Integrative and comparative genomics analysis of early hepatocellular carcinoma differentiated from liver regeneration in young and old.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related deaths worldwide. It is often diagnosed at an advanced stage, and hence typically has a poor prognosis. To identify distinct molecular mechanisms for early HCC we developed a rat model of liver regeneration post-hepatectomy, as well as liver cells undergoing malignant transformation and compared them to normal liver using a microarray approach. Subsequently, we performed cross-species comparative analysis coupled with copy number alterations (CNA) of independent early human HCC microarray studies to facilitate the identification of critical regulatory modules conserved across species. RESULTS: We identified 35 signature genes conserved across species, and shared among different types of early human HCCs. Over 70% of signature genes were cancer-related, and more than 50% of the conserved genes were mapped to human genomic CNA regions. Functional annotation revealed genes already implicated in HCC, as well as novel genes which were not previously reported in liver tumors. A subset of differentially expressed genes was validated using quantitative RT-PCR. Concordance was also confirmed for a significant number of genes and pathways in five independent validation microarray datasets. Our results indicated alterations in a number of cancer related pathways, including p53, p38 MAPK, ERK/MAPK, PI3K/AKT, and TGF-beta signaling pathways, and potential critical regulatory role of MYC, ERBB2, HNF4A, and SMAD3 for early HCC transformation. CONCLUSIONS: The integrative analysis of transcriptional deregulation, genomic CNA and comparative cross species analysis brings new insights into the molecular profile of early hepatoma formation. This approach may lead to robust biomarkers for the detection of early human HCC.

Dietary lipoic acid supplementation can mimic or block the effect of dietary restriction on life span.

Dietary restriction feeding extends survival in a range of species but a detailed understanding of the underlying mechanism is lacking. There is interest therefore in identifying a more targeted approach to replicate this effect on survival. We report that in rats dietary supplementation with alpha-lipoic acid, has markedly differing effects on lifetime survival depending upon the dietary history of the animal. When animals are switched from DR feeding to ad libitum feeding with a diet supplemented with alpha-lipoic acid, the extended survival characteristic of DR feeding is maintained, even though the animals show accelerated growth. Conversely, switching from ad libitum feeding a diet supplemented with alpha-lipoic acid to DR feeding of the non-supplemented diet, blocks the normal effect of DR to extend survival, even after cessation of lipoic acid supplementation. Unlike the dynamic effect of switching between DR and ad libitum feeding with a non-supplemented diet where the subsequent survival trajectory is determined by the new feeding regime, lipoic acid fixes the survival trajectory to that established by the initial feeding regime. Ad libitum feeding a diet supplemented with lipoic acid can therefore act as mimetic of DR to extend survival.

High level expression of N-acetylgluosamine-6-O-sulfotransferase is characteristic of a subgroup of paediatric precursor-B acute lymphoblastic leukaemia.

Microarray analysis is a powerful technology, but its impact on routine diagnosis for the near future maybe in revealing individual genes, which are useful diagnostic markers. Recently microarray analysis has identified a novel subgroup of childhood precursor-B acute lymphoblastic leukaemia (ALL) from a unique gene expression profile of over 30 genes. We have evaluated the four most highly expressed genes from this profile, by quantitative real time RT-PCR, to determine whether any of these genes by itself could be useful as a diagnostic indicator. The levels of expression of N-acetylglucosamine-6-O-sulfotransferase (GN6ST), protein tyrosine phosphatase receptor M (PTPRmu), G protein-coupled receptor 49 (HG38) and KIAA1099 protein were determined in childhood precursor-B ALL samples from a cohort of 116 Indian patients. In nine cases, three or four of these genes exhibited very high expression levels, but only GN6ST was consistently over-expressed. We suggest that very high level expression of GN6ST is a useful diagnostic marker for a subgroup of previously unclassified ALL.

Genes, telomeres and mammalian ageing.

