REVIEW ARTICLE

STUDYING LIVER REGENERATION BY MEANS OF MOLECULAR BIOLOGY:

HOW FAR WE ARE IN INTERPRETING THE FINDINGS?
David Rychtrmoc1,2, Antonín Libra2, Martin Bunček2, Tomáš Garnol1, Zuzana Červinková1

Charles University in Prague, Faculty of Medicine in Hradec Králové, Czech Republic: Department of Physiology1;
GENERI BIOTECH Ltd., Hradec Králové, Czech Republic2

Summary: Liver regeneration in mammals is a unique phenomenon attracting scientific interest for decades. It is a valu-
able model for basic biology research of cell cycle control as well as for clinically oriented studies of wide and heteroge-
neous group of liver diseases. This article provides a concise review of current knowledge about the liver regeneration,
focusing mainly on rat partial hepatectomy model. The three main recognized phases of the regenerative response are de-
scribed. The article also summarizes history of molecular biology approaches to the topic and finally comments on
obstacles in interpreting the data obtained from large scale microarray-based gene expression analyses.

Key words: Liver Regeneration; Hepatectomy; Gene Expression; Microarray, Hepatocytes; Rat

Introduction Concerning the structure the liver is an organ with

a complex architecture, with several cell populations taking
The liver regeneration attracts scientific attention for part. The parenchymal cells – hepatocytes build up sixty
decades as a unique phenomenon in mammalian physiolo- per cent of the total cell count and eighty per cent of the
gy. The interest does not decline in recent years. It rather overall organ volume. The remaining twenty per cent is
grows as its relevancy to clinical situations and discovery shared by lining cells of liver sinusoids and several types of
potential for basic research are well recognised. immune system cells, Ito cells and epithelial cells of intra-
New methods employed in this field yield important and hepatic bile ducts. Kupffer cells and dendritic cells belong
useful knowledge which in turn shapes design of further stu- among the resident immune system cells, while various
dies. The literature dealing with liver regeneration is replete subpopulations of lymphocytes rank among the migrating
and arises from various scientific branches including but not ones. Ito cells, called also stellate (or fat-storing) can pro-
limited to physiology, genomics, radiology and imaging dis- duce several paracrine factors and thus play a regulatory
ciplines, tissue engineering and liver surgery and trans- role in extracellular matrix remodelling and eventual deve-
plantation fields. The aim of this article is to provide a brief lopment of liver fibrosis (28, 37).
review of current understanding of the mechanisms of liver Basically, the liver parenchyma is organised into hexa-
regeneration, list molecular biology approaches employed in gonal lobules each having a centrally located hepatic vein,
this field and to bring a discussion of challenges arising terminal branch of systemic venous vasculature draining
when interpreting the results of recent microarray gene ex- blood into the inferior vena cava. Each structural lobule is
pression analyses. surrounded by usually six peripherally located portal spaces,
containing mainly triads of following structures: terminal
On the normal architecture and function branch of portal vein, terminal branch of hepatic artery and
of the liver interlobular bile duct. Besides these lymphatic vessels, nerves
and small amount of interstitial connective tissue can be
The liver is a vital organ performing plenty of essential found in portal spaces (Fig. 1). Only 3 % of liver volume is
body functions. It synthesizes and secretes plasma proteins constituted by extracellular matrix (ECM), making the liver
and majority of clotting factors; maintains blood glucose highly cell dense organ.
and ammonia levels critical for central nervous system Despite its small proportion the ECM can importantly
function. Creation of bile and urea and many more roles in influence the signal transduction by binding or release of
intermediate metabolism and elimination of both endoge- regulatory substances either in active or inactive form.
nous and exogenous substances make liver the main deto- ECM compounds can even perform cleavage of the humo-
xifying organ of the body. ral factors to increase or limit their function (54).

