The burden of HIV-associated neurocognitive disorder (HAND) in post-HAART era: a multidisciplinary review of the literature.

OBJECTIVE: The purpose of the present multidisciplinary review is to give an updated insight into the most recent findings regarding the pathophysiology, diagnosis and therapeutics of HIV-associated neurocognitive disorder (HAND). MATERIALS AND METHODS: We performed a comprehensive search, through electronic databases (Pubmed - MEDLINE) and search engines (Google Scholar), of peer-reviewed publications (articles and reviews) and conferences proceedings on HAND pathophysiology, diagnosis, and therapy, from 1999 to 2016. RESULTS: It seems to be increasingly clear that neurodegeneration in HIV-1 affected patients is a multi-faceted disease involving numerous factors, from chronic inflammation to central nervous system (CNS) compartmentalization of HIV. Diagnosis of HAND may benefit from both laboratory analysis and advanced specific neuroimaging techniques. As regards HAND therapy, modified HAART combinations and simplification strategies have been tested, while novel exciting frontiers seem to involve the use of nanoparticles with the ability to cross the Blood-Brain Barrier (BBB). CONCLUSIONS: Albeit highly active antiretroviral therapy (HAART) allowed a major decrease in morbidity and mortality for AIDS patients, CNS involvement still represents a challenge in HIV patients even today, affecting up to 50% of patients with access to combination antiretroviral therapy (cART). Future studies will have to focus on CNS compartmentalization, drugs' ability to penetrate and suppress viral replication in this compartment, and on new approaches to reduce HIV-associated neuroinflammation.

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value.

BACKGROUND: The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. METHODS: DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. RESULTS: Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. CONCLUSION: DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC.

BACKGROUND AND AIMS: Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G(1)-S transition in corresponding lesions from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed. METHODS AND RESULTS: Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2-cyclin B1 complexes (implying the highest G(2)-M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding non-tumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients' length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines. CONCLUSIONS: FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.

Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition.

The different frequency of hepatocellular carcinoma (HCC) in humans at risk suggests a polygenic predisposition. However, detection of genetic variants is difficult in genetically heterogeneous human population. Studies on mouse and rat models identified 7 hepatocarcinogenesis susceptibility (Hcs) and 2 resistance (Hcr) loci in mice, and 7 Hcs and 9 Hcr loci in rats, controlling multiplicity and size of neoplastic liver lesions. Six liver neoplastic nodule remodeling (Lnnr) loci control number and volume of re-differentiating lesions in rat. A Hcs locus, with high phenotypic effects, and various epistatic gene-gene interactions were identified in rats, suggesting a genetic model of predisposition to hepatocarcinogenesis with different subset of low-penetrance genes, at play in different subsets of population, and a major locus. This model is in keeping with human HCC epidemiology. Several putative modifier genes in rodents, deregulated in HCC, are located in chromosomal segments syntenic to sites of chromosomal aberrations in humans, suggesting possible location of predisposing loci. Resistance to HCC is associated with lower genomic instability and downregulation of cell cycle key genes in preneoplastic and neoplastic lesions. p16(INK4A) upregulation occurs in susceptible and resistant rat lesions. p16(INK4A)-induced growth restraint was circumvented by Hsp90/Cdc37 chaperons and E2f4 nuclear export by Crm1 in susceptible, but not in resistant rats and human HCCs with better prognosis. Thus, protective mechanisms seem to be modulated by HCC modifiers, and differences in their efficiency influence the susceptibility to hepatocarcinogenesis and probably the prognosis of human HCC.

Frequent loss of heterozygosity at the Hcr1 (hepatocarcinogenesis resistance) locus on chromosome 10 in primary hepatocellular carcinomas from LFF1 rat strain.

