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A B Pardee

Publications and source records attributed to A B Pardee.

At least 199 records · Page 11Linked to original sources
PubMed1980

Quiescent cells but not cycling cells exhibit enhanced actin synthesis before they synthesize DNA.

Major proteins synthesized by Swiss 3T3 cells at different stages of the cell cycle have been analyzed using double isotope labeling and one-dimensional SDS-polyacrylamide slab gels. The synthesis of actin was previously shown to be markedly enhanced a few hours after quiescent cells initiated growth following addition of serum. In contrast, the synthesis of actin remained at a constant rate, similar to that in quiescent cells, relative to synthesis of other proteins during the entire cell cycle. We conclude that enhanced actin synthesis is a process specific for the G0 to S transit, and may serve as a marker event during this interval. In contrast, three other proteins (90,000, 57,000, and 33,000 daltons) were synthesized throughout the cell cycle at higher rates than in G0 cells, and thus, are markers characteristic of cells traversing the cell cycle. A transient increase, such as seen for actin synthesis, by cells emerging from quiescence, may represent a process that these cells must perform before they can enter the G1 portion of the cell cycle. A transient event such as this need not be a periodic event that occurs during each cycle.

Actins↗
PubMed1980

Synergistic effects of epidermal growth factor and thrombin on the growth stimulation of diploid Chinese hamster fibroblasts.

The serum supplement used in the culture of a variety of mammalian cells can be replaced by known growth factors. Diploid Chinese hamster fibroblasts (CHEF/18) will grow for several days in a medium (4F) supplemented with four growth factors: epidermal growth factor (EGF), insulin, fibroblast growth factor (FGF), and transferrin. The growth rate is only about 50% as fast as when fetal calf serum is added. This difference is eliminated by thrombin (10--100 ng/ml; 0.3--3 nM). The CHEF/18 cell line is unique in that no other cell line responds to thrombin in this concentration range. Thrombin acts synergistically with other growth factors to stimulate CHEF/18 cell growth. By itself, thrombin is only mitogenic at elevated concentrations. Thrombin can largely compensate for the absence of EGF and partly for the absence of insulin in serum-free media. Chemically and "spontaneously" transformed cell lines related to CHEF/18 have lost requirements for both EGF and thrombin, and have retained requirements for insulin and transferrin expressed by CHEF/18. No CHEF cells in this work required FGF. These results suggest that the mechanisms by which EGF and thrombin stimulate cells to grow are related.

Animals↗
PubMed1980

Multienzyme complex for metabolic channeling in mammalian DNA replication.

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.7), were largely localized in the nuclear region (karyoplasts). By contrast, in quiescent and G1 phase cells these enzymatic activites were mainly absent from the nucleus and were recovered in the cytoplasmic portion (cytoplasts). These nuclear (but not cytoplasmic) enzymatic activities cosedimented rapidly on sucrose density gradients. Further, the rapidly sedimenting enzyme activities were unique to cells in S phase. An organized supramolecular structure that allows channeling of metabolites into DNA was demonstrated by kinetics of nucleotide incorporation. "Permeabilized" cells selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates. Deoxyribonucleoside triphosphate incorporation occurred when ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) activity was abolished by hydroxyurea. Our interpretation is that during DNA replication, the nucleus contains a complex of DNA precursor-synthesizing enzymes juxtaposed with the "replication apparatus" comprising DNA polymerase, other enzymes, and structural proteins. Functional integrity of this structure is impaired when one of its essential components is inactivated. We propose the name "replitase" for this multienzyme complex for DNA replication and suggest that it incorporates precursors rapidly and efficiently. Possibly its assembly signals the initiation of the S phase of the cell cycle.

Animals↗
PubMed1980

Prevalent deficiency in tumor cells of cycloheximide-induced cycle arrest.

Mammalian cell growth is regulated by a process that is completed at a restriction point in the late G1 part of the cycle. This process is highly sensitive to serum concentration and to moderate inhibition of protein synthesis by cycloheximide (CHM) or other agents. We have proposed that a cell must accumulate a labile protein in a critical amount before events related to its DNA synthesis can start. The accumulation of this protein requires conditions suitable for growth, including sufficient amounts of serum-derived factors. An important criterion for attributing a major role to such a regulatory mechanism is that cells whose growth control is modified--e.g., by mutation--should be defective in this process. Cells of this kind are produced by tumorigenic transformation. We show here that mouse 3T3 cells, human fibroblasts, and Chinese hamster CHEF/18 cells have stringent G1 growth control by CHM. In contrast, tumorigenic lines obtained from these cells by transformation with varius agents (DNA tumor virus, RNA tumor virus, chemical carcinogens) or spontaneously all showed relaxed growth control under the influence of CHM. In these lines, growth control was relaxed to different degrees; some lines were held in G1 by a combination of low serum concentration and CHM, but others were not. Serum concentration showed a synergistic effect with CHM. Low serum concentrations did not limit growth only by affecting the rate of protein synthesis. The labile-protein mechanism is likely to be basic to growth control by serum factors. Transformed tumorigenic cells in general may have relaxed this mechanism.

