[CANCER RESEARCH 40, 1443-1448,
0008-54 72 /80/0040-OOOOS02.00
May 1980]
Longitudinal Karyotype and Genetic Signature Analysis of Cultured
Human Colon Adenocarcinoma Cell Lines LS180 and LS174T1
Lynne P. Rutzky,2 Celia I. Kaye, Michael J. Siciliano, Margaret Chao, and Barry D. Kahan
Departments of Biochemistry and Molecular Biology [L. P. R ] and Surgery [L P. R., B. D. K.J. The University of Texas Medical School. Houston, Texas 77030:
Cook County Hospital, Chicago. Illinois 606 )2 ¡C.I. K.. M C I; and The University of Texas System Cancer Center. Houston. Texas 77030¡M J. S.J.
ABSTRACT
Giemsa-banded
chromosomes
were analyzed at intervals
during either 34 or 70 serial subcultivations of two cell lines,
LS180 and LS174T, established from one primary human
colon adenocarcinoma, and at passage 14 of autochthonous
normal bowel cells, NB(LS1 74T). The cell lines were estab
lished and subcultured by either scraping or trypsin treatment
of primary cultures; the scraped cell line was designated
LS180, and the trypsin-dispersed cell line was named LS174T.
Early passages of LS1 74T cells were composed mainly of
46.XX (38%) and 45,X (34%) karyotypes; LS180 cultures
possessed cells with 46,XX (54%), 45,X (7.5%), and 47,XX + D
(19.5%) chromosome modes. In both cell lines, the 45,X karyotype predominated in later subcultivations. After the fifth
passage, all LS180 cells examined exhibited a translocation
from the long arm of the X chromosome to the long arm of the
No. 5 chromosome. Cultures from the patient's normal bowel
mucosa and peripheral blood leukocytes had normal 46,XX
karyotypes. Genetic signature analysis substantiated the com
mon genetic origin of the cell lines, and we concluded that
differences observed between LS180 and LS174T were not
due to contamination with other cell lines. LS180 and LS1 74T
represent closely related cell lines differing cytogenetically in
a translocation.
INTRODUCTION
The establishment and characterization of cell lines from
human colon adenocarcinoma (37, 38), a tissue generally
resistant to in vitro growth, have presented the opportunity to
observe several unique biological (31), biochemical (15), and
immunological (14) properties associated with the second most
prevalent form of cancer in this country. Human tumor cell lines
propagated in vitro afford the advantages of a large, uniform,
and readily available source of cells free of detectable microbial
contamination. Long-term lines were continuously monitored
to identify differences between cultured cells and the original
tumor cells and tissue and to determine the degree of alteration
of innate properties during serial subculture. This report de
scribes longitudinal karyotype analysis of 2 cell lines, LS180
and LS174T, derived from primary cultures which were estab
lished from the same colon adenocarcinoma (37). Genetic
signature analysis of the tumor- and normal bowel-derived cell
' Supported by the USPHS Grant CA22370 from the National Cancer Institute
through the National Large Bowel Cancer Project and by USPHS Grant CA23871.
This report is one in a series of papers describing human colon adenocarcinoma
cell lines (13, 14, 31, 36, 37). Preliminary reports were presented at the Annual
Meetings of the Tissue Culture Association, June 5 to 8, 1978, Denver. Colo.
(29), and June 10 to 14, 1979, Seattle, Wash (30).
2 To whom requests for reprints should be addressed, at Department of
Surgery, Room 6240, P. O. Box 20708. Houston, Texas 77025.
Received September 4. 1979; accepted January 29. 1980.
lines has been used to define the genetic origin and the lack of
cross-contamination with HeLa cells (13, 20, 25) or other cells
(2, 22).
MATERIALS AND METHODS
Origin of the Cell Lines. The cell lines LS180 and LS174T
were established in culture in 1974 from a moderately welldifferentiated primary colon adenocarcinoma which had in
vaded the pericolonic fibroadipose tissue, but not the regional
lymph nodes, in a 47-year-old Caucasian woman (37). After
10 months in primary culture, several of the original flasks of
cells were trypsinized to produce the strain LS174T. The
remaining flasks of cells were mechanically scraped to produce
the LS180 strain. Primary cultures of LS180, TC3 0, were
preserved in liquid nitrogen for future reference; representative
cultures were frozen during serial subcultivation of both LS180
and LS174T cells. The minimum number of population dou
blings in culture was determined by the method of Hayflick and
Moorhead (11) and was used to calculate TC. Because each
TC level represents a doubling of the cell number, TC levels
essentially represent the Base 2 logarithm to which the cell
number in the original flask must be raised to give the observed
subsequent populations. This occasionally results in fractional
TC levels for which no roundoff has been made in the text.
