establishment and characterization of a chicken ... · fig. 1. trypsin produced g-banding...
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(CANCER RESEARCH 47, 4460-4464, August 15, 1987]
Establishment and Characterization of a Chicken Hepatocellular CarcinomaCell Line, LMH1
Tokuichi Kawaguchi, Kimie Nomura, Yoko I lirayama, and Tomoyuki Kitagawa2
Department of Pathology ¡T.Ka., K. N., T. Ki.J and Department of Viral Oncology [Y. H.J, Cancer Institute, Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan
ABSTRACT
A hepatocellular carcinoma cell line, LMH, has been established froma hepatocellular carcinoma induced in a male leghorn chicken by dieth-ylnitrosamine. The cell line is characterized by well-differentiated morphological and biochemical features including the expression of glucose-6-phosphatase and canalicular ATPase activities and triploid karyotypewith six marker chromosomes.
The cells have been continuously propagated in culture for 5 yr andare now at about the 120th passage. Morphological change occurred inculture associated with gradual increase in growth rate at about the 40thpassage. However, the biochemical and chromosomal features remainedconstant.
This is the first established domestic fowl epithelial cell line and willallow comparative investigation of a number of parameters relevant tochicken hepatocarcinogenesis.
INTRODUCTION
Cell lines provide useful tools for a variety of studies includingcellular metabolism and regulation, virus replication, and cur-cinogenesis under standardized conditions. For these purposesnumerous cell lines from different species have been established.The difficulty of obtaining permanent culture cell lines fromchicken tissues has long been known (1-5), and it is onlyrelatively recently that lymphoblastoid cell lines from avianvirus-induced lymphomas (6, 7) or leukemias (8, 9) and fibro-blastic cells demonstrating long-term growth from normal,Rous sarcoma virus-treated or carcinogen-treated chicken embryo cells were established (10-13). Chicken epithelial cell lineshave hitherto not been reported. We describe here the establishment of a hepatoma cell line from diethylnitrosamine-inducedprimary hepatocellular carcinoma in a male leghorn. The cellsretain a number of differentiated phenotypic traits of chickenhepatocytes and are of potential usefulness for comparativeinvestigation of biological parameters.
MATERIALS AND METHODS
Animals and Carcinogen Treatment. Male leghorn LM strain chickens, 20 days of age, were purchased from Nisseiken Co., Tokyo, Japan.They were maintained in aluminum cages in an air-conditioned room.Starting from July 3, 1980, animals were given drinking water containing 100 ppm die ih.vlniirosa mint' (Tokyo Kase i Co., Tokyo, Japan) for
9 wk and then weekly or biweekly i.m. injections of 20 to 100 mgdiethylnitrosamine/kg for 30 wk. About 24 wk after discontinuation ofcarcinogen treatment, an animal became moribund with a hepatic tumorand was sacrificed on Oct. 9, 1981. The liver was greatly enlarged andgranular with multiple, up to bean-sized tan nodules. No morphologicaldistinction could be made between the original and tumor-intrahepaticmetastasis grossly.
Cell Culture. Tumorous tissues were taken from several nodules ofthe liver separately, transferred onto plastic plates (Nunc; 60 mm)
Received 1/27/87; revised 5/6/87; accepted 5/13/87.The costs of publication of this article were defrayed in part by the payment
of page charges. This ¡iniciemust therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by a grant-in-aid for cancer research from the Ministry of Educa
tion, Science, and Culture of Japan.2To whom requests for reprints should be addressed.
containing 5 ml of Waymouth's medium supplemented with 10% fetalcalf serum and kanamycin (6 ¿ig/ml),minced, and cultured at 37*C in
a CO; incubator. The medium was changed the next day and twice awk thereafter.
Morphological Observation. The original hepatic tumors and tumorswhich developed in nude mice after inoculation of cells were fixed in4% formalin and used for routine histológica!observation. The culturedcells were examined under inverted phase-contrast microscopy.
Tumorigenicity Test. Athymic nude mice were inoculated s.c. with 1x 107/ml passage 30 cells suspended in Waymouth's MB 752/1 me
dium and then maintained in isolated sterile cages.Histochemistry. Culture cells and frozen sections of the tumors which
developed in nude mice after inoculation of the cells were stained forGGTase3 activity by the method described by Rutenburg et al. (14) and
for GóPase and ATPase activity by the methods of Wachstein andMeisel (IS). Fibronectin was detected by immunological reaction inculture fluid using anti-fibronectin serum (Bethesda Research Laboratories, Inc., Philadelphia, PA).
