Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10159

RESEARCH ARTICLE

Study of DNA synthesis and mitotic activity of hepatocytes and itsrelation to angiogenesis in hepatectomised tumour bearing miceLaura B. Andrini1*, Marcela N. García1, Ana María Inda1,2 and Ana Lía Errecalde1

1 Cátedra de Citología, Histología y Embriología “A,”, Facultad de Ciencias M�edicas, UNLP, La Plata, Argentina2 CIC, Provincia de Buenos Aires, La Plata, Argentina

Abstract

Partial hepatectomy (PH) alters serum concentrations of substances involved in cellular proliferation, leading to thecompensatory liver hyperplasia. Furthermore, angiogenesis is mainly stimulated by vascular endothelial growth factor (VEGF)and is a fundamental requirement either in liver regeneration or in tumours growth. This study looks at the expression of VEGF,DNA synthesis (DNAs) and mitotic activity (MA) in hepatectomised (H) and hepatectomised-tumour bearing (HTB) micethroughout a 24 h period. Adult male mice were sacrificed every 4 h from 26 to 50 h post-hepatectomy. Hmice show a circadianrhythm in VEGF expressionwith a maximum value of 2.6� 0.1 at 08/46 h of day/hours posthepatectomy (HD/HPH); in DNAs,the maximum value was 3.4� 0.3 at 16/30 (HD/HPH) and in MA it was 2.3� 0.01 at 12/50 (HD/HPH). In HTB animals thepeak of VEGF expression appears at 16/30 (HD/HPH) with a maximum value of 3.7� 0.1, the peak of DNAs was at 00/38 (HD/HPH) with a value of 4.6� 0.3 and the maximum value of MA of 08/46 (HD/HPH) with a value of 3.01� 0.3. We can concludethat the presence of the tumour induces modifications in the intensity and the temporal distribution of the circadian curves ofVEGF expression, DNAs and MA of hepatectomised animals.

Keywords: cancer; cell cycle; growth factors; immunochemistry; liver/hepatocytes

Introduction

Partial hepatectomy (PH) alters the serum concentrations ofsubstances involved in cellular proliferation, leading to thecompensatory liver hyperplasia (Fausto et al., 2012). In liverregeneration, the development of new capillary blood vesselsby sprouting from existent microvessels, is a fundamentalrequirement for cellular proliferation (Furnus et al., 2003;Ribatti and Crivellato, 2012). These processes produce asignificant increase in plasma levels of a number of growthfactors (Michalopoulos, 2011). Some of these factors can“cross-talk” between the growing tumour and the regenerat-ing liver (Andrini et al., 2010). Particularly in mice, andunder controlled conditions of light-dark standardisation,this hyperplasia process involves a circadian rhythm ofhepatocytes, in both MA and DNAs, of the hepatocytes(Garcia et al., 2008).

On the other hand, angiogenesis is mainly promoted byVEGF, the most potent and specific growth factor for bothangiogenesis and vasculogenesis (Dufour and Clavien, 2010),

the mRNA of which is expressed, after PH, in bothhepatocytes and non-parenchymal cells (Mochidaet al., 1996).

The effect of hepatectomy on the proliferation and growthof the different tumours is well known (Ono et al., 1986; Sunet al., 1996; Altun and Ozalpan, 2004). Many cellularneoplastic populations have circadian variations in their MA,such as primary human and murine tumours (Colomboet al., 2000), hepatomas and hepatocellular carcinomas andin DNAs of some hepatomas and hepatocellular carcinomas(Garcia et al., 2008). This is very important not only in cellbiology, but in clinical oncology because hepatectomy is theprimary treatment for hepatic tumours (Wood andHrushesky, 1996). Tumour-based signals, acting upon thetumour bed stroma, could mimic normal signalling, and,among others things, produce a reciprocal cue beneficial tothe proliferating tumour cell (West and Van de Rjin, 2007).The interaction between tumour cells and their supportivestroma plays a crucial role in tumour development andprogression (Zhang and Zhao, 2006), in which angiogenesis

�Corresponding author: e-mail: landrini@med.unlp.edu.ar

1233Cell Biol Int 37 (2013) 1233–1237 � 2013 International Federation for Cell Biology

is the essential process since solid tumours cannot growthbeyond 1–2mm in diameter without neovascularization(Folkman et al., 1989).

