RESUMO
Phosphatidylethanolamine N-methyltransferase(PEMT) is an enzyme in liver that catalyzes the stepwise methylation of phosphatidylethanolamine to phosphatidylcholine, in addition to the main pathway that synthesizes phosphatidylcholine directly from choline. We have reported that PEMT is permanently inactivated in liver cancer induced by the Solt and Farber model. Here we studied, (i) whether similar changes also occur in the progression of hepatocarcinoma triggered by aflatoxin B(1) (AFB(1)) in rats; (ii) whether the hepatoma phenotype could be reversed by over-expression of PEMT2. We found that PEMT2 protein decreased in pre-neoplastic nodules and virtually disappeared in hepatocellular carcinoma induced by AFB(1) due to decreased levels of mRNA without any deletion or mutation of the DNA sequence. PEMT activity, which reflects the function of both PEMT1 and PEMT2, was lower in nodules and negligible in the tumor, consistent with its regulation at the level of gene transcription. McArdle hepatoma cells transfected with PEMT2 failed to form anchorage-independent colonies in soft agar, while the vector-transfected control line grew efficiently. Moreover, PEMT2-transfected cells were also poorly tumorigenic in vivo in athymic mice, as shown by the lower tumor incidence, the longer cancer-free-time and the lower tumor volume and weight. Together, these data indicate that the loss of PEMT function may contribute to malignant transformation of hepatocytes.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Neoplasias Hepáticas Experimentais , Metiltransferases/genética , Aflatoxinas/toxicidade , Animais , Transformação Celular Neoplásica/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/terapia , Masculino , Camundongos , Fosfatidiletanolamina N-Metiltransferase , Ratos , Ratos WistarRESUMO
Previous studies have implicated phosphatidylethanolamine N-methyltransferase-2 (PEMT2) in the regulation of non-neoplastic liver growth [Tessitore,L., Cui,Z. and Vance,E. (1997) Biochem. J., 322, 151-154]. We have now investigated whether or not PEMT2 is also involved in the control of proliferation of hepatoma cells growing in an animal and cell death by apoptosis in the liver of tumor-bearing rats. PEMT activity was barely detectable and PEMT2 protein was absent in hepatoma cells growing exponentially in vivo whereas CTP:phosphocholine cytidylyltransferase (CT) activity and expression were high. The lack of PEMT2 corresponded with the absence of its mRNA. Both PEMT2 protein and mRNA appeared when cells entered the stationary phase of tumor growth and, in parallel, CT expression decreased. The host liver first became hyperplastic and exhibited a slight increase in CT activity and decrease in PEMT2 expression. During the stationary phase of hepatoma growth the host liver regressed and eventually became hypoplastic following induction of apoptosis. The appearance of apoptosis in the host liver was associated with a marked reduction in both CT activity and expression as well as an enhancement of PEMT activity and PEMT2 expression. McArdle RH7777 hepatoma cells underwent apoptosis when transfected with cDNA for PEMT2. The evidence supports the proposal that PEMT2 may have a role in the regulation of 'in vivo' hepatoma and hepatocyte cell division as well as hepatocyte cell death by apoptosis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Isoenzimas/biossíntese , Neoplasias Hepáticas Experimentais/enzimologia , Fígado/enzimologia , Metiltransferases/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Apoptose , Ascite/patologia , Divisão Celular , Indução Enzimática , Hiperplasia , Isoenzimas/genética , Isoenzimas/fisiologia , Fígado/citologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Metiltransferases/genética , Metiltransferases/fisiologia , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Fosfatidiletanolamina N-Metiltransferase , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Ratos , Ratos Wistar , Transfecção , Células Tumorais CultivadasRESUMO
In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a noninitiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 +/- 0.50 vs. 2.64 +/- 0.20, p < or = 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer.
