Cardnogenesis vol.13 no. 12 pp.2287-2291, 1992

Inhibition of DNA synthesis by phenobarbital in primary cultures of hepatocytes from normal rat liver and from hepatic nodules

S.Manjeshwar, P.M.Rao, S.Rajalakshmi and D.S.R.Sarma Department of Pathology, University of Toronto, Medical Sciences Building, Toronto, Ontario, Canada M5S 1A8

Introduction The phenobarbital (PB*) model, developed by Peraino and coworkers, was the first experimental model with an initiationpromotion protocol for liver carcinogenesis. When exposed for several weeks, PB promoted hepatocarcinogenesis in the rat initiated with a variety of liver carcinogens including diethylnitrosamine (1,2). Although several hypotheses exist, the mechanism by which PB promotes liver carcinogenesis is still unknown. The continuous administration of PB to rats has a wide range of effects on the rat liver. Short-term exposure results in hypertrophy, increase in the amount of smooth endoplasmic reticulum, increase in enzymes such as cytochrome P450 and 7-glutarnyltranspeptidase (3). Initially, it also induces transient hyperplasia in vivo (3) and in primary hepatocytes in vitro at concentrations < 3 mM (4,5). •Abbreviations: PB, phenobarbital; OA, orotic acid; EGF, epidermal growth factor, TGF-a, transforming growth factor-a; LDH, lactate dehydrogenase; acidic FGF, acidic fibroblast growth factor; BM, basal medium. © Oxford University Press

Materials and methods In vitro cell culture studies Hepatocytes were isolated from male Fischer 344 rats weighing 150-180 g by the coUagenase perfusion technique as described previously (13). Briefly, 2 x 103 viable hepatocytes were cultured on 35 mm dishes coated with collagen (Vrtrogen, 60 (ig/dish; Collagen Corp., Palo Alto, CA) in modified William's E medium containing fetal bovine serum (10% v/v), insulin (20 U/l), L-glutamine (2 mM), HEPES (10 mM), penicillin (100 U/ml) and streptomycin (100 ^g/ml). After an attachment period of 3 h at 37°C in air/carbon dioxide (95:5) the medium and non-attached cells were removed. At this time, medium was changed to serumfree modified William's E medium supplemented with L-proline (2 mM) and sodium pyruvate (10 mM). Appropriate dishes also contained epidermal growth factor (EGF; 20 ng/ml) or transforming growth factor-a (TGF-a; 40 ng/ml); PB ( 3 - 6 mM) and [3H]thymidine (5 jiCi/dish). At the time of harvest, the cells were washed in cold PBS and then processed for DNA synthesis either by determining labelling index or by determining acid precipitable radioactivity. For determining labelling index, cells, after washing in cold PBS, were fixed in 10% buffered formalin, processed for autoradiography and then counterstained with Giemsa (Fisher Scientific, Canada). The acid-precipitable radioactivity was measured as described previously (16). Briefly, the cells were harvested with 1.5 ml of 0.33 N NaOH and an aliquot was taken and the DNA precipitated with an ice-cold solution of 40% TCA and 1.2 N HC1. The precipitate was then

2287

Downloaded from http://carcin.oxfordjournals.org/ at University of Michigan on April 18, 2015

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-Initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80—90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from nonnodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocyes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.

When given to rats bearing foci of enzyme-altered hepatocytes, the foci responded with a higher labelling index than the nonnodular surrounding hepatocytes (6). These studies led to the hypothesis that PB may act as a mitogen and promote hepatocarcinogenesis by differential stimulation of initiated hepatocytes as compared to the surrounding non-initiated hepatocytes. In contrast, studies with chronic administration of PB as required in initiation—promotion experiments have shown PB significantly to inhibit hepatocyte proliferation in vivo in response to two-thirds partial hepatectomy (7; E.Laconi and E.Farber, unpublished observations). In addition, hepatocytes from rats chronically exposed to PB exhibited a lower labelling index in response to growth factors in vitro (8) and PB inhibited DNA synthesis in normal hepatocytes when added in vitro at concentrations > 3 mM (9,10). In light of these studies, it was hypothesized that PB, like the resistant hepatocyte model, may achieve tumor promotion by differential mitoinhibition of surrounding hepatocytes while permitting the initiated hepatocytes that were resistant to the mitoinhibitory effects of the promoter to respond to growth stimuli and form foci and nodules. Our interest in PB first started when we noted the structural simiUarities between PB and orotic acid (OA), another liver tumor promoter (11) and a mitoinhibitor to hepatocytes (12,13). Studies in our laboratory have shown that one of the target sites at which OA exerts its mitoinhibitory effects is beyond die growth factor receptor and receptor mediated early events (14). In addition, hepatic nodules appear to be relatively resistant to the mitoinhibitory effects of OA (15). Given the similarity in structure of the two promoters OA and PB, it became of interest to determine whether (i) PB, like OA, exerts its mitoinhbitory effect at a site beyond the growth factor receptor and the growth factor receptor mediated early events; and (ii) to determine whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated but not the initiated hepatocytes in vitro.