ACTA MEDICA (Hradec Králové) 2009;52(3):91–99 91

 tion feature is seen in all vertebrates from fish to humans
(38).
Liver functions can not be omitted during the regenera-
tion. Hepatocytes do not dedifferentiate during the regene-
ration and are capable of readily bearing increased functional
demands after loss of part of the organ volume (5, 13). It is
truly amazing that the regenerating liver despite the re-
duced mass serve the rest of the body by all of its homeo-
static functions (38).
The minimal volume of the liver remnant capable of
Fig. 1: Simplified representation of liver parenchyma mic- such functioning and simultaneous recovery from an insult
roarchitecture. A) View of several neighbouring structural has been subject to research and both in animals and hu-
lobules, each having a centrally located vein. Triads of mans survivable 90 per cent liver resections were reported
structures stand for portal triads in each corner of the app- (16, 49). In such a large scale hepatectomies additional
roximately hexagonal lobule. B) Closer look at the single lo- measures are employed to increase the survival rate. These
bule structure. Portal triads are again simply represented in include high energy diets or portal vein arterialization in ex-
its corners. We see also several sinusoids, spanning from perimental and clinical settings (9, 40, 42).
the terminal branches of portal vein to the central vein. The liver regeneration after partial hepatectomy is not
Individual hepatocytes are shown on the left, while the only a biological model for study of tissue homeostasis but
right part of the picture implies the spatial organisation of it may also bring better insight into the regeneration of the
the lobule. For details see text. donor liver remnant in the living donor liver transplanta-
tion. Surgeons could then provide safer care for donors who
Functional division of the liver parenchyma follows dif- are preoperatively healthy volunteers and harm to them
ferent principles, main factor being the quality of blood should be avoided or minimised (24). Donor complications
supply to the hepatocytes. From this point of view portal are nowadays reported with varying methodology, which
triads are central structures to the functional hepatic acini. leads to largely different results among the transplantation
Each acinus has three zones numbered I to III from the centres (1). Other clinically relevant settings are liver resec-
centre to the periphery with oxygen and nutrients levels de- tions due to tumour metastases or liver injury. Developers
creasing with the distance from the portal triad (48, 55). of artificial liver support devices also seek knowledge about
The differences in the characteristics of supplied blood are the mechanisms of regeneration in effort to increase their
responsible for so called functional heterogeneity of hepa- efficiency (5, 43).
tocytes among the zones of acini, impacting the scope of It is worth emphasizing that unless specifically pre-
metabolic activities and susceptibility or resistance to oxy- vented, the liver volume restoration is accomplished by pro-
gen or nutrient deprivation or chemical intoxication. liferation of mature parenchymal cells – hepatocytes, unlike
wound repair seen in skin or small intestine (38, 56). A re-
Importance and description of the liver cord number of 12 sequential hepatectomies performed in
regenerative capacity rats without failure of the regenerative potential of the liver
was reported (51). Overturf and colleagues (1997) have
Referring to liver regenerative capacity would be in- shown even more striking results with adult mice hepato-
complete without mentioning the ancient Greek myth of cytes. Increase in telomerase activity may contribute to this
Prometheus, who brought people the secret of fire from the almost endless capacity of hepatocytes to divide in vivo.
divine seat mount Olympus and was punished by chaining Moderate (up to twofold) increase in telomerase activity
to a rock. Every day an eagle arrived to batten on his liver, was found in regenerating rat hepatocytes, but there is a de-
which amazingly regrew its volume again. tectable telomerase activity in quiescent rodent livers which
The liver is a central organ in above mentioned inevit- is not the case in humans (17). A study in pig model, which
able functions and at the same time it is due to its localisa- better fits human telomerase biology with no activity in
tion first exposed to antigenic and toxic substances ingested quiescent livers, revealed even fivefold increase in telo-
by an organism. These enter the liver via its functional merase activity after two thirds partial hepatectomy (63).
blood supply by portal vein. Portal vein collects blood from
all other azygous abdominal organs, thus presenting vast In vivo models of liver regeneration
entrance gateway of exogenous substances (and endoge-
nous catabolites as well) into the internal environment of The need of in vivo models of liver regeneration is em-
an organism. It is hypothesized that the unique regenerative phasized by complexity of the process. Significance of the
capacity of the liver is an adaptation to this enormous load interactions among all liver cell populations is nowadays
with an intention to enable survival of a being, which for in- undoubted and the action of circulating modulators of ex-
stance tried a previously unknown feed. The liver regenera- trahepatic origin can not be simulated in vitro. Moreover

