Analysis of novel apolipoprotein B mutations using a modified U937 cell line LDL binding assay.
The U937 myelomonocyte proliferation assay can be used to detect patients with familial defective apolipoprotein B-100 (FDB). Previous studies have employed electronic cell counting to assess cell proliferation. We simplified the assay using 3H-thymidine incorporation DNA analysis to measure cell growth. We tested the modified method by analyzing the effects of different concentrations of native low density lipoproteins (LDL), methylated LDL, as well as LDLs obtained from patients with FDB on cell growth. Methylation of LDL to various degrees reduced cell proliferation correspondingly, and LDLs obtained from FDB patients decreased cell growth confirming that the modified method was able to detect binding defective species of LDL. We applied this method to analyze three novel apoB polymorphisms recently characterized in this laboratory (apoB His1896-->Arg, apoB Asn1887-->Ser, apoB Ala4454-->Thr), which did not significantly alter U937 cell proliferation. Our results show that this simplified assay can be used for screening for LDL variants with defective binding.
Meng
Q H
QH
Department of Medicine, University of Helsinki, Finland.
Pajukanta
P
P
Ilmonen
M
M
Schuster
H
H
Schewe
C K
CK
Andersson
L C
LC
Tikkanen
M J
MJ
D000483:Alleles; D001055:Apolipoproteins B / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology*; D001671:Biochemistry / Q000379:methods*; D005260:Female; D006801:Humans; D008077:Lipoproteins, LDL / Q000187:drug effects / Q000378:metabolism*; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism*; D008297:Male; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D008077:Lipoproteins, LDL