA on protein synthesis in synchronized cells were determined with [ 35S]-methionine incorporation and SDS-PAGE. Decreased protein synthesis in both G 1- and S-phase MG-63 cells was present. In S-phase HeLa cells, proteins of ⋍ 40, 92 and 150 kD were markedly expressed. Signalling mechanisms involved in cell proliferation and cell death may be differently affected in MG-63 and HeLa cells.

Effects of ganglioside G M1 on DNA synthesis in isolated nuclei and on the activity of DNA polymerase α derived from S-phase HeLa cells

Ganglioside G M1 inhibited either DNA synthesis in isolated nuclei or the activity of DNA polymerase α fractionated from S-phase HeLa cells. The concentrations of G M1 necessary for 50% inhibition were about 5 μM and 10 μM for nuclei and DNA polymerase α, respectively. The G M1 inhibition of the enzyme activity was suppressed by the addition of 0.05% Triton X-100. Neither gangliotetraosylceramide (asialo-G M1) nor free N-acetylneuraminic acid inhibited the enzyme activity. These facts suggest that G M1, probably in the form of micelles, could influence the enzyme activity by behaving as a polyanionic macromolecule. The kinetic studies indicate that the G M1 inhibition of the enzyme activity was not competitive with the substrate, deoxythymidine triphosphate, but rather with the template DNA. Binding of G M1 and DNA polymerase α was suggested by the cocentrifugation of G M1 and the enzyme fraction after their preincubation. It was also observed that other acidic glycolipids, i.e., brain sulphatide and seminolipid, also inhibited the enzyme activity, whilst neutral galactosylceramide did not. The inhibitory influences of these sulphate esters of glycolipids were, similarly to G M1, suppressed by the addition of 0.05% Triton X-100.

Distinct transcription factors bind specifically to two regions of the human histone H4 promoter.

Two proteins specifically binding to separate regions of the human histone H4 promoter were identified in nuclear extracts prepared from synchronized S-phase HeLa cells. Competition experiments with H4 promoter mutants and DNase protection assays ("footprinting") demonstrate that these factors bind to regions of the H4 promoter that are essential for maximal expression in vitro. One of these factors (H4TF-1) binds to sequences between -80 and -110 base pairs upstream of the H4 cap site, whereas the other (H4TF-2) binds to the H4 subtype-specific sequence element immediately upstream from the "TATA" homology. Neither of these activities can efficiently bind to any of the other histone gene subtypes or simian virus 40 DNA. Binding of H4TF-1 to the distal region of the pHu4A histone H4 promoter is inhibited competitively with varying efficiency by four of six human histone H4 genes cloned in this laboratory, whereas efficient competition for binding of H4TF-2 is exhibited by five of the six H4 genes. Since both of these factors bind to significant regions of the pHu4A histone H4 promoter and can be bound by several different human H4 genes, we believe that they are important for maximal transcription of the gene and that they may be involved in its regulated expression during the cell cycle.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro.

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro.

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

Early initiation of DNA synthesis in G1 phase HeLa cells following fusion with red cell ghosts loaded with S-phase cell extracts

A modification of polyethylene glycol-mediated cell fusion procedure has been described and standardized for red blood cell mediated microinjection of proteins into cells in suspension. Using this procedure, proteins are routinely introduced into 50 % of the target cells. We have applied this microinjection procedure to introduce cytoplasmic extracts obtained from S-phase HeLa cells into HeLa cells synchronized in G1. This experiment resulted in an accelerated entry of the G1 cells into S phase as measured by the incorporation of [ 3H]thymidine. This technique may provide a means to identify and characterize the S-phase factors responsible for the induction of DNA synthesis.

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [ 3H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.

Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei

Cytosol obtained by centrifugation of cytoplasm from synchronized S-phase HeLa cells at 200 000 × g for 30 min had a stimulatory effect on the rate and extent of DNA synthesis in isolated nuclei. The cytosol preserved the ability of isolated nuclei to initiate early nascent intermediates (primary DNA pieces). The stimulatory activity was partially separated from the DNA polymerase activity present in the cytosol.

Partial purification and characterization of individual histone messenger RNAs from HeLa cells

A polyribosomal 5–15 S poly(A)(−)-RNA preparation from S-phase HeLa cells, the only in vitro translation products of which were the five histones H1, H3, H2b, H2a and H4, was subjected to a new method of preparative polyacrylamide gel electrophoresis. With this method the mRNA for histone H4 (apparent molecular weight 1.4 × 10 5 daltons) was separated from the mRNAs coding for histones H3, H2b and H2a (m.w. 1.6 – 1.8 × 10 5 daltons) and from two different mRNAs coding for two histones H1 (m.w. 2.4 × 10 5 daltons). The mRNAs were identified by translation in a reticulocyte lysate.

Dephosphorylation of nonhistone proteins specifically alters the pattern of gene transcription in reconstituted chromatin.

A nonhistone phosphatase devoid of detectable protease activity has been purified from nuclear sap by chromatography on DEAE-Sephadex. After linkage to an insoluble agarose matrix, this enzyme was used to dephosphorylate nonhistone proteins obtained from S-phase HeLa cells. Chromatin reconstituted with these dephosphorylated proteins exhibited roughly a 50% reduction in the overall number of initiation sites available for transcription when compared to controls. Specific measurements of transcription of histone genes with use of a complementary DNA probe showed that genes coding for histones are preferentially inhibited after nonhistone dephosphorylation. These results provide the first direct support for the theory that phosphorylation of nonhistone proteins is involved in regulating the availability of individual genes for transcription.

Studies on the translational control of histone synthesis: I. Translation of histone messenger RNA by heterologous cell-free systems prepared from cells inactive in DNA synthesis

The translation of histone mRNA has been studied in a cell-free system prepared from mouse ascites cells treated with cytosine arabinoside to inhibit DNA synthesis. No difference in the efficiency of translation was observed with respect to an extract of control untreated cells. Moreover, a rabbit reticulocyte cell-free system can translate histone mRNA; thus, cells inactive in DNA synthesis can translate this mRNA. A search for an inhibitor of translation of histone mRNA was carried out by adding postribosomal supernatant from S-phase-synchronized HeLa cells treated with cytosine arabinoside, to the ascites cell-free system. No effect on the translation of histone mRNA was detected. Even when the cell-free system was reconstituted with ascites ribosomes and postribosomal supernatant from S-phase HeLa cells blocked in DNA synthesis, no effect on translation of histone mRNA was observed. These results suggest that the regulatory mechanism which couples histone to DNA synthesis does not involve synthesis of a factor specifically necessary for the translation of histone mRNA, nor the formation of a stable inhibitor. We cannot exclude, however, that an inhibitor of translation may be formed in amounts corresponding exactly to those of the histone mRNA present at the time of the block in DNA synthesis and that for this reason we have failed to find it in the postribosomal supernatant.