We have analysed MA, DNA and VEGF expression inhepatocytes from hepatectomised and hepatectomised-tumour bearing mice through a circadian cycle, with theobjective of knowing whether carrying a solid tumourchanges the normal parameters of cell proliferation involvedin hepatic regeneration, since hepatic surgery (partialextirpation of the organ) is the primary treatment forprimary liver tumours.

Materials and methods

Animals

Two hundred and fifty-two C3H/S adult (90 days old) malemice were used. Conditions concerning animal managementfully respected the policy and mandates of the Guide forthe Care and Use of Laboratory Animal Research Council.They were given the following standardization conditions:caged singly with water and food available ad libitum in anambient with temperature maintained at 22� 28C, withalternating light and dark periods restricted to 12 heach with illumination by fluorescent lamps beginning at06:00 h.

Experimental groups

The animals were divided in three experimental groups:Group I: hepatectomised and hepatectomised-tumourbearing mice will be used to study the expression ofVEGF; Group II: hepatectomised and hepatectomised-tumour bearing mice for the study of DNAs; and GroupIII: hepatectomised and hepatectomised-tumour bearingmice for the study of MA.

After an appropriate period of synchronization (15 days),C3H/S-histocompatible ES2 hepatoma was grafted into thesubcutaneous tissue of each animal´s flank for the tumour-bearing mice of each group. This fast growing undifferenti-ated hepatocellular carcinoma is maintained in our labora-tory by subcutaneous serial transplant in male adult mice.After 15 days all the mice (with and without tumour) werepartially hepatectomised (70%) at 10:00 h. The tumour-bearing mice had similar sized tumours at the end time-point,�1.8� 0.2 cm. Each group was divided into seven lotsof 5–7 animals each. The mice were housed for further2 weeks under standardization conditions before the lots wereseparated. The animals were killed at 12:00/26, 16:00/30,20:00/34, 00:00/38, 04:00/42, 08:00/46, 12:00/50 (HD/HPH).Samples were removed promptly and processed for theanalysis of MA and immunohistochemistry (determinationof DNAs and study VEGF expression).

BrdU immunoreaction

The mice were injected i.p. with BrdU (Sigma) at 50mg/kgbody weight 1 h before decapitation. Liver samples forimmunochemistry obtained at necropsy were fixed andprocessed. Sections were deparaffinised in xylene, rehydratedthrough graded alcohols and washed in Tris buffer saline(TBS) pH 7.4. After blocking endogenous peroxidase with3% H2O2 for 15min, heat-induced epitope retrieval (HIER)was used (Shi et al., 1991; Cattoretti et al., 1993; Furnuset al., 2003). Briefly, slides were washed in TBS and placed in amicrowavable plastic container filled with 250mL of 0.01Mof citrate buffer pH 6 and irradiated in a householdmicrowave oven, with a rotating carousel, at 750W for10min (two cycles of 5min each). Distilled water was used torefill the container after each cycle to maintain the bufferconcentration. After microwaving the slides were washed inTBS and incubated with primary antibody (Bu 20a, 1/100;Dako) for 1 h at room temperature (23–258). Envision wasused as a detection system with 3,30-diaminobenzidine(Sigma) as the chromogen. The sections were lightlycounterstained with Mayer's hematoxylin. In each section,positive and negative nuclei were determined at 450�magnification counting �3000 cells per animal. DNAs indexwas calculated and expressed as labeled nuclei/100.