S.Manjeshwar et al. filtered onto 0.45 /un Millipore filters and washed with ice-cold 5% TCA. The air dried filters were dissolved in 1.0 ml Cellosolve (Fisher Scientific, Canada) and after adding 10 ml Universol (ICN Biochemicals, USA) the radioactivity was determined in a LKB liquid scintillation spectrometer. Experiments using primed hepatocytes In these experiments, after the 3 h attachment, the medium was changed to a serum-free medium and in appropriate dishes the growth factor was added to the medium. Twenty-four hours later, the medium was washed off, and fresh serum-free medium containing diiferent concentrations of PB and [3H]thymidine was added and the dishes were incubated for another 24 h. At the end of the experiment cells were processed for DNA synthesis by measuring the acidprecipitable radioactivity as described above.

000

BM

TGF-a (40ng/ml)

PB (3mM)

Results

Experiments with hepatocytes from normal rats The approaches taken to determine the site of mitoinhibition by PB in normal hepatocytes in vitro were twofold. The first was to use different growth factors, which use different receptors to stimulate DNA synthesis in the primary hepatocytes. Figure 1 shows that PB inhibited the DNA synthesis induced by TGF-a in a dose-dependent manner. PB also inhibited DNA synthesis induced by acidic fibroblast growth factor (FGF), though the extent of stimulation of DNA synthesis by acidic FGF varied from one experiment to the next (data are not presented). The second approach taken to determine the role of the growth factor receptor and receptor-mediated early events in the inhibition of DNA synthesis by PB, was first to prime the hepatocytes with the growth factor for 24 h, then wash it off and add PB at which time presumably the early signal transduction events have been completed. Figure 2 shows the effect of PB on TGF-a-induced DNA synthesis in primary hepatocytes when it was added 24 h after the initial priming with the growth factor. 2288

PB (5mM)

PB (6mM)

Fig. 1. Dose-dependent effect of PB on TGF-a-induced DNA synthesis in normal hepatocytes in vitro. Isolated hepatocytes were incubated with basal medium (BM), basal medium containing TGF-a (40 ng/ml) or basal medium containing TGF-a (40 ng/ml) together with different concentrations of PB. All dishes contained 5/iCi [3H]thymidine. After incubation at 37°C for 48 h the hepatocytes were harvested and processed for acid-precipitable radioactivity as described in Materials and methods. Values are expressed as the mean ± SD of three dishes. The experiment was repeated at least three times with a similar pattern of results. PB at all doses was significantly different (P < 0.05) from TGF-a alone (40 ng/ml).

