PROCEEDINGS OF THE BALKAN SCIENTIFIC CONFERENCE OF BIOLOGYIN PLOVDIV (BULGARIA) FROM 19TH TILL 21ST OF MAY 2005

(EDS B. GRUEV, M. NIKOLOVA AND A. DONEV), 2005 (P. 34–40 )

BIOLOGICAL CHARACTERIZATION OF CELL LINESESTABLISHED FROM Mc29-VIRUS INDUCEDTRANSPLANTABLE CHICKEN HEPATOMA

R. Alexandrova1*, W. Tsenova2, P. Jordanova1

1Institute of Experimental Pathology and Parasitology, Bulgarian Academyof Sciences, Acad. Georgi Bonchev Str., Block 25, Sofia 1113, Bulgaria

rialexandrova@hotmail.com2Tzaritza Ioanna Hospital, 8 Bialo More Str., Sofia 1527, Bulgaria

ABSTRACT. The cell lines E7 and G9B4 were obtained after cloning of thepermanent cell line LSCC-SF-Mc29, established from a transplantable chickenhepatoma induced by the myelocytomatosis virus Mc29. It was found that these cellsdiffer from each other in morphology, karyotype, in vitro and in vivo growthproperties. The results obtained revealed some causal relationship between alterationsin chromosomal number, on one hand, and the tumorigenicity of the cells on theother. The correlation was – the higher percentage of the threeploidy, the lowertumorigenicity of the cells. Our data suggest that these cell lines could be consideredas a model for tumor heterogeneity investigations and could be useful in the attemptsto get a better understanding of the mechanisms involved in (virus-induced)cancerogenesis.

KEY WORDS. myelocytomatosis virus Mc29; virus-induced transplantable chickenhepatoma, cell lines, tumor heterogeneity

INTRODUCTIONDespite the advances in diagnosis and therapy, morbidity and mortality from cancercontinue to represent a major health burden. The main obstacle preventing successfultreatment of malignant neoplasms is the biological heterogeneity of tumor cells.Tumors and cell cultures established from them are generally composed ofsubpopulations of cells that differ in many characteristics such as morphology,growth rate, metastatic potential, karyotype, biochemical properties, sensitivity tochemotherapeutic agents and radiation etc. The isolation and comparative study ofsubpopulations/cell sublines derived from the same tumor/tumor cell line would help

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scientists to reveal the mechanisms of important biological phenomena like malignantproliferation, tumor progression, incidence of metastases, resistance to chemo- andradiotherapy (Heppner, 1984; Fleuren et al., 1995; Aabo, 1996; Heppner, Miller,1998; Alexandrova, 2001). That is why there exist a need to establish suitableexperimental tumor models for tumor heterogeneity investigations. We suggest thatthe transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29(LSCT-BPO) is a very promising candidate for this purpose, because: 1) It has a shortlatent period; 2) It can metastasize (low metastatic potential); 3) It shows an abilityfor spontaneous regression; 4) This is a virus (retrovirus) induced hepatoma. Thegreat part of the hepatomas that are currently in use have been originated fromgrowths induced by chemical carcinogens; 5) v-myc oncogene that is responsible forthe oncogenic potential of the myelocytomatosis virus Mc29 belongs to the mycfamily oncogenes (c-myc, N-myc, L-myc). The cellular myc protooncogenes areproved to be involved in ethiology and pathogenesis of different kinds of humancancer (Lapis et al., 1975; Mladenov, 1974; Nazerian, 1987; Nesbit et al., 1999).

The aim of the study presented here was to determine some of the mainbiological characteristics of cell lines established from a transplantable chickenhepatoma induced by the myelocytomatosis virus Mc29.