VEGF immunoreaction

Liver samples were fixed in 10% buffered formalin; 5mmsections were cut, placed on silanised (3-aminopropyltrie-thoxylane, Sigma) slides, and dried overnight. The sectionswere deparaffinised in xylane, rehydrated through gradedalcohols, and washed in Tris-buffered saline (TBS) at pH 7.4.EndogenousperoxidasewasblockedwithH2O23%for15min.Thesectionsweremicrowavedfor10min inbuffercitrate atpH6 before the slides were washed in TBS. The primary antibodyagainst VEGF-A (mouse monoclonal antibody; Santa CruzBiotechnology, CA,USA1:100 dilution)was incubated, for 1 hat roomtemperature.BoundprimaryantibodywasdetectedbyEnvision Systen (Dako) for 30min and the reaction wasdeveloped using 3,30-diaminobenzidine in TBS with 0.03%H2O2, and counterstained with Mayer's hematoxylin. Thepositive controlwasa sectionof liverwithahighVEGFcontent.VEGF staining was seen in the hepatocyte cytoplasm.

The liver sections were examinedmicroscopically under anoil-immersion objective (at 1500�) to score the total numberof labelled cells within a minimun of 3000 cells. Theexpression of VEGF was assessed according to the percentageof immunoreactive cells (quantitative analysis).

Mitotic activity

Colchicine (2mg/g body weight) was given i.p. to each animal4 h before being killed. Liver samples obtained during

Study of DNA synthesis and mitotic activity of hepatocytes L. B. Andrini et al.

1234 Cell Biol Int 37 (2013) 1233–1237 � 2013 International Federation for Cell Biology

necropsy were processed for histological analysis. Metaphasesand total nuclei were counted in each slide.MAwas expressedas colchicine metaphases/100 nuclei.

Statistical analysis

The results are expressed as mean� standard error (SE).Differences among experimental groups were analysed withANOVA and Student-Keuls Multiple Comparisons test andTuckey as a post-test, with P< 0.05 being consideredsignificant.

Results

Hmice show a circadian rhythm in their proliferation. VEGFexpression gave a maximum value of 2.6� 0.1 at 08/46 (HD/HPH) and a minimum value of 1.6� 0.3 at 12/50 (HD/HPH;Table 1). DNAs gave a maximum value of 3.4� 0.3 at 16/30(HD/HPH) and a minimum value of 1.8� 0.2 (Table 2).Finally, MA gave a maximum value of 2.3� 0.01 at 12/50(HD/HPH) and a minimum value of 0.1� 0.02 at 20/34 and04/42 (HD/HPH; Table 3).

In HTB animals the above parameters were altered. VEGFexpression had maximum value of 3.7� 0.1 at 16/30 (HD/HPH) and a minimum value of 1.2� 0.2 (Table 1). DNAsgave a maximum value of 4.6� 0.3 at 00/38 (HD/HPH;Table 2). Finally, MA had a maximum value of 3.01� 0.3 at08/46 (HD/HPH) and a minimum value of 0.05� 0.05 at 12/26 (HD/HPH; Table 3).

Discussion and conclusions

The liver has a remarkable capacity to proliferate after a PH,and can regulate precisely its growth and mass to adjust itssize, which is due to the great replicative capacity of thehepatocytes capable of repopulating the liver (Fausto

et al., 2012). For it to take place, it is necessary thatangiogenesis occurs, which involves endothelial cell migra-tion, capillary bredding, neovascular remodelling, in additionto endothelial cell proliferation (Ribatti and Crivellato, 2012).In this process, VEGF and its receptors represent one of thebest-validated signalling pathways (Ferrara, 2010). Severalauthors (Kraizer et al., 2001; Bockhorn et al., 2007;Michalopoulos, 2011), showed that VEGF is one of themost potent angiogenic factors that plays an important roleduring liver regeneration in mice. Our results show that thehighest expression of VEGF in mice hepatectomised occurswithin 46 h posthepatectomy; cf. Kraizer et al. (2001) whoshowed an increase after PH in the expression of VEGFmRNA in hepatocytes at 24 and 72 h after the surgery.Moreover, Shimizu et al (Shimizu 2001) and Michalopoulos

