Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

Tea polyphenols can restrict benzo[a]pyrene-induced lung carcinogenesis by altered expression of p53-associated genes and H-ras, c-myc and cyclin D1

The modulatory influence of tea polyphenols (epigallocatechin gallate, epicatechin gallate and theaflavin) on benzo[a]pyrene (B[a]P)-induced lung carcinogenesis in mice was analyzed using histopathological and molecular parameters. Progression of lung lesions was restricted at the hyperplastic stage by tea polyphenols. A significant reduction in cellular proliferative index and an increase in apoptotic index were noted in the restricted lung lesions. High expression of H-ras, c-myc, cyclin D1 and p53 genes was seen at the inflammatory stage (9th week) and in subsequent premalignant lesions, but down-regulation of H-ras at the hyperplastic stage (17th week). Expression of bcl-2 was high in hyperplastic lesions, whereas the expression of mdm2 and bcl-xl increased only at the moderately dysplastic stage (36th week). The tea polyphenols inhibited inflammatory response in the lung lesions on the 9th week, when decreased expression of H-ras and c-myc and increased expression of bax were noted. Prolonged treatment (>9th week) with tea polyphenols resulted in changes in the expression of some additional genes, such as reduced expression of cyclin D1 (from the 17th week), bcl-2 (from the 26th week; mild dysplasia) and p21 (on the 36th week), and high expression of p53 (from the 17th week) and p27 (on the 36th week). These observations indicate that the tea polyphenols can restrict B[a]P-induced lung carcinogenesis by differential modulation of the expression of p53 and its associated genes such as bax, bcl-2, mdm2, p21 and p27, along with H-ras, c-myc and cyclin D1, at different time points.     

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

Immunohistochemical study of alpha-fetoprotein in the postnatal ontogeny of mice with a varying sensitivity to liver carcinogens]

The ontogenesis of alpha-fetoprotein (AFP) was studied in the C57BL, CC57BR, C3H/He, A/He and DD mice during the first 4 weeks of life. The AFP hepatocytes were determined by indirect immunofluorescent staining of liver cells isolated with sodium tetraphenyl borate; the content of AFP in blood was determined by the method of rado immunodiffusion. The methods utilized allowed to obtain the quantitative characteristics of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood. In the newborn mice over 90% hepatocytes contain AFP, the intensity of luminescence being heterogenous. The number of bright-liminescent cells equals to 50% during the first day of life rapidly decreases and none of them are found by 9--11 days. The number of average- and weak-luminescent hepatocytes does not decrease during the first 10 days, but then gradually decreases and none of them are found by 23 days in the CC57BR and by 27 days in the DD and A/He mice. A comparison of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood has shown that the bright-luminescent hepatocytes are the main producent of this protein in the early postnatal ontogenesis.     

The Cdk inhibitor p21 is required for necrosis,but it inhibits apoptosis following toxin-induced liver injury

Liver injury and repair were examined in wild type, p21Waf1/Cip1, and p27Kip1-deficient mice following carbon tetrachloride (CCl4) administration. In wild type liver, p21 expression is induced in a biphasic manner following injection of CCl4, with an early peak of p21 expression occurring in pericentral hepatocytes at 6 h, prior to evidence of injury, and a second peak succeeding regenerative proliferation. In contrast, p27 is present throughout the quiescent liver, but its expression decreases following CCl4 injection. Surprisingly, p21-deficient animals were resistant to CCl4-induced necrotic injury, indicating that rapid induction of p21 in pericentral hepatocytes following CCl4 injection contributes to subsequent necrosis. Expression of cytochrome P450 2E1, which plays an essential role in CCl4-induced necrotic injury, was not affected in p21-deficient mice. Although they had the least injury, p21-deficient mice had the highest levels of hepatic proliferation that correlated with increases in hyperphosphorylated retinoblastoma protein and Cyclin A gene expression. Increased replication in p21-deficient livers was counteracted by an increase in hepatocyte apoptosis as detected by caspase-3 activation. p21 plays distinct and opposing roles regulating hepatocyte survival during injury and subsequent repair, with early induction of p21 contributing to necrotic injury and later expression to cessation of proliferation and hepatocyte survival.     

