Study of Ex Vivo Growth Characteristics of N-Nitrosomorpholine Treated Rat Hepatocytes / 대한암학회지

PURPOSE: The early carcinogenic effect of N-nitrosomorpholine (NNM) on acquisition of increased survival and growth was investigated using ex vivo culture of 5th to 10th week rat liver hepatocytes after NNM treatment. MATERIALS AND METHODS: Rats were fed with NNM (200 mg/l). Hepatocytes were isolated by two step perfusion techniques and grown on tissue culture. These ex vivo hepatocytes were then subjected to analysis of growth related signal molecules resided in nucleoli. RESULTS: One of the most characteristic differences of the NNM-treated liver from normal liver was genesis of megahepatocytes. These megahepatocytes survived approximately 2~3 times as long as normal hepatocytes in ex vivo conditions. There was also a significant increase in various nucleolar proteins, including Erk1/2, p38, hsp72 and nucleophosmin (B23). CONCLUSION: At promotion stage of tumorigenesis induced by NNM, it was possible to isolate and characterize abnormal hepatocytes. These abnormal hepatocytes showed increased survival in in vitro (ex vivo) than normal hepatocytes, although they were not immortal.

N-nitrosomorpholine Directed Preneoplastic Reprogramming of Nuclear Metabolism of Rat Hepatocytes / 대한암학회지

PURPOSE: An attempt was made to investigate N-nitrosomorpholine (NNM) induced carcinogenic processes in rat liver. MATERIALS AND METHODS: Rats were fed with NNM (200 mg/l) for 7 weeks, after then stopped. Length of telomere and activity of telomerase were analyzed. Hepatocytes were isolated and grown on tissue culture. Heat shock was treated at 43oC, and patterns of cell death ere evaluted by fluorescent study. Nuclei and nucleoli were isolated for analysis of various signal molecules. RESULTS: Shortening of telomere length presented in NNM treated liver, but induction of telomerase was not found. Ex vivo hepatocytes from 10~12th week showed increased heat shock resistance at 43oC. NNM-treated hepatocytes exhibited heat shock induced cell death (necrosis) after 7 hours, whereas the control showed necrosis after 3 hours. The signal molecules related to nucleolar growth revealed increased expression which included B23, C23, p38, Erk1/2 and p120. Partial degradation of B23 and Erk2 was noted in necrosis of NNM treated hepatocytes induced by heat shock. CONCLUSION: The hepatocytes at the stage of 10~12th week in the stop experiment of NNM are situated in the tumour promotion. Those cells showed various metabolic alterations. We found that the increased growth related signals were accompanied with increased heat shock resistance, telomere shortening but no induction of telomerase.

Nucleolus contains signal molecules that constitute membrane-nucleolus linked pathway

Since there have been very few studies on nucleolar signaling, an attempt was made to establish nucleolar signal pathways which link the cell membrane to the nucleolus for the transfer of extracellular signals. Two pathways were studied. One was the G alpha s mediated cAMP pathway where two signal molecules were yielded, including RII and protein kinase A. The other was the G alpha q mediated DAG/IP3 pathway which yields two signals including protein kinase C and IP3/Ca2+. By the studying isolated nucleoli from resting liver, regenerating liver or weak carcinogen thioacetamide treated liver, it was possible to detect protein kinase A (PKA), protein kinase C (PKC) and RII subunits. In addition, CK2 was detected. It was found that external signals transmitted through G protein coupled receptors could reach into the nucleolus and that physical translocation of signal molecules was an integral step involved in membrane-nucleolus linked pathways. When an in vitro assay of the above signal molecules was carried out using [gamma-32P]-ATP, most kinase dependent phosphorylation was via the major CK2 (more than 95%). Therefore, it is suggested that the major CK2 dependent pathway is involved in 'house keeping' for nucleolar integrity and the minor pathways, dependent on PKA, PKC and others, are involved in subtle regulatory mechanisms such as 'extra-house-keeping' activities by nucleolar chromosomal remodeling.