HEPATOPROLIFERIN A GROWTH FACTOR FROM HEPATOCYTES REVEALED THE STATUS OF A COMPLETE HEPATOMITOGEN

Abstract

P667 Quiescent hepatocytes in liver regeneration need to be primed by factors such as TNF-α and IL-6. To reach competence, by entering into the cell cycle’s G1-phase, they also require the complete mitogens HGF, TGF-α and EGF. The latter together with the co-mitogen norephinephrin (NE) augment proliferation. For the hepatocytes to progress fully into DNA synthesis the replication of hepatocytes also require potentiating factors such as insulin. Termination of liver regeneration needs TGF-β and activin. Liver regeneration is controlled by two kinds of factors, viz. priming or initiating- (IFs) and progressing or potentiating factors (PFs). The IFs are thought to be paracrinal and organ specific, while the PFs are mainly endocrinal and originate from the blood or other organs. The PFs lack organ specificity. Rat hepatoproliferin (HPF) was purified to SDS-PAGE homogeneity into two active species of 14 and 18.5 kDa. In an AA sequencing attempt the two species were found to be molecular diverse containing only a few amino acids. Through several manipulations it became clear that HPF aggregate to larger molecular sizes (big-HPF). By using de-aggregating conditions HPF was purified to homogeneity as small-HPF with a mol size of 1.03 kDa. With solid-phase ion-exchange, we established that small-HPF was an ionic molecular complex; containing three catecholamines and one disulphonated disaccharide. The two ionic-species had exceptionally high affinity for each other and a large tendency to aggregate to 14, 30 and 62 kDa. The structure of the amine was elucidated by HPLC and EC detection; while that of the disaccharide by (i) DEAE-HPLC with ABEE derivatization and (ii) RP-HPLC with OPA derivatization. Radioactive tracing aided in elucidating some of the sub-structures. Separate the ionic-species had no bio-activity, but after recombination activity was restored. Aim: The aim of this study was to assess the mitogenic status of HPF in human cells. Methods: HPF which stimulates rat hepatocyte on its own was assessed for its ability to stimulate the human hepatoma cell line (PLC/PRF-5) towards replication. The ability of HPF, whether acting on its own as a complete mitogen, was determined by investigating its sole ability to stimulate the incorporation of 3H-TdR into DNA of PLC/PRF-5. The co-mitogenic potential of HPF was also determined by assessing its ability to augment DNA synthesis through combining it with TNF-α, TGF-α or EGF respectively. Results: The hepatoma cell line’s response to the latter growth factors (GFs) when used alone showed the same kind of response for all, namely a bi-phasic bell-shaped dose-dependent response i.e. stimulation at low levels and inhibition at higher levels. However, HPF showed only a linear dose-response at the levels used for GFs (i.e. 1.0 - 15 ng/5 x 105 cells). The hepatoma cells unexpectedly did not respond to HGF at any level tested (0.5 – 6 ng/5 x 105 cells), although it is 10 x more potent than the other complete mitogens. Also the inhibition of cell proliferation by TGF-β started only at 15 ng/5 x 105 cells. Conclusion: Since HPF was able to stimulate DNA synthesis on its own, we conclude that HPF was a complete mitogen for human hepatoma cells. HPF did not act as a co-mitogen since it did not cause an augmentation when combined with the said GFs but showed instead an additive effect on DNA synthesis when used with some of these complete mitogens.