92
 months the recovery of original mass is complete. Trans-
plant clinician Manzarbeitia (2002) in clinical setting of
living donor liver transplantations witnesses almost com-
plete restoration of both the donors’ and recipients’ livers
in 6–8 weeks, with most of the recovery occurring in the
first two weeks. Older report by Jansen (1990) found out
maximal 75±2 % size recovery after a post partial hepatec-
tomy follow-up period of 50 days. Taken together it is not
simple to determine the time needed for ultimate recovery
in humans and the comprehensive discussion of this issue
is above the frame of this article.

Overview of liver regeneration principles

Response of the liver to a damaging insult which is not
immediately lethal to the organism is commonly called “re-
generation“ although it does not fulfil criteria for doing so.
Fig. 2: Animal models of liver regeneration. Adapted with The principle is different from what is seen in amphibians
kind permission from Ref. 43. TAA = thioacetamide; CCl4 regrowing lost limbs. It is not a new creation of lost tissue
= tetrachlormethane. in its original localisation. In fact, the remaining mature
cells undergo replication, and thus enlargement of unaf-
the regulation by body functional demands is only possible fected organ remnant underlies the volume recovery of the
in the whole organism (43). Studies in humans present liver. Therefore the phenomenon should be named com-
ethical obstacles and also the heterogeneity of conditions pensatory hyperplasia (12, 56). Nevertheless the term liver
leading to liver injury in clinical settings complicates eva- regeneration is already well established and is commonly
luation of such studies (44). used even in scholarly literature. That is why we keep the
Animal in vivo studies are a reasonable compromise in term also in this article.
terms of availability, affordability, reproducibility and stan- The liver regeneration occurs after a variety of different
dardised conducting and evaluation. But of course evidence insults including the mechanic tissue loss (injury, resec-
exist about differences in physiology of regeneration among tion) or necrosis of some portion of the liver cells without
smaller and higher animals and situation in humans (43). reduction in organ volume (leading to functional deficien-
The already established animal models are summarised in cy) caused e.g. by ischemia, acute or chronic intoxication
Fig. 2. eventually by infection (typically viral hepatitis).
The time frame of liver regeneration depends on the It is generally accepted, that the liver regeneration pro-
kind of organ damage and its severity and extent. Rats or cess after the loss of functional mass consists of three fun-
mice are the most frequently used model organisms because damental phases: a) initiation or priming, progression of
the liver regeneration is especially rapid in small rodents. In the quiescent hepatocytes to repeated division, b) prolifera-
both species two thirds partial hepatectomy first introduced tive phase, restoring the liver tissue to its volume before an
by Higgins and Anderson in 1931 (19) is the prevalent in- insult and c) termination of growth and balancing the re-
tervention. The anatomical differences necessitate adjust- sultant size of the liver to just comply with functional de-
ment of surgical procedure for the actual species, but both mands of the organism (14).
techniques are widely spread. While rat model gives more
reproducible results, mice are valuable for the possible use Priming phase
of genetically engineered strains (13). The full organ size
restoration is reported within 5 to 10 days in most of the ro- Hepatocytes the most numerous liver cells are long lived
dent studies. elements which rarely divide. Their replication rate is one
In humans reported times necessary for liver regenera- in twenty thousand (50). Under normal conditions hepa-
tion vary greatly among the articles. It is of course due to tocytes are irresponsive to growth stimuli. This changes
the different insults studied and varied methods used for amazingly after an appropriate triggering event, partial he-
measuring the organ size. But there is even no consensus patectomy being among the most efficient ones.
about the completeness of the response. While Michalopou- Other triggering events are necrosis after blunt injury,
los (2007) states that normal liver weight is re-established metabolic stress imposed e.g. by toxins, or any phenome-
in 8–15 days in humans, followed by a slow lobular reorga- non leading to disruption of intercellular contacts (10, 25,
nization taking place for several weeks (62), Court et al., 43) or digestion of the extracellular matrix (31).
(2002) states, that under normal circumstances, the liver This change from quiescent to cycling state is called
regeneration in humans is initiated within 3 days and by 6 priming and is generally ascribed to cytokine-dependent sig-