Hepatocarcinogenesis sensitivity (Hcs1, 2) and resistance (Hcr1-3) loci have been identified by linkage analysis on rat chromosomes 7 and 1, and 10, 4, and 8, respectively. Cytogenetic studies documented deletions on chromosomes 3 and 6 of neoplastic rat hepatocytes. Hepatocellular carcinomas (HCCs) were produced in F1 hybrid rats between Long-Evans (LE) and Fisher 344 (F344) rats. Scanning of the above chromosomes for loss of heterozygosity (LOH) showed allelic imbalance (AI) at multiple regions on chromosomes 6, 7, and 10q. Detailed deletion mapping of chromosome 10 localized a putative suppressor Hcr1 gene to within a 3.2-cM interval flanked by D10Rat51 and D10Rat121. Two other distinct regions with frequent AIs were found inside the Hcr1 locus, at marker loci including DNaseI and Mrp genes, and in a segment including 4 consecutive markers (D10Rat64, D10Rat182, D10Rat113, D10Rat216). In 40% of HCCs, AI was seen at the p53 locus. AI on chromosome 7 occurred at the Hcs1 locus, where is located c-myc, which is amplified in HCCs, suggesting allelic gain. Most AIs occurred in poorly/moderately differentiated carcinomas, and a few events were seen in well-differentiated tumors on chromosomes 7 and 10. These data suggest that alteration of a cluster of oncosuppressor genes on 10q is important for HCC progression. The existence of AI on segments of rat chromosomes 6, 7, and 10, syntenic to chromosomal segments of human HCCs where chromosomal gains or deletions occur, suggests a commonality of some molecular events in the pathogenesis of HCCs in rats and humans. Our map provides information toward cloning putative oncosuppressor genes associated with this carcinoma.

5'-Methylthioadenosine administration prevents lipid peroxidation and fibrogenesis induced in rat liver by carbon-tetrachloride intoxication.

BACKGROUND: 5'-Methylthioadenosine (MTA), a product of S-adenosylmethionine (SAM) catabolism, could undergo oxidation by mono-oxygenases and auto-oxidation. MTA and SAM effects on oxidative liver injury were evaluated in CCl4-treated rats. METHODS: Male Wistar rats were killed 1-48 h after poisoning with a single intraperitoneal CCl4 dose (0.15 ml/100 g) or with the same dose twice a week for 14 weeks. Daily doses of MTA or SAM (384 micromol/kg), started 1 week before acute CCl4 administration or with chronic treatment, were continued up to the time of sacrifice. RESULTS: Acute and chronic CCl4 intoxication decreased MTA and, to a lesser extent, SAM and reduced glutathione (GSH) liver levels. MTA administration increased liver MTA without affecting SAM and GSH. SAM treatment caused complete/partial recovery of these compounds. MTA and, to a lesser extent, SAM prevented an increase in liver phospholipid hydroperoxides in acutely and chronically intoxicated rats and in prolyl hydroxylase activity and trichrome-positive areas in chronically treated rats. MTA prevented upregulation of Tgf-beta1, Collagen-alpha1 (I) and Tgf-alpha genes in liver of chronically intoxicated rats, and TGF-beta1-induced transdifferentiation to myofibroblasts and growth stimulation by platelet-derived growth factor-b of stellate cells in vitro. CONCLUSIONS: MTA and SAM protect against oxidative liver injury through partially different mechanisms.

Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies.

In this review, genetic changes known to occur in human and experimental animal hepatocarcinogenesis are evaluated comparatively, with the aim of identifying genes that could potentially be targets of new preventive and therapeutic strategies, albeit the fact that although a step-by-step analysis of the premalignant stages has been largely accomplished in experimental hepatocarcinogenesis, this goal is still elusive in the case of humans. Overexpression of several of the genes implicated in the MAPK signaling cascade and cell cycle control appears to be most likely responsible for initiated cells acquiring a proliferating phenotype that facilitates the accumulation of structural changes in additional genes, resulting in the generation of autonomously growing preneoplastic and neoplastic lesions. Several gene abnormalities seen in precancerous lesions of rodents also occur in human hepatocellular carcinomas, suggesting that at least some of them could be present also in human precancerous lesions. Furthermore, there are reports that epigenetic events, such as abnormal DNA methylation, may be critical in hepatocarcinogenesis. DNA hypomethylation is an early event, both in human and experimental hepatocarcinogenesis, and its role in the activation of various genes, has been postulated. In recent years, linkage analysis studies have led to the identification of susceptibility/resistance loci that influence the progression stage of hepatocarcinogenesis in mice and rats. The relevance of these findings, though, will depend on the identification of the genes, and on whether in humans there are genes ortholog with rodent's susceptibility/resistance genes. It is proposed that rodent hepatocarcinogenesis represents a promising model for the identification of genes implicated in the early stages of the process, and that many of these genes may represent key targets for the application of gene therapy in the prevention and treatment of liver cancer.