Determination of lactate dehydrogenase (LDH) activity The LDH activity was determined colorimetrically using the LDH kit (Sigma Diagnostics, cat. no. 500). Hepatocytes were harvested at 24 and 48 h after the addition of EGF or EGF + PB (5 mM). The medium was collected separately and the hepatocytes were subsequently lysed with 1 % Triton X-100 and the LDH activity in both the medium and the lysate was measured colorimetrically as described by the supplier. The data obtained were expressed as a percentage of the total LDH present in the medium. Effect of PB on hepatocytes from hepatic nodules Hepatic nodules were generated by initiating male Fisher 344 rats, 110-120 g (fed a basal semisynthetic diet, no. 101; Dyets Inc., Bethlehem, PA) with 1,2 dimethylhydrazine—2HC1 (100 mg/kg) given i.p. 16 h after a two-thirds partial hepatectomy. Two weeks later, they were started on the basal diet containing 1 % OA (11). Eight to nine months later, when persistent nodules had developed, the rats were taken off the OA diet and maintained on the basal diet for 2—5 weeks, to remove the effects of OA before use in the experiment. Nodular and non-nodular hepatocytes were isolated by collagenase perfusion and cultured (2 x 105 cells/dish) in 35 mm dishes as outlined above. The percentage viability was - 8 0 - 9 0 % in hepatocytes from both nodular as well as surrounding nonnodular liver. Appropriate dishes contained the growth factor TGF-a and TGFa + PB. At the end of the experiment (48 h), the dishes were processed for autoradiography as described above. On an average 200 cells in 4—6 different fields were counted per dish and the labelling index was determined as a percentage of cells labelled.

PB (4mM)

X i

BM

TGF-a (40ng/ml)

(3mM)

PB (4mM)

PB

PB

(5mM)

(6mM)

Fig. 2. Inhibition of TGF-a-induced DNA synthesis by PB when added 24 h after priming the hepatocytes with the growth factor. Experimental details are identical to that described in the legend to Figure 1 except that the hepatocytes were incubated with TGF-a (40 ng/ml) for 24 h. The cells were then washed and incubated with fresh medium for another 24 h with different doses of PB and [3H]thymidine. The cells were harvested and processed as described in Materials and methods. Values are the mean ± SD of three dishes. The experiment was repeated two or three times with a similar pattern of results. PB at doses of 5 and 6 mM was significantly different (/> < 0.05) from TGF-a alone (40 ng/ml).

As can be seen from the figure, the pattern of inhibition is similar to that seen when PB was added at the beginning. Similar results were obtained with EGF (Figure 3) and acidic FGF (data not shown), suggesting that there may be other site(s) for the mitoinhibition by PB which was beyond the growth factor receptor and receptor-mediated early events. It was important at this point to determine whether the PBinduced mitoinhibition at concentrations > 3 mM was not a reflection of a lethal effect. To examine this aspect experiments were done wherein the hepatocytes were first incubated with EGF + PB (5 mM) for 24 h. The EGF and PB were washed off, the hepatocytes incubated with fresh medium and their recovery was monitored by measuring thymidine incorporation into DNA as a function of time as described in Materials and methods. The results presented in Figure 4 show that PB inhibited EGF-induced DNA synthesis at 24 h. However, after the PB and EGF were washed off, the hepatocytes were able to recover and incorporate thymidine with a time course similar to EGF-stimulated hepatocytes.

Downloaded from http://carcin.oxfordjournals.org/ at University of Michigan on April 18, 2015

Recovery of DNA synthesis in normal hepatocytes To monitor the recovery in DNA synthesis following withdrawal of PB, primary hepatocytes were cultured as described above. Dishes were incubated with either EGF or EGF + PB (5 mM) for 24 h. To determine the kinetics of DNA synthesis following withdrawal of PB, some dishes were harvested at 24 h; others were washed to remove the growth factor and PB. Dishes were then harvested at diiferent time points. [3H]Thymidine was added 1 h prior to harvest. In a second experiment the cumulative labelling index was measured at 0, 24 and 48 h after withdrawal of PB. For this purpose, after washing off the EGF and PB, some dishes were harvested (0 h), while others were incubated in fresh medium containing 5 lid [3H]thymidine/dish. Dishes were harvested at 24 and 48 h thereafter. The dishes were washed in cold PBS and then fixed overnight in 10% buffered formalin and then processed for autoradiography (13).

1 20-1

Inhibition of DNA synthesis by PB • ^

Basal Medium TGF-a (40ng/ml) TGF-a (40nfl/ml) + PhenobarblmKPB)

P8 PB PB 3mM 4mM SmM Surrounding Hepatocytes

Phenobarbital (mM)

Nodular Hepatocytej

Fig. 5. Effect of PB on TGF-a-induced DNA synthesis in hepatocytes isolated from nodular and non-nodular surrounding liver. Details for the generation of hepatocyte nodules and the isolation of hepatocytes from them and from the non-nodular surrounding liver and the measurement of labelling index are given in the text. In most experiments hepatocytes from a single nodule 1 cm or larger were used. The values are the mean ± SD of three dishes. The experiment was repeated two or three times with a similar pattern of results. 60 50

