The effect of two periods of short-term fasting during the promotion stage of hepatocarcinogenesis in rats: the role of apoptosis and cell proliferation

The loss of body and liver weight caused by chronic caloric restriction and its effects on carcinogenesis are well known; however, the effects of acute fasting on carcinogenesis have not been intensively investigated. We have studied some parameters of rat liver during short- term fasting and its effect on the stage of promotion in hepatocarcinogenesis in rats. During two fasting periods, body and liver weight decreased remarkably. Bromodeoxyuridine (BrdU) labeling indices (LI) decreased, and cell density increased prominently in liver sections. Hematoxylin and eosin-stained and nick end labeling (TUNEL)- stained sections showed an increase of apoptotic bodies in the absence of necrosis during the fasting period. Moreover, gel electrophoresis of DNA isolated from whole liver revealed ladder formation indicative of nucleosomal DNA cleavage. At the beginning of the fasting period livers exhibited a small but definite number of altered hepatic foci (AHF) expressing glutathione S-transferase, placental form (GST-P), but at the end of the fasting period no AHF were discernible in all livers of animals subjected to the fasting period. After refeeding, cell density and the incidence of apoptotic bodies decreased prior to a transient increase of BrdU LI. The percentage volume of liver occupied by AHF of fasted rats was significantly greater than that of control rats at 140 days after initiation. These results suggest that both the liver weight loss and the complete loss of discernible AHF from short-term fasting was caused by (i) decrease of cell volume, (ii) cell loss by apoptosis, and (iii) a decrease of hepatocyte proliferation. Furthermore, this relatively transient liver weight loss enhanced the promotion of hepatocarcinogenesis, possibly by enhanced cell proliferation compensatory to the fasting cycles.      

DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion

Carcinogenesis was initiated in female rat liver by a single dose of N-nitrosomorpholine; subsequently phenobarbital (PB) was administered via the diet at a daily dose of 50 mg/kg body weight for up to 49 weeks. Subgroups of rats were left untreated after 10 or 28 weeks on PB. PB produced the following changes: (a) accelerated appearance of neoplastic nodules and hepatocellular carcinoma (from 28 weeks onwards); (b) phenotypic changes in altered foci such as a shift from clear to eosinophilic appearance, enhanced expression of gamma-glutamyltranspeptidase and other markers, and more distinct borders from surrounding liver; (c) an increase in foci number; and (d) accelerated foci enlargement. The increase in foci number was found to be due to increased phenotypic expression of foci. DNA synthesis was measured by [3H]thymidine labeling at multiple time points. The rate of DNA synthesis was always approximately 10-fold higher in foci than in surrounding liver tissue. Despite this, after N-nitrosomorpholine alone foci grew little before 18-24 weeks. Continuous treatment with PB did not produce a persistent further increase of DNA synthesis in foci, although it accelerated foci growth. Furthermore, at early stages small and larger foci showed similar DNA synthesis activity. These findings indicate that the rate of cell replication as measured by DNA synthesis is not the only determinant of the growth rate of foci. Further studies showed that foci with indistinct borders (reflecting weak expression of the altered phenotype) grew much less than foci with distinct borders; this was at least in part due to an increased rate of cell death by apoptosis found in foci with indistinct borders. In conclusion, besides cell replication, apoptosis and the extent of phenotypic expression (remodeling) determine the growth rate of foci. Foci with weak phenotypic expression predominated after N-nitrosomorpholine alone; in these, a high incidence of apoptosis counterbalanced cell replication. In contrast, during PB treatment foci with strong phenotypic expression predominated; in these, apoptotic activity was lower and the high replicative activity could manifest itself. Finally, all effects of PB on foci were largely although not completely reversible upon cessation of treatment; as a result phenotypic expression declined, and "remodeling" foci with high apoptotic activity predominated again.     

c-fos gene expression in rat liver is induced by phenobarbital

In the present study we investigated c-fos expression in rat livers, that was initiated with the three arylamines, 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene. The tumor promoter phenobarbital was applied chronically for 26, 52 and 100 weeks. Gene expression, determined by the mRNA level, and FOS protein were increased after 52 weeks of treatment in arylamine initiated as well as in phenobarbital only treated animals. Expression of c-fos seems to be a phenobarbital induced effect that is independent of additional initiator treatments. This finding was supported by immunohistochemical studies demonstrating increased FOS levels to be localized around the central vein. The results indicate that phenobarbital, a widely used tumor promoter, induces c-fos expression. In addition, we demonstrated enhanced FOS in GST-P-positive foci and in tumors.     

