Radiation-stimulated DNA synthesis in cultured mammalian cells

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H³-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G₁ and G₂ cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.     

Anticlastogenicity in cultured mammalian cells.

Basic and applied research on anticlastogenicity has not only revealed valuable evidence on the mechanisms governing the induction of chromosomal aberrations by environmental mutagens, but also contributed effective ideas on a practical employment of this knowledge for the protection of individuals at risk. Considering the basic role played by chromosomal anomalies in oncogenesis, additional weight must be attributed to studies on anticlastogenicity. The employment of human cells in this kind of study dates back to 1969/70, while classical mammalian cell systems were used only later on. Various modes of application of both clastogens and anticlastogens (AC) were examined, but simultaneous addition to the cultures of both reagents was the most favored way. A wide spectrum of cytogenetic endpoints can be studied, but differences can be demonstrated with regard to efficacy of inhibitors on different types of cytogenetic changes, e.g., open breaks vs. rearrangements, but also vs. SCEs. Depending on their mode of influence on this spectrum, ACs can be arranged in various categories which are of practical importance, for instance, with regard to their oncogenic potential. A wide variety of factors was shown to influence AC action, e.g., time and mode of application of the test substances, physiologic and metabolic features of the cell types studied, type and mechanism of the clastogen used, etc. The addition of S9 mix can drastically change the patterns of efficacy of the ACs. The combined application of two or more ACs, as far as investigated, apparently neither potentiates nor even merely adds their effects.     

Mechanisms of acquired resistance to acute heat shock in cultured mammalian cells

A clonal strain of mammalian cells with increased resistance to acute heat shock at 46° was compared with the heat-sensitive parental line from which it was derived for possible differences that might account for this acquired resistance. Studies on synchronized populations of the two cell strains did not reveal any differential heat sensitivities in the various parts of the cell cycle (G₁, S, G₂) within either the sensitive or resistant populations. During thermal stress both cell types exhibited marked inhibition of ability to incorporate isotopically labeled precursors into macromolecular RNA, DNA, and protein. However, significant differences were observed between the sensitive and resistant cells in the amount of leakage of materials from the two cell types during heat stress and in their relative rates of recovery after stress. Sensitive cells pre-labeled with ³H-uridine released considerably more acid-soluble (cold, 5% TCA) label-containing materials during heat stress than did pre-labeled resistant cells. This differential release of uridine-containing materials was not paralleled by a generalized differential leakiness to other compounds. In addition, the resistant cells were found to regain the capacity to synthesize vital macromolecules sooner, and at initially faster rates, than the sensitive cells after stress. These results suggest that permeability changes causing decreased leakage of uridine-containing materials during heat stress combined with accelerated rates of recovery of synthesis of essential macromolecules after stress may be important cellular mechanisms in resistance to heat shock.     

Mechanisms and cellular roles of local protein synthesis in mammalian cells

After the export from the nucleus it turns out that all mRNAs are not treated equally. Not only is mRNA subject to translation, but also through RNA-binding proteins and other trans-acting factors, eukaryotic cells interpret codes for spatial sorting within the mRNA sequence. These codes instruct the cytoskeleton and translation apparatus to make decisions about where to transport and when to translate the intended protein product. Signaling pathways decode extra-cellular cues and can modify transport and translation factors in the appropriate cytoplasmic space to achieve translation locally. Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function.     

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

Regulation of tubulin synthesis and cell cycle progression in mammalian cells by gamma-tubulin-mediated microtubule nucleation

We have previously shown that gamma-tubulin, the third member of the tubulin family that functions in microtubule nucleation, when overexpressed, accumulates throughout the cytoplasm and forms numerous ectopic microtubule nucleation sites in mammalian cells (Shu and Joshi [1995] J. Cell. Biol. 130:1137-1147). We now show that overexpression of gamma-tubulin differentially upregulates the synthesis of alpha- and beta-tubulins in mammalian cells. Surprisingly, despite a dramatic increase in the level of gamma-tubulin protein in transfected cells, there is no obvious alteration in the level of endogenous gamma-tubulin mRNA, suggesting that synthesis of gamma-tubulin might employ a regulatory mechanism other than the autoregulatory pathway shared by alpha- and beta-tubulins. Interestingly, a significant number of mammalian cells transfected with gamma-tubulin fail to form normal bipolar mitotic spindle during mitosis; instead, numerous microtubules occur in the cytoplasm intermingled with the condensed chromosomes. In addition, they reduplicate their DNA after an abnormal mitotic exit. These results thus suggest that the number of microtubule nucleation sites, or even gamma-tubulin itself, might play an important role in the regulation of tubulin synthesis as well as cell cycle progression.     

