HeLa Cells In Tissue Culture Research Articles

Evolution of Increased Survival in RNA Viruses Specialized on Cancer-Derived Cells

Viruses and other pathogens can diverge in their evolved host-use strategies because of exposure to different host types and conflicts between within-host reproduction and between-host survival. Most host-pathogen studies have emphasized the role of intrahost reproduction in the evolution of pathogen virulence, whereas the role of extra-host survival has received less attention. Here, we examine the evolution of free-living virion survival in RNA virus populations differing in their histories of host use. To do so, we used lineages of vesicular stomatitis virus (VSV) that were experimentally evolved in laboratory tissue culture for 100 generations on cancer-derived cells, noncancerous cells, or alternating passages of the two host types. We observed that free-living survival improved when VSV populations specialized on human epithelial carcinoma (HeLa) cells, whereas this trait was not associated with selection on noncancer cells or combinations of the cell types. We attributed this finding to shorter-lived HeLa monolayers and/or rapid cell-to-cell spread of viruses on HeLa cells in tissue culture, both of which could select for enhanced virus stability between host-cell replenishment. We also showed evidence that increases in virion survival were associated with decreases in virulence, which suggests a trade-off between survival and virulence for the VSV populations on one cell type. Our results shed new light on the causes and consequences of "sit and wait" infection strategies in RNA viruses.

Behavior of Yersinia enterocolitica in Foods.

Yersinia enterocolitica are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can survive and grow at refrigeration temperatures. The ability to propagate at refrigeration temperatures is of considerable significance in food hygiene. Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture. Two chromosomal genes, inv and ail, were identified for cell invasion of mammalian. The pathogen can cause diarrhoea, appendicitis and post-infection arthritis may occur in a small proportion of cases. The most common transmission route of pathogenic Y. enterocolitica is thought to be fecal-oral via contaminated food. Direct person-to-person contact is rare. Occasionally, pathogenic Y. enterocolitica has been detected in vegetables and environmental water; thus, vegetables and untreated water are also potential sources of human yersiniosis. However, the isolation rates of pathogenic Y. enterocolitica have been low, which may be due to the limited sensitivity of the detection methods. To identify other possible transmission vehicles, different food items should be studied more extensively. Many factors related to the epidemiology of Y. enterocolitica, such as sources, transmission routes, and predominating genotypes remain obscure because of the low sensitivity of detection methods.

Non-homologous end joining, but not homologous recombination, enables survival for cells exposed to a histone deacetylase inhibitor

Non-homologous end joining (NHEJ) and homologous recombination (HR) are pathways that repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, the repair of these breaks is influenced by histone acetylation. Therefore, we tested mammalian cells deleted for NHEJ (Ku80 or DNA Ligase IV) or altered for HR (breast cancer associated gene, Brca2, or Bloom's syndrome, Blm) for sensitivity to trichostatin A (TSA), a histone deacetylase inhibitor that is being investigated as an anti-cancer therapeutic. We show that cells mutated for Ku80 (ku80−/−) or DNA Ligase IV (lig 4−/−), but not cells mutated for Brca2 (brca2lex1/lex2) or Blm (blmtm3Brd/tm4Brd), are hypersensitive to TSA in a dose-dependent manner. TSA-induced toxicity stimulates apoptosis and cell cycle checkpoint responses independent of p53, but does not increase phosphorylated histone H2AX (γ-H2AX) as compared with a clastogenic agent, camptothecin, indicating that the quantity of DSBs is not the primary cause of TSA-induced cell death. In addition, we show that potential anti-cancer drugs (LY-294002 and vanillin) that inhibit the family of phosphatidylinositol 3 kinases that include the NHEJ protein, DNA–PKCS act in synergy with TSA to reduce the viability of HeLa cells in tissue culture presenting the possibility of using the two drugs in combination to treat cancer.

Inhibition of Shiga-like toxins by brefeldin A.

