The carcinostatic and proapoptotic potential of 4-hydroxynonenal in HeLa cells is associated with its conjugation to cellular proteins.
BACKGROUND
Previous studies have shown that the lipid peroxidation product 4-hydroxynonenal (HNE) acts as a cell growth modulator if used at low, physiological concentrations being strongly cytotoxic at higher concentrations for a number of cells. These effects of HNE also appeared to be mutually dependent on the effects of serum growth factors. The aim of this investigation was to study the concentration-dependent response of human cervical carcinoma (HeLa) cells in vitro with respect to the intracellular uptake of exogenous HNE, the cellular energy metabolism, DNA synthesis, overall gene expression and susceptibility to apoptosis.


METHODS
MTT assay was applied as an index of energy metabolism and the replicative activity was quantitated by the 3H-thymidine incorporation assay. The occurence and intracellular distribution was studied with monoclonal antibodies directed against HNE-protein conjugates. Binding of HNE to serum proteins was determined with the same antibodies by Western blotting. Differential gene expression was studied by differential display RT-PCR while a novel photometric assay, denoted Titer-TACS, was used for in situ detection and quantitation of apoptosis in monolayer cell cultures.


RESULTS
A physiological concentration of HNE (1 microM) had hardly any effect on the parameters of the replicative activity and the energy metabolism. No morphological changes were observed and the number of HNE-positive cells was not significantly different when compared to the untreated control cells, while most of the aldehyde appeared to be bound to serum proteins (albumin fraction). A ten-fold higher concentration (10 microM) was found to be cytostatic. Spindle-shaped cells with a picnotic nucleus were observed occasionally, as well as membrane blebs, which were HNE-positive. The number of HNE-positive cells was significantly increased compared both to the control cells and cells treated with 1 microM HNE, but in the presence of serum the effects of 10 microM HNE were negated due to its binding to the serum proteins. Finally, 100 microM HNE was cytotoxic for the HeLa cells. Most of the cells were picnotic, together with a few spindle-shaped or oval cells. The staining for HNE was diffuse and strong (90% of the cells were HNE-positive) while even binding of the aldehyde to serum proteins did not prevent its cytotoxic effects. This concentration of HNE caused acute stress response of the cells resulting in the decreased expression of several as yet unidentified genes. The altered pattern of gene expression was followed by programmed cell death, i.e. an increased number of apoptotic cells after treatment with low (1 and 10 microM) concentrations of HNE. A rebound effect was observed, i.e. a decrease of apoptotic cells after 24 hours followed by an overshooting increase after 48 hours.


CONCLUSIONS
For HeLa carcinoma cells there appears to be a concentration range of HNE where it does not cause necrosis but preferentially apoptosis. At this concentration range HNE is cytochemically detectable within the cells as a protein conjugate. It is proposed that a possible differential sensitivity of cancer cells and their normal counterparts to the cytostatic activity of HNE should be explored.
Sovic
A
A
Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Austria.
Borovic
S
S
Loncaric
I
I
Kreuzer
T
T
Zarkovic
K
K
Vukovic
T
T
Wäg
G
G
Hrascan
R
R
Wintersteiger
R
R
Klinger
R
R
Zurak
N
N
Schaub
R J
RJ
Zarkovic
N
N
D000447:Aldehydes; D017209:Apoptosis; D015153:Blotting, Western; D002273:Carcinogens; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors; D004305:Dose-Response Relationship, Drug; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology
D000447:Aldehydes; D002273:Carcinogens; D015853:Cysteine Proteinase Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; C027576:4-hydroxy-2-nonenal