The enhancing effect of fasting/refeeding on the growth of nodules selectable by the resistant hepatocyte model in rat liver

Caloric restriction causes a generalized decrease in growthrate and has been shown to delay the development of both spontaneousand induced neoplasia. In contrast to chronic food restriction,the extreme condition of fasting/refeeding is associated withan overall increase in cell turnover in several organs, includingliver, compared with regular feeding. The present study wastherefore designed to investigate the effect of complete foodwithdrawal followed by refeeding on the growth of hepatocytenodules in initiated rat liver. Male Fischer 344 rats were givena single dose of diethylnitrosamine (DEN, 200 mg/kg i.p.) andthen, starting 1 wk later, they were exposed to one or threecycles of fasting (3 days) followed by refeeding (11 days).The control group was fed continuously. Seven weeks after DENadministration all rats were subjected to the resistant hepatocytemodel (2-acetylaminofluorene coupled with CCl₄) and 2 weekslater 2/3 partial hepatectomy (PH) was performed. All animalswere killed 2 weeks after surgery. At PH rats given one cycleof fasting/refeeding had significantly larger glutathione S-transferase7–7-positive hepatic lesions compared with controls (meanarea 0.73 ± 0.04 versus 050 ± 0.05 mm², P <0.025; mean percent area 25.6 ± 3.2 versus 12.4 ±0.9, P < 0.005), while no significant change was observedin their number. The observed differences were more pronouncedwith three cycles of fasting/ refeeding. A similar pattern ofresults was obtained at the time of killing. It is concludedthat fasting/refeeding can exert a positive effect on the growthof rat hepatocyte foci and nodules, in contrast to the generalinhibitory effect on carcinogenesis caused by food restriction.     

Effect of rat developmental stage at initiation on the expression of biochemical markers during liver tumor promotion.

The phenotypic response of rat liver to a carcinogenic protocol involving initiation/selection and promotion with and without phenobarbital (PB) feeding was studied in pubertal and adult male rats. Considering the early presence of preneoplastic nodular areas, it appeared that pubertal rats, initiated at 6-7 weeks, presented a higher susceptibility to the protocol than adult rats initiated at 9-10 weeks. Altered liver phenotype was characterized by: (1) gamma-glutamyl-transpeptidase (GGT) and glutathione S-transferase (GST) activities; (2) the expression of two forms of cytochrome P-450; de novo PB-inducible P-450 II B 1,2 and P-450 II C 7 normally expressed in 45-day-old rats and PB-inducible, and (3) the expression of albumin and alpha-fetoprotein cDNAs. In the absence of PB, the susceptibility of pubertal rat liver to hepatocarcinogenesis was related to a special metabolic phenotype enriched in GGT and GST activities by comparison with the quasi-normal expression of both P-450s. Adult rat liver presented a less altered pattern closer to that of noninitiated rat liver. During PB promotion, the loss of PB inducibility of P-450 II C 7 in pubertal rat liver suggested that the hormonal status of the animals could interact with initiation to modulate specific gene expression. The late phase of PB promotion revealed the loss of highly differentiated functions (P-450s, albumin), whereas enzymatic markers associated with preneoplastic foci showed a persistent high expression.     

Molecular profiling of genes up-regulated during promotion by phenobarbital treatment in a medium-term rat liver bioassay

In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(-), PH(-) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.     

Dose-response relationship of phenobarbital promotion of diethylnitrosamine initiated tumors in rat liver

The dose-response relationship was determined in rats for the enhancement by phenobarbital of diethylnitrosamine (DENA)-initiated neoplastic nodules and hepatocellular carcinomas. Male Sprague-Dawley rats received a single oral dose of either 80 mg/kg DENA or water. Seven days later, the animals were divided into groups that started to receive 0, 62.5, 125, 250, 500 or 1000 ppm sodium phenobarbital in the drinking water. Animals from each group were killed at 48 and 70 weeks after the DENA. No significant difference was observed in the low response of neoplastic nodules among the DENA-initiated groups. The incidence of DENA-initiated hepatocellular carcinoma was enhanced at 70 weeks by 250, 500 and 1000 ppm sodium phenobarbital but not by 62.5 or 125 ppm sodium phenobarbital. Equal enhancement of the incidence of hepatocellular carcinomas was obtained with 250, 500 and 1000 ppm sodium phenobarbital. In non-DENA-initiated rats, phenobarbital did not induce neoplastic nodules or hepatocellular carcinomas. Our results suggest that a daily dose of at least 250 ppm sodium phenobarbital is required in order for it to exert tumor promoting activity.     

