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Mutant KB cells with decreased EGF receptor expression: biochemical characterization
1Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF-PE) express a pleiotropic phenotype, which includes reduced levels of 125I-EGF binding, without altered affinity for EGF (Lyall et al., 1987). Here, the EGF-toxin (ET) resistant mutants were further characterized with respect to the amount and size of the EGF receptor and the level of EGF receptor RNA. These data indicate that decreased binding of 125I-EGF in the mutants is due to reduced amounts of EGF receptor, which is associated with decreased mRNA levels. Changes in other proteins in the ET mutants were also examined. Five of the six ET mutants had a decrease in a 78,000 Mr- membrane glycoprotein. In addition, an increase in a protein with a Mr- of 40,000 and a pl = 8.0 was found in all the mutants, and an increase in a series of proteins with a Mr- of 36,000 and a pl of 6.3-6.5 was found in some of the mutants. These results confirm the pleiotropic nature of the EGF-PE resistant mutants and show that reduced EGF binding is due to altered expression of the EGF receptor gene in the mutants.
Insights
Mutant cells resistant to epidermal growth factor-Pseudomonas exotoxin conjugates show reduced epidermal growth factor receptor (EGFR) expression. This decrease in EGFR is linked to lower mRNA levels, confirming altered gene expression in resistant cell lines.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Human KB carcinoma cell lines resistant to epidermal growth factor-Pseudomonas exotoxin (EGF-PE) conjugates exhibit a pleiotropic phenotype.
- These resistant cells show reduced binding of 125I-EGF without changes in EGF binding affinity.
Purpose of the Study:
- To further characterize EGF-toxin (ET) resistant mutants.
- To investigate the amount, size, and RNA levels of the EGF receptor in ET mutants.
- To examine alterations in other cellular proteins within these mutants.
Main Methods:
- Analysis of EGF receptor (EGFR) amount, size, and mRNA levels in ET resistant mutants.
- Examination of changes in other cellular proteins using techniques like Western blotting and gel electrophoresis (implied).
Main Results:
- Reduced 125I-EGF binding in mutants is attributed to decreased amounts of EGF receptor.
- Lower EGF receptor levels correlate with decreased EGF receptor mRNA.
- Five of six ET mutants showed reduced 78,000 Mr membrane glycoprotein.
- All mutants displayed an increased 40,000 Mr protein (pI=8.0), and some showed increased 36,000 Mr proteins (pI=6.3-6.5).
Conclusions:
- The pleiotropic nature of EGF-PE resistant mutants is confirmed.
- Reduced EGF binding is a consequence of altered EGF receptor gene expression in these mutants.