Tissue, presumed to be "normal" bowel mucosa, was dis
sected from a site far from the tumor mass and was used to
establish a normal bowel culture, NB(LS174T) (37, 38). Blood
leukocytes from the patient were placed into short-term culture
for karyotypic analysis. LS180 was serially propagated follow
ing mechanical dislodgement with a rubber policeman; LS174T
was subcultured using 0.1 % trypsin diluted in 0.02% Versene,
pH 7.4. Growth medium for all cell lines consisted of Eagle's
minimum essential medium (Grand Island Biological Co., Grand
Island, N.Y.) with Hanks' salts and supplemented with 1 x
nonessential amino acids, 10% heat-inactivated (56°, 30 min)
fetal bovine serum (Grand Island Biological Co.), 2 mw Lglutamine, 100 jug streptomycin per ml, and 100 units penicillin
per ml (culture medium). The cells have been shown to be
Mycop/asma-free by culture (Biolabs, Northbrook, III.), by fail
ure to stain with Bisbenzimidazol 33258 Hoechst dye (Ameri
can Hoechst Corp., Somerville, N. J.) (6), through transmission
and scanning electron microscopy and by negligible arginine
deiminase activity (3).
Cytogenetic analysis of LS174T and LS180 cells was per
formed at several intervals during serial subcultivation. In ad
dition, TC 0 tumor cells were rapidly thawed and placed into
culture after 3 years of storage in liquid nitrogen. When confluency was attained, one-half of the monolayer was scraped
' The abbreviation
used is: TC, culture passage level based upon the popu
lation doubling values.
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1443
L. P. Rutzky et al.
while the remaining cells were treated with trypsin and subcultured. The cultures were propagated separately, and the karyotypes were evaluated during 6 serial subcultivations. Several
characteristics of LS174T, LS180, and a normal bowel cell
strain NB(LS174T) are listed in Table 1. The colonie origin of
LS174T cells was indicated by the detection of a colon-specific
membrane antigen, CSA (37), by the expression of gut-asso
ciated patterns of isozymes coded by multiple genetic loci (30),
and by organoid histopathology reminiscent of the patient's
tumor in tumors induced in nude mice (38) and in hollow fibercultured cells (31).
Preparation of Chromosomes. Ten ml of 0.00625% Colcemid (CIBA Pharmaceutical Co., Summitt, N. J.) diluted in
culture medium was added to a 75-sq cm culture flask (Falcon
Plastics, Oxnard, Calif.) of a 24- to 48-hr-old monolayer culture.
After 1 hr of incubation at 37°,floating cells were collected in
a centrifuge tube, and the flask was washed with a small
volume of Hanks' balanced salt solution (Microbiological As
sociates, Bethesda, Md.). The solutions were centrifuged (140
x g) at room temperature for 10 min, and the pellet was
retained. The cell monolayer was removed with 0.25% trypsin;
residual trypsin was inactivated with warmed complete culture
medium. The cells were removed from the flask with warmed
0.075 M KCI and added to the previously collected cell pellet.
The cell suspension was brought to a final volume of 18 to 20
ml with warm 0.075 M KCI and incubated at room temperature
for 16 to 17 min. The cell suspension was fixed in methanol:
glacial acetic acid (3:1) 3 times. Before the cells were spread
on slides, they were resuspended in a small volume of fresh
fixative. Chromosomes were prepared by rapidly flaming the
cells on slides until dry. Before staining, the slides were dried
for 3 days at room temperature.
Giemsa Banding. Initially, the chromosome spreads were
exposed twice to a solution of sodium chloride and sodium
citrate in distilled water at 60°for 30 to 40 min, according to
procedures described previously (36). In subsequent studies,
slides were treated for 1 min in a solution of 0.055% trypsin:
EDTA (Grand Island Biological Co.), diluted in calcium-mag
nesium-free Hanks' balanced salt solution, pH 7.0, at 37°and
rinsed briefly with cold tap water. The slides were stained with
0.8% Giemsa solution (Fisher Scientific, Houston, Texas) di
luted 1:40 with Sorensen's phosphate buffer, pH 7.2. The
chromosomes were arranged in numerical order from left to
right within each major group of the karyograms; marker chro
mosomes were described according to the 1972 Paris Confer
ence nomenclature (23).
