Metabolic Regulation in the Cell Cycle

Klevecz, Robert R.; Forrest, Gerald L.

doi:10.1016/b978-0-12-598303-7.50011-4

Cited by 8 publications

(3 citation statements)

References 200 publications

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“…5, lane a). Lanes [2][3][4][5][6][7][8][9][10] show the synthesis ofproteins during 30-min labeling at hourly points through the first 10 hr of the cell cycle. The fluorograph shows a period of low DHFR synthetic activity through the first 5 hr.…”

Section: Resultsmentioning

confidence: 99%

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells.

Mariani¹,

Slate²,

Schimke³

1981

Proc. Natl. Acad. Sci. U.S.A.

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We investigated the cell cycle modulation ofdihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase, 7,8-dihydroxyfolate:NADP+ oxidoreductase, EC 1.5.1.3) levels in methotrexate-resistant Chinese hamster ovary cells synchronized by mitotic selection. DNA content and DHFR concentration were analyzed throughout the cell cycle by standard biochemical techniques and by double fluorescence staining utilizing the fluorescence-activated cell sorter. We found an S phase-specific period of DHFR biosynthetic activity. Commencing within hour 2 of S phase and continuing throughout the duration of S phase, there is a 90% increase in DHFR specific activity. This results from an "=2.5-fold increase in the level of DHFR, while total soluble protein increases 50% during the same period. This increase is the result of new synthesis of DHFR molecules initiated after the cell is physiologically committed to DNA replication. This increase in DHFR activity through S phase parallels the increasing rate of[3H]thymidine incorporation during the same interval. The maximum peak of DHFR activity is coincident with the maximum rate ofDNA synthesis, both activities occurring during the bulk ofDNA replication within the last stages of the 6.5-hr S phase.Control of specific protein synthesis in the framework ofthe cell cycle represents a fundamental form of regulation. Numerous enzyme activities have been studied as a function of S phase in mammalian cells. The activities of DNA polymerases (review in ref. 1) and the enzymes necessary for the provision ofdeoxyribonucleoside triphosphates (review in ref.2)-i.e., thymidine kinase (3), thymidylate kinase (4), thymidylate synthetase (5, 6), dihydrofolate reductase (7), ribonucleotide reductase (8), and deoxycytodine monophosphate deaminase (9)-follow a general pattern of increasing through S phase and attaining a maximum near the S/G2 interface. We investigated one enzyme involved in the integrative process of growth regulation:dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase, 7,8-dihydroxyfolate:NADP+ oxidoreductase, EC 1.5.1.3).DHFR is necessary for the production of tetrahydrofolate, a key intermediate in one-carbon transfer reactions. Thus, DHFR activity is temporally coupled with the maintenance of sufficient thymidylate pools necessary to support DNA synthesis. Normally, the intracellular concentration of a "housekeeping" enzyme such as DHFR is extremely low-0. 1% of total protein (7). The low concentration of DHFR limits any study exploring the biochemical parameters involved in regulation. This study takes advantage of a methotrexate (MTX)-resistant Chinese hamster ovary cell line, K1B110.5, which contains elevated levels of DHFR corresponding to an amplified number of genes encoding the information for DHFR production, the target enzyme for methotrexate inhibition (10).A previous report (11) has centered on the modulation of DHFR content in MTX-resistant mouse 3T6 fibroblasts when serum-deprived cells were induced to reenter the cell cycle as a result of serum ...

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Section: Resultsmentioning

confidence: 99%

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells.

Mariani¹,

Slate²,

Schimke³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…alter normal cellular metabolism (8). Therefore, it is difficult to distinguish between results representing the properties of a particular cell virus combination and changes caused by the synchronization protocol.…”

Section: P30gagmentioning

confidence: 99%

Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells

Balazs¹,

Caldarella²

1981

J Virol

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Synchronized mouse cells (JLS-V9) chronically infected with Rauscher murine leukemia virus were used to study virus production, the synthesis of gag and env precursor proteins, and the expression of env protein on the cell surface during the cell cycle. The amount of virus released into the medium by synchronized cells during a 30-min interval was determined by using the XC plaque assay and by measuring reverse transcriptase activity. The results show that virus production occurs during mitosis. Labeling of the cell surface of synchronized cells with 125I or with fluorescein-conjugated antiserum shows that the amount of gp 70env on the cell surface parallels cellular growth. Therefore, the cell cycle-dependent release of virus is not accompanied by similar variations in the amount of viral envelope protein on the cell surface. Immunoprecipitation of cells labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was used to measure viral protein synthesis during the cell cycle. The rate of synthesis of gag precursor proteins show three maximums corresponding to the G1, middle S, and late S to G2 phases of the cell cycle. The rate of synthesis of env precursor proteins does not change, suggesting that in these cells the synthesis of these two gene products is controlled separately.

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“…empirically by numerous observations of oscillations in enzyme activity, in enzyme synthesis, and in rates of RNA and DNA synthesis (Klevecz and Forrest, 1977).…”

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confidence: 99%

Phase response to heat shock as evidence for a timekeeping oscillator in synchronous animal cells

Klevecz

Kros

King³

1980

Cytogenet Genome Res

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Synchronous animal cells given heat shocks at 0.5-h intervals through an 8.5-h cell cycle display a biphasic pattern of advances and delays in subsequent cell divisions. This phase-response curve to perturbation by heat shock has a period of approximately 4 h and cannot readily be explained by commonly used models of cell cycle traverse. We suggest that this outcome is the consequence of timekeeping by a macromolecular oscillator with limit cycle properties.

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Metabolic Regulation in the Cell Cycle

Cited by 8 publications

References 200 publications

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells.

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells.

Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells

Phase response to heat shock as evidence for a timekeeping oscillator in synchronous animal cells

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