Table 1 Vascular Endothelial Growth Factor expression in twohepatectomizedgroups: controls and tumour-bearingmice, along acircadian period

Lot HD/HPH H (X� ES) n HTB (X� ES) n P

1 12/26 1.8� 0.2 6 2.1� 0.1 6 ns

2 16/30 1.9� 0.2 5 3.7� 0.1 6 0.0013 20/34 1.7� 0.1 6 2.2� 0.2 6 ns

4 00/38 1.9� 0.3 6 1.2� 0.2 5 ns

5 04/42 2� 0.1 6 2� 0.2 5 ns

6 08/46 2.6� 0.1 6 1.8� 0.1 6 0.057 12/50 1.6� 0.3 5 1.7� 0.2 6 ns

X 1.9� 0.1 2.1� 0.1

H, hepatectomised mice group (controls); HTB, hepatectomisedtumour-bearing mice group; n, number of animals; HD/HPH, hourof day/hour posthepatectomy; X, mean; P, probability.H: Lots 6–7: P< 0.05.HTB: Lots 2-1, 3, 4, 6 and 7: P< 0.001.

Table 2 DNA synthesis in two hepatectomized groups: controls andtumour-bearing mice, over a circadian period

Lot HD/HPH H (X� ES) n HTB (X� ES) n P

1 12/26 2.4� 0.1 5 1.6� 0.1 6 ns

2 16/30 3.4� 0.3 6 1.1� 0.3 6 0.013 20/34 3.3� 0.4 6 2.8� 0.4 5 ns

4 00/38 1.8� 0.2 6 4.6� 0.3 6 0.0015 04/42 2.5� 0.4 5 2.9� 0.3 5 ns

6 08/46 2.1� 0.1 6 2.4� 0.2 5 ns

7 12/50 3.2� 0.1 6 2.3� 0.3 6 ns

X 2.7� 0.2 2.5� 0.4

H, hepatectomisedmice group (controls); HTB, hepatectomised andtumour-bearing mice group; n, number of animals; HD/HPH, hourof day/hour posthepatectomy; X, mean; P, probability.H: Lots 2–4: P< 0.01 and 2–6: P< 0.05.HTB: 4-1, 2, 5, 6 and 7: P< 0.001.

Table 3 Mitotic activity in two hepatectomised groups: controlsand tumour-bearing mice, over a circadian period

Lot HD/HPH H (X� ES) n HTB (X� ES) n P

1 12/26 0.2� 0.1 5 0.05� 0.05 5 ns

2 16/30 0.4� 0.008 5 0.3� 0.1 5 ns

3 20/34 0.1� 0.02 5 0.1� 0.06 5 ns

4 00/38 0.2� 0.02 5 0.1� 0.1 5 ns

5 04/42 0.1� 0.02 5 0.6� 0.1 5 ns

6 08/46 0.8� 0.2 6 3.01� 0.3 6 0.0017 12/50 2.3� 0.01 6 2.1� 0.3 6 ns

X 0.6� 0.3 0.9� 0.4

H, hepatectomisedmice group (controls); HTB, hepatectomised andtumour-bearing mice group; n, number of animals; HD/HPH, hourof day/hour posthepatectomy; X, mean; P, probability.H: 7-3 and 5: P< 0.001.HTB: 6-1, 2, 3, 4 and 5: P< 0.001.

L. B. Andrini et al. Study of DNA synthesis and mitotic activity of hepatocytes

1235Cell Biol Int 37 (2013) 1233–1237 � 2013 International Federation for Cell Biology

et al (Michalopoulos 2011) argue that endothelial cellsproliferate the third day posthepatectomy. In ES2 tumour-bearing mice. VEGF expression was seen beforehand, withhigher values in comparison with that found in mice withouttumours.