Reversible alteration in the expression of the gap junctional protein connexin 32 during tumor promotion in rat liver and its role during cell proliferation

Although numerous biochemical markers can identify putative preneoplastic altered hepatic foci (AHF) in rat liver, no consistent pattern of expression during hepatocarcinogenesis has emerged. Using quantitative stereologic analyses we demonstrated that decreased expression of the major hepatocyte gap junction protein, connexin 32 (Cx32), in rat AHF is a consistent observation in several protocols of multistage hepatocarcinogenesis. This change was observed after initiation by either ethylnitrosourea (ENU) or diethylnitrosamine (DEN), followed by promotion with phenobarbital (PB), dioxin, chlorendic acid, C.I. Solvent Yellow, or tamoxifen. AHF generated by Wy-14,643, ciprofibrate, and a choline/methionine-deficient dietary regimen also showed decreased Cx32 expression. The decrease of Cx32 in AHF was rapidly reversible after withdrawal of PB, and this change preceded a reduction in placental isozyme of glutathione-S-transferase (GST) expression in the same AHF. Within 20 days of withdrawal, fewer than 4% of GST-positive AHF were Cx32 deficient, while the volume of total AHF decreased 30%. Chronic PB treatment also resulted in a reversible decrease in Cx32 specifically in mid- and centro-lobular hepatocytes. Continuous thymidine labeling demonstrated that Cx32 could be uncoupled from the cell cycle, suggesting that some liver promoters may act directly to alter the expression of Cx32. These observations suggest that a decrease in Cx32 content was a relatively common epigenetic change in AHF induced during hepatocarcinogenesis by a number of initiating and promoting agents but that this change was not sufficient for carcinogenesis. This change, however, may be necessary for the mechanism(s) of tumor promotion, since Cx32-positive AHF did not proliferate as readily as Cx32-deficient AHF.     

Early stimulation of polyamine biosynthesis during promotion by phenobarbital of diethylnitrosamine-induced rat liver carcinogenesis. The effects of variations of the S-adenosyl-L-methionine cellular pool

A decrease of S-adenosyl-L-methionine liver content was observedbetween the 14th and the 35th day after the start of 2-acetylaminofluorenefeeding in diethylnitrosamine-initiated rats according to the‘resistant-hepatocyte’ model of hepatocarcinogenesis.The decrease was enhanced by phenobarbital given to the animalsafter the end of 2-acetylaminofluorene feeding. These changeswere associated with an increase in ornithine decarboxylaseactivity and the spermidine: spermine ratio. S-adenosyl-L-methionineadministration to rats caused a great fall in the percentageof -glutamyl-transpeptidase-positive liver as well as in polyaminesynthesis. An increase in ornithine decarboxylase activity,associated with a decrease in the liver S-adenosyl-L-methioninepool, also occurred in normal animals on the first day followinga partial hepatectomy and was enhanced by phenobarbital. Theassociation of 2-acetylaminofluorene feeding with partial hepatectomyresulted in a slower liver regeneration, while the decreasein S-adenosyl-L-methionine level and the increase in polyaminesynthesis were observed over a longer period of time after partialhepatectomy. These changes were further prolonged in diethylnitrosamine-initiatedrats in which -glutamyltranspeptidase-positive foci developed.In these animals a high level of polyamine synthesis was stillpresent when liver regeneration was complete. At this stageof the observation period the labeling index was very low insurrounding liver, but still high in the -glutamyltrans-peptidase-positiveareas. Phenobarbital stimulated polyamine synthesis and cellgrowth and further prolonged the period of time during whicha high ornithine decarboxylase activity and labeling index werepresent. These results indicate that the liver lipotrope contentcould be a rate-limiting factor for cell growth and liver neoplasiapromotion and this could depend on the modulation of polyaminebiosynthesis.     

Ploidy and specific karyotypic changes during promotion with phenobarbital, 2,5,2',5'-tetrachlorobiphenyl, and/or 3,4,3'4'-tetrachlorobiphenyl in rat liver.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.     