X

^ 40000-

30-

I |

20 -

10 -

30 42 48 Time In hours

54

66

Fig. 4. Recovery of DNA synthesis in hepatocytes upon withdrawal of PB. Hepatocytes were incubated with basal medium containing EGF (20 ng/ml) ( • — D ) or EGF (20 ng/ml) plus PB (5 mM) ( • — • ) . At 24 h, a few dishes were harvested while the others were washed and fresh basal medium was added. At different time periods thereafter, cells were harvested for acid precipitable radioactivity. [3H]Thymidine (5 /xCi/dish) was added 1 h before harvesting the cells. Values are the mean ± SD of three dishes. This experiment was repeated two or three times with a similar pattern of results.

In addition, the LDH activity was determined in the medium at 24 and 48 h after stimulating the hepatocytes with EGF in the presence or absence of PB (5 mM). The results indicate that there was no significant difference in the percent release of LDH activity in the different groups. The percentage release of LDH in the medium varied between 15 and 20% at 24 h and 20 and 27% at 48 h. These results indicate that at this dose, PB is merely mitoinhibitory to the hepatocytes and not lethal to any significant extent to the hepatocytes. Meyer et al. (17) have reported that PB down-regulated EGF receptors when added to hepatocytes 1 h prior to the addition of the growth factor EGF. Accordingly, in one experiment PB was added 1 h prior to the addition of EGF and then incubated for 24 h. The hepatocytes were subsequently washed free of PB and the growth factor and the kinetics of DNA synthesis was monitored as described in Materials and methods. The data (not shown) indicate that the hepatocytes are able to recover in a manner similar to that presented in Figure 4, suggesting that down-regulation of EGF receptors may not be a major mechanism by which PB inhibits DNA synthesis in hepatocytes. Experiments with hepatic nodules Implication of differential mitoinhibition as a mechanism for tumor promotion requires that the initiated hepatocytes be resistant

Surrounding Hepatocytes

Nodular Hepatocytes

Fig. 6. Recovery of DNA synthesis in hepatocytes from nodular and nonnodular surrounding liver upon withdrawal of PB. Hepatocytes isolated from nodular or surrounding liver were incubated with TGF-a (40 ng/ml) or TGF-a + PB (5 mM). Groups TGF-a (H) and TGF-a + PB ( • ) were incubated with 5 /iCi/dish [3H]thymidine for 0 - 2 4 h and then harvested. A second set TGF-a + PB (D), was incubated for 24 h, then washed and incubated for another 24 h with fresh medium containing 5 ^Ci/dish [3H]thymidine. A third set TGF-a + PB (E)', contained [3H]thymidine for the full 48 h period. All dishes were processed for autoradiography as described in the text. The labelling index of 4 - 6 different fields was determined as a percentage of cells labelled. Values are the mean of two dishes. The individual values for the different groups are as follows: Surrounding hepatocytes: TGF-a ( 3 ) 58.8, 60.4; TGF-a + PB ( • ) 3.8, 5.9; TGF-a + PB (Q) 45.5, 47.2; TGF-a + PB (H) 4.2, 5.3. For the nodule hepatocytes: TGF-a (B) 49.7, 51.6; TGF-a + PB ( • ) 16.4, 17.7; TGF-a + PB ( • ) 42.8, 44.0; TGF-a + PB (H) 4.8, 5.4. The experiment was repeated twice with a similar pattern of results.

to the mitoinhibitory effects of the promoter. Since there is no method to isolate initiated hepatocytes, the next series of experiments were performed using hepatocytes from nodules to determine whether they are resistant to the mitoinhibitory effects of PB. Results presented in Figure 5 indicate that PB inhibited DNA synthesis induced by TGF-a in surrounding as well as nodular hepatocytes even at a dose of 3 mM as compared to growth factor alone. This observation is rather surprising in that it suggests that perhaps nodular hepatocytes are not completely resistant to the mitoinhibitory effects of PB. When the same batch of hepatocytes were tested for the mitoinhibitory effects of OA it was observed that OA inhibited TGF-a-induced DNA synthesis in the surrounding hepatocytes while the nodular hepatocytes were almost completely resistant to the mitoinhibitory effects of OA (unpublished observations). 2289

Downloaded from http://carcin.oxfordjournals.org/ at University of Michigan on April 18, 2015

Fig. 3. Inhibition of EGF-induced DNA synthesis in primary hepatocytes in vitro by PB when added either together with ( D — • ) or 24 h after ( • — • ) the growth factor. Experimental details are described in the legend to Figure 2 except that at the end of the experiment the dishes were fixed in 10% buffered formalin and processed for autoradiography as described elsewhere (13). The labelling index was determined as a percentage of cells labelled.

