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A B Pardee

Publications and source records attributed to A B Pardee.

At least 145 records · Page 8Linked to original sources
PubMed1988

Identification of a novel stress-inducible glycoprotein in Saccharomyces cerevisiae. I. Preliminary characterization.

We have identified a novel stress-inducible protein in Saccharomyces cerevisiae by pulse-labeling with [35S]methionine and two-dimensional gel analysis. The protein was characterized biochemically to gain further insight into mechanisms regulating the stress response. It has a Mr = 118,000 and exists in two forms of pI = 4.2 (p118A) and pI = 4.3 (p118B). p118A and p118B are modified by N-glycosylation. Tunicamycin treatment revealed the presence of precursor proteins of Mr = 105,000, pI = 4.1 (p105A) and pI = 4.25 (p105B). The synthesis of p118A and p118B was almost completely shut off in cycling cells and was increased 11-fold following a mild heat shock. Both forms of p118 decayed in a biphasic manner under induced conditions. A tight correlation was observed in the kinetics of thermotolerance induction and p118A synthesis. Other forms of stress such as sulfur starvation which lead to arrest in the unbudded phase also resulted in enhanced synthesis of both p118A and p118B. However, in cell division cycle mutants blocked at various stages at the restrictive temperature, p118A and p118B had different synthetic patterns. Taken together, these data imply a role for induced p118 in proliferation arrest in the unbudded state.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine.

Newly synthesized mRNA from hamster cells was labeled in vivo with the thionucleoside analogs 4-thiouridine (4-TU) and 6-thioguanosine (6-TG). The thio-substituted RNA was selectively recovered by Affi-Gel 501 phenylmercury affinity chromatography. Following a 1-h labeling period, enrichment for newly transcribed RNA after a single round of chromatography ranged between 10- and 15-fold when compared with total RNA. Exposure of CHO UrdA- cells, a uridine auxotrophic line, to 50 microM 4-TU allowed for optimal recovery of newly transcribed RNA. Increasing the concentration of 4-TU to 100 microM or labeling with 6-TG at concentrations of 3 microM or greater resulted in similar recoveries from uridine-prototrophic hamster cell lines. For shorter term labeling, exposure of prototrophic cells to 500 microM 4-TU or 100 microM 6-TG for 15 min allowed newly synthesized RNA to be selectively recovered. As a specific test case, enrichment for histone H3.2 mRNA was analyzed after hamster cells were labeled with 4-TU under conditions in which the gene was highly transcriptionally active. Northern blot analysis and the specific activity of thio-substituted RNA revealed a 15-fold enrichment when compared to total RNA. In vivo labeling of cellular RNA with 4-TU or 6-TG should provide a useful method for studying inducible gene expression and for isolating and cloning specific mRNAs from mammalian cells.

Affinity Labels↗
PubMed1988

Anticarcinogenic potential of DNA-repair modulators.

Effects of compounds that inhibit repair of DNA lesions in cells have been reported frequently. The consequences include altered incidence of carcinogenicity in vivo, tumorigenic transformation of cultured cells, mutations, and increased lethality as well as sister-chromatid exchanges and chromosome aberrations. This literature is reviewed here, with major emphasis on methylxanthines (caffeine in particular) and nicotinamide analogs. Existing information is also summarized on a novel potent repair inhibitor, beta-lapachone. Compounds that inhibit both DNA replication and repair are not discussed in detail since they have been reviewed often, but miscellaneous inhibitors of repair are summarized in a table. The relatively small number of experiments performed on the anticarcinogenic effects of methyl-xanthines and nicotinamide analogs gave very conflicting results. Some investigators report decreased carcinogenicity of DNA-damaging agents when caffeine was provided, but others obtained the opposite effect. The three studies with nicotinamide analogs all reported enhanced tumorigenicity of carcinogens. The data are too few to enable firm conclusions to be drawn regarding the possibility of using repair inhibitors to prevent cancer in humans. Variations of experimental conditions, carcinogens, cells, etc. have provided conflicting results. The possibility of cancer prevention is, nevertheless, so important that further investigations with DNA-repair inhibitors, particularly with human cells, seem very well justified.