MATERIAL AND METHODSCell line establishment and cultivation. The permanent cell line LSCC-SF(Mc29)was established from a transplantable chicken hepatoma induced by themyelocytomatosis virus Mc29 in 15 I White Leghorn chicken. The tumor wascarefully excised, minced, and treated with a mixture of 0.05% trypsin (Gibco) –0.02% ethylendiamino tetraacetic (EDTA). The resulting cell suspension wascentrifuged for 10 min at 390 g. The cells were washed two times in serum-freeculture medium at the same conditions and pellet was resuspended in culturemedium. The cell sublines E7 and G9B4 were obtained after cloning was performedin 96-well microplates (Nunclon) with LSCC-SF(Mc29) cells seeded at aconcentration of 1 cell/wall. Cultures were monitored every day by inverted lightmicroscope to ensure that clones observed were the progeny of single cells. Theparental cell line as well as both sublines were grown as monolayer cultures in acombination of E-199 and Iscove’s modified Dulbeco’s media suplemented with 10%bovine serum and antibyotics. The cells were cultivated at 37oC in a CO2 incubator.Staining of cells. Cells grown on cover slips were fixed with methanol and stainedwith hematoxiline – eosine.Growth rate in vitro. For determination of cell doubling time, replicate culturedishes (35 nm) received inocula of 3, 5 and 10 x 104 cells/ml. Cells from three disheswere harvested separately each day during 5 day interval and counted with a Burker’scamera after staining with a solution of 0.1 % trypan blue. Doubling time wascalculated from the growth curves.Karyological analysis. Chomosome analysis of the initial cell line and the twosublines was performed according to the standard procedure. Briefly, the cells werearrested in methaphase with 0.02 µg/ml Colcemide (Ciba Pharmaceutical Co) for 2 h.

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Chromosome spreads were prepared by hypotonic treatment, fixation and Giemsastaining. Fifty well spread metaphases from each tumor cell line were analyzedfollowng the standard nomenclature for chicken chromosomes.Determination of tumorigenic potential in vivo. To investigate the ability of thecell lines investigated to induce tumor growth in vivo, the cells from each cell linewere resuspended in phosphate buffered saline (PBS) and injected s.c. into the kneeflexure of 7-8 day old 15I White Leghorn chickens (4 to 7 chickens in each group) inconcentration of 2.5, 5.0 and 7.5 x 106 viable cells/chicken. Animals were observedthree times per week for tumor growth by palpation.

RESULTSGrowth properties in vitro. Cultured at the same conditions the cell lines investigatedwere found to differ in their growth properties in vitro. The doubling time of E7 cellswas calculated be the shortest whereas G9B4 cells were found to be the most slowlyproliferating (Table 1).

Cell morphology. 24 h cultures of cells from the parental cell line LSCC-SF(Mc29)and both sublines – E7 and G9B4, are presented in Fig.. 1.

Karyotypical analysis. Karyological analysis confirmed the presence of cells ofGallus domesticus. Up to eight chromosome pairs (autosomes) and the sexchromosomes were identified in every metaphase. The microchromosomes werecount, if possible. Diploid chromosome number was observed in 87% of the cellsfrom the parent cell line and in subline E7, and in 59% of the cells from the sublineG9B4. The rest of the cells had threeploid chromosome numbers. In both sublines –E7 and G9B4, endoreduplication was observed in about 7% of the cells analyzed.

Tumorigenic potential in vivo. Tumor cells (2.5; 5; 7.5 x 106) were implantedsubcutaneously into the knee flexure of 8-9 day old 15I White Leghorn chickens. Theresults showed that the cell lines examined differed in their growth properties in vivo.The E7 cells were found to be the most tumorigenic – 85-100% of the birdsinoculated developed tumors at the site of inoculation after 5-8 day latent period. 60-100% of the animals received LSCC-SF(Mc29) tumor cells developed tumors within5 – 9 days after the treatment. G9B4 cells exhibited the lowest tumorigenic potential– tumor growth appeared only in 0-16.5 % of the implanted chickens after aprolonged latent period (10 days) (Table 2).

DISCUSSIONThe term “tumor heterogeneity” means the existence of distinct subpopulations

of tumor cells with specific characteristics within a single neoplasm. Suchheterogeneity is also found in cell cultures established from tumors. The leadinghypothesis for the origin of tumor subpopulations is the genetic instability of cancercells. This process is a fundamental for malignant neoplasms and has importantbiological and clinical consequences among which augmentation of tumor

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progression and development of resistance to treatment are the most crucial for thehost (Heppner, 1984; Fleuren et al., 1995; Aabo, 1996; Heppner, Miller, 1998;Alexandrova, 2001).

Tumor heterogeneity is a well-documented phenomenon. Subpopulations havebeen isolated from cancers of every major histological type and organ site both fromexperimental and human neoplasms. Clonal heterogeneity has been demonstrated intumors induced by chemicals, by physical agents, by viruses as well as inspontaneously transformed clones in vitro (Heppner, Miller, 1998; Alexandrova,2001).

Cell lines derived from Mc29-induced transplantable hepatoma in chickenwere described earlier by Langlois et al., (1974) and Sovova et al., (1981). To ourknowledge, in this study we report for the first time about the isolation of cell lineswith different biological characteristics from this tumor model. The sublines G9B4and E7 obtained from the cell line LSCC-SF(Mc29) were found to differ significantlyfrom each other in morphology, karyotype, in vitro and in vivo growth properties.The cells from the E7 subline seemed to be similar to those from the parental cellline. Our findings suggest the presence of some causal relationship between alterationin chromosomal numbers, on one hand, and the tumorigenicity on the other. Thecorrelation was: the higher percentage of the threeploidy, the lower tumorigenicity ofthe cells.