93
nalling pathways action in short time after an insult. It is During the proliferative phase almost all of the hepato-
concluded that the triggering factors must be in place faster cytes undergo mitosis. This percentage is approximately
than de novo proteosynthesis could occur and that they are 95 % in young rats and decreases to about 70 % in old ani-
substances already present in the body. One of the sug- mals (3, 46). The mechanism of replicative senescence of
gested early initiators is lipopolysaccharide (LPS), pro- the hepatocytes is not clarified so far. In humans there are
duced by the gut flora and released from the intestines as less observations of such kind available, but in liver trans-
a consequence of deteriorated intestinal barrier due to sur- plantation grafts from younger donors generally carry bet-
gical stress. LPS reaches liver with the portal blood and can ter outcome (67).
readily influence its cells. The LPS activates the non-pa- Not all the hepatocytes that passed the S phase finally
renchymal cells (including Kupffer cells and stellate cells) replicate. The proportion of binucleate cells within the liver
and increases their production of tumour necrosis factor increases, and some hepatocytes become polyploid but un-
alpha (TNFα) and interleukin 6 (IL-6) (56). Another sub- divided. The first mitotic wave concerning the vast majori-
stances acting as rapidly as LPS are activated complement ty of parenchymal cells is followed by a second one, about
compound C3a and C5a, showing that the response is not 48 hours after PH, involving smaller percentage of hepato-
isolated to the liver itself, but that the circulating factors cytes. In total there is about 1.6 cycles of replication in all
take part as well (35, 53). cells to fully restore the liver (56).
Cytokine signals including TNFα and IL-6 cause tran-
scription factors NFκB, STAT3, AP-1 and CEBPβ to bind Termination phase
DNA rapidly by means of their posttranslational modifica-
tions (6, 12). Subsequently, in 20 to 30 minutes after the Cessation of the regenerative response is the least eluci-
PH, derepressed transcription factors increase expression dated phase of the process. It is still not clear whether it
of so called immediate early genes. These include the pro- is onset of inhibitory agents or withdrawal of stimulatory
tooncogenes c-fos, c-jun, c-myc and c-met, the latter being substances that finally stops regeneration. The interplay
the HGF receptor (52). among the liver cell subpopulations and probable parti-
Cytokine mediated pathway is responsible for G0/G1 cipation of the recovered extracellular matrix complicate
transition, after which the action of growth factors is ine- revealing the mechanisms of observed precise liver size re-
vitable. Complete hepatic mitogens recognised in vivo and gulation. Nonparenchymal cells may mediate the inhibition
also in primary hepatocyte cultures are HGF, TGFα, and of regrowth during structural reorganisation of the liver
EGF. These allow cells to overcome the G1 restriction which comes after the volume regain is finished. Reap-
point and continue towards mitosis (32). pearing extracellular matrix may play an important role by
Extrahepatic body organs also have a role to play in re- renewed binding of pro-HGF (35).
action to liver injury. Cooperative priming signals come Mechanisms that link the replicative activity of hepato-
from the pancreas (insulin), duodenum or salivary glands cytes in the regenerating liver with body functional demands,
(EGF; epidermal growth factor), thyroid gland (T3, triiod- have been sought and mammalian target of rapamycin
thyronine) and adrenal glands (norepinephrine). These co- (mTOR) and its downstream effectors belong to proposed
mitogens help initiate the recovery of the liver (7, 8, 30, 56). candidates (11).
Factors with known inhibitory effects on hepatocyte
Proliferative phase proliferation in cultures such as TGF-β1 (in vivo normally
synthesized by Ito cells) have been subject of research.
Proliferative phase is characterised by mitotic waves of Some observations suggest that disappearance of (detect-
hepatic cells. The well obvious marker of the cell cycle pro- able) tissue TGF-β1 from periportal to pericentral region
gression is the rate of DNA synthesis observed to appear in of lobule enables progression of hepatocyte mitotic wave
a coordinated fashion in individual liver cell subpopula- in the same direction at the onset of regeneration. TGF-β1
tions. Hepatocytes reach the S phase first, with the DNA released in the plasma shortly after PH is probably inac-
synthesis beginning to rise 12 hours after the PH and peaking tivated by binding to α2 macroglobulin. Hepatocytes them-
at about 24 hours after the operation. The S phase in the selves are transiently resistant to mito-inhibitory effects
nonparenchymal cells occurs later; at 48 hours for Kupf- of TGF-β1 during the proliferative phase. After this re-
fer and biliary cells and around 96 hours for endothelial fractory period, TGF-β1 could play a role in ending the re-
cells (12, 56). After an insult causing necrosis or apopto- generation, however in transgenic mice overexpressing
sis of hepatocytes, the course of the cell priming is similar TGF-β1 regeneration is slowed down but it finally comple-
as after the PH, but the replicative waves are less coordi- tes (5, 37). Related TGFβ family members, such as activin,
nated. act similarly.
Functional heterogeneity of hepatocytes applies also to Several negative feedback loop interactions related to
the onset of DNA synthesis within the liver acini, starting priming phase stimuli were recognised. To name some of
around the portal vein (zone I) and gradually proceeding these: plasminogen activator inhibitor (PAI) induced by
towards the central vein (zone III). IL-6, blocks the HGF action by preventing the cleavage of