Inhibitory effect of UFT on rat hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene and phenobarbital promotion]

Inhibitory effect of UFT on hepatocarcinogenesis in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and phenobarbital (PB) promotion was studied. Donryu male rats were divided into four groups. Group A was fed a diet containing 0.06% 3'-MeDAB for 3, 5, or 7 weeks, and then fed normal diet for 2 weeks, subsequently received a diet containing 0.05% PB. Group B was given UFT (20 mg/kg/5 days a week) simultaneously with feeding 3'-MeDAB. Group C was given UFT simultaneously with feeding PB. Group D was given 3'-MeDAB alone. In all groups, the development of hepatocellular carcinoma was investigated 37 weeks later and the number and area per mm2 of induced glutathione S-transferase placental form (GST-P) positive foci were measured using an image processor. The number and area of GST-P positive foci in group B and group C were markedly decreased as compared with those in group A. These results seem to show that the administration of UFT inhibited the production of GST-P positive foci and that stronger inhibitory effect of UFT was observed by simultaneous administration of an initiator than by that of a promoter.     

Altered expression of cell cycle and apoptotic proteins in human liver pathologies

Τhe expression of cell cycle (P53, Ki-67, P21, and P27) and apoptotic proteins (BCL-2 and BAX) was investigated by immunohistochemistry in paraffin-embedded formalin-fixed tissues of normal and pathologic liver. An increased frequency of expression of P21 in cirrhosis and hepatocellular carcinoma (HCC) (p=0.003 and p=0.001 respectively) was found; P27 protein expression was more frequent in hepatitis (p=0.001) and HCC (p=0.003) when compared with normal tissue. BCL-2 protein was markedly more frequent in steatohepatitis (p<0.05) as compared to normal liver, in hepatitis cases (p=0.002) and in metastases (p<0.033). The expression of BAX was more frequent in hepatitis (p=0.001) and cirrhosis (p<0.001). We demonstrated in our study the expression of these proteins at different levels in liver pathologies. These findings have implications for understanding the evolution from liver inflammation to cirrhosis and associated carcinogenesis.     

Tyrosine aminotransferase gene expression in a temperature-sensitive adult rat liver cell line

Regulation of tyrosine aminotransferase gene expression was studied in an adult rat hepatocyte line (RALA255-10G) which is temperature sensitive for the maintenance of the differentiated liver phenotype. Glucocorticoid hormones such as cortisol were necessary for expression of the aminotransferase gene. In the absence of these steroids, enzyme synthesis, activity, and mRNA accumulation were virtually abolished. In the presence of cortisol at 33 degrees C, RALA255-10G cells showed characteristics of malignant transformation and contained little tyrosine aminotransferase activity, synthesized low levels of this enzyme, and produced low levels of enzyme mRNA. At 40 degrees C, cells maintained in the presence of cortisol regained the normal, differentiated phenotype, and tyrosine aminotransferase synthesis and mRNA accumulation were greatly increased. This increase in aminotransferase synthesis paralleled the increase in the enzyme mRNA. However, after a temperature shift-up, tyrosine aminotransferase activity was increased only for the first 2 days, probably due to thermal inactivation of this enzyme at 40 degrees C. Dibutyryl cAMP alone was not sufficient to induce expression of the tyrosine aminotransferase gene, but it enhanced the induction caused by cortisol. Immunocytochemical studies revealed that the enhanced expression of the tyrosine aminotransferase gene at 40 degrees C and in the presence of cortisol or cortisol plus dibutyryl cAMP resulted from an increase in both the number of cells producing this enzyme and the quantity of tyrosine aminotransferase synthesized per cell.     

Effect of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma in the rat.

Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 +/- 1.1 to 16.5 +/- 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 +/- 2.3 versus 4.6 +/- 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.     

Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) oncell division and cell death (apoptosis) in glutathione S-transferase(GST-P)-positive liver foci were analyzed in diethylnitrosamine-initiatedfemale Wistar rats that were treated with TCDD, either acutelyfor 3 days or chronically for 115 days. Apoptotic bodies werequantitated in liver sections simultaneously stained for GST-Pexpression and H&E using a novel fluorescence microscopicdetection method which greatly facilitates recognition of apoptoticbodies due to their high level of eosin fluorescence. WhileTCDD treatment only marginally affected cell division in GST-P-positiveliver foci, as estimated by 5-bromo-2'-deoxyuridine-labelling,apoptotic indices were decreased to     

In situ hybridization studies on expression of albumin and alpha-fetoprotein during the early stage of neoplastic transformation in rat liver