Implication of Bcl-2 family genes in basal and D-amphetamine-induced apoptosis in preneoplastic and neoplastic rat liver lesions.

Molecular mechanisms of basal and D-amphetamine (AMPH)-induced apoptosis were studied in rat liver nodules, 12 (N12) and 30 (N30) weeks after initiation, and in hepatocellular carcinoma (HCC) induced by diethylnitrosamine in rats subjected to resistant hepatocyte model. Basal apoptosis in hematoxylin/eosin- and propidium iodide-stained sections was higher in nodules and HCC than in normal livers. It sharply increased in all tissues 4 hours after AMPH treatment (10 mg/kg), and declined to basal levels at 8 to 12 hours in liver and N12, but remained high up to 18 hours in N30 and HCC. c-myc, Tgf-alpha, p53, and Bcl-X(S) messenger RNA (mRNA) levels were higher, and Bcl-2 mRNA was lower in N12 and/or N30 and HCC than in normal liver. Four hours after AMPH injection, increase in c-myc and decreases in Bcl-2 and Bcl-X(L) mRNAs occurred in all tissues, whereas p53, Bax, and Bcl-X(S) mRNAs increased in N30 and HCC. These changes disappeared in liver and N12 at 18 hours, but persisted in N30 and HCC. c-Myc, P53, Bcl-2, and Bax proteins in normal liver and HCC +/- AMPH showed similar patterns. Tgf-beta1, Tgf-beta-RIII, CD95, and CD95L mRNA levels underwent slight or no changes in any tissue +/- AMPH. Basal Hsp27 expression was high in nodules and HCC, and was stimulated by AMPH in liver and N12, but not in N30 and HCC. These data suggest a role of dysregulation of Bcl-2 family genes and, at least in atypical lesions, of p53 overexpression, in basal and AMPH-induced apoptosis in nodules and HCCs. Hsp27 does not appear to sufficiently protect atypical lesions against apoptosis.

Identification of genetic loci controlling hepatocarcinogenesis on rat chromosomes 7 and 10.

Neoplastic liver nodules and hepatocellular carcinomas (HCCs) were induced, by "resistant hepatocyte" model, 32 and 70 weeks after initiation with diethylnitrosamine, respectively, in F344 Brown Norway (BN), and (BNxF344)F1 rats. Nodule number/liver (N) did not significantly differ among rat strains, whereas nodule mean volume (V) and nodule volume fraction (VF) were higher in susceptible F344 than in resistant BN and BFF1 strains and were predictive of subsequent development of HCCs. Genomic scanning of 157 backcross BFF1xF344 rats with 190 polymorphic microsatellites, and linkage analysis, revealed two quantitative trait loci (QTL) on chromosomes 7 and 10, which showed significant linkage with VF, and two QTL on chromosomes 4 and 8, which showed suggestive linkage with V and VF. On the basis of phenotypic patterns of homozygous and heterozygous backcross progeny and of allelic distribution pattern, QTL on chromosomes 10, 8, and 4 were tentatively identified as resistance loci, and QTL on chromosome 7 was identified as susceptibility locus for rat hepatocarcinogenesis. An analysis of interactions allowed us to identify additional putative QTL on chromosomes 5 and 8 and suggested an additive effect of loci on chromosomes 10, 8, and 4 for VF and V. These data are the first to identify chromosomal regions containing putative susceptibility/resistance loci for rat hepatocarcinogenesis, which seems to be highly complex in terms of the number of genetic factors involved.

Correlation of c-myc overexpression and amplification with progression of preneoplastic liver lesions to malignancy in the poorly susceptible Wistar rat strain.