Animals↗
PubMed1988

Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

The posttranscriptional regulatory mechanism(s) underlying thymidine kinase (TK) mRNA accumulation was investigated in BALB/c 3T3 cells during their progression from G0 into S phase of the cell cycle. Very little TK mRNA could be detected in either the nuclear or the cytoplasmic compartment from cells harvested in G0 or G1. At the onset of S phase, however, the level of nuclear TK mRNA precursors and mature TK mRNAs increased dramatically. The high molecular weight TK heterogeneous nuclear RNA species detected in the nuclei of S-phase cells were polyadenylylated and hybridized to intron sequences derived from the TK gene. A series of high molecular weight precursors could be chased to lower molecular weight species in the presence of actinomycin D, suggesting an ordered removal of intron sequences with the kinetics of a precursor-product relationship. These results demonstrate a striking change in the nuclear posttranscriptional processing of TK heterogeneous nuclear RNA at the G1-S boundary and, furthermore, define a model system for the examination of RNA-processing events in vivo.

Animals↗
PubMed1987

Potentiation of halogenated pyrimidine radiosensitizers in human carcinoma cells by beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran- 5,6-dione), a novel DNA repair inhibitor.

3,4-Dihydro-2,2-dimethyl-2H-naptho[1,2,-b]pyran-5,6-dione (beta-lapachone) is a novel DNA repair inhibitor. It was tested for synergistic X-ray-induced lethality in combination with several halogenated pyrimidine radiosensitizers. Logarithmic-phase growing human epidermoid laryngeal carcinoma (HEp-2) cells were allowed to incorporate pyrimidine analogues for 48 h (approximately two cell doublings) and then were X-irradiated and subjected to various posttreatments. beta-Lapachone synergistically increased the dose enhancement ratios (DERs) of all analogues screened, with the exception of the 2'-chloro derivative of 5-bromodeoxyuridine. For example, following 5-bromodeoxycytidine sensitization an X-ray DER value of 1.87 +/- 0.04 at 1% survival was increased to 3.51 +/- 0.42 due to a 4-h post-X-irradiation exposure to 4 microM beta-lapachone. Do and Dq values for halogenated pyrimidine-sensitized human epidermoid laryngeal carcinoma cells were decreased 1.4- to 5.4-fold and 1.4- to 4.0-fold, respectively. beta-Lapachone had little effect upon the cytotoxicities of unirradiated human epidermoid laryngeal carcinoma cells whether or not they were previously exposed to any of the halogenated pyrimidine radiosensitizers. beta-Lapachone treatment following X-irradiation of cells that had not incorporated a pyrimidine analogue exhibited DER values of 1.38 +/- 0.05 and 1.40 +/- 0.01 at 10 and 1% survival levels, respectively. beta-Lapachone enhanced the radiosensitization of deoxycytidine analogues to a greater extent than the structurally related deoxyuridine analogues. Greater DERs and lower Do and Dq values were found for deoxycytidine than for deoxyuridine analogue radiosensitizers following beta-lapachone treatment. This agent may improve presently used radiation therapies and enhance proposed strategies which utilize deoxycytidine analogue radiosensitization together with protection of normal tissues by tetrahydrouridine to achieve tumor-selective radiotherapy.

Bromodeoxycytidine↗
PubMed1987

Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events.

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.

Animals↗
PubMed1987

Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts.

Chinese hamster embryo fibroblast cells (CHEF/18) possess a plasma membrane-associated, amiloride-sensitive Na+/H+ antiporter that affects intracellular pH (pHi) and is activated by growth factor addition. Our results using 14C-benzoic acid distribution indicate that both epidermal growth factor (EGF) and thrombin are capable of causing rapid rises in the pHi of CHEF/18 cells. The maximal shift induced by these factors is 0.20 to 0.25 pH units above the basal unstimulated level. Distinctive differences were observed between the modes of action of these two growth factors. Sequential additions revealed that the rise in pHi due to EGF was additive with that caused by diacylglycerols (DAG), while that of thrombin was not. Furthermore, exposure of cells to the phorbol ester PMA for a prolonged period of time in order to down-regulate protein kinase C (pkC), or treatment with the pkC inhibitor H-7, abolished the pHi response to thrombin but not to EGF. In contrast, incubation of cells in nominally calcium-free medium or with the calmodulin antagonists W-7 or trifluoperazine (TFP) decreased only the ability of EGF to cause changes in pHi. These data suggest that there are two distinct mechanisms for activation of the Na+/H+ antiporter in CHEF/18 fibroblast cells and thus provide an example of the use of alternative modes for the modulation of intracellular processes.

