**6. The role of cholesterol**

Albi and Viola-Magni (2002) have demonstrated the presence of CHO in hepatocyte chromatin. Previous researchers have attributed many functions to CHO metabolism (Luskey 1988) and an increase in its concentration has been demonstrated in both cancer and proliferating cells (Rao 1986). In liver regeneration, the amount of chromatin CHO changes during the first 24 h. Two fractions were demonstrated in the chromatin fraction, one of which is a free fraction and the other that is only extractable after SMase or proteinase K digestion (Albi and Viola-Magni, 2002).

After partial hepatectomy, the bound CHO increased reaching a peak after six hours whereas the free CHO reached a peak only after 18 h. This may be explained as being due to the increased SMase activity and to the block of SM synthesis which favours the transformation of the bound CHO fraction to the free fraction.

At 24 h, SM synthase activity increased and the ratio between bound and free CHO returned to the normal value seen in the non-dividing hepatocytes.

It was demonstrated that inhibition of the melavonate-CHO pathway with nitrogen bisphosphate arrested the cells in S-phase with a reduction in the expression of cdk2 and cdk4, whereas the expression of cdk21 increased (Reszka et al. 2001). The role of CHO in liver regeneration may be linked to the stimulation of the activities of cyclin-dependent kinases. In order to clarify this point the analysis of cyclin behaviour, especially that of A and B1, during liver regeneration may be of interest.

### **7. The cell cycle**

#### **7.1 The behaviour of cyclin A and B1**

Cyclin A is present in normal liver and in sham operated rats. During liver regeneration, the behaviour of cyclin A and B1 expression was analysed by Splewak &.Thorgeirsson (1997). They showed an increased amount of both cyclin A and B1 between 12 h and 22 h post hepatectomy when the hepatocytes entered the G1-S phase transition as shown by the ten-

This enzyme system may also be involved with gene expression by controlling the transfer of RNA to the cytoplasm. After enzymatic digestion with DNase and RNase, it was possible to isolate a complex containing a small amount of DNA, RNA, proteins and PLs. The RNA is RNase insensitive and behaves as double-stranded RNA. There are only two PLs invloved,

The enzymes SMase, SM synthase and reverse SM-synthase are present. If the complex is treated with SMase, the undigested RNA becomes RNase sensitive. Therefore, the presence of SM appears to protect the RNA from digestion. SMase aids the digestion by causing a decrease of SM that returns to the normal value through the activation of SM-synthase that exploits the PC derived from phosphorylcholine. The amount of PC may be restored

The products of PL metabolism may act also as internal signals by activating other nuclear proteins such as PKC or favouring the synthesis of polymerases through the presence of PS

Albi and Viola-Magni (2002) have demonstrated the presence of CHO in hepatocyte chromatin. Previous researchers have attributed many functions to CHO metabolism (Luskey 1988) and an increase in its concentration has been demonstrated in both cancer and proliferating cells (Rao 1986). In liver regeneration, the amount of chromatin CHO changes during the first 24 h. Two fractions were demonstrated in the chromatin fraction, one of which is a free fraction and the other that is only extractable after SMase or proteinase K

After partial hepatectomy, the bound CHO increased reaching a peak after six hours whereas the free CHO reached a peak only after 18 h. This may be explained as being due to the increased SMase activity and to the block of SM synthesis which favours the

At 24 h, SM synthase activity increased and the ratio between bound and free CHO returned

It was demonstrated that inhibition of the melavonate-CHO pathway with nitrogen bisphosphate arrested the cells in S-phase with a reduction in the expression of cdk2 and cdk4, whereas the expression of cdk21 increased (Reszka et al. 2001). The role of CHO in liver regeneration may be linked to the stimulation of the activities of cyclin-dependent kinases. In order to clarify this point the analysis of cyclin behaviour, especially that of A

Cyclin A is present in normal liver and in sham operated rats. During liver regeneration, the behaviour of cyclin A and B1 expression was analysed by Splewak &.Thorgeirsson (1997). They showed an increased amount of both cyclin A and B1 between 12 h and 22 h post hepatectomy when the hepatocytes entered the G1-S phase transition as shown by the ten-

through the activation of the enzyme reverse SM-synthase (Micheli et al. 1998).

namely, SM and PC (Albi et al. 1996; Micheli et al.1998).

(Albi et al. 1991).

**7. The cell cycle** 

**6. The role of cholesterol** 

digestion (Albi and Viola-Magni, 2002).

transformation of the bound CHO fraction to the free fraction.

to the normal value seen in the non-dividing hepatocytes.

and B1, during liver regeneration may be of interest.

**7.1 The behaviour of cyclin A and B1** 

fold rise in the cyclin mRNA level, its level remaining high between 24 and 48 h and returning to the normal value only after 72 h.

During S-phase, cyclin A is associated with p32cdk2 kinase, whereas during the transition G2-M it forms a complex with p34cdc2 (Pagano et al. 1992).

The mRNA levels of cyclin B1 and p34cdc2 behave in a similar manner remaining low for the S-phase period and increasing only after 20 h to reach a peak at 26 h. This is followed by a decline at 34 h with a new peak forming between 38 h and 44 h. The two peaks correspond to the G2-M phases of the first wave and second waves of the hepatocyte cell cycle.