Chromosome Studies. Logarithmic phase cells were treated for 2 hwith Colcemid and harvested by ordinary 0.2% trypsin treatment. Theywere then hypotonically treated with 0.075 M KC1 for 20 min at 37'C
and fixed with met hum >lacetic acid (3:1) for 30 min, and ordinary air-dried chromosome spreads were made on clean slides. Chromosomenumber counts were carried out on enlarged photographs of metaphaseplates. Chromosomes were also examined by the trypsin-Giemsa banding technique (16).
Electron Microscopy. The cells obtained were seeded in a 100-mmplastic dish (Corning) and allowed to proliferate. The dishes were rinsedwith Dulbecco's phosphate-buffered saline, and the cells were scraped
gently with a rubber policeman from culture vessels, fixed in 2%glutaraldehyde in 0. l M phosphate buffer (pH 7.4) for 30 min at roomtemperature, and then postfixed in 2% osmium tetroxide-0. l M phosphate buffer (pH 7.4) for 30 min at room temperature. They weredehydrated in series of alcohol and propylene oxide and embedded inEpon-812. 11Itrut hin sections were cut on an 1KB microtome with aglass knife. Observations and photography were performed using aHitachi I It'-12 electron microscope, after contrasting sections with
uranyl acetate.Reverse Transcriptase Activity. The cell-free medium of the culture
was assayed for endogenous reverse transcriptase activity according tothe method described by Bauer and Temin (17).
RESULTS
Establishment of the Cell Line. Within a few days cellsforming small foci began to grow slowly. From all platescontaining cells from different nodules, very similar sheets ofepithelial-looking cells were obtained. The first subpassage ofone of the largest epithelial foci was carried out after 2 mowhen the size reached 20 mm in diameter. The cells havesubsequently been continuously subcultured for 5 yr, approximately once a mo for the initial 3.5 yr and once a wk now, atabout the 120th passage level.
Fibroblastic cells and macrophages were observed as contaminating elements among epithelial-type cells in the initial fewpassages, but later they disappeared completely.
Chromosome Analysis. Chromosome analysis was performed
3The abbreviations used are: GGTase, ->glutamyl transpeptidase; G6Pase,glucose-6-phosphatase.
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CHICKEN HEPATOMA CELL LINE
large chromosomes
Bli««li il il i»2
small and micro chromosomes
ft it W M M «4* «••••«*•*•t •»••*•••*•»(MM»*»»è
•'
sex chromosomesmarker chromosomes
fu
txio*468
culture time (days)
Fig. 1. Trypsin produced G-banding karyotypes of chicken hepatocellular carcinoma cell line LMH with 116 chromosomes showing 6 marker chromosomes (30thpassage). The chromosome number was determined by counting 39 metaphase cells.
morphology associated with gradual increase in the growthrate. At present, 5 yr from the beginning and at about the 120thpassage, the cells are growing much faster, requiring subpassageonce a wk. Growth curves of the cells at passage 120 are shownin Fig. 2. The population doubling time during the logarithmicphase was 25 h. The LMH cells proliferate well in serum-containing medium but also grow in serum-free medium withan approximate halving of the growth rate (Fig. 2).
Morphological Characteristics. All the hepatic nodules examined and the tumors which developed in the subcutis of nudemice after inoculation demonstrated a very similar histologicalappearance, i.e., well-differentiated trabecular hepatocellularcarcinoma with occasional transition to adenocarcinomatousstructures (Fig. 3, a and />).
Until about the 40th passage, the LMH cells grew in a typicalcobblestone-like pattern, and cells were polygonal with abundant cytoplasm and large round nuclei with a few prominentnucleoli (Fig. 4). After this time, the cells began to adopt adendritic-like appearance, and the typical cobblestone-like pattern was observed only in occasional high cell density areas ofdishes (Fig. 5). This morphological change appeared irreversibly fixed. Electron microscopy at the 30th passage revealed alarge number of mitochondria, prominent Golgi apparatus,abundant rough endoplasma- reticulum, free ribosomes, and
glycogen granules in the cytoplasm (Fig. 6). The plasma membrane possessed numerous well-developed microvilli. Biliarycanaliculus-like structures (Fig. 6), desmosomes, and tight junctions were formed between adjacent cells. No virus particleswere identified.
Fig. 2. Growth curves of the LMH cell line at passage 120 with 10% fetal calfserum (O). Medium was changed every third day. Growth curve in serum-freemedium (•).Medium was changed every 2 days. Bars, SE.
twice. The first analysis was carried out at about the 30thpassage and the second at the 120th passage. The results ofboth analyses were essentially identical. The avian origin of thecells was confirmed by the presence of numerous microchromosomes.
The number of chromosomes ranged from 88 to 124, themodal number being 116 (normal diploid 2n = 78) (18-20). Allthe cells commonly possessed 6 marker chromosomes (Fig. 1),clearly indicating a clonal origin for the cells.
Concluding the establishment of a new cell line, we designated the culture line LMH.