Mechanisms responsible for the limited lifespan and immortal phenotypes in cultured mammalian cells

Normal mammalian cells have a limited lifespan in culture and hypotheses explaining cellular senescence usually fall into one of two categories. One of these postulates that random errors or damage accumulate in essential macromolecules and eventually outstrip the cell's capacity for resynthesis and repair. The second considers the changes when immortal clones are produced from normal cells and in particular the lifespans of hybrids when cells of differing growth potentials are fused. These data can be explained by postulating that the mortal phenotype is dominant and that trans-acting growth inhibitors are involved in limiting lifespan. But the results do not indicate if the inhibitors are the primary cause of senescence or a secondary effect induced by quite different initial events. We suggest that normal cells possess proof-reading mechanisms which monitor the accuracy of chromosome segregation and replication and which can induce the synthesis of growth inhibitors when they detect major errors in chromosome metabolism. It is further postulated that random damage accumulates during the growth of normal cells and eventually leads to detectable chromosome changes and the synthesis of inhibitors. Our hypothesis predicts that the emergence of immortal clones will be linked to the absence of active inhibitors and therefore to a loss in the fidelity of chromosome metabolism. Data are quoted which show that in contrast to normal cells, immortal clones have highly irregular karyotypes, amplify segments of their chromosomes, integrate exogenous DNA efficiently, maintain a constant level of 5-methylcytosine residues and have high frequencies of chromosomal aberrations. The mechanism of the proof-reading is unknown, but it may monitor changes in the patterns by which chromosome domains are attached to the nuclear matrix.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Mechanisms regulating epidermal stem cells

The skin epidermis contains different appendages such as the hair follicle and the sebaceous glands. Recent studies demonstrated that several types of stem cells (SCs) exist in different niches within the epidermis and maintain discrete epidermal compartments, but the exact contribution of each SC populations under physiological conditions is still unclear. In addition, the precise mechanisms controlling the balance between proliferation and differentiation of epidermal SC still remain elusive. Recent studies provide new insights into these important questions by showing the contribution of hair follicle SC to the sebaceous lineage and the importance of chromatin modifications and micro-RNAs (miRs) in regulating epidermal SCs renewal and differentiation. In this review, we will discuss the importance of these papers to our understanding of the mechanisms that control epidermal SC functions.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

Ribonuclease of cultured mammalian cells

KB cell ribonuclease has been purified 260-fold and the fundamental properties have been studied. Though the enzyme is concentrated in the lysosomal fraction, appreciable quantities are present in the cell sap and nuclear fractions. Comparison of the optimal temperature and pH for activity, and the heat stability of enzyme from these three fractions suggests that only one species of this enzyme exists in these cells. The enzyme behaves as an endonuclease, cleaving synthetic pyrimidine polynucleotides to smaller oligonucleotides with cyclic 2′:3′ end-groups. The final product is pyrimidine nucleoside 3′ monophosphate. Polyadenylic acid is not hydrolyzed. Of the properties examined in this study only two differences were noted between KB cell and pancreatic ribonuclease. KB cell enzyme acts optimally at pH 6 as opposed to an optimum at pH 7 to 8 for pancreatic enzyme. In addition ribonuclease from KB cells is definitely less stable to heating at 100°C than is the enzyme isolated from pancreas.     

Calmodulin-microtubule association in cultured mammalian cells

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.     

T-tubes in cultured mammalian myocardial cells

Summary T-tubes are among the last structural elements of the mammalian myocyte to develop in vivo. We were able to identify T-tubes in early cultures of neonatal rat myocytes. Ventricles were excised from 3- to 4-day-old neonatal rats, incubated overnight in cold trypsin, and treated with sequential changes of collagenase-hyaluronidase. Fractions of cells isolated in this manner were pooled and cultured in plastic petri dishes. In cells prepared for transmission electron microscopy, T-tubes were observed at the cell periphery of cultured myocytes, but were more difficult to identify as the cultures aged and became overgrown by fibroblasts. T-tubes were identified by virtue of their continuity with the sarcolemma, their relatively large diameter, and their regular entry at the level of the Z line. Even at optimal culture ages, T-tubes were not present in every myocyte. At the times T-tubes could be located, myocytes were beating and had begun to establish intercalated discs and gap junctions. The de novo formation of T-tubes in cultured myocytes of neonatal rat heart reflects a duplication of in vivo differentiation by the cultured myocyte. The appropriateness of cultured myocytes in the study of the development and physiology of the heart is emphasized by the in vitro formation of T-tubes.Supported by research grants from the Muscular Dystrophy Association, Inc., The Schlieder Foundation, and USPH-Training Grant HL 07098-04. The authors are indebted to Philip Constantin for assistance in dissociating and culturing heart tissue.