94
pro-HGF into an active HGF. Suppressor of cytokine sig- sion patterns, which is based on assumption that common
nalling-3 (SOCS3) upregulated downstream to IL-6, causes activation or suppression indicates functional relationships
downregulation of phosphorylated signal transducer and among transcripts. Much database searching necessarily
activator of transcription 3 (STAT3) ultimately terminating follows when interpreting the findings (15, 39, 59, 64).
the original IL-6 signal (56). Extensive use of public data repositories and bioinformatic
Apoptosis, as a possible mechanism correcting the re- platforms fosters their rapid development.
sultant size of the liver was studied in normal and IL-6 -/- Real time PCR gene expression analyses usually com-
mice, by Sakamoto et al (1999). This work suggested greater plement high throughput methods. Either as a confirmative
role of apoptosis in normal than in IL-6 deficient liver in approach for selected genes or as a preliminary method for
eliminating the superfluous hepatocytes generated by rege- more precise focusing of laborious and expensive advanced
neration. techniques. Moreover the real time PCR provides wider
linear dynamic range of transcript quantitation and thus re-
Molecular biology methods to study fines the results. Work by Cimica et al. (2007), who used se-
mechanisms of the liver regeneration rial analysis of gene expression (SAGE) for one post partial
hepatectomy interval based on previous real time PCR and
Given the long history of research interest in the liver immunostaining results, is a recent example of such combi-
regeneration, it is not surprising, that it has been studied by nation of methods.
molecular biology methods as well. Early works, in the Interesting novel approach was used by Juskeviciute et
1980’s, assessed the presence and relative abundance of the al. (2008), who were able to identify previously unknown
specific mRNAs by autoradiography measurements fol- transcription factors participating in liver regeneration,
lowing membrane-based hybridisation techniques like Nor- thanks to bioinformatic analysis of shared transcription fac-
thern blot and Dot blot (21, 29, 58). These approaches tor binding sites in the clustered microarray data.
were eventually complemented by histochemistry and cyto-
genetic methods (36). These works mostly dealt with seve- Interpreting recent microarray data
ral genes of interest focusing on narrowly specified aspects
of hepatocyte growth. Experiments by Taub and co-workers Majority of so far published papers is focused on early
outstand by the number of genes analysed and the time in- changes after the liver insult, based on the notion, that the
tervals studied (18). reactivation from quiescence and subsequent entry into the
Experiments using the genetically engineered mice S phase of the cell cycle is the most exceptional feature of
strains gained wider use in 1990’s. The ability to perma- liver regeneration model. Propagation of the proliferative
nently or transiently overexpress or suppress (knock out) response seems to follow the same principles as seen in
a gene of choice has enabled much progress in studying the other extrahepatic cells or tissues, making later phases of
involvement of individual factors or whole transcription regeneration less scientifically attractive. This general fact
pathways in the process (2, 13). applies to microarray-based analyses as well.
Advent of microarray platforms, introduced in 1995, al- As seen in Tab. 1, summarising several features of seven
lowed for wider scope of gene expression analyses. Simul- recently published studies using microarrays, the sample ac-
taneous gene expression analyses of thousands of genes quisition is usually denser in shorter intervals and becomes
raised a new challenge. It is now demanding to sort out ge- notably looser after 24 hours after the resection. Thus it
nes truly related to the observed functional or morpholo- would seem logical; that the molecular mechanisms under-
gical changes among many detected by high throughput lying the first 12 or 24 hours of liver regeneration should be
microarray experiments. Usually time course of expression well described and understood in good agreement among
alterations after the intervention is used as a criterion for the researchers. Unfortunately the situation is more com-
creating clusters, subgroups of genes with similar expres- plicated. Anyone trying to interpret the findings of large