Persistent liver nodules (PNs) and hepatocellular carcinomas (HCCs) induced in F344 rats by the resistant hepatocyte (RH) model exhibit c-myc overexpression and amplification. The role of these changes in progression of PN was investigated in nodules with different propensities to evolve to HCC in resistant Wistar rats and, for comparison, in susceptible F344 rats. Initiation of rats with diethylnitrosamine was followed by selection with 2-acetylaminofluorene (AAF) plus partial hepatectomy (RH groups). Two additional Wistar rat groups received a second AAF treatment without (RH+AAF) and with a necrogenic dose of CCl4 (RH+AAF/CCl4) 15 d after selection. The number to liver ratio and volume of glutathione-s-transferase placental form-positive lesions were lower in the Wistar than the F344 RH groups 9 and 32 wk after initiation and increased after a second AAF cycle treatment with and without CCl4. DNA synthesis in glutathione-s-transferase placental form-positive lesions was low in Wistar RH group at 9 wk and was stimulated by additional AAF treatments. HCCs developed at 57-60 wk in F344 RH, Wistar RH+AAF, and RH+AAF/CCl4 rats. Tumor incidence and multiplicity were lower in RH+AAF rats than in RH+AAF/CCl4 and F344 rats. At 32 wk, PN exhibited c-myc overexpression that increased from RH to RH+AAF rats and to RH+AAF/CCl4 Wistar rats. This was associated with c-myc amplification in Wistar RH+AAF/CCl4 rats. These results showed correlation of c-myc overexpression and amplification with nodule propensity to progress to HCC in poorly susceptible Wistar rats and suggested a possible genetic mechanism for susceptibility to hepatocarcinogenesis. The experimental system used in this work may be a valuable tool for studies on molecular mechanisms underlying liver growth and tumorigenesis supported by c-myc overexpression.

Transferrin and transferrin receptor gene expression and iron uptake in hepatocellular carcinoma in the rat.

Iron plays an important role in cell growth and metabolism. In preneoplastic liver nodules, a rise in the number of transferrin receptors (Tf-R) is associated with decreased endocytosis of the Fe2-Tf/Tf-R complex. Because nodules are precursors of hepatocellular carcinoma (HCC), the question arises whether changes in iron uptake by nodules persist in HCC. Current work showed up-regulation of Tf messenger RNA (mRNA) production in preneoplastic nodules, 12 to 37 weeks after initiation, and down-regulation in atypical nodules (at 45 and 50 weeks) and HCCs, induced in rats by the "resistant hepatocyte" model. Tf-R gene expression increased in nodules and HCCs. Tf-R numbers increased, without changes in affinity constant, in HCC. Iron uptake was higher in HCC than in normal liver, 5 to 40 minutes after injection of 59Fe2-Tf, with preferential accumulation in cytosol of tumor cells and in microsomes of normal liver. Purification through Percoll gradient of mitochondria plus lysosomes allowed the identification in liver and HCC of an endosomal compartment sequestering injected 125I-Tf. This subfraction was not seen when 59Fe2-Tf was injected into rats, and 59Fe was found in particulate material of both tissues. Liver and HCC exhibited comparable basal activities of plasma membrane NADH oxidase, an enzyme involved in iron uptake and cell growth. Stimulation of this activity by Fe2-Tf was higher in HCC than in normal liver. These results indicate that Tf expression may be a marker of preneoplastic liver progression to malignancy. Differently from nodules, HCC may sequester relatively high iron amounts, necessary for fast growth, both through the endocytic pathway and the reduced form of nicotinamide adenine dinucleotide (NADH) oxidase system.

c-myc amplification in pre-malignant and malignant lesions induced in rat liver by the resistant hepatocyte model.

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the "resistant hepatocyte" protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the beta-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy.

The BN rat strain carries dominant hepatocarcinogen resistance loci.

