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

Abstract

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 ...

Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status.

“…DNA synthesis in AG1522 cells immediately after reoxygenation occurred both in cells emerging from G1 phase and also in cells that had a greater than GI-phase content of DNA, indicating that under hypoxia, some cells in S phase stopped synthesizing DNA and, upon reoxygenation, restarted where they had stopped. This is consistent with previously published results and is thought to reflect a lack of replicon initiation under hypoxia (20,62). GM02184B cells differed from AG1522 cells in that they lacked any DNA synthesis in the first 4 h following reoxygenation (Fig.…”

Anoxic Fibroblasts Activate a Replication Checkpoint That Is Bypassed By E1a

“…Some studies have suggested that DNA initiation is affected by hypoxia and is most apparent at low oxygen tensions (21,42,43,46,47). Alternatively, deoxynucleotide depletion, which would be expected to affect DNA initiation, as well as elongation, has also been suggested to occur in hypoxic cells (9,37).…”

p21Cip1 and p27Kip1Regulate Cell Cycle Reentry after Hypoxic Stress but Are Not Necessary for Hypoxia-Induced Arrest

“…In fact, most cells are able to resume DNA synthesis within 10 min to 3 h after reoxygenation (38). However, little information is known about the genetic determinants that govern the reentry of hypoxic cells into the cell cycle except that recovery of DNA synthesis is blocked by cycloheximide addition in Ehrlich ascites cells (11,32) and is associated with the appearance of hyperphosphorylated pRb in T-47D breast cancer cells (2).…”

“…Studies using alkaline sedimentation analysis and fiber autoradiography have revealed that replicon initiation is suppressed by severe oxygen deprivation, but chain elongation and maturation continue normally for at least a few hours (11,29,32). While the persistence of chain elongation under hypoxic conditions implies that the lack of precursors is not directly responsible for the failure to synthesize DNA, it has been proposed that the nucleotide imbalance caused by hypoxia can act as a signal leading to cessation of replication (32).…”