Tab. 1: Recently published microarray-based works on liver regeneration and their sampling intervals.
First Model
PY Post partial hepatectomy sampling intervals
author organism
Mortensen 2008 pig 1 m 30 m 90 m 3h 4h 6h
Fukuhara 2003 rat 6h 12 h 18 h 24 h 48 h 72 h 168 h
Togo 2004 mouse 60 m 3h 6h 12 h 24 h
Xu 2005 rat 2h 4h 8 h 12 h 16 h 24 h 36 h 48 h 72 h 96 h 144 h
Shao 2007 rat 30 m 60 m 2h 4 h 6 h 8 h 12 h 24 h 36 h 54 h 66 h 72 h 120 h144 h168 h
Yokoya 2004 rat 60 m 6h 24 h 48 h 168 h
Xu 2007 rat 30 m 60 m 2h 4 h 6 h 8 h 12 h 16 h 18 h 24 h 30 h 36 h 42 h 48 h 54 h 60 h 66 h 72 h 96 h 120 h144 h168 h
PY = year of publication; m = minute(s); h = hours(s)

95
Tab. 2: Differentially expressed genes common to at least two of the three compared microarray-based studies recently
published on liver regeneration after partial hepatectomy in rodents.
Published in article
Gene name and its alternatives (including symbols)* – Ref. No.
64 47 66
Cytochrome P450, family 7, subfamily a, polypeptide 1, Cyp 7a1, P-450 cholesterol X X
7-alpha-hydroxylase, Cholesterol 7-alpha-monooxygenase
Connective tissue growth factor precursor (Connective tissue growth-related protein). Ctgf X X
Dual specificity protein phosphatase 1 (EC 3.1.3.48) (EC 3.1.3.16) (MAP kinase phosphatase 1) X X
(MKP-1) (Protein-tyrosine phosphatase CL100) (Protein-tyrosine phosphatase non-receptor
type 16).
Early growth response factor 1 (Egr1) (KROX-24 PROTEIN) (ZIF268) (NERVE GROWTH X X
FACTOR-INDUCED PROTEIN A) (NGFI-A)
Plasminogen activator inhibitor 1 precursor (PAI-1) (Endothelial plasminogen activator inhibitor) X X X
(PAI) Serine protease inhibitor 1(Serpin 1) (Serpine1)
Cytochrome P450 15-beta (Cyp2c12) X X
Alpha-2-macroglobulin precursor (Alpha-2-M). A2m X X X
Bile acid CoA:amino acid N-acyltransferase (EC 2.3.1.65) (BAT) (BACAT) (Glycine X X
N-choloyltransferase) (Kan-1) (Long-chain fatty-acyl-CoA hydrolase) (EC 3.1.2.2). (Baat)
(Hepatic) Flavin-containing monooxygenase 1 (Fmo1) X X
*found in Ensembl Genome Browser and Entrez Gene databases (23, 33)