The effects of Shiga-like toxins on the morphology and protein synthetic capability of HeLa cells in tissue culture could be prevented by brefeldin A, an inhibitor of certain intracellular Golgi functions. Brefeldin A was without effect on the binding of Shiga-like toxin to cells. These results provide evidence that the Shiga-like toxins are first processed by the Golgi before moving to their 60S ribosome target site.

Transient cold shock induces the heat shock response upon recovery at 37 degrees C in human cells.

We evaluated the effects of a transient cold shock followed by recovery and incubation at 37 degrees C on the regulation of heat shock gene expression in the IMR-90 human diploid fibroblasts and HeLa cells in tissue culture. We showed that preincubation of cells at 4 degrees C induced the synthesis and accumulation of the heat shock proteins (HSPs) upon recovery at 37 degrees C, and the degree of this induction was directly related to the time that the cells spent at 4 degrees C. Assays on the abundance of the hsp 70 transcript, the hsp 70 gene promoter activity, and the trimerization and activation of heat shock factor (HSF) to bind to its consensus heat shock element (HSE) provided evidence that this induction of the heat shock response in cells recovering from a transient cold shock is attributable to a transcriptional event mediated by the activation of HSF. Further, the induction was a response to the temperature upshift from 4 to 37 degrees C as opposed to the 4 degrees C treatment itself; quantitation of the HSE-binding activity of cells incubated at 4 degrees C without recovery and incubation at 37 degrees C gave no evidence of an activated response. Analysis of the effects of protein synthesis inhibitors demonstrated that neither cycloheximide nor puromycin was effective in blocking the induction of HSE-binding activity in cells recovering from a transient cold shock. Experiments on the time course and temperature dependence of this induction of HSE-binding activity showed that the onset, magnitude, and duration of this induction were directly proportional to the severity of the cold stress (measured by time and temperature). We discuss the possible mechanism(s) involved in this induction of the heat shock genes at 37 degrees C by a transient cold shock and the biological implications of this observation.

Influence of the spermicidal compound nonoxynol-9 on the adhesion of E. coli to human epithelial cells

Two type I piliated strains of Escherichia coli were cultured in medium supplemented with physiologically used concentrations of the spermicidal compound nonoxynol-9. Their ability to adhere to HeLa cells in tissue culture was found to increase significantly (p<0.05). For strain 12313, there was a 2.3- and 1.9-fold increase when cultured in 5% and 12.5% (w/v) nonoxynol-9 respectively, and adhesion of strain 12269 increased by 1.7 times after growth in 12.5% (w/v) nonoxynol-9. The increased adhesion was accompanied by loss of mannose-sensitive yeast agglutination and hemagglutination. The absence of type I piliation was confirmed by electron miscroscopy and was accompanied by a decrease in cell surface hydrophobicity. The ability of E. coli to grow in high concentrations of nonoxynol-9, combined with increased adhesion to human epithelial cells in vitro, may contribute to the increased incidence of E. coli urinary tract infection seen in women using diaphragms and spermicidal preparations for contraception.

Influence of the spermicidal compound nonoxynol-9 on the growth and adhesion of urogenital bacteria in vitro

Lactobacilli and uropathogenic bacteria isolated from the female urogenital tract were tested for their susceptibility to nonoxynol-9. Nonoxynol-9 is a spermicidal compound, generally used at a concentration of 5% in cream and 12.5% in foam. The growth of 67% of fresh, vaginal lactobacillus isolates was inhibited by concentrations of nonoxynol-9 between 0.1% and 1.0%; these were termed sensitive. Of a total of 47 lactobacilli from various sources, 55% were found to be sensitive to nonoxynol-9, being bacteriostatic for 42% of these isolates and bactericidal for the remaining 58% at N-9 concentrations ⩾1.0%. The remaining lactobacilli and 96% (48/50) of uropathogenic organisms had minimal inhibitory concentrations of ⩾25% for nonoxynol-9. Inhibition of the lactobacilli did not appear to be species specific nor related to the source of the lactobacilli. The adhesion of Gram-positive bacteria, namely lactobacilli and enterococci, to HeLa cells in tissue culture was significantly increased over 60 min in the presence of physiologically used concentrations of nonoxynol-9; however, adhesion ofEscherichia coli was not affected. We believe that nonoxynol-9 has the potential to increase susceptibility to urinary tract infection in women using spermicidal preparations for contraception by inhibiting the growth of lactobacilli, which are believed to have a protective function in the vagina, and allowing overgrowth of uropathogenic bacteria.

