Abstract
The current studies of the growth altering effects of plasma lipoproteins on cultured cells, such as vascular endothelial and smooth muscle cells, have shown conflicting results in various experimentals models. No informations are available about the effects of plasma lipoproteins on the growth of cultured tumor cells. In the present study, the human tumor cells used were HeLa cells, which were cultured with human lipoproteins and the growth of tumor cells was investigated.
The human lipoproteins, low density lipoproteins (LDL: 1.019<d<1.050g/dl) or high density lipoproteins (HDL: 1.063<d<1.210g/dl), each was prepared by sequential ultracentrifugation and dialyzed exhausively against 0.154M NaCl at 4°C. Each of the lipoprotein fractions was concentrated by ultrafiltration and filtrated through a Millipore filter (pore size=0.22μm) for cell culture experiment.
HeLa cells were obtained from the department of virology in our institute, and they have been maintained by passage twice weekly in plastic flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum under 5% CO2/95% air at 37°C.
Approximately 1.5×105 of the cells were harvested and cultured in DMEM with various concentrations of HDL (80, 200μg protein/ml) or LDL (35μg protein/ml). The cells were dissociated by trypsinization and the number of cells was counted every day for 4 days. Morphology of the cells was examined under a light microscope.
Phase contrast photographs of the cells cultured with HDL on day 3 showed almost all the cells were flattened in shape and formed a monolayer sheet partly. In the medium without lipoproteins, the cells were round in shape and small in size and the plating efficiency was very low. An addition of HDL resulted in significant proliferation in the number of cells per dish for 4 days. The number of cells on day 4 was inversely proportional to concentration of triglyceride (r=0.698) and of cholesterol (ester) (r=0.538) in the medium. While neither concentration of phospholipid nor of cholesterol (free) was correlated with cell proliferation. As the concentration of HDL increased in the medium, the incorporation of 3H-Tdr into DNA increased after the cells were cultured with HDL for 3 days. An increase of HDL concentration above 1, 500μg protein/ml, however, showed no change in the level of DNA synthesis compared with control value.
These results suggested that LDL and HDL, especially HDL, has growth promoting effect on HeLa cells at a low concentration in vitro.