scale gene expression studies, as these listed, soon encoun- should unravel the temporal pattern of their involvement.
ters several principal difficulties. Are we able to answer these questions then? Well, it is not
Firstly, the overlap between the results of microarray- rare that authors group their explored differentially expres-
based studies is commonly much smaller than would be sed genes into categories based on functional relationships
expected. As noted by van Bakel and Holstege (2008), com- (e.g. 57), but the problem again occurs when trying to clas-
paring independently obtained lists of differentially expres- sify genes from more studies together. Still, it is the uncer-
sed genes from several experiments on the same topic may tainty in describing the genes, what complicates this task.
end up in very limited number of shared matches. In the Fortunately, sophisticated and moreover user-friendly tools
model example shown in their work, three gene lists con- were developed to ease such an effort. What we strive to get
taining 115, 160 and 250 genes respectively, only 15 genes is the functional classification of genes and there are bioin-
were common to all the lists. We made a similar experience formatic instruments available to do that. One of the pos-
comparing three published gene lists from references 63, 47 sible ones is DAVID database accessible free of charge via
and 66 and yielding mere nine genes common to at least the internet (22; this work also lists some alternative tools
two of the three works (see Tab. 2). This is caused most for functional annotation and principles they utilize in its
probably by varied microarray platforms and methodology Supplementary Data 1).
of working with the data, e.g. different cut off values for fold With help of DAVID database we searched through gene
change in gene expression level. lists derived from the seven papers listed in Tab. 1. Doing the
Interpreting effort is moreover complicated by nonuni- same comparison among the gene lists „manually“ without
form way of presenting results, regarding the gene names, such an aid is obviously laborious and thus discouraging.
symbols or database annotations. Not having the raw data, To avoid that, we wanted to begin the work with well
reader of the papers is dependent on some of the compre- characterised background. This we derived from Gene
hensive databases such as Entrez Gene (33), Ensembl Ge- Ontology database (57). We chose the biological process
nome Browser (23) or Rat Genome Database (RGD) (60) GO:0006953~acute-phase response, surely involved in ear-
in finding the missing descriptors to decide whether the ly phases of liver regeneration after surgical resection. For
gene results from different studies do or do not overlap. The Rattus norvegicus species there were 23 gene products an-
authors list their gene results with various level of exact- notated under this term in GO database release 2009-09-24.
ness, not always using unambiguous names or codes like We consequently used these genes as a user defined Gene
Entrez Gene ID, Ensembl Gene ID or RGD Gene ID. List for upload into the DAVID database. There, using the
The most desired information from large scale studies is ID conversion tool, we were able to obtain unambiguous
not about several individual genes confirmed among thou- descriptors for all the 23 genes. We decided for conversion
sands under investigation, but about functional classes or into following four categories: RGD ID, GENE SYMBOL,
even better cellular pathways involved in the particular ENTREZ GENE ID and UNIGENE. Results of the con-
steps of liver regeneration. Time course experiments also version are summarized in the Tab. 3.