Assuntos
Azoximetano , Carcinógenos , Neoplasias do Colo/induzido quimicamente , Ingestão de Alimentos , Lesões Pré-Cancerosas/induzido quimicamente , Neoplasias Retais/induzido quimicamente , Animais , Apoptose , Divisão Celular , Neoplasias do Colo/patologia , Jejum , Masculino , Ratos , Ratos Endogâmicos F344 , Neoplasias Retais/patologia , Redução de PesoRESUMO
The effect of fasting-refeeding during the promotion phase of dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis has been investigated. Female Sprague-Dawley rats were given 130 mg/kg of DMBA and divided into four groups: Group 1 was fed ad libitum; Group 2 was fed ad libitum and, in addition, received daily subcutaneous injections of beta-estradiol and haloperidol for eight days, beginning two weeks after DMBA administration; Group 3 was exposed to three days of fasting one week after carcinogen injection, then fed ad libitum until the end of the experiment; Group 4 was treated as Group 2, except the animals were fasted for three days beginning one week after DMBA administration. Treatment with beta-estradiol and haloperidol enhanced the development of DMBA-induced mammary tumors. Rats fasted after DMBA administration showed increased genesis and growth and reduced latency of mammary tumors. Fasting before beta-estradiol and haloperidol injection resulted in a more pronounced effect on tumor yield and latency than hormones or fasting alone. The data indicate that, in rats fasted during the promotion phase, tumor growth is enhanced and the permissive effects of hormones on mammary carcinogenesis are potentiated.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Jejum , Alimentos , Neoplasias Mamárias Experimentais/induzido quimicamente , Animais , Estradiol/administração & dosagem , Estradiol/farmacologia , Feminino , Haloperidol/administração & dosagem , Haloperidol/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this work was to investigate the effect of fasting on the induction and growth of chemically-induced mammary carcinogenesis. Female Sprague-Dawley rats were given methylnitrosourea (MNU) i.p. (50 mg/kg) at 50 days of age; a group of rats were exposed to 4 day fasting followed by 1 day of refeeding before the administration of the carcinogen, while another group was exposed to three cycles of 3 days fasting in 10 days, beginning 1 week after MNU injection. Fasting enhanced the development of mammary tumours only in rats fasted after carcinogen damage, while it did not affect the induction of tumours in rats fasted before MNU, if compared with full-fed controls. The enhanced growth of mammary tumours sustained by fasting during promotion was observed in the cervical-thoracic region. In addition, exposure to fasting made rats susceptible to the development of MNU-induced extra-mammary cancers. Different from the preventive effect of caloric restriction on tumor development, these data demonstrate that fasting affects the promotion phase of carcinogenesis by enhancing the growth of MNU-induced mammary tumours.
Assuntos
Adenocarcinoma/etiologia , Jejum/fisiologia , Neoplasias Mamárias Experimentais/etiologia , Adenocarcinoma/induzido quimicamente , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/etiologia , Linfoma não Hodgkin/induzido quimicamente , Linfoma não Hodgkin/etiologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia , Neoplasias Ovarianas/induzido quimicamente , Neoplasias Ovarianas/etiologia , Ratos , Ratos Sprague-Dawley , Neoplasias Uterinas/induzido quimicamente , Neoplasias Uterinas/etiologiaRESUMO
We have recently shown that fasting before initiation markedly stimulated the growth of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the rat medial colon. Here we investigated the mechanisms by which fasting enhanced the growth of ACF. Rats were exposed to 4 day-starvation, then they were given AOM (20 mg/kg) on the first day of refeeding. 4 day-fasting depressed cell proliferation as shown by the decreased mitotic index and enhanced cell death by apoptosis. On the first day of refeeding, apoptotic index remained higher than control values, while mitotic index markedly increased in the colonic epithelium of fasted/ refed rats. The administration of AOM induced an apoptotic wave, that was higher in controls, and a transient drop in the mitotic index that recovered quickly in the fasted/refed group. These data suggest that starvation-induced apoptosis represents the mitogenic stimulus to increase the rates of cell proliferation responsible for the enhanced growth of ACF in fasted/refed rats.
