Related Experiment Videos
Cross-linkage of nucleophosmin in tumor cells by nitrogen mustard
1Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
Mouse leukemia (P388) cells were incubated in cell culture medium containing nitrogen mustard [2-chloro-N-(2-chloroethyl)-N-methylethanamine] for 4 h. The nucleophosmin immunoband with a molecular weight of 37,000 (p37; other molecular weights are similarly designated) was observed in both control and nitrogen mustard-treated cells. Three additional immunobands with molecular weights of 80,000 (p80), 120,000 (p120), and 230,000 (p230) were identified in the drug-treated cells. The same results were observed with melphalan, but were not detected when mitomycin C, cis-platinum, Adriamycin, or actinomycin D were used. Treatments with DNase and RNase did not alter the molecular weights of these immunobands. These results indicate that the cross-linked products of nucleophosmin were not linked to DNA or RNA. The pI of p80, p120, and p230 is 5.1, which is the same as that of nucleophosmin (p37). The iodinated tryptic peptide map of p80 is identical to that of nucleophosmin. This result indicates that p80 is a dimer cross-linked by nitrogen mustard. The p80 and p120 immunobands were observed in Novikoff hepatoma and in hypertrophic rat liver, but were not detected in normal liver under the same conditions. These results indicate that tumor or proliferating cells have hexameric nucleophosmins which can be cross-linked by nitrogen mustards.
Insights
Nitrogen mustards cross-link nucleophosmin in mouse leukemia cells, forming higher molecular weight forms. These cross-linked nucleophosmins, indicative of proliferating cells, were not observed with other chemotherapeutics.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Nucleophosmin (NPM) is a multifunctional nucleolar phosphoprotein.
- Altered NPM expression and localization are observed in various cancers.
- Nitrogen mustards are alkylating agents used in chemotherapy.
Purpose of the Study:
- To investigate the effect of nitrogen mustards on nucleophosmin.
- To identify potential cross-linked products of nucleophosmin induced by nitrogen mustards.
- To determine if these cross-linked products are specific to certain cell types or conditions.
Main Methods:
- Incubation of mouse leukemia (P388) cells with nitrogen mustard.
- Analysis of nucleophosmin immunobands using Western blotting.
- Characterization of immunobands through molecular weight, isoelectric point (pI), and peptide mapping.
- Comparison with other chemotherapeutic agents and enzymatic treatments (DNase, RNase).
Main Results:
- Nitrogen mustard treatment induced novel nucleophosmin immunobands (p80, p120, p230) in P388 cells.
- These higher molecular weight forms were not observed with other tested drugs (mitomycin C, cis-platinum, Adriamycin, actinomycin D).
- Enzymatic treatments confirmed that cross-linked nucleophosmin products were not linked to DNA or RNA.
- p80 was identified as a dimer of nucleophosmin.
- Higher molecular weight nucleophosmin forms (p80, p120) were detected in Novikoff hepatoma and hypertrophic rat liver, but not in normal liver.
Conclusions:
- Nitrogen mustards, specifically melphalan, induce cross-linking of nucleophosmin.
- The formation of cross-linked nucleophosmin is associated with tumor or proliferating cells.
- These findings suggest that cross-linked nucleophosmin could serve as a biomarker for cellular proliferation or tumor status.