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Nuclear chromatin condensation of mouse lymphoma (L-1210) cells by methylnitrosourea. An electron microscopic and
Abstract:
When the mouse lymphoma (L-1210) cells are treated with methylnitrosourea (MNU) at 37 degrees C for 30 min and then cultured for 4 h in a normal medium nuclear structure and functions of the cells are changed. We have investigated the mechanism as to how nuclear structure and functions are changed by MNU. In MNU-treated cells euchromatin area diminishes and chromatin condensation occurs. [3H]thymidine and [3H]uridine uptakes of the MNU-treated cells decrease. In contrast, when the MNU-treated cells are cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) synthetase changes of nuclear structure do not appear. [3H]Thymidine and [3H]uridine uptakes are partially and almost completely recovered, respectively. Autoradiographs of the cells labelled with [3H]NAD, a substrate of poly(ADP-ribose) synthetase show that silver grains due to [3H]ADP-ribose are densely located only in the cells where chromatin condensation occurs. Chromatin-bound proteins of molecular masses 20-25 X 10(3) daltons are specifically poly(ADP-ribosyl)ated in the MNU-treated cells. These results suggest that MNU-induced chromatin condensation is caused by poly(ADP-ribosyl)ation of chromatinbound proteins.
Insights
Methylnitrosourea (MNU) causes nuclear changes in L-1210 cells by inducing chromatin condensation. This process is linked to poly(ADP-ribosyl)ation of chromatin-bound proteins, which can be inhibited by 3-aminobenzamide.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Methylnitrosourea (MNU) is a chemical mutagen known to induce DNA damage.
- Nuclear structure and function are critical for cellular processes.
- L-1210 mouse lymphoma cells are a common model for studying cytotoxic and genotoxic effects.
Purpose of the Study:
- To investigate the mechanism by which MNU alters nuclear structure and function in L-1210 cells.
- To determine the role of poly(ADP-ribose) synthetase in MNU-induced nuclear changes.
- To identify specific proteins involved in the MNU-induced cellular response.
Main Methods:
- Treatment of L-1210 cells with MNU and subsequent culture.
- Assessment of nuclear structure and chromatin condensation using microscopy.
- Measurement of [3H]thymidine and [3H]uridine uptake to evaluate DNA and RNA synthesis.
- Inhibition studies using 3-aminobenzamide, a poly(ADP-ribose) synthetase inhibitor.
- Autoradiography with [3H]NAD to visualize poly(ADP-ribose) synthesis.
- Analysis of chromatin-bound proteins using gel electrophoresis.
Main Results:
- MNU treatment led to diminished euchromatin and chromatin condensation in L-1210 cells.
- [3H]thymidine and [3H]uridine uptakes decreased significantly in MNU-treated cells.
- 3-aminobenzamide treatment prevented nuclear structure changes and partially/fully restored nucleic acid uptakes.
- Poly(ADP-ribose) synthesis, visualized by [3H]ADP-ribose, was localized to condensed chromatin.
- Specific chromatin-bound proteins (20-25 x 10(3) daltons) were poly(ADP-ribosyl)ated following MNU treatment.
Conclusions:
- MNU-induced chromatin condensation in L-1210 cells is mediated by the poly(ADP-ribosyl)ation of specific chromatin-bound proteins.
- Poly(ADP-ribose) synthetase plays a crucial role in the cellular response to MNU-induced damage.
- Inhibition of poly(ADP-ribose) synthetase can mitigate MNU-induced nuclear alterations.