Assuntos
Colo/patologia , Jejum/efeitos adversos , Animais , Azoximetano/toxicidade , Carcinógenos/toxicidade , Morte Celular , Divisão Celular , Cocarcinogênese , Neoplasias do Colo/etiologia , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
PEMT and CT activities were reciprocally regulated during the perinatal period. Consistently, PEMT2 expression was undetectable before birth when CT was highly expressed. Surprisingly, PEMT2 was relatively highly expressed at birth when the cell division and CT expression were still high. During development liver cell growth was associated with enhanced levels in the activity of beta, zeta and, particularly, alpha PKC. The activity of delta PKC was lower in foetal, higher in the newborn and again slightly lower than adult liver 10 days after birth. These data show that CT expression and alpha, beta and zeta PKC activities are positively, whereas PEMT2 expression and delta PKC activity are negatively associated with the liver cell division during development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/enzimologia , Metiltransferases/metabolismo , Nucleotidiltransferases/metabolismo , Proteína Quinase C/metabolismo , Animais , Colina-Fosfato Citidililtransferase , Ativação Enzimática , Feminino , Isoenzimas/metabolismo , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Masculino , Fosfatidiletanolamina N-Metiltransferase , Ratos , Ratos WistarRESUMO
Aberrant crypt foci (ACF) are putative preneoplastic lesions of colon cancer which are being utilized currently as a biological end point to evaluate the induction and modulation of colon carcinogenesis. In rodents, caloric restriction reduces carcinogen-induced colon cancer incidence. The present study was designed to investigate the effect of fasting followed by refeeding on the development of ACF. Male Fisher 344 rats were fasted for 4 days and they were given a single injection of azoxymethane (AOM) at the dose of 20 mg/kg body weight on the first day of refeeding, and killed 3 months later. Controls were fed ad libitum and received the same dose of AOM. The number and crypt multiplicity (number of crypts/focus) of ACF were measured on the medial colon. No effect of fasting/refeeding was observed in the total number of foci/medial colon. On the contrary, rats fasted for 4 days and refed developed foci with higher number of crypts than fed controls. Our results are of particular interest because the crypt multiplicity, rather than the number of foci, is a consistent predictor of tumor incidence. As a consequence these data suggest a possible role of fasting/refeeding in enhancing the colon tumor outcome.
Assuntos
Neoplasias do Colo/etiologia , Jejum , Alimentos , Animais , Azoximetano/farmacologia , Carcinógenos/farmacologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Endogâmicos F344RESUMO
The effect of fasting/refeeding on MNU-induced mammary carcinogenesis was investigated. Female Sprague-Dawley rats were given i.p. a single dose of MNU (50 mg/Kg body weight) and beginning 1 week after MNU administration were exposed to 3 cycles of 3 days fasting followed by refeeding (10 days). Rats were palpated twice a week and killed when tumor diameter was about 2 cm. Tumors palpated were registered by location. The exposure to fasting/refeeding after initiation increased the total number of mammary tumors about 2-fold compared to full-fed control group. In addition, fasted rats developed about 3-fold mammary tumors in the cervical-thoracic gland chains versus the abdominal-inguinal gland chains, while no difference in tumor distribution was observed in controls. The present study reports that fasting/refeeding enhances the development of MNU-induced mammary tumors.
Assuntos
Carcinógenos/toxicidade , Cocarcinogênese , Ingestão de Alimentos , Jejum , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia/toxicidade , Animais , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
Previous data of our laboratory showed that female rats regenerated earlier than males and choline shifted the female growth pattern toward that of males. We investigated if the effect of choline on the liver compensatory growth was associated to a modulation of the expression and methylation pattern of an early cell cycle dependent proto-oncogene, c-myc. The peak of DNA synthesis was 22 h after 2/3 partial hepatectomy in female regenerating liver, while it was delayed to 30 h when female rats received choline for 3 weeks before liver surgery. Partial hepatectomy induced the expression of c-myc that was already maximal at 1 h. Choline reduced the c-myc expression and it shifted the maximum increase at 2 h. The methylation pattern of c-myc was studied with the Hpa II restriction enzyme. The delay in c-myc expression was not due to hypermethylation of the gene.