The colonization of Peyer's patches by a strain of Salmonella typhimurium cured of the cryptic plasmid.

We cured a strain of Salmonella typhimurium of its cryptic plasmid and confirmed that orally administered cured strains lost virulence for mice. Loss of the cryptic plasmid rendered the S. typhimurium strain sensitive to the bactericidal action of normal human serum. However, loss of the plasmid did not change the ability of the strain to associate with HeLa cells in tissue culture. Furthermore, when administered orally to mice, both the plasmid-containing and plasmid-free strains invaded the Peyer's patches of the small intestine to the same extent, and both were capable of inducing resistance to oral challenge with virulent S. typhimurium. When injected intraperitoneally, the cured strain was eliminated rapidly, whereas the parental strain persisted. We also showed that the cured strain did not contain a plasmid copy in the chromosome. We propose that although the plasmid-cured strain of S. typhimurium is able to colonize Peyer's patches, it cannot survive when administered intraperitoneally because it is susceptible to elimination by macrophages.

Transformation of a plasmid encoding an adhesin of Staphylococcus aureus into a nonadherent staphylococcal strain.

Plasmid DNA was isolated and purified from a strain of Staphylococcus aureus demonstrating adherence to human epithelial cells, as determined by an assay quantitating adherence of radiolabeled S. aureus to HeLa cells in tissue culture. Other phenotypic characteristics of the donor strain are resistance to ampicillin and absence of hemolysis. A 23.5-kb (kilobase) plasmid was transformed into a nonadherent, ampicillin-sensitive, beta-hemolytic recipient of S. aureus, rendering it both ampicillin-resistant and adherent to a degree approaching that of the donor strain. Plasmid analysis of the donor and transformant strains revealed three identical EcoRI fragments of 7.6 kb, 6.5 kb, and 2.2 kb, together with a 3.6-kb EcoRI fragment in the transformant that demonstrated homology with the last 7.2-kb fragment in the donor. We conclude that an adhesin of S. aureus is encoded by this 23.5-kb penicillinase-encoding plasmid and that techniques of molecular genetics may be utilized to clarify the mechanisms of adherence of S. aureus.

Enzymatic synthesis of nucleoside antibiotics. Part IV. Microbial synthesis of purine 2'-amino-2'-deoxyribosides.

The microbial synthesis of some purine 2′-amino-2′-deoxyribonucleosides from purine bases and 2′-amino-2′-deoxyuridine is described. Various bacteria, especially Erwinia herbicola, Salmonella schottmuelleri, Enterobacter aerogenes and Escherichia coli, were able to transfer the aminoribosyl moiety of 2′-amino-2′-deoxyuridine to purine bases (transaminoribosylation) in the presence of inorganic phosphate. The optimum conditions for the reaction were pH 7.0 and 63°C. No reaction was observed in the absence of inorganic phosphate and the optimum concentration of it was around 30 mm. Adenine, guanine, 2-chlorohypoxanthine and hypoxanthine were transformed to the corresponding 2′-amino-2′-deoxyribonucleosides by the catalytic activity of the wet cell paste of Enterobacter aerogenes AJ 11125. The enzymatically synthesized purine 2′-amino-2′-deoxyribonucleosides were isolated and identified by physicochemical means. 2′-Amino-2′-deoxyadenosine strongly inhibited the growth of Hela cells in tissue culture, and the...