Although it is not known whether the subpopulations (E7 and G9B4)described by us existed in the initial tumor/tumor cell line or appeared during thecultivation procedures in vitro, we suggest that these cell lines would be a suitablemodel for tumor heterogeneity investigations. Comparative study of such cell lineswith one and the same origin and different biological behaviour could help scientiststo clarify important biological phenomena like appearance of metastases, drugresistance, spontaneous regression and could improve cancer prevention, diagnosisand therapy. In addition, selection and studying of such cell variants are useful in theunderstanding of the relation between in vitro and in vivo properties of the cancercells.

Acknowledgement: This study was supported by Grants CC 1/2000 and CC1402/2004, National Science Fund, Bulgarian Ministry of Education and Science.

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REFERENCESAABO, K. 1996. Cellular heterogeneity in malignant tumors. – Dannish Medical

Buletin, 43: 336-349.ALEXANDROVA, R. 2001. Tumour cell heterogeneity. Experimental Pathology and

Parasitology, 4/6: 57-67.FLEUREN, G.J., A. GORTER, P.J.K. KUPPEN, S. LITVINOV, S.O. WARNAAR.

1995.Tumor heterogeneity and immunotherapy. – Immunol. Rev., 145: 91-121.HEPPNER, G.H. 1984. Tumor heterogeneity. – Cancer Res., 44: 2259-2265.HEPPNER, G., F.R. MILLER. 1998. The cellular basis of tumor progression. – Int. J.

Cytol., 177: 1-56.LANGLOIS, A.J., K. LAPIS, R. ISHIZAKI, J.W. BEARD, D.P. BOLOGNESI. 1974. Isolation

of a transplantable cell line induced by the Mc29 avian leukosis virus. CancerRes., 34: 1457-1464.

LAPIS, K., D. BEARD, J.W. BEARD. 1975. Transplantation of hepatomas induced inthe avian liver by Mc29 leukosis virus. Cancer Res., 35: 132-138.

MLADENOV, Z. 1974. Comparative Pathology of Avian Leukoses. Sofia, PublishingHouse of the Bulgarian academy of Sciences, (In Russian).

NAZERIAN, A.K. 1987. An updated list of avian cell lines and transplantable tumors.Avian Path., 16: 527-544.

NESBIT, C. E., J. M. TERSAK, E. V. 1999. Prochownik. MYC oncogenes and humanneoplastic disease. – Oncogene, 18: 3004-3016.

SOVOVA, V., I. HLOZANEK, H. CERNA, D. DOSTALOVA, H. SAINEROVA. 1981. Analy-sis of a chicken hepatoma induced by virus Mc29. Avian Pathol., 10: 461-469.

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Table 1. In vitro growth properties of cell lines established from a transplantable chickenhepatoma induced by the myelocytomatosis virus Mc29

Cell Line Initial concentration of thecells seeded, x 104

Doubling time(h)

LSCC-SF(Mc29)

3510

28.824.021.3

E7 3510

22.518.019.4

G9B4 3510

40.532.729

Table. 2 Tumorigenic potential in vivo of cell lines isolated from a tranplantable chickenhepatoma induced by the myelocytomatosis virus Mc29

Cell line Age(Days)

No of cellsinoculated,

x 106

Latentperiod(Days)

No of chickenswith tumours/TotalNo of chickensinoculated

LSCC-SF(Mc29)

77

5.07.0

6-95-8

5/7 (71.4%)7/7 (100%)

E7 778

2.55.07.0

85-85-8

5/5 (100%)7/7 (100%)6/7 (85.7%)

G9B4 778

2.55.07.0

-1010

0/4 (0%)1/6 (16.7%)1/6 (16.7%)

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A B

C

1A. Cells of subline E7 cultured for 24 h.Formation of numerous density-stainedepithelial cell clusters. Presence ofrectangular cells and few fibroblasts.H&E. Orig. 10X

1B. A 24 h culture of pleomorphic cellsof the G9B4 subline. H&E. Orig. 10X

1C. Cells from the initial cell line LSCC-SF(Mc29) cultured for 24 h. Presence ofrectangular cells, epitheloid cells,forming acinar-like structures and fewfibroblasts. H&E. Orig. 10X