Growth Characteristics. The LMH cells grew relatively slowlyduring the initial 3.5 yr requiring subpassage once a mo when2 x 105/ml cells were plated onto 60-mm dishes. After about
40 passages, the cell line began to demonstrate change in cell4461
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CHICKEN HEPATOMA CELL LINE
Fig. 3. a, photomicrograph of the original liver tumor demonstrating a well-differentiated trabecular hepatocellular carcinoma. H & E, x60. b, light microscopicappearance of a tumor that developed in a nude mouse after transplantation of LMH cells. The histológica!appearance is indistinguishable from the original tumorshown ino. H& E, xl40.
Fig. 4. Morphological features of the LMH cells in culture at passage 5.Polygonal cells with abundant cytoplasm and large round nuclei with prominentnucleoli are arranged in a typical cobblestone-like pattern. Phase contrast, x 14.
Fig. 6. An electron micrograph of LMH cells. Well-developed cell organelles,desmosomes, tight junctions, and biliary canaliculus-like structures are shown(30th passage). X9360.
Fig. 5. LMH cells in culture at passage 120. Note dendritic-like appearancetogether with sheet-like pattern. x30.
Enzyme Markers. In spite of the change in the morphologyand growth rate, the biochemical features of the LMH cells atthe 30th and 120th passages were essentially the same. GoPaseactivity could be clearly demonstrated histochemically both inthe cultured cells (Fig. la) and in tumor tissue which developedin nude mice (Fig. Ib).
Weak Ca2+, Mg2+-dependent, formalin-resistant ATPase ac
tivity was also evident in cultured cells (Fig. 8) and tumor tissue.No GGTase activity could be demonstrated histochemically ineither cultured cells or tumors in nude mice. Fibronectin wasdetected in the culture fluid.
Reverse Transcriptase Activity. This was negative in the culture medium.
Tumorigenicity. Tumorigenicity was demonstrated in nudemice twice, utilizing 30th and 120th passage cells. When IO7
cells at the 30th passage were inoculated into the subcutis ofthe back, tumors reaching 2 cm x 1 cm developed after 7 mo.In the second inoculation experiment, similar tumors developedafter 3 mo.
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CHICKEN HEPATOMA CELL LINE
Fig. 7. a. cytochemical demonstration ofG6Pase activity in cultured cells (passage 6). Diffuse cytoplasmic activity is evident. x80. b, histochemical demonstrationof strongly positive G6Pase activity in a tumor developed in a nude mouse after transplantation of cells. xl2.
Fig. 8. Cytochemical reaction for ATPase in the culture cells. The enzymeactivity is demonstrated on the outer cellular membrane between the neighboringcells. x30.
DISCUSSION
To our knowledge this is the first paper describing the establishment of an epithelial cell line of domestic fowl. This linewas derived from a hepatocellular carcinoma induced by dieth-ylnitrosamine after long-term treatment. Although not originally cloned, clonality of the cells comprising the LMH line isindicated by the presence of 6 marker chromosomes in all thecells.
The hepatocellular nature of the LMH line is evident fromthe morphology of the tumors developing in nude mice afterinoculation and the expression of GoPase activity. Morphological features including only mild cellular atypism, possession ofabundant cellular organelles, and formation of biliary canalic-ular-like structures together with positive GoPase and ATPaseactivities indicate a relatively high grade differentiation of thecarcinoma cells, even after the morphological change and increased growth rate.
Since many important avian sarcoma and leukemia viruseshave been isolated from mesenchymal malignancies of chickenand extensively contributed to oncogene studies, the availability
of this cell line might be useful for studying chemical carcino-genesis, especially that of epithelial cells, in comparison withviral sarcomagenesis.
In addition to the problems of determining ideal cultureconditions, one of the reasons for the difficulty in establishingepithelial cell lines of fowls has been the difficulty of inducingprimary neoplasms in this animal by carcinogen treatment. Ascompared with the situation in rodent systems, much largerdoses of carcinogenic agents and much longer latent periodshave been necessary for inducing carcinomas of parenchyma!organs in the domestic fowl (21-23). In the present experiment,the total dose of diethylnitrosamine and the latent period required to induce a hepatocellular carcinoma were roughly 4 to5 times those required in the rat system. The presently describedLMH cell line might prove useful for elucidating mechanismsof resistance to carcinogenesis of domestic fowl cells.
ACKNOWLEDGMENTS
We thank Dr. M. Yoshida, Chief, Department of Viral Oncology,for his help in assaying the reverse transcriptase activity and Dr. T.Utakoji, Chief, Department of Cell Biology, for his help in chromosomeanalysis.
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1987;47:4460-4464. Cancer Res Tokuichi Kawaguchi, Kimie Nomura, Yoko Hirayama, et al. Carcinoma Cell Line, LMHEstablishment and Characterization of a Chicken Hepatocellular
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