Different responses of DD/He and CC57BR/Mv mice to fasting

Reaction to fasting of 2 mice strains differing in their sensitivity to spontaneous and induced hepatocarcinogenesis, has been investigated. It was shown that mice of both strains displayed similar stress reaction after 3-day fasting manifested in increase in blood corticosterone level; and decrease in testosterone level. At the same time, both strains demonstrated opposite changes at tissue- and enzyme levels in the liver. It was shown that DD/He mice, highly sensitive to induction of liver tumors, were characterized by significant increase in tyrosine aminotransferase (TAT) activity and reduction of lipid droplets in hepatocytes. CC57BR/Mv mice, demonstrating high frequency of spontaneous hepatomas and insensitive to induction of such tumors, were characterized by a decrease in the TAT activity and fatty infiltration of the liver.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Immunoperoxidase labelling of alpha1-fetoprotein (AFP) in normal and regenerating livers of a low and a high AFP producing mouse strain

Summary Serum AFP concentrations in normal adult BALB/c/J and in normal adult C3H/He mice were in the order of 0.6 g/ml and 0.1 g/ml, respectively. In BALB mice, AFP was localized in the cytoplasm of differentiated mono- and binucleated hepatocytes in centrolobular and intermediate zones of normal adult liver. No cellular AFP could be detected in liver sections of normal adult C3H mice.CCl₄ intoxication was accompanied by increase of serum AFP levels. A maximum was reached on day 4. Afterwards, concentrations declined. In sera of BALB/c/J mice, AFP levels reached values 10-fold higher and more than in sera of C3H/He mice.From day one after CCl₄ intoxication, cellular AFP was detected in hepatocytes of portal and periportal areas including intermediate zones adjacent to the necrosis. The intensity of AFP staining reached a maximum between the days 3 and 4. Hepatocytes in front of the necrotic areas usually contained the strongest AFP reactions. In both mouse strains, cellular AFP pattern was comparable, but strongest immunoreactivity was observed in liver sections of BALB/c/J mice.Liver injury and subsequent regeneration occurred to the same extent in both studied strains. The much higher serum AFP levels and the stronger AFP immunolocalizations in BALB mice were thought not due to increased numbers of AFP producing and releasing cells during liver regeneration. Additional mechanisms must play a role in increased AFP synthesis per single cell. C3H/He was a low AFP-inducible and BALB/c/J was a high AFP-inducible mouse strain.Supported by the Deutsche Forschungsgemeinschaft (Ku 257/3) Bonn. Federal Republic of Germany     

Failure of autoresuscitation in weanling mice: significance of cardiac glycogen and heart rate regulation.

"Autoresuscitation" (AR) is the spontaneous recovery from hypoxic apnea by gasping. We examined aspects of heart function in two situations: 1) the maturationally acquired failure of AR that is characteristic of SWR, but not BALB/c, weanling mice and 2) AR failure in BALB/c mice induced by repeated exposures to anoxia. We determined maturational changes in heart and liver glycogen. Unlike liver glycogen levels, heart glycogen levels in SWR mice differed from those in BALB/c mice. They were consistently much lower throughout maturation and reached a nadir during the brief period when SWR weanling mice are vulnerable to AR failure. Also, rate of cardiac glycogen utilization in vulnerable SWR mice was lower than that of same-aged BALB/c mice and was nil during the latter one-half of the gasping stage when heart function is critical for AR success. Therefore, because glycogen utilization reflects cardiac work, heart failure could explain AR failure in SWR weanlings. Additionally, the increase in hypoxic heart rate that occurs with maturation is developmentally delayed in SWR mice, and this may contribute to their AR failure. Cardiac glycogen was not fully depleted in BALB/c mice during repeated anoxic exposures, indicating other reasons for AR failure. We view these findings as a potential model for the age-related peak in incidence of sudden infant death syndrome.     

Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.     

Evidence that cyclin D1 mediates both growth and proliferation downstream of TOR in hepatocytes

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regeneration in vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.