96
Tab. 3: Genes involved in acute phase response and their coverage in the seven compared microarray-based studies.
Ref No. 64 47 66 39 59 15 65
Gene Name RGD ID GENE ENTREZ UNIGENE
SYMBOL GENE ID
BILE ACID-COENZYME A: AMINO ACID 2190 BAAT 29725 RN.11129 X X X
N-ACYLTRANSFERASE
LIPOPOLYSACCHARIDE BINDING 61865 LBP 29469 RN.48863
PROTEIN
TRANSIENT RECEPTOR POTENTIAL 628841 TRPV1 83810 RN.3073
CATION CHANNEL, SUBFAMILY V,
MEMBER 1
TRANSFERRIN 3845 TF 24825 RN.91296
ENDOTHELIN RECEPTOR TYPE B 2536 EDNRB 50672 RN.11412
PROSTAGLANDIN E RECEPTOR 3 3435 PTGER3 24929 RN.10361 X
(SUBTYPE EP3)
INTERLEUKIN 1 BETA 2891 IL1B 24494 RN.9869
SIGNAL TRANSDUCER AND ACTIVATOR 3772 STAT3 25125 RN.10247 X
OF TRANSCRIPTION 3
ALPHA-2-MACROGLOBULIN 2004 A2M 24153 RN.780 X X X X
TUBEROUS SCLEROSIS 2 3908 TSC2 24855 RN.5875
PHOSPHOLIPASE A2, GROUP IVA 67366 PLA2G4A 24653 RN.10162
(CYTOSOLIC, CALCIUM-DEPENDENT)
TRANSFERRIN RECEPTOR 70488 TFRC 64678 RN.98672
SIMILAR TO SINGLE IG IL-1 RECEPTOR 1306732 SIGIRR 309106 RN.16525
RELATED PROTEIN
INSULIN 2 2916 INS2 24506 RN.989
CCAAT/ENHANCER BINDING PROTEIN 2326 CEBPA 24252 RN.22163
(C/EBP), ALPHA
EPH RECEPTOR A3 68389 EPHA3 29210 RN.10713
ARGININOSUCCINATE SYNTHETASE 2163 ASS 25698 RN.5078
CHEMOKINE (C-C) RECEPTOR 5 620596 CCR5 117029 RN.10736
INTERLEUKIN 1 RECEPTOR 621159 IL1RN 60582 RN.85806 X
ANTAGONIST
MACROPHAGE INFLAMMATORY 708446 CCR1 57301 RN.34673
PROTEIN-1 ALPHA RECEPTOR GENE
ALPHA-2-HS-GLYCOPROTEIN 2075 AHSG 25373 RN.32083
SIGNAL TRANSDUCER AND ACTIVATOR 3774 STAT5B 25126 RN.54486
OF TRANSCRIPTION 5B
CHEMOKINE (C-C MOTIF) LIGAND 5 69069 CCL5 81780 RN.8019

Thanks to this prerequisite, searching for the genes in- any of the Gene Ontology functional categories, allowing
volved in the acute phase response among all the seven large anyone interested to focus on the field of their study.
scale studies became significantly more efficient. Resultant
findings are also in the Tab. 3. Although there are again only Conclusion
scarce matches to the gene products we were looking for,
confirming small overlap between independent papers, we This article deals with liver regeneration, the unique abi-
want to emphasize the benefit of such interpreting proce- lity of the liver to recover from serious damages. The artic-
dure. It lies mainly in removing uncertainty from the gene le describes significance of this interdisciplinary study topic
specifications and thus allowing comparison of the results and summarizes current knowledge in the field. Particular
originally presented using different ways, e.g. only gene names attention is paid to molecular biology methods utilized in li-
or its symbols vs. giving the database descriptors which un- ver regeneration studies. Finally, authors provide a discus-
fortunately may turn obsolete from the time of publication. sion of possible obstacles in interpreting the recent findings
The latter being frequent impediment given the perpetually from microarray-based studies and propose their way of cir-
ongoing refinements and updates to the internet-based geno- cumventing them as a solution for others experiencing si-
mic databases. Finally, the similar approach may be used for milar difficulties.

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Received: 03/06/2009.
Accepted in revised form: 20/09/2009.

Corresponding author:

David Rychtrmoc, MD, Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Physiology,
Šimkova 870, 500 38 Hradec Králové 1, Czech Republic; e-mail: rychtrmocd@lfhk.cuni.cz

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