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Studies on the penetration of mammalian cells by deoxyribonucleoside-5'-phosphates
Abstract:
We have tested the ability of [5'-32P]-deoxyribonucleoside monophosphates (dNMPs) to penetrate living mouse fibroblast L cells and human HeLa cells. Under the conditions of our experiments, small numbers of apparently intact dNMP molecules appeared to penetrate into the interior of L cells and be incorporated into DNA. This incorporation was not due to mycoplasma contamination nor to extracellular hydrolysis of the dNMPs followed by resynthesis inside the cell. Under these same conditions, penetration of HeLa cells by intact dNMPs did not occur to a significant extent. However, HeLa cells were capable of hydrolyzing extracellular dNMPs to Pi and deoxyribonucleosides at a much faster rate than L cells. These experiments provide a starting point for attempts to specifically label the DNA in intact, living eukaryotic cells with [32P]-dNMPs.
Insights
Researchers investigated if [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) could enter living cells. Intact dNMPs were incorporated into mouse L cells
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Specific labeling of DNA in living eukaryotic cells is crucial for various biological studies.
- Understanding the mechanisms of nucleoside monophosphate uptake and incorporation is essential for developing targeted labeling strategies.
Purpose of the Study:
- To evaluate the ability of [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) to penetrate and be incorporated into living mouse fibroblast L cells and human HeLa cells.
- To investigate the differences in dNMP uptake and metabolism between L cells and HeLa cells.
Main Methods:
- Incubation of L cells and HeLa cells with [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) under controlled experimental conditions.
- Analysis of dNMP penetration into cells and incorporation into DNA using radiolabeling techniques.
- Assessment of extracellular hydrolysis of dNMPs by both cell types.
Main Results:
- Intact [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) were observed to penetrate L cells and be incorporated into their DNA.
- HeLa cells showed limited significant penetration of intact dNMPs but exhibited a much higher rate of extracellular dNMP hydrolysis to inorganic phosphate (Pi) and deoxyribonucleosides.
- Mycoplasma contamination and extracellular hydrolysis followed by intracellular resynthesis were ruled out as causes for dNMP incorporation in L cells.
Conclusions:
- Mouse fibroblast L cells can incorporate intact [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) into their DNA, suggesting a potential for direct DNA labeling.
- Human HeLa cells possess a more robust mechanism for hydrolyzing extracellular dNMPs, hindering the direct incorporation of intact molecules.
- These findings provide a foundation for developing methods to specifically label DNA in intact, living eukaryotic cells using [32P]-dNMPs.