The expressions of albumin and alpha-fetoprotein (AFP) genes were studied in early preneoplastic liver lesions produced by the Solt-Farber protocol using "in situ" hybridization with single stranded RNA probes. In normal rat liver, albumin was expressed at a lower level in the centrilobular than in the periportal areas of the liver acinus, whereas the bile duct epithelium did not show any expression. Five weeks after initiation with diethylnitrosamine, islands of hepatocytes were present which showed heterogeneous expression of albumin and were surrounded by cells comprised of albumin negative hepatocytes and oval cells. gamma-Glutamyltranspeptidase positive foci of enzyme altered cells were located in albumin positive areas. Albumin expression gradually decreased in permanent nodules but increased in the hepatocytes outside the nodules during the first five months after initiation with diethylnitrosamine. Remodeling nodules, which were partly gamma-glutamyltranspeptidase and albumin positive, were also present. However, no consistent correlation was found between gamma-glutamyltranspeptidase positive and albumin negative areas during the first 5 months after initiation. Occasionally, cells showing an elevated expression of albumin were found in permanent nodules. These cells were located in the vicinity of oval type cells, which also showed a weak expression of albumin. AFP was expressed at high level in oval cells 5 weeks after the initiation. However, oval cells observed at later time points, either around the neoplastic nodules or inside the nodules showed only low expression of AFP. Hepatocytes in the enzyme-altered foci and in neoplastic nodules were always negative for AFP. The presence of strongly albumin positive cells inside the neoplastic nodules in close proximity to oval type cells suggests that these cells may be derived from primitive "stem-cell"-like oval cells.     

BHRF1表达对鼻咽癌细胞凋亡能力的影响

目的 了解ＥＢ病毒（Ｅｐｓｔｅｉｎ－Ｂａｒｒｖｉｒｕｓ）基因ＢＨＲＦ１（ＢａｍＨＩ－ＨｒｉｇｈｔｗａｒｄｒｅａｄｉｎｇｆｒａｍｅＩ）表达对鼻咽癌细胞抗凋亡能力的影响。方法 构建表达ＢＨＲＦ１重组载体并转入鼻咽癌细胞株ＣＮＥ２细胞中,以检测癌细胞在＾６０Ｃｏ照射后生物学行为的改变。结果 ＢＨＲＦ１表达可抑制增殖细胞核抗原的表达,促使癌细胞的细胞周期重新分布,癌细胞对辐射的敏感性下降,生存能力增强,结     

Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamine-initiated rats

The effec t of the chronic feeding of methionine or cholineon liver tumor promotion by phenobarbital (PhB) or 1, 1 bis(p-chlorophenyl)-2,2, 2-trichloroethane (DDT) was studied in rats receiving aninitiating dose of diethylnitrosamine (DEN). Male weanling ratswere injected i.p. with DEN (200 mg/kg body wt). Control ratswere injected with saline. Five days after the injection, therats were placed on different diets containing 0.05% PhB or0.05% DDT with or without added 1.5% DL-methionine or 1.0% cholinechloride. Each diet was administered for 72 weeks, when theanimals were placed on the unsupplemented chow diet for an additional30 weeks. Rats treated with DEN and then fed PhB or DDT developedan 85–100% incidence of hepatocellular carcinomas (HCCs).Single injection of DEN alone produced a 60% incidence of HCCs.Dietary feeding of methionine and choline either alone or incombination with PhB or DDT did not have any significant effecton the incidence of HCCs. Liver tumor formation was negligiblein uninitiated rats. Lung metastases developed in 42% and 46%of the DEN + PhB-and DEN + DDT-treated groups, respectively.Supplementation of methionine in the diet lowered the incidenceof lung metastases to 14% in the DEN + PhB-treated rats andto 19% in DEN + DDT-treated rats. Choline was not effectivein inhibiting the development of lung metastases in either case.The injection of DEN alone produced a 54% incidence of lungtumors. PhB and DDT feeding lowered the DEN-induced lung tumorincidence to 23% and 14% respectively. Further, when the datafrom different diet groups were combined it was found that singleinjection of DEN also doubled the incidence of leukemia normallyseen in F344 rats. The present report constitutes the firstevidence that a single injection of DEN induces lung tumorsand enhances the incidence of leukemia in rats.     

Morphological alterations and DNase deficiency in phenobarbital promotion of N-nitrosomorpholine initiated rat hepatocarcino-genesis

Tumorigenic effect in rat liver was increased when phenobarbitalwas given chronically after N-nitrosomorpholine. In these ratsthe liver parenchyma surrounding pre- and neo-plastic lesionsdemonstrated distinct, mainly centrilobular zones of hypertrophichepatocytes with abundant eosinophilic, filamentous cytoplasm,increase in nucleic acids and decrease in DNase activity. Thesealterations might be considered as signs of tumor-promotingactivity of phenobarbital.