Although there appear to be several influences, which contribute to the ageing of mammals, the role of DNA appears to be pivotal. There is increasing evidence that oxidative damage is an important factor in producing mutations in genes, shortening telomeres and damaging mitochondrial DNA. Accumulation of mutations in genomic DNA could result in the gradual decline in cellular function, which is exhibited in a variety of tissues. The random nature of these mutations, could also offer an explanation for differences in the degree and time of onset of age-related changes, exhibited by different individuals. Shortening of telomeres, caused by oxidative damage or the end-replication problem, could result in the accumulation of post-mitotic cells in-vivo during ageing. This might impair certain aspects of physiology, such as wound healing. Mutation of mitochondrial DNA may also be important in causing loss of cells in post-mitotic tissues such as muscle or brain. In addition changes in the redox state during the life of an animal may alter transcription factor activities, leading to consistent changes in the gene expression profiles of mammalian tissues. The latter could explain consistent age-related changes that have been observed in cell structure and physiology. Although all of these mechanisms may make a contribution to ageing, it is likely that it is the interplay between them that produces the most prominent effects.

Induction of cell proliferation in old rat liver can reset certain gene expression levels characteristic of old liver to those associated with young liver.

During the past decade, it has become increasingly clear that consistent changes in the levels of expression of a small cohort of genes accompany the aging of mammalian tissues. In many cases, these changes have been shown to generate features that are characteristic of the senescent phenotype. Previously, a small pilot study indicated that some of these changes might be reversed in rat liver, if the liver cells became malignant and were proliferating. The present study has tested the hypothesis that inducing proliferation in old rat liver can reset the levels of expression of these age-related genes to that observed in young tissue. A microarray approach was used to identify genes that exhibited the greatest changes in their expression during aging. The levels of expression of these markers were then examined in transcriptomes of both proliferating hepatomas from old animals and old rat liver lobes that had regenerated after partial hepatectomy but were again quiescent. We have found evidence that over 20 % of the aging-related genes had their levels of expression reset to young levels by stimulating proliferation, even in cells that had undergone a limited number of cell cycles and then become quiescent again. Moreover, our network analysis indicated alterations in MAPK/ERK and Jun-N-terminal kinase pathways and the potential important role of PAX3, VCAN, ARRB2, NR1H2, and ITGA5 that may provide insights into mechanisms involved in longevity and regeneration that are distinct from cancer.

Increased expression of the S25 ribosomal protein gene occurs during ageing of the rat liver.

BACKGROUND: It appears that consistent changes in the levels of activity of a small cohort of genes (probably <1% of all active genes) occur in all mammalian cells during ageing. Identification of such genes could provide valuable insights into the ageing process. OBJECTIVE: We have studied age-related changes in gene expression profiles in the rat liver. One of the genes which exhibited clear and consistent increases in expression is characterised. METHODS: Analysis of the gene expression profile was carried out using a combination of an optimised form of differential display and single-strand conformational polymorphism (SSCP) gel analysis. Gene expression levels were quantified by Northern blot analysis. The gene's identity was determined by comparing its nucleotide sequence to DNA databases. RESULTS: During this investigation we observed one gene which exhibited an increase in expression in livers from young adult (6 months) to old adult (24 months) rats. The differential expression of this gene was confirmed by SSCP gel analysis and Northern blotting. Densitometry of the latter indicated that expression increased by 165% with age. Characterisation of the isolated polymerase chain reaction fragment demonstrated it to code for the ribosomal protein S25 (RPS25). CONCLUSIONS: This increase in RPS25 expression is further evidence that the composition of ribosomes may alter with age and as a result could have significant effects on protein synthesis.

Decline in the expression of C4 binding protein alpha-chain gene during ageing of the rat liver.

It appears that consistent changes in the levels of activity of a small cohort of genes (probably less than 1% of all active genes) occur in all mammalian cells during ageing. We have studied this phenomenon in rat liver using an optimised form of differential display. During this investigation we observed one gene which exhibited a decline in expression in livers from young adult (6 months) to aged adult (24 months) animals. The differential expression of this gene was confirmed by single strand conformational polymorphism (SSCP) gel analysis and Northern blotting. Densitometry of the latter indicated that there was a decline of 35% in its expression with age. Characterisation of the isolated PCR fragment demonstrated it to code for the alpha subchain of the complement 4 binding protein (C4BP). The C4BP is a key regulatory protein of the complement system and this observation therefore indicates that a decline in the efficiency of the complement system may be an important factor in the overall decline in immune function that has been observed during ageing.