Occurrence of cell division is not exponentially distributed: differences in the generation times of sister cells can be derived from the theory of survival of populations.

The Eyring-Stover survival theory has been applied to the kinetics of the distribution of intermitotic intervals of mammalian cells and by inference to the transition from the G1 phase into DNA synthesis (S phase). The theory faithfully fits experimental data acquired by time-lapse cinemicrography of cloned HeLa cells in tissue culture and also suggests the existence of a labile initiator substance which mediates the G1-S phase transition. This theory provides an alternative to the transition probability model proposed by Smith and Martin [Smith J. A. & Martin, L. (1973) Proc. Natl. Acad. Sci. USA 70, 1263-1267; see also Shields, R. & Smith, J. A. (1977) J. Cell Physiol. 91, 345-355 and Brooks, R. F., Bennett, D. C. & Smith, J. A. (1980) Cell 19, 493-504].

Reactions of 2-acyloxyisobutyryl halides with nucleosides. 6. Synthesis and biological evaluation of some 3'-acyl derivatives of 2,2'-anhydro-1-(.beta.-D-arabinofuranosyl)cytosine hydrochloride

The reactions of cytidine with 22 different 2-O-acyloxyisobutyril chlorides lead to the isolation of the corresponding 2,2'-anhydro-1-(3'-O-acyl-beta-d-arabinofuranosyl)cytosine hydrochlorides 9. These compounds, which all show cytotoxicity against HeLa cells in tissue culture, have been examined for antiviral and antileukemic activity. Activity against DNA viruses (vaccinia and Herpes) in tissue culture is maximal in compounds containing acyl groups with 8--12 carbon atoms. Activity against L1210 leukemia in mice varies markedly according to the length of the acyl groups, and high activities were observed in the case of long-chain (C16--C22) esters. The reaction between cytidine and O-acetylsalicyloyl chloride provides an alternate route for the synthesis of 3'-O-Ac cycloC hydrochloride.

Reactions of 2-acyloxyisobutyryl halides with nucleosides. 7. Synthesis and biological evaluation of some 2,2'-anhydro-1-(3',5'-di-O-acyl-.beta.-D-arabinofuranosyl)cytosine hydrochlorides

The direct acylation of 2,2'-anhydro-1(beta-D-arabinofuranosyl)cytosine hydrochloride (cycloC) with a homologous series of saturated and unsaturated acyl chlorides in dimethylacetamide has been investigated. Such acylation reactions have made available a considerable number of 3',5'-diesters of cycloC that have been examined for biological activities. The compounds all show cytotoxicity against HeLa cells in tissue culture, and with the exception of the highly insoluble long-chain diesters (C16--C22), show pronounced activity against vaccinia and Herpes simplex viruses. Against L1210 leukemia in mice the compounds show varied activities, the C12--C14 saturated diesters and the C18--C22 unsaturated diesters being highly effective. Other diesters, varying by only a few methylene groups, show dramatically different results.

Improved Method for the Purification of Biologically Active Transcobalamin II

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.

The effect of mannitol-myleran and two new dibromo-hexitols on the metabolic activities of nucleic acids and proteins—I.

The effects of dibromo-dulcitol (DBD), dibromo-manitol (DBM) and mannitol-myleran (MM) on the DNA-, RNA and protein synthesis of bone marrow cells in vitro and in HeLa cells in tissue culture were studied. The inhibition of DNA synthesis assessed by 14C-formate incorporation displays time and concentration dependence. Forty to fifty μg/ml of DBD were sufficient to produce 50 per cent inhibition in normal rabbit bone marrow cells after 7 hr incubation. Two to two and a half times more of DBM and 4–5 times more of MM are required to produce the same degree of inhibition. Bone marrow cells from rats with Shay leukemia reacted practically to a similar extent. Incorporation of 14C labelled precursors was used to compare the effects exerted on the syntheses of the three biopolymers in bone marrow and HeLa cells. In bone marrow cells DNA synthesis is inhibited to the highest extent, RNA synthesis less and protein synthesis the least. In HeLa cells this order was modified inasmuch as RNA synthesis is inhibited to the same or even to a higher extent than DNA synthesis. The effect exerted on the protein synthesis is ‘indirect’ since even high concentrations of the compounds failed to inhibit amino acid incorporation, into the cell-free ribosomal system. Nucleic acid synthesis or some metabolic process closely related seems to be the point of attack of these cytotoxic agents.