DNAs of hepatocytes in regenerating mouse liver showscircadian variations (García et al., 2010). We detectedmaximum synthesis between 30 and 34 h posthepatectomy,in accordance with Michalopoulos (2011) who described theincrease of the starting DNAs in hepatectomised rats at 12 hafter surgery with a peak at 24 h. Fujito et al. (2001) foundincreased incorporation of BrdU in regenerating hepatocytesbetween 36 and 48 h after surgery. In contrast in tumour-bearing mice, we found that DNAs had very low values in thefirst 34 h, but a peak at 38 h posthepatectomy.

Compensatory hyperplasic response of hepatocytes afterhepatectomy show circadian variations, with maximummitotic activity in the resting phase of the animal. Thisrhythmicity is maintained until 10 days after surgery (Echaveet al., 1985). We found that the MA increased from 16/30 to12/50 HD/HPH in adult intact mice, and in hepatectomisedtumour-bearing mice the MA peak was higher and moreadvanced than the control.

Comparing the three main parameters during liverregeneration in intact adult mice, we have shown a sequenceof events starting with a peak of DNAs between 30 and 34 hposthepatectomy, followed by a peak of VEGF expression12 h later and ending with the MA peak at 50 h.

The presence of grafted hepatocellular carcinoma in adulthepatectomised mice produces detectable changes in thecircadian curves of these parameters. Thus, the maximumVEGF expression appears first at 30 h after hepatectomy, andwe detected the DNAs peak 8 h later and finally the MA peak8 h later.

We can conclude that tumoural cells from hepatocellularcarcinoma ES2 produces substances that are released into thegeneral circulation and recognise the hepatocytes ofregenerating liver, altering the normal sequence of events(rythmicity of DNAs, VEGF expression and MA) in adultC3HS mice.

Acknowledgements and funding

We thank Susana Formoso for her surgical technique andJaviera Marini for her technical assistance. This work wassupported by the Incentive Programme for Professors Researchof the National Education Ministry [code 11M/129].

Author contribution

Laura Andrini performed the VEGF and MA experimentsand wrote the manuscript. Marcela Garcia performed theDNA synthesis experiments. Ana María Inda wrote the

manuscript. Ana Lía Errecalde was the research projectdirector.

References

Altun S, Ozalpan A (2004) Interactive regeneration of liver and

growth of Ehrlich ascites tumor in mice. Biología, Bratislava 59:

375–82.

Andrini L, García M, Inda AM, Errecalde AL (2010) Circadian

rhythm of VEGF expression in the liver of hepatectomized

tumor bearing mice. Biol Rhythm Res 41: 57–62.

Bockhorn M, Schóllmann S, Opitz B, Sotiropoulos GC, Sheu SY,

Niehaus E, Trippler M, Frilling A, Broelsch CE, Schlaak JF

(2007) Vascular endothelial growth factor does not improve

liver regeneration and survival after 90% subtotal liver

resection. Hepatol Res 37: 353–9.

Cattoretti G, Pileri S, Parravicini C (1993) Antigen unmasking on

formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:

83–98.

Colombo LL, Mazzoni EO,Meiss RP (2000) The time of tumor cell

division and death depends on the site of growth. Oncol Rep 7:

1363–6.

Dufour J, Clavien P (2010) VEGF signaling. Signaling pathways in

liver diseases. 421–437. doi: 10.1007/978-3-642-00150_28

Echave L, Lanos J, Moreno F, Badrán A (1985) The growth of

hepatocytes and sinusoid litoral cells during liver regeneration.

Com Biol 4: 151–8.

Fausto N, Campbell JS, Riehle KJ (2012) Liver regeneration. J

Hepatol 57: 692–4.

Ferrara N (2010) Pathways mediating VEGF-independent tumor

angiogenesis. Cytokine Growth Factor Rev 21: 21–6.

Folkman J, Watson K, Ingiber D, Hanahan D (1989) Induction of

angiogenesis during the transition from hyperplasia to neopla-

sia. Nature 339: 58–61.