PB PB PB SmM 4mM 5mM

S.Manjeshwar et al.

To eUminate the possibility that the susceptibility of hepatocytes from nodules is not a reflection of PB-induced lethal effect, the hepatocytes were incubated with TGF-a + PB (5 mM) for 24 h. The growth factor and PB were subsequently washed off and fresh medium containing [3H]thymidine was added. They were then incubated for another 24 h and then processed for autoradiography as described in Materials and methods. The data in Figure 6 indicate that when the PB is washed off, the surrounding as well as nodular hepatocytes are able to recover and incorporate thymidine, suggesting that hepatocytes from surrounding non-nodular as well as those from hepatic nodules are not killed to any significant extent by PB.

2290

Thus in summary, the present study demonstrates that PB inhibits DNA synthesis in hepatocytes from normal rat liver and one target site for the PB induced mitoinhibitory effect is beyond the growth factor receptor or growth factor receptor-mediated early events. In addition, this study demonstrated that PB not only inhibited the DNA synthesis in the non-nodular surrounding liver but also inhibited DNA synthesis in hepatocytes from the nodules. More quantitative in vivo data are needed to determine whether differential mitoinhibition is a major contributing factor in the PB-mediated liver tumor promotion. Acknowledgements This work was supported by USPHS grant CA 37077 and in part by funds from National Cancer Institute, Canada.

References l.Peraino,C.,Fry,R.J.M. and Staffeldt.E. (1971) Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res., 31, 1506-1512. 2. Pitot,H.C, Barsness.L., GoWsworthy,T. and Kitagawa.T. (1978) Biochemical characterization of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature, 111, 456-458. 3. Peraino,C, Fry.RJ.M., Staffddt^E. and ChristopherJ.P. (1975) Comparative enhancing effects of phenobarbital, amobarbital, diphenylhydantoin and dichlorodiphenyltrichloroethane on 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res., 35, 2884-2890. 4. Edwards.A.M. and Lucas.C.M. (1985) Phenobarbital and some other liver tumor promoters stimulate DNA synthesis in cultured rat hepatocytes. Biochem. Biophys. Res. Common., 131, 103-108. 5. Armato,U., Andreis.P.G. and Ramano.F. (1985) The stimulation by the tumor promoters 12-CMetradecarioyl-phorbol-13-acetate and phenobarbital of the growth of primary neonatal hepatocytes. Carcinogenesis, 6, 811 -822. 6. Schulte-Hermaim.R., Ohde.G., SchuppterJ. and Timmermann-Trosiener,I. (1981) Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital.

Downloaded from http://carcin.oxfordjournals.org/ at University of Michigan on April 18, 2015

