Identification, purification and characterization of a putative novel growth-inhibitory and/or apoptotic protein from rat liver.
OBJECTIVE
The existence of endogenous growth inhibitors was postulated in 1914 by Boveri. However, most regretfully, progress in the isolation, characterization and mechanisms of actions of endogenous growth-inhibitory proteins is scanty compared to the information available on growth-stimulatory proteins. Accordingly, the major purpose of the present study was to isolate and characterize an endogenous growth-inhibitory protein from normal rat liver so that its role during liver carcinogenesis could be evaluated.


METHODS
For protein purification, a combination of alcohol precipitation, gel permeation chromatography and ion exchange chromatography techniques was utilized. For characterization and mechanisms, the methods utilized were DNA synthesis, immunoblotting, immunohistochemistry, protein sequencing, DNA-agarose electrophoresis and Hoechst staining.


RESULTS
The purified protein inhibited the growth of several cell lines in culture as measured by the rate of DNA synthesis using 3H-thymidine. In SDS-PAGE stained by the silver staining method, the molecular weight of the polypeptide was found to be 14 kD. Polyclonal antiserum was raised against this 14 kD polypeptide in rabbit. Immunoblotting experiments showed that the antibody recognizes specifically the 14 kD polypeptide and immunolocalization studies showed that the polypeptide is predominantly a cytoplasmic protein. Addition of antibody and inhibitory polypeptide simultaneously to the cultures more or less abolished the inhibitory activity of the polypeptide. Sequencing of the N-terminal 17 amino acids of the growth-inhibitory polypeptide showed Val-Leu-Leu-Ala-Glu-Ala-Glu-Thr-Ala-Ile-Val-Asn-Gly-Leu-Asp-Lys-Ile. Comparing this sequence using a BLAST protein data base indicated that there was no significant homology between the sequence of the growth-inhibitory polypeptide and protein sequences deposited with the data bank, suggesting that this could be a novel growth-inhibitory polypeptide. The mechanisms of growth inhibition appeared to be apoptosis as determined by electrophoretic analysis of DNA fragmentation and staining of the cells with the dye Hoechst 33342.


CONCLUSIONS
A growth-inhibitory protein of 14 kD can be isolated from normal rat liver. The physiologic role of the protein in liver appears to be either growth regulatory or apoptotic.
Dadke
S S
SS
Cellular Carcinogenesis Laboratory, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay, India.
Rao
K V
KV
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002845:Chromatography; D006224:Cricetinae; D053938:DNA Fragmentation; D004591:Electrophoresis, Polyacrylamide Gel; D006131:Growth Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D008099:Liver / Q000737:chemistry*; D008114:Liver Neoplasms, Experimental / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D008647:Mesocricetus; D051381:Rats
D006131:Growth Inhibitors; D008565:Membrane Proteins; C031717:growth inhibitory proteins