The great majority of p34cdc2 is linked to cyclin B1 whereas only 25% is linked to cyclin A (Loyer et al. 1994). The presence of the phosphorylated form of cyclin A may represent an inactivation of this cyclin during the G2-M transition.

### **7.2 Cell cycle regulation by chromatin-associated phospholipids (Table 2)**

The increase in chromatin-bound CHO during the first six hours activates the kinases with PS favouring DNA polymerase alpha synthesis and PIP. At 12 h post-hepatectomy a transient increase of DAG is due to the hydrolysis of PI followed the activation of the chromatin enzyme PI-PLC.

The increase of DAG favours the translocation of PKC into the nucleus (Divecha et al. 1991). The synthesis of cyclin A increases as it complexes with p32cdk2 kinase. When the S-phase starts, a more consistent DAG peak is evident due to the hydrolysis of PC by the enzyme PC-PLC. At the same time, there is a decrease in SM due to the activation of SMase and consequently an increase in the free CHO present in the nucleus.


Table 2. Molecular events in relation to the time after hepatectomy

Possible Roles of Nuclear Lipids in Liver Regeneration 73

from external stimuli and may function as an internal balance. In fact, the increase in DAG due to PC hydrolysis stimulates SMase with the liberation of ceramide that may stimulate reverse sphingomyelin synthase to form new PC so favouring the liberation of DAG. On the

This internal clock appears to control cell activity favouring proliferation, differentiation when the cells remain in Go or apoptosis when the ceramide present is transformed into

It is clear from this that the role of phospholipids must be considered for a role in cell

Albi, E., Viola Magni, M.P., Lazzarini, R. & Gahan, P.B. (1991). *Chromatin phospholipid changes* 

Albi, E., Mersel, M., Leray, C., Tomassoni, M.L. & Viola Magni, M.P. (1994). *Rat liver* 

Albi, E., Micheli, M. & Viola Magni, M.P. (1996). *Phospholipids and nuclear RNA*. Cell Biol.

Albi, E. &Viola Magni, M.P. (1997a). *Chromatin neutral spingomyelinase and its role in hepatic* 

Albi, E. & Viola Magni, M.P. (1997b). *Choline base exchange activity in rat hepatocyte nuclei and* 

Albi, E. & Viola Magni, M.P. (1999a). *Sphingomyelin-synthase in rat liver nuclear membrane and* 

Albi, E. & Viola Magni, M.P. (1999b). *Phosphatidylcholine-dependent phospholipase C in rat liver* 

Albi, E. & Viola Magni, M.P. (2002). *The presence and the role of chromatin cholesterol in rat liver* 

Albi, E., Pieroni, S., Viola Magni, M.P. & Sartori, C. (2003a). *Chromatin sphingomyelin changes* 

Albi, E., Rossi, G., Maraldi, N.M., Viola Magni, M.P., Cataldi, S., Solimando, L.& Zini, N.

*cycle progression during rat liver regeneration*. J. Cell. Physiol. 197, 181-188. Albi, E. & Viola-Magni M.P. (2003c). *The metabolism of nuclear phospholipids in cell function and* 

Albi, E. & Viola-Magni, M.P. (2004). *The role of intranuclear lipids.* Biology of the Cell 96, 657-

Albi E., Cataldi S. & Rossi G. (2008). *The nuclear ceramide/diacylglicerol balance depends on the* 

Albrecht, J.H., Poon R.Y., Ahonen C.L., Rieland B.M., Deng C. & Crary C.S. (1998).

*in cell proliferation and/or apoptosis induced by ciprofibrate.* J. Cell. Physiol. 196, 354-361

(2003b). *Involvement of nuclear phosphatidylinositoldependent phospholipases C in cell* 

*physiological state of thyroid cells and changes during UV-C radiation induced apoptosis*.

*Involvement of p21 and p27 in the regulation of CDK activity and cell cycle progression in* 

other hand, the increase in SM reduces the ceramide present.

duplication regulation favouring the regeneration process in liver.

*chromatin phospholipids*. Lipids 29, 715-719.

*nuclear membrane*. Cell Biol. Intern. 21, 217-221.

*chromatin*. FEBS Lett. 460, 369-372.

*regeneration*. J. Hepatol. 36, 395-400.

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*the regenerating liver.* Oncogene 16, 2141-2150

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*during rat liver development.* Cell Biochem. Funct. 9, 119-123.

*regeneration.* Biochim. Biophys. Res. Commun. 236, 29-33.

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*regulation*. Recent Res.Devel.Biophys.Biochem., 3, 45-63

Albi, E. (2011) *Role of intranuclear lipids in health and disease* Clin. Lipidol. 6,59-69

sphingosine.

**9. References** 

667

However, the complexing of cyclin A with p32cdk2 kinase increased up to 22h i.e. the end of S-phase. After 18h, SM synthase activity increased with the increase in SM reducing the CHO free fraction and hence the activity of cyclinA.

Cyclin B complexing with p34cdc2 increased its activity so favouring the transition of G1-M phase. The phosphoinositides also decrease during S-phase leading to an inhibition of DNA synthesis through an increase in the activity of dephosphorylating enzymes (York and Majerus 1994). The small fraction of cyclin A complexed with p32 cdc2 is inactivated by phosphorylation, the cells progressing to G2-M (Splewak & Thorgeirsson 1997). The complex B-p34cdc2 also decreases followed by a second peak between 38-44 h posthepatectomy corresponding to the second mitotic peak.