Discussion The results of this study demonstrate that PB inhibits DNA synthesis in normal hepatocytes as well as in hepatocytes from hepatic nodules. Further, the mitoinhibition is not a reflection of any lethal or irreversible effect of PB because washing off the PB permitted the hepatocytes to enter S phase. Although PB has been shown to inhibit DNA synthesis in hepatocytes both in vitro (8 —10) and in vivo (7), the mechanism by which it inhibits is not yet clear. Studies by Eckl et al. (8) and by Hwang et al. (18) further suggested that surface binding of EGF to isolated hepatocytes continuously declined with increasing time of exposure to PB in vivo. However, results presented in this study, using normal hepatocytes in vitro, indicate that there may be an additional site(s) beyond the growth factor receptor and receptormediated early events for the mitoinhibition by PB. Taken together, these studies indicate that PB might have multiple sites for the inhibition of DNA synthesis. It is interesting to note that OA, another liver tumor promoter, also inhibits DNA synthesis in hepatocytes both in vivo (19) and in vitro (12 — 14). One of the target sites is beyond the growth factor receptor and growth factor receptor mediated early events. This conclusion is based on the fact that OA inhibited DNA synthesis in hepatocytes induced by several growth factors including EGF/TGF-a, hepatocyte growth factor and acidic FGF (14). These growth factors are known to use different receptors. Further, OA also inhibited DNA synthesis when added 24 h after priming the hepatocytes by EGF or TGF-a(14). Although the PB model was the first to be described in rat liver carcinogenesis with an initiation—promotion sequence, molecular mechanisms by which it may promote have not yet been elucidated in great detail. However, several hypotheses were put forward to explain the mechanism of tumor promotion by PB. For example, it was postulated that PB exerted a differential stimulatory effect on nodular hepatocytes as compared to the surrounding non-nodular liver (6). In addition, studies by Bursch and Schulte-Hermann suggested that PB inhibits single cell death (apoptosis) in hepatocyte nodules which could result in their continued growth (20). Nimms et al. (21) have reported that the ability of PB and PB-like compounds to promote hepatocarcinogenesis generally correlated with their ability to induce phase I drug metabolizing enzymes such as cytochrome P450 Iffll. However, these hypotheses were formulated based on the effects observed following an acute administration of PB. On the other hand, upon chronic exposure, a condition required for liver tumor promotion, PB actually inhibits liver DNA synthesis (7). Further, it also inhibits DNA synthesis in hepatocytes in vitro (8,10). In addition, recently, it was shown that hepatocytes of rats exposed to PB exhibited increased concentrations of TGF-/3, which is known to inhibit DNA synthesis in hepatocytes (9,22). In contrast to normal liver, a

subset of PB-promoted preneoplastic nodules do not contain TGF/31 (9,22). These considerations raised the possibility that PB may act as a differential mitoinhibitor. The results presented in this communication, however, suggest that although PB is a mitoinhibitor of surrounding non-initiated hepatocytes, it also inhibited the nodular hepatocytes from responding to TGF-a. It is interesting to note that Jirtle and Meyer (9) have also found putative preneoplastic hepatocytes to be less sensitive to the mitoinhibitory effect of PB as compared to normal hepatocytes in primary culture. Hence, although one cannot rule out the possibility that small differences may yet exist between surrounding and nodular liver in vivo in their response to the mitoinhibitory effects of PB, the results of this study indicate that PB may not create as strong a differential between surrounding and nodular hepatocytes as is evident with 2-acetylaminofluorene in the resistant hepatocyte model and with OA. In the resistant hepatocyte model, 2-acetylaminofluorene exerts a differential mitoinhibitory effect and permits selective amplification of resistant hepatocytes (23). In the OA model, OA also exerts a differential mitoinhibitory effect on the non-initiated hepatocytes (14, 19). However, it has not yet been demonstrated that OA promotes liver carcinogenesis by the differential mitoinhibitory mode. If these results can be extrapolated to an in vivo situation, one wonders how the foci of enzyme-altered hepatocytes grow in a mitoinhibitory environment created by PB. Is it likely that the initiated hepatocytes lack, or are less responsive to, the negative regulatory mechanisms? If this is true, such a situation could confer some selective advantage for their growth. It is also conceivable that the lack of a strong differential may be one reason why PB takes so long to promote hepatocarcinogenesis as compared to the resistant hepatocyte model.

Inhibition of DNA synthesis by PB

Downloaded from http://carcin.oxfordjournals.org/ at University of Michigan on April 18, 2015

