Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Probst, Hans; Gekeler, Volker; Helftenbein, Elke

doi:10.1016/0014-4827(84)90157-5

Cited by 31 publications

(20 citation statements)

References 21 publications

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“…In earlier reports (17)(18)(19)(22)(23)(24), we demonstrated that replicon initiation in Ehrlich ascites cells is specifically and reversibly suppressed by transient hypoxia. Other steps of replication, such as chain growth and DNA maturation, are not affected in these cells.…”

supporting

confidence: 57%

“…We labeled reoxygenated G1 cells at 0, 60, 120, 180, and 360 min after reoxygenation (with and without the addition of CHX) according to a 20/20 min "hot/warm" schedule. The autoradiographs usually exhibited, as in earlier work of our laboratory with the same ascites cells (16,19,24), evaluable stretches of 150 to 600 ,um in length representing two to seven replicons in tandem arrangement (clusters). Adjacent replicons were mostly found in similar stages, thereby confirming the coordination of neighboring initiations (6,16,19).…”

Section: Methodsmentioning

confidence: 72%

“…The autoradiographs usually exhibited, as in earlier work of our laboratory with the same ascites cells (16,19,24), evaluable stretches of 150 to 600 ,um in length representing two to seven replicons in tandem arrangement (clusters). Adjacent replicons were mostly found in similar stages, thereby confirming the coordination of neighboring initiations (6,16,19). Figure 4 shows examples of autoradiographs representing three basically different conditions of the cellular replication machinery generated in this experiment (commented on below in the respective context).…”

Section: Methodsmentioning

confidence: 72%

“…In 1986, we prepared a large frozen stock of pooled in vivo tumor cells which we have used since. Extensive studies characterizing growth and cell cycle parameters under different conditions in vivo and in vitro were published (18,19,22). According to reference 13, the cells are near tetraploid.…”

Section: Methodsmentioning

confidence: 99%

“…Details of the following procedures, leading to documentation of the tracks generated in a 6-month exposure on photographs (21 by 30 cm), were described in detail before (16). Evaluation of photographs, essentially according to criteria defined by Hand and Tamm (5, 7), was performed in the same way as in earlier investigations by our group (16,19,24) but was now greatly facilitated and sped up by a special computer program written in ELAN. Human activities were restricted to locating, in well-spread regions, continuously aligned arrays of tracks surmised to belong to the same fiber and then converting evaluable arrays into lines of the program input file by using a simple special language.…”

Section: Methodsmentioning

confidence: 99%

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Selective and synchronous activation of early-S-phase replicons of Ehrlich ascites cells.

Gekeler¹,

Epple²,

Kleymann³

et al. 1993

Mol. Cell. Biol.

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Twelve-hour exposure of G1 Ehrlich ascites cells to controlled hypoxia (200 ppm of 02 at 1 bar) suppressed replicon initiation. Synchronous cycling, beginning with a normal S phase, was released by reoxygenation immediately. The addition of cycloheximide at reoxygenation largely resuppressed, after a short initial burst, succeeding replicon initiations. Alkaline sedimentation analysis of growing daughter strand DNA, DNA fiber autoradiography, and analysis of the newly formed DNA demonstrated that normal chain growth and DNA maturation (replicon termination) in the initially activated replicons continued in the presence of cycloheximide. After 2 to 3 h, a low level of cycloheximide-insensitive background replication emerged out of the then-ebbing single surge of activity of the initially released replicons.In earlier reports (17-19, 22- (24). The regulation occurs when the oxygen tension in the cellular environment is lowered to the range between about 200 and 2,000 ppm (relative to 1 bar of total pressure). These 02 tensions are distinctly above those depressing mitochondrial respiration (2) and do not alter the cellular adenylate energy charge (22). Furthermore, we have shown that this regulation takes place during ascites tumor growth in vivo (18,22). There it may represent a major determinant of tumor cell propagation by adapting growth to the supply of nutrients (not only of 02) which is increasingly impaired when the tumor mass in the peritoneal cavity grows. Possibly, it also influences cell propagation in poorly vascularized regions of solid tumors. Repeatedly (18, 22-24), we pointed to the possible significance of 02-dependent replication regulation in critical situations during embryonic development of mammals before placentation. A further function in nonmalignant cell growth may be associated with certain stages of healing of a wound. When hypoxia hits cells in the G1 phase they obviously proceed in the cycle up to the point at which normally the first replicons of the S phase are activated. Reoxygenating such hypoxic G1 cells triggers, within a few minutes, entry into the S phase and succeeding synchronous cycling (3,24).In detailed experiments, we investigated the effects of cycloheximide (CHX) and puromycin on replicon action in aerated cells and in transiently hypoxic cells (3, 24). The results suggested (i) that replicon initiation in Ehrlich ascites cells depends on one or several short-lived protein(s) whose formation is not affected by the hypoxia and (ii) that inhibition of protein synthesis affects in Ehrlich ascites cells, unlike in many other cell lines (24), and in analogy to hypoxia, only replicon initiation. * Corresponding author.Thus, hypoxia and CHX provide two independent tools for specifically suppressing replicon initiation in Ehrlich ascites cells. These tools are differently suited for quickly releasing and establishing, respectively, the suppression. Establishing hypoxia in a cell culture requires 1 to 2 h (22), but a normal partial 02 pressure can be restored instantaneously ...

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supporting

confidence: 57%