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Enhanced synthesis of specific proteins, RNA, and DNA caused by hypoxia and reoxygenation
1Department of Biophysics, University of Rochester, NY 14642.
Abstract:
When Chinese Hamster ovary cells were exposed to severe hypoxia they responded with an enhanced synthesis rate of a small group of specific proteins, which we have termed the oxygen regulated proteins (ORPs). The five major ORPs had molecular weights of 33, 80, 100, 150, and 260 kD. ORP260 was the first ORP to be synthesized at enhanced rates after the induction of hypoxia, reaching its maximal synthesis rate after only a few hours. The other ORPs responded slower, but were being synthesized at maximal rates by 8 hr of hypoxia. Upon reoxygenation, the synthesis rates of the ORPs declined rapidly with ORP260 again responding most rapidly. They all reached control levels by 12 hr of recovery. The induction of enhanced synthesis of these proteins required a very severe degree of hypoxia. They were not seen at oxygen levels higher than 0.03%. While enhanced synthesis of the ORPs was turned on and off quickly with induction of, and recovery from hypoxia, the proteins were not degraded at high rates once synthesized. All five major ORPs have apparent half-lives in excess of 24 hr. When cells were exposed to hypoxia for 20 hr and then allowed to recover for 15 hr, a population of cells was generated that had, on average, at least twice the cell volume as aerobic control cells. These large cells also contained greater than 4C DNA content as well as enhanced amounts of RNA.
Insights
Severe hypoxia triggers enhanced synthesis of specific oxygen-regulated proteins (ORPs) in Chinese Hamster ovary cells. These ORPs, with long half-lives, accumulate and lead to significant cell enlargement and increased DNA/RNA content upon recovery.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Hypoxia, a state of low oxygen, significantly impacts cellular function and protein expression.
- Chinese Hamster ovary (CHO) cells are a common model system for studying cellular responses to environmental stress.
Purpose of the Study:
- To identify and characterize proteins whose synthesis is upregulated under severe hypoxic conditions in CHO cells.
- To investigate the kinetics of protein synthesis and degradation in response to hypoxia and reoxygenation.
- To examine the downstream cellular effects of hypoxia-induced protein changes.
Main Methods:
- Exposure of CHO cells to severe hypoxia (oxygen levels < 0.03%).
- Analysis of protein synthesis rates using radiolabeling techniques.
- Determination of protein half-lives through pulse-chase experiments.
- Cellular analysis including volume, DNA content (e.g., flow cytometry), and RNA quantification.
Main Results:
- Identification of five major oxygen-regulated proteins (ORPs) with molecular weights of 33, 80, 100, 150, and 260 kD.
- ORP260 showed the most rapid induction and decline in synthesis rates upon hypoxia and reoxygenation, respectively.
- All identified ORPs exhibited long half-lives (>24 hours), indicating stable accumulation.
- Prolonged hypoxia followed by recovery resulted in significantly enlarged cells with increased DNA (>4C) and RNA content.
Conclusions:
- Severe hypoxia induces a rapid and specific upregulation of ORP synthesis in CHO cells.
- The stability of ORPs contributes to their accumulation and subsequent cellular alterations.
- Hypoxia-induced ORP synthesis is linked to significant changes in cell size and genetic material content, suggesting a role in cellular adaptation or preparation for division.