hexachlorocyclohcxane, steroid compounds and nafenopin. Cancer Res., 41, 2556-2562. 7.Barbason,H., Rassenfosse,C. and Betz.E.H. (1983) Promotion mechanism of phenobarbital and partial hepatectomy of DENA hepatocarcinogenesis cell kinetics effect. Br. J. Cancer, 47, 517-525. 8. Ecld.P.M., Meyer.S.A., Whitcombe.W.R. and Jirtle.R.L. (1988) Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation. Carcinogenesis, 9, 479-483. 9. Jirtle.R.L. and Meyer.S.A. (1991) Liver tumor promotion: Effect of phenobarbital on EGF and protein Irinase C signal transduction and transforming growth factor-/31 expression. Dig. Dis. Sci., 36, 659-668. 10. Manjeshwar.S., Sheikh.A., Rao.P.M., Rajalakshmi.S., Michalopoulos.G., Pediaditakis.P. and Sarma.D.S.R. (1991) Inhibition of DNA synthesis by orotic acid and phenobarbital in hepatocytes in vitro. Proc. Am. Assoc. Cancer Res., 32, 157. ll.Laurier.C, Tatematsu.M., Rao.P.M., Rajalakshmi.S. and Sarma.D.S.R. (1984) Promotion by orotic acid of liver carcinogencsis in rats initiated by 1,2-dimethylhydrazine. Cancer Res., 44, 2186-2191. 12. Laconi.E., Li,F., Semple.E., Rao.P.M., Rajalakshmi.S. and Sarma.D.S.R. (1988) Inhibition of DNA synthesis in primary cultures of hepatocytes by orotic acid. Carcinogenesis, 9, 675—677. 13. Pkhiri-Coni.G., Coni,P., Lacotu.E., Schwarze.P.E., Seglen.P.O., Rao.P.M., Rajalakshmi.S. and Sarma.D.S.R. (1990) Studies on the mitoinhibhory effect of orotic acid on hepatocytes in primary culture. Carcinogenesis, 11,981-984. 14. Manjeshwar.S., Sheikh,A., Pichiri-Coni.G., Coni.P., Rao.P.M., Rajalakshmi.S., Pediatditakis.P., Michalopoulos.G. and Sarma.D.S.R. (1992) Orotic acid, nucleotide-pool imbalance and liver tumor promotion: a possible mechanism for the mitoinhibitory effects of orotic acid in isolated rat hepatocytes. Cancer Res., 52, 2078s-2081s. 15. Laconi.E. (1988) Studies on rat liver tumor promotion by orotic acid. Ph.D. thesis, University of Toronto, Toronto, Canada. 16. Michalopoulos.G., Houck.K.A., Dolan.M.L. and Luetteke.N.C. (1984) Control of hepatocyte replication by two serum factors. Cancer Res., 44, 4414-4419. 17. Meyer.S.A., Gibbs.T.A. and Jirtle.R.L. (1989) Independent mechanisms for tumor promoters phenobarbital and 12-0-tetradecanoylphorbol-13-acetate in reduction of epidermal growth factor binding by rat hepatocytes. Cancer Res., 49, 5907-5912. 18.Hwang,D.L., Rohman.A., Lev-Ran,A. and Carr.B.I. (1986) Chronic treatment with phenobarbital decreases the expression of rat liver EGF and insulin receptors. Biochem. Biophys. Res. Common., 135, 501-506. 19. Sheikh,A., Yusuf.A., Laconi.E., Manjeshwar,S., Rao.P.M., Rajalakshmi.S. and Sarma.D.S.R. (1991) Orotic acid inhibits DNA synthesis in rat liver following partial hepatectomy. Proc. Am. Assoc. Cancer Res., 32, 154. 20. Bursch.W. and Schulte-Hermann.R. (1989) Cell death (apoptosis) in normal and preneoplastk liver tissue. In Robcrfroid.M.B. and Preat.V. (eds), Experimental Hepatocarcinogenesis. Plenum Press, New York, pp. 143-159. 21. Nimms.R.W., Dcvor.D.E., HennemanJ.R. and Lubet.R.A. (1987) Induction of alkoxyresorufin O-dealkylases, epoxide hydrolase, and liver weight gain: Correlation with liver tumor promoting potential in a series of barbiturates. Carcinogenesis, 8, 67—71. 22. Haukins.G.R. and Jirtle.R.L. (1992) The role of growth factors in liver regeneration and tumor promotion. FASEB J., 6, A1029. 23. Solt.D. and Farber.E. (1976) New principles for the analysis of chemical carcinogenesis. Nature, 263, 701 - 7 0 3 . Received on June 12, 1992; revised on August 11, 1992; accepted on August 20, 1992

2291

Inhibition of DNA synthesis by phenobarbital in primary cultures of hepatocytes from normal rat liver and from hepatic nodules.

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhib...
531KB Sizes 0 Downloads 0 Views