Animals↗
PubMed1987

Cell cycle and growth factor-dependent phosphoprotein of 78kD differently regulated in normal and transformed mouse fibroblasts.

A novel 78kD phosphoprotein (pp78) of BALB/c 3T3 mouse fibroblasts is reported. Its properties of appearance in late G1 phase, dependence on specific growth factors, and altered constitutive kinetics in benzo(a)pyrene-transformed (BPA31) cells suggest its role in growth and transformation. Pp78 phosphorylation is in a dynamic state and is stabilized by inhibitors of protein and mRNA synthesis, possibly because of inhibition of a labile phosphatase or protease. Its disappearance in S phase and its low level in exponential cells also indicate a dynamic control that is dependent on growth conditions. Enhancement by phorbol myristate acetate indicates that phosphorylation of pp78 is a consequence of protein kinase C activation, but it appears much later than does an 80kD phosphoprotein (pp80), which is a recognized substrate of kinase C. No simple relation between the appearance of pp78 and mitogenesis was found. Two other phosphoproteins varied with growth conditions. One is the pp80 kinase C substrate which was found in the untransformed (A31) 3T3 cells early after stimulation but was absent in BPA31 cells. The other is an 18kD phosphoprotein that appeared shortly after quiescent 3T3 cells were stimulated.

Animals↗
PubMed1987

The Yang and Yin of cell proliferation: an overview.

Cell proliferation is a regulated process. The major events that determine production of new cells, from growth factors stimulation to cell division, are briefly summarized. Regulation of cell growth is exerted on specific cell cycle events. It depends upon specially evolved biochemical mechanisms, based on molecules additional to the functional molecules which control catalysis, or genetic sequences which code for proteins (as for regulation of bacterial enzyme and gene activities). The principle of Yang and Yin--the balance between opposing natural forces--is emphasized as a fundamental property of growth regulation. Such a balance creates a dynamic state that is far more responsive to effectors than is a process modified by a unidirectionally controlling agent. Derangements of dynamically controlled processes can seriously upset metabolic balances in a cell. Our understanding of growth control and its derangements in cancer depends on discovery of these regulatory molecules and processes.

Animals↗
PubMed1987

Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene.

Binding Sites↗
PubMed1987

Periodic mitotic events induced in the absence of DNA replication.

We have discovered and report here a means of separating a mitotic "subcycle" from the G1- and S-phase events of the mammalian cell cycle. Time-lapse videomicroscopy of Syrian hamster fibroblast (BHK) cells revealed that caffeine could induce multiple entries into mitosis while cells were blocked in DNA synthesis. As with normal mitoses, the abundance of mitosis-specific phosphoproteins was coupled with the condensation of chromatin. The BHK temperature-sensitive mutant tsBN2 also completed multiple entries into mitosis while arrested during DNA replication and raised to the restrictive temperature. Periodic mitotic events occurred even when BHK cells were exposed to low concentrations of serum or cycloheximide, conditions that prevent the cycling of BHK cells by blocking their entry into S phase. These results suggest that an oscillation governing the activation and inactivation of mitotic factors can be generated in mammalian cells and uncoupled from the G1 and DNA replication events of the normal cell cycle. This system will be useful for examining the molecular nature of mitotic factors.

Animals↗
PubMed1987

Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts.

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pHi) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. This increase of pHi in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na+/H+-antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pHi regulation in the tumorigenic CHEF/18 derivatives lies in the Na+/H+ antiporter itself. Further studies to determine the biological significance of an increased pHi show that the external pH (pHo)-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pHi in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pHi can contribute to the acquisition of altered growth properties.

Animals↗
PubMed1987

Effects of ciprofloxacin on eucaryotic pyrimidine nucleotide biosynthesis and cell growth.