The phylogenetically distant F344 and BN rat strains and their (BN x F344) F1 hybrids were compared for susceptibility to hepatocarcinogenesis using the 'resistant hepatocyte' model. Quantitative stereological analysis of frequency (number/liver) and size (mean volume and volume fraction) of placental form glutathione S-transferase (GST-P)-positive lesions was carried out at 8, 15 and 32 weeks after diethylnitrosamine initiation. The number/liver of GST-P-positive lesions at any time point was slightly higher in BN and (BN x F344) F1 rats than in F344 rats, but not statistically different. However, mean volume and volume fraction of GST-P positive lesions were much higher in F344 than in both BN and (BN x F344) F1 rats at any time point, with a difference of up to > 10-fold. GST-P-positive lesions exhibited a significantly higher labeling index and much lower remodeling in male F344 than in BN and (BN x F344) F1 rats. HCCs were present at 54-57 weeks after initiation in 77% of male F344 and in no (BN x F344) F1 rats and at 70 weeks HCCs were observed in 100% of male F344 and in 23% of (BN x F344) F1 rats. These results suggest that the BN rat strain is resistant to hepatocarcinogenesis and that its resistance is genetically transmitted as a dominant character to F1 hybrids of the BN strain with the F344 susceptible strain.

Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats.

S-Adenosyl-L-methionine (SAM) is a strong chemopreventive agent of rat liver carcinogenesis. Examination was made to determine whether inhibition by SAM of the development of preneoplastic liver lesions persists to SAM withdrawal in diethylnitrosamine-initiated F344 rats promoted with thiobenzamide (TB). The rats were subjected, 2 weeks after initiation, to 5 weeks feeding with a 0.1% TB diet followed by a TB-free diet for 6 weeks and then a second TB treatment for 3 weeks. SAM (384 micromol/kg/day) was injected i.m. during the first TB cycle (treatment A) or for 6 weeks after the first TB cycle (treatment B). Many gamma-glutamyltranspeptidase (GGT)-positive lesions developed in initiated rats after the first TB cycle. They decreased in number after TB withdrawal, while partial recovery of lesion number and a great increase in volume occurred after the second TB cycle. Liver ornithine decarboxylase (ODC) activity and c-myc and c-Ha-ras mRNAs increased during the TB cycles and returned to normal liver values after TB withdrawal. Number and size of GGT-positive lesions, DNA synthesis of GGT-positive cells, liver ODC activity and c-myc and c-Ha-ras mRNA levels decreased as a consequence of SAM treatment A. The recovery of these parameters, induced by a second TB cycle in rats not treated with SAM, was prevented by SAM treatment B. These results suggest that SAM causes a persistent decrease in growth capacity of preneoplastic liver lesions in rats subjected to a diethylnitrosamine/TB protocol.

Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity and gene expression by dehydroepiandrosterone in preneoplastic liver nodules.

Previous work has demonstrated that dehydroepiandrosterone (DHEA) strongly inhibits growth and de novo cholesterol (CH) biosynthesis in preneoplastic rat liver. Administration of a mixture of 4 ribo- or deoxyribonucleosides of adenine, guanine, cytosine and uracil/thymine, prevents growth inhibition but not inhibition of CH synthesis. The purpose of this paper was to identify the site of inhibition of CH synthesis by DHEA. Persistent nodules (PNs) were induced, in diethylnitrosamine-initiated male F344 rats, by 'resistant hepatocyte' protocol. Fifteen weeks after initiation, nodule bearing rats and normal controls received a diet containing 0.6% DHEA for 3 weeks. They were then killed. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and mRNA levels were 18- and 14-fold higher, respectively in nodules than in normal liver. DHEA strongly inhibited HMGR activity in both tissues in vivo, but had a slight effect on HMGR activity, when added in vitro to the reaction mixture for determination of this activity. In vivo DHEA treatment caused a 65% decrease in the level of HMGR mRNA in PNs, which, however, does not seem to completely account for the decrease in HMGR activity (83%). Low density lipoprotein receptor (LDL-R) mRNA level underwent a slight decrease in PNs, with respect to control liver, which did not lead to a significant decrease in 125I-LDL binding to LDL-R. DHEA treatment caused 30% and 24% increases in LDL-R expression and 125I-LDL binding, respectively, in nodules. These observations indicate that in addition to HMGR gene expression, increased influx of LDL into preneoplastic cells may contribute to the deregulation of mevalonate synthesis by DHEA. The observation that HMGR activity and gene expression were still 3- to 5-fold higher in PNs of DHEA-treated rats than in control liver, and previous findings of preneoplastic liver cell growth in the presence of relatively low CH synthesis, suggest that even relatively low levels of mevalonate are sufficient for the growth of preneoplastic liver cells.

Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.

Chemoprevention of liver carcinogenesis by S-adenosyl-L-methionine (SAM) was studied in F344 male rats. The rats were given 1,2-dimethylhydrazine (1,2-DMH) 2 HCl (100 mg/kg, i.p.) 18 h after two-thirds hepatectomy. One week later they were fed a semisynthetic basal diet containing 1% orotic acid (OA) for 29 weeks. At this time the rats were transferred to the basal semisynthetic diet and were killed 3 weeks later. SAM treatment (384 mumol/kg/day, i.m.), was started 1 week after 1,2-DMH and was continued up to the end of the experiment. Controls received solvent alone. SAM exerted an inhibitory effect on the induction of preneoplastic and neoplastic lesions. For example, nodules with diameters of 1-2 and 2-6 mm exhibited a decrease in both incidence and number per liver, while no such inhibitory effect was seen in the category of larger nodules. Furthermore, hepatocellular carcinoma (HCC) also exhibited a decrease in the SAM-treated group. The number/liver and incidence were 0.04 and 4.8% respectively in the SAM-treated group, compared to 0.38 and 37.8% in the control group. Microscopic examination showed the presence of well-differentiated carcinomas and atypical nodules in control rats, while only one small, well-differentiated tumor and one nodule with patterns of initial transformation were seen in SAM-treated rats. No patchy staining of glutathione-S-transferase, indicative of remodeling, was observed in nodules of both SAM-treated and control rats. Nodules and HCCs developing in SAM-treated rats exhibited a relatively high number of apoptotic bodies. Apoptotic bodies count showed 2.8- and 1.8-fold increases in nodules and HCCs of SAM-treated rats with respect to controls. These results indicate that SAM exerts a chemopreventive effect on hepatocarcinogenesis induced by the OA model. SAM seems to be more effective in inhibiting nodule to HCC progression than on the growth of nodule per se. The inhibitory effect is associated with an increase in cell loss by apoptosis in nodules and HCC.

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Effect of S-adenosyl-L-methionine on the development of preneoplastic foci and the activity of some carbohydrate metabolizing enzymes in the liver, during experimental hepatocarcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive foci and glutathione-S-transferase, placental (GST-P)-positive lesions occupied 36% and 54% of liver parenchyma, respectively, in Wistar rats 8 weeks after initiation with diethylnitrosamine, followed by selection. The administration of S-adenosyl-L-methionine (SAM, 384 mumol/kg/day) caused 77% and 42% falls in the percentage of GGT-positive and GST-P-positive lesions, respectively. There also occurred a 46% decrease in labeling index of GGT-positive foci, in SAM-treated rats. These changes were associated with decrease in liver pyruvate kinase (PK), lactate dehydrogenase and glycerol-3-phosphate dehydrogenase. SAM did not affect these enzymatic activities in normal and uninitiated controls, but it caused a consistent increase in initiated rats. Enolase, fructose-biphosphatase and malic enzyme (ME) activities increased in the liver of initiated rats. SAM did not modify significantly these enzymatic activities, either in control or in initiated rats. Glucose-6-phosphate dehydrogenase (G6PDH) was 113% higher in the liver of initiated rats than in uninitiated controls. SAM treatment did not significantly affect this enzymatic activity in uninitiated rats, but caused a great decrease in initiated ones. As expected, there occurred a marked rise in GGT activity in the liver of initiated rats, with respect to controls. SAM caused an increase in GGT activity in normal and uninitiated controls, but it caused a 77% fall in GGT activity in initiated rats, coupled with a 380% rise in remodeling of GGT-positive lesions. Histochemical determination of G6PDH and ME activities showed that in the absence of SAM many preneoplastic lesions expressed higher G6PDH and ME activities than surrounding liver. SAM did not affect ME-positive lesions, while it caused a decrease in the number of G6PDH-positive lesions. Immunohistochemical determination of PK activity, isoenzyme L, showed a decrease in GST-P-positive lesions. Many of these lesions were no longer recognizable as lesions expressing a low PK activity, in SAM-treated rats. However, a relatively small number of GST-P-positive lesions expressing a low PK activity were still present in these rats. These data suggest that glucose channelled into triacylglycerol and pyruvate synthesis decreases in rat liver, during the development of preneoplastic foci, while the production of reducing equivalents and pentose phosphates increases, thus favoring DNA synthesis and detoxification reactions. Decrease in DNA synthesis, in SAM-treated rats, is paralleled by a partial reversion of carbohydrate metabolic features to those present in normal liver.