EFFECTS OF MAGNETIC FIELDS ON GROWTH OF HELA CELLS IN TISSUE CULTURE.

MACCLEAN1,2 has indicated that magnetic fields were effective in arresting carcinoma growth in patients, and Barnothy3 stated that magnetic fields, in some way, retarded the ageing (hence growth) process in mice.

The Effects of Nitrogen Mustard on HeLa Cells in Tissue Culture

The Effects of Nitrogen Mustard on HeLa Cells in Tissue Culture

Effect of α-particle and X-ray irradiation onDNAsynthesis in tissue cultures

The effects of both α-particle and X-ray in radiation, on theDNAsynthesis rate in mouse fibroblast and Hela cells in tissue culture is described. Tritiated thymidine autoradiography was used to indicate the rate ofDNAsynthesis in the single-layer cultures used. The results of these experiments show that: (1) The fraction of cells in a culture synthesizingDNAis not markedly affected by α-particles and X-rays in the dose used in the experiment. (2) The effect of either type of radiation is to reduce the rate of synthesis ofDNAof the irradiated cells in synthesis. (3) The effect of a given dose of either type of radiation is to reduce the rate of synthesis of all the cells to a constant fraction of what it was in the unirradiated cells. (4) The rate ofDNAsynthesis is reduced to 37 % (1/e) by a dose ofca. 25 α/μ2or an X-ray dose of 14000 rads for mouse fibroblast cultures. In Hela cell cultures a dose ofca. 90000 rads is needed to reduce the rate ofDNAsynthesis to 37 % of the initial value. (5) The reduction in synthesis occurs not more than half an hour after irradiation and may be an immediate effect. From (4) above the target shape can be roughly calculated and if it is assumed to be cylindrical it appears to have dimensions 16 Å in one direction and 160000 Å in the other, i. e. a long thin thread with a mol. wt. ofca. 5 x 107in the case of the mouse fibroblast experiments. In the case of the Hela cell experiments the target volume gives a mol. wt. ofca. 107. These results are consistent with the view that the target may possibly be theDNAtemplate (or maybeDNPbecause of the high value for the molecular weight in one case). If the effects described reflect damage to theDNA(orDNP) template during the exponented phase of synthesis then observations, (1) (2), and (3) above follow as obvious corollaries.

The effect of vitamin B 12 and two vitamin B 12 antagonists on the growth of HeLa cells in tissue culture and of Ochromonas malhamensis

1. 1. Multiplication of HeLa cells in tissue culture is stimulated to some extent by small quantities of vitamin B 12. 2. 2. The methylamide and anilide analogues of vitamin B 12 show some inhibition of HeLa cell multiplication. 3. 3. The inhibitory effect of the methylamide analogue is reversed by vitamin B 12 at a concentration of approx. 1% of that of the inhibitor. 4. 4. The methylamide and anilide are more effective inhibitors of Ochromonas malhamensis than of HeLa cells. 5. 5. At low concentrations, the analogues have a limited vitamin B 12-like activity on O. malhamensis.

The Role of Glycolysis in the Growth of Tumor Cells: II. THE EFFECT OF OXAMIC ACID ON THE GROWTH OF HELA CELLS IN TISSUE CULTURE

The Role of Glycolysis in the Growth of Tumor Cells: II. THE EFFECT OF OXAMIC ACID ON THE GROWTH OF HELA CELLS IN TISSUE CULTURE