Animals↗
PubMed1979

Role of nuclear size in cell growth initiation.

Swiss 3T3 cells arrested in B0 (quiescent state) by reducing serum content of the medium all contain the same amount of DNA but vary in nuclear volume over approximately a twofold range. By use of flow microfluorimetry, scatterplots of nuclear volume versus DNA content were obtained in intervals after serum stimulation. The earliest cells to enter DNA synthesis were those with the largest nuclei, whereas cells with the smallest nuclei were among the latest. Regulation of cellular transit from G0 to the S phase was therefore, at least in part, deterministic, since all G0 cells did not have equal probabilities of entry into S at a given moment. All cells having the same nuclear volume did not initiate DNA synthesis at the same moment; therefore, factors other than nuclear volume must also influence this timing. Nuclear volume correlated with the maximum rate at which cells could enter S. The kinetic model of the cell cycle postulating a probabilistic event as solely responsible for entry into S thus appears too simple.

Animals↗
PubMed1979

Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product.

DNA is more extensively degraded after it is damaged in recA mutants of E. coli than in wild type cells. All data presented here are consistent with the recA gene product, protein X, being an inhibitor of nalidixic acid induced degradation of the bulk DNA (but not of newly replicated DNA). Production of protein X also is correlated with appearance of various "S.O.S." repair functions. Evidence was obtained by comparing the rates of protein X synthesis and solubilization of uniformly-labeled DNA in intact cells, incubated in the presence of nalidixic acid. A set of mutants at the lexA locus produced protein X at different rates and degraded their DNA at rates which were inversely correlated to their rates of protein X production. A low concentration of rifampicin quite specifically inhibited protein X production by wild type E. coli, and allowed more rapid DNA degradation. After the DNA was damaged by the incubation of cells in the presence of nalidixic acid, cells preloaded with protein X degraded their DNA more slowly. We propose that protein X could protect DNA against degradation by binding to single-stranded regions, thereby inhibiting nuclease action.

Bacterial Proteins↗
PubMed1979

Growth control of normal and transformed cells.

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G1 period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentrations of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.

Animals↗
PubMed1979

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from G0 to S phase. Its synthesis rose to a maximum 4--6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remained high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.

Actins↗
PubMed1979

Loss of epidermal growth factor requirement and malignant transformation.

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells. The medium's four growth factors-epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin-were added or omitted as desired. A decreased requirement for EGF was most closely related to tumorigenicity of chemically (ethyl methanesulfonate) transformed cells in nude mice. All lines examined retained their requirement for transferrin, which is needed throughout the growth cycle, in contrast to the other factors, which are needed primarily in G(1) phase. Lines that had lost their EGF requirement but had retained their insulin requirement were arrested in G(1) by insulin deficiency, indicating that their growth control system remained. Mutagenesis with ethyl methanesulfonate can also create requirements of the transformed cells for unknown factors in serum. We conclude that an initial step that reduces the serum requirement in culture, and in tumorigenesis, is relaxation of the growth-regulatory function of EGF.

Blood↗
PubMed1979

Synthesis of labile, serum-dependent protein in early G1 controls animal cell growth.

We present a model to account for several major observations on growth control of animal cells in culture. This model is tested by means of kinetic experiments which show that exponentially growing animal cells whose ability to synthesize total protein has been inhibited with cycloheximide (by up to 70%) grow at rates approximately proportional to their rates of protein synthesis. However, virtually the entire elongation of the cell cycle occurs in the part of the G(1) phase that depends on a high concentration of serum in the medium. This part of the cycle has earlier been suggested to lie prior to the restriction point-i.e., the point beyond the main regulatory processes of G(1). The remainder of the cycle, from restriction point to mitosis, is markedly insensitive to these concentrations of cycloheximide as well as to growth regulation. We quantitatively account for the specific lengthening of that part of the cycle involved in growth regulation by assuming that cells must accumulate a specific protein in a critical amount before they can proceed beyond the restriction point. The lability of this protein (half-life about 2 hr) makes its accumulation unusually sensitive to inhibition of total protein synthesis by cycloheximide. Its production appears to depend on growth factors provided by serum. The model can also account for greater variations of G(1) durations as the growth of cell populations is made slower. It also predicts two sorts of quiescence: one of cells slowly traversing G(1), in slightly suboptimal conditions; the other of cells that enter G(0) under inadequate conditions. Transformation of different sorts could create cells with altered variables for initiation, synthesis, or inactivation of the regulatory protein or could altogether eliminate the need for the protein.

Animals↗