Fujito J, Marino M, Wada H, Jungbluth A, Mackrell P, Rivadeneira

D, Stapleton P, Daly J (2001) Effect of TNF gene depletion on

liver regeneration after partial hepatectomy in mice. Surgery

129: 48–54.

Furnus C, Inda A, Andrini L, García M, García L, Badrán A,

Errecalde A (2003) Chronobiology of the proliferative events

related to angiogenesis in mice liver regeneration after partial

hepatectomy. Cell Biol Int 27: 383–6.

Garcia M, Andrini L, Errecalde A, Cerutti R, Barbeito C (2008)

Changes in DNA circadian rythms in a transplantable

hepatocellular carcinoma alter and hepatectomy. Biol Rhythm

Res 39: 1–11.

García M, Andrini L, Inda AM, Ronderos J, Hijano J, Errecalde AL

(2010) Changes in VEGF expression and DNA synthesis in

hepatocytes from hepatectomized and tumour-bearing mice.

Cell Biol Int 34: 283–6.

Kraizer Y, Mawasi N, Seagal J, Paizi M, Assy N, Spira G (2001)

Vascular endothelial growth factor and angiopoietin in liver

regeneration. Biochem Biophys Res Commun 287: 209–15.

Michalopoulos G (2011) Liver regeneration: alternative epithelial

pathways. Int J Biochem Cell Biol 43: 173–9.

Study of DNA synthesis and mitotic activity of hepatocytes L. B. Andrini et al.

1236 Cell Biol Int 37 (2013) 1233–1237 � 2013 International Federation for Cell Biology

Mochida S, Ishikawa K, Inao M, Shibuya M, Fujiwara K (1996)

Increased expression of vascular endothelial growth factor and

its receptors, flt-1 and KDR/flk-1, in regenerating rat liver.

Biochem Biophys Res Commun 226(1): 176–9.

Ono M, Tanaka N, Orita K (1986) Complete regression of mouse

hepatoma transplanted after partial hepatectomy and the

immunological mechanism of such regression. Cancer Res 46:

5049–53.

Ribatti D, Crivellato E (2012) Sprouting angiogenesis, a

reappraisal. Dev Biol 372: 157–65.

Shi S, Key M, Kalra K (1991) Antigen retrieval in formalin-fixed,

paraffin-embedded tissues: an enhancement method for

immunohistochemical staining based on microwave oven

heating of tissue sections. J Histochem Citochem 39: 741–8.

Shimizu H,MiyazakiM,Wakabayashi Y,Mitsuhachi N, Kato A, Ito

H, Nakagawa K, Yoshidome H, Kataoka M, Nakajima N (2001)

Vascular endothelial growth factor secreted by replicating

hepatocytes induces sinusoidal endothelial cell proliferation

during regeneration after hepatectomy in rats. J Hepatol 34:

683–9.

Sun J, Toshinori I, Zhang F (1996) Enhancement of tumor growth

after partial hepatectomy and blood transfusion. Chung hua

Shung Liu Tsa Chic 18: 113–5.

West RB, Van de Rjin M (2007) Experimental approaches to the

study of cancer stroma interactions: recent findings suggest a

pivotal role for stroma in carcinogenesis. Lab Invest 87: 967–70.

Wood P, HrusheskyW (1996) Circadian timing of cancer chemotherapy.En: critical reviews in eukariotic gene expression. 2nd edition.

Baltimore: Willims and Wilkins, 177–202.

Zhang L, Zhao Z Ru G, Ma J, (2006) Correlative studies on uPA

mRNA and uPAR mRNA expression with vascular endothelial

growth factor, microvessel density, progression and survival

time of patiens with gastric cancer. World J Gastroenterol 12:

3970–6.

Received 17 April 2013; accepted 4 July 2013.Final version published online 19 August 2013.

L. B. Andrini et al. Study of DNA synthesis and mitotic activity of hepatocytes

1237Cell Biol Int 37 (2013) 1233–1237 � 2013 International Federation for Cell Biology