Several of the new 4-quinolones significantly increase the incorporation of [3H]thymidine into the DNA of mitogen-stimulated human lymphocytes. This study suggests that ciprofloxacin inhibits de novo pyrimidine biosynthesis, thereby resulting in a compensatory increase in the uptake of pyrimidine precursors through salvage pathways, and that additional effects may affect eucaryotic cell growth. Incorporation of deoxyuridine, uridine, and orotic acid as well as thymidine was increased in the presence of ciprofloxacin, one of the antibacterially most active of the new 4-quinolones. In contrast, the uptake was decreased in very high concentrations of the drug. Culture in HAT (hypoxanthine, aminopterine, thymidine) medium, which blocks de novo thymidylate synthesis, abrogated the increase in [3H]thymidine incorporation induced by ciprofloxacin. Ciprofloxacin also failed to increase the uptake of [14C]hypoxanthine or leucine, indicating a selective effect on pyrimidine and not on purine nucleotide biosynthesis. N-(Phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine nucleotide biosynthesis, also increased [3H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes in a fashion similar to ciprofloxacin. The growth of several cell lines was partially inhibited by ciprofloxacin at 20 micrograms/ml and completely inhibited at 80 to 160 micrograms/ml. Growth inhibition by ciprofloxacin could not be restored by the addition of uridine to the medium. Chromosome breaks, gene amplification, or other genetic alterations could not be detected in human lymphocytes incubated with up to 25 micrograms of ciprofloxacin per ml.

Animals↗
PubMed1987

Control of thymidine kinase mRNA during the cell cycle.

To investigate the mechanism which controls the onset of DNA synthesis, we examined the regulation of thymidine kinase (TK) and its mRNA in the cell cycle. TK activity provides a useful marker for the onset of the S phase in mammalian cells. The present analysis of regulation of TK mRNA in BALB/c 3T3 cells showed that (i) the increase in TK activity depended on the availability of TK mRNA, (ii) the level of TK mRNA between G0 and S increased more than 20-fold, (iii) the rate of run-on TK transcription increased at most 2- to 4-fold between the G0 and S phases, (iv) the half-life of TK mRNA was greater than 8 to 12 h in the S and M phases and decreased as cells entered quiescence, (v) the TK mRNA increase was fully blocked by inhibition of protein synthesis by only 60%, (vi) this inhibition was completely effective for up to about 10 h following serum addition and progressively much less effective when the drugs were added later. These results suggest that the appearance of TK mRNA at the beginning of the S phase in serum-stimulated 3T3 cells is controlled not only by the rate of gene transcription but importantly also by the decreased rate of mRNA degradation. Similar mechanisms may be involved in regulation of the onset of DNA synthesis and the increase in TK mRNA since both are controlled in a manner consistent with a requirement for a labile protein.

Animals↗
PubMed1986

C-myc expression is dissociated from DNA synthesis and cell division in Xenopus oocyte and early embryonic development.

The combined use of a human c-myc probe and of an antibody raised against the human c-myc gene product demonstrated that the Xenopus cells contained a 2.5-kb c-myc transcript and synthesized a c-myc immunoreactive 65-kd polypeptide. In full-grown oocytes, p65c-myc was predominantly located in the nucleus. In non-dividing Xenopus oocytes c-myc mRNA was present at a steady-state level 10(4) times higher than that of growing somatic A6 cells. This very high level of c-myc transcript was reached early in oogenesis and remained constant thereafter. The rate of p65c-myc synthesis also reached high levels, but only in vitellogenic oocytes, suggesting a post-transcriptional control. Although the cell cycle is resumed at a very fast pace in developing embryos, no further increase in total embryonic content of c-myc RNA could be demonstrated up to the swimming tadpole stage. Furthermore, in embryos the rate of synthesis of p65c-myc decreased to a level markedly lower than that of cell cycle-arrested vitellogenic oocytes. This observation suggests that the function of the c-myc gene in the cell cycle may not be implicated directly in sustaining DNA synthesis or mitosis.

Animals↗
PubMed1986

Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells.

Caffeine was shown to induce mitotic events in mammalian cells before DNA replication (S phase) was completed. Synchronized BHK cells that were arrested in early S phase underwent premature chromosome condensation, nuclear envelope breakdown, morphological "rounding up," and mitosis-specific phosphoprotein synthesis when they were exposed to caffeine. These mitotic responses occurred only after the cells had entered S phase and only while DNA synthesis was inhibited by more than 70 percent. Inhibitors of protein synthesis blocked these caffeine-induced events, while inhibitors of RNA synthesis had little effect. These results suggest that caffeine induces the translation or stabilizes the protein product (or products) of mitosis-related RNA that accumulates in S-phase cells when DNA replication is suppressed. The ability to chemically manipulate the onset of mitosis should be useful for studying the regulation of this event in mammalian cells.

Animals↗
PubMed1986

Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts.

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.

Animals↗