Genomic abnormalities in hepatocarcinogenesis. Implications for a chemopreventive strategy.

Carcinogenesis is a complex process characterized by the cumulative activation of various oncogenes and the inactivation of suppressor genes. Epigenetic mechanisms are also involved. Mutational activation of ras family genes occurs in most spontaneous or carcinogen-induced liver tumors, in susceptible mice, and less frequently in preneoplastic lesions. This suggests a pathogenetic role of these changes in hepatic carcinogenesis, in the mouse. Overexpression of various growth-related genes occurs in preneoplastic tissue during rat liver carcinogenesis, but mutational activation of protooncogenes, notably of ras family genes, seems to be a late and rare event, while c-myc amplification is a late but frequent event in both rodent and human carcinogenesis. However, mutation of the suppressor p53 gene has been found in relatively early preneoplastic lesions in rat liver, and it may be frequently seen in human hepatocellular carcinomas. The possibility that this mutation is involved in the initiation stage of liver carcinogenesis is an attractive hypothesis which needs further evaluation. DNA hypomethylation is involved in carcinogenesis, but the mechanisms underlying this effect are still elusive. Hypomethylation of growth-related genes is associated with their overexpression and this could favor overgrowth of preneoplastic liver tissue. Decrease in S-adenosyl methionine/S-adenosylhomocysteine (SAM/SAH) ratio occurs in the liver of rats fed a methyl deficient diet, which is a carcinogenic treatment, and in preneoplastic liver tissue, developing in initiated/promoted rats fed an adequate diet. The role of low SAM/SAH ratio in carcinogenesis is substantiated by the tumor chemopreventive effect of lipotropic compounds. Treatment with exogenous SAM prevents the development of preneoplastic and neoplastic lesions in rat liver. This is associated with recovery of SAM/SAH ratio, DNA methylation and inhibition of growth-related gene expression. SAM effect on prenoplastic cell growth is abolished by 5-azacytidine, a hypomethylating agent, indicating the involvement of DNA methylation. The possibility that in SAM-treated rats, methylation and inhibition of the expression of growth-related genes is implicated in growth restraint is attractive and should be further evaluated. Modulation of rat liver carcinogenesis by influencing gene expression through DNA methylation or other epigenetic mechanisms could be a new approach to chemoprevention of these tumors.

l-5-formyltetrahydrofolate and l-5-methyltetrahydrofolate rescue in L1210 leukemia treated with high methotrexate doses.

Highly purified 5-l-methyltetrahydrofolate (m-THF) and 5-l-formyl-THF (f-THF) preparations were compared for rescuing from methotrexate (MTX) toxicity in DBA2 mice transplanted with L1210 leukemia. Mice received two doses of reduced folates (2 mg/kg, s.c.) 16 and 24 h after a single s.c. MTX dose. f-THF was 1.8 time more effective than m-THF in protecting tumor cells from MTX (800 mg/kg). This MTX dose caused a 57% fall in circulating polymorphonucleates, which was prevented by both reduced folates. Treatment with 800 mg/kg of MTX plus m-THF was 1.5 fold more effective than the same MTX dose plus f-THF in increasing survival time of tumor-bearing mice. These data suggest a higher selectivity and efficacy of l-m-THF with respect to l-f-THF in rescuing from MTX toxicity.