Assuntos
Colina/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes myc , Regeneração Hepática/efeitos dos fármacos , Animais , Northern Blotting , DNA/biossíntese , Feminino , Hepatectomia , Poli A/análise , Ratos , Ratos WistarRESUMO
We have analyzed the expression pattern of epsilon protein kinase C (PKC) in normal liver tissue, in hyperplastic liver nodules and in hepatocellular carcinomas generated in the rat with the Solt-Farber protocol. A progressive increase in PKC epsilon expression was observed in nodules and carcinomas compared to normal liver tissue, suggesting that the expression level of this PKC isoenzyme could be associated with increased malignancy. To test this hypothesis, the well differentiated, poorly tumorigenic MH1C1 rat hepatoma cell line was stably transfected with a full length epsilon PKC cDNA. No increase in growth rate, saturation density, soft agar growth or in vivo tumorigenicity was observed in transfected cells, compared to parental or mock-transfected cells. These results indicate that epsilon PKC does not seem to participate in signaling pathways involved in neoplastic transformation or malignant progression in our liver cell model. The fact that epsilon PKC overexpression is tumorigenic in several other cell types suggests that this effect might be strictly cell- and tissue-specific.
Assuntos
Biomarcadores Tumorais , Isoenzimas/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Proteína Quinase C/metabolismo , Animais , Divisão Celular , Proteína Quinase C-épsilon , Ratos , Células Tumorais CultivadasRESUMO
Caloric restriction causes a generalized decrease in growth rate and has been repeatedly associated with an inhibitory effect on cancer development in several systems. In contrast, exposure to complete fasting followed by refeeding is a metabolic condition associated with increased cell turnover in different organs, including the liver. The present study examines whether such condition is able to sustain the induction of initiated hepatocytes following a subnecrogenic dose of diethylnitrosamine (DENA). Male Fisher-344 rats were fasted for 4 days and 1 day after refeeding they were given a single dose of DENA (20 or 200 mg/kg body wt, i.p.). Negative and positive control groups were fed ad libitum and injected with 20 and 200 mg/kg of DENA, respectively. One week later all animals were subjected to the resistant hepatocyte model for the selection of hepatocyte nodules and they were killed 2 weeks thereafter. Results indicated the presence of gamma-glutamyltransferase (GGT) positive foci and nodules (38 +/- 7/cm2) in rats regularly fed and given 200 mg/kg of DENA, while virtually no focal lesions (< 1/cm2) were found in the group receiving 20 mg/kg of DENA and fed throughout the experiment. However, a significant number of GGT positive foci/nodules (14 +/- 7) also developed in rats exposed to fasting and given 20 mg/kg of DENA 24 h after refeeding. No evidence of hepatocellular necrosis was found in the latter group following DENA administration. No effect of fasting was observed when rats received 200 mg/kg of DENA. It is concluded that fasting/refeeding provides conditions which are able to sustain initiation in rat liver by a subnecrogenic dose of a carcinogen. These findings are in contrast with the commonly reported inhibitory effect of chronic food restriction on various stages of carcinogenesis, including initiation.
Assuntos
Carcinógenos/toxicidade , Dietilnitrosamina/toxicidade , Ingestão de Alimentos , Jejum , Neoplasias Hepáticas Experimentais/induzido quimicamente , Animais , Carcinógenos/administração & dosagem , Dietilnitrosamina/administração & dosagem , Indução Enzimática/efeitos dos fármacos , Glutationa Transferase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Masculino , Ratos , Ratos Endogâmicos F344 , gama-Glutamiltransferase/metabolismoRESUMO
A sexual dimorphism occurs in liver cell proliferation following partial hepatectomy, female liver regenerating faster than male, while a continuous excess of choline to females shifts their growth pattern toward that of males (L. Tessitore, P. Pani and M.U. Dianzani, Carcinogenesis, 13 (1992) 1929). In this study we have investigated (a) if the same sexual modulation occurs in a different type of liver growth, hyperplasia induced by a direct mitogen and (b) if the pre-administration of choline to females is able to modulate this dimorphism. Liver hyperplasia induced by lead nitrate, a potent mitogen, has also shown a peculiar sexual dimorphism in all phases of the proliferative process. In contrast with liver regeneration after partial hepatectomy, the mitogenic action of lead nitrate was less effective and was delayed in females as compared with males, by evaluating liver weight, protein accumulation, DNA synthesis and mitotic index. These results were also confirmed by the trend of liver regression by apoptosis. The apoptotic index was higher in males than in females. A prolonged administration of an excess of choline has partially filled these sexual differences, since choline has moved, in females, all the observed parameters (liver weight, protein accumulation, DNA synthesis, mitotic and apoptotic indexes) to values closer to those observed in males.