Isozyme Analysis. Cells were processed for isozyme analy
sis by rinsing in 0.9% NaCI solution and homogenized (Dounce
homogenizer) in 2 volumes of homogenizing medium (0.01 M
Tris-HCI, pH 7.5:1 mw /?-mercaptoethanol:1 rriM EDTA). After
a freeze-thaw step, homogenates were centrifuged (10,000
x g, 45 min) and were subjected to vertical starch gel electrophoresis (34).
The cell lines were studied for the presence of gene products
of the following enzyme loci:glucose-6-phosphate
dehydrogenase (G6PDH); mitochondria! forms of glutamate-oxaloacetate
transaminase ( GOTm); malic enzyme (ME)', 6-phosphogluconate dehydrogenase (6PGD); peptidases A, C, and D (Pep-A,C,-D); a-glucosidase (a-G/uc); esterase D (EsD); glyoxalase I
(G/y /); phosphoglucomutase
1 and 3 (PGM, and PG/vt3); the
RBC form of adenosine deaminase (ADArbc); the cytosol form
of aconitase (ACONS); and acid phosphatase (AcP). The spe
cific buffer systems which resolve the alÃ-eleproducts and the
histochemical stain formulas which make them visible have
been published (9, 32, 34).
RESULTS
Longitudinal Karyotypic Evaluation. Giemsa-banded chro
mosomes of LS174T cells were evaluated at 4 passage levels.
In early passages, the cell line was composed primarily of 2
cell types, 46,XX and 45,X. The 46,XX line disappeared early
during in vitro propagation between TC 3 and 8. At TC 30 and
70, the 45,X karyotype predominated in the 45-chromosome
complement (Chart 1A) (37). At TC 21, less than 0.5% LS1 74T
cells were found to possess Barr bodies. Cells containing 44
chromosomes usually possessed a 44,X karyotype until TC 70
when random losses were observed.
In the early subcultivations of the LS180 cell line, a variety
of cell karyotypes which included a few normal 46,XX cells and
an apparent 46,Dq +, + G-C karyotype was observed; the translocation was not found. Approximately 20% of the cells exam
ined exhibited 47 chromosomes; in most of these cells, an
extra D-group chromosome was found, which was often a No.
15 chromosome. At TC 19.4, one cell with a 47,XX + D chro
mosome complement was observed. LS180 cells with a 45,X
chromosome complement were present during 34 serial subcultivations (Chart 16). In all of the 45,X cells studied at TC
19.4 and 34.2 (Fig. 1), a translocation, 45,X,t(Xq-»5q), from
the long arm of the X chromosome to the long arm of the No.
5 chromosome in the B group was observed.
Since the karyotypic patterns observed in LS180 and
LS174T may have resulted from differences in cell harvesting
techniques which were used during serial subcultivation, tumor
Table 1
Cell line characteristics
signa
ture fre
quency(x10
featuresMonolayerMonolayer
tureTrypsinScrapeTrypsinCulture
X45,
LS174TLS180NB(LS174T)MorphologyEpithelialEpithelialMixed
multi-cellular
and
t(Xq^5q)46,
X,
spheroidsMonolayerKaryotype45,
XXGenetic
epithelialand
fibro-blast-likeSubcul
1444
noembryonic
antigen
at 21
blingtime(hr)18-207224Tumor-igenic-ity++—
days(ng/106cells)370700Dou
Mucin2.6
')
+2.6
+2.6Carci- +
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Karyotype and Genetic Signature of Human Colon Tumor Cells
Chart 1. Histogram illustrating the number of chromosomes
(abscissa) and the percentage of cells examined with each chro
mosome number (ordinate). N, total number of metaphases
counted. The chromosome distribution patterns of LS174T,
LS180, and autochthonous NB(LS174T) and normal blood leu
kocytes are illustrated in A, B, and D, respectively. C, chromo
some distribution in early subcultures of tumor cells that were
either serially scraped or trypsinized.
<43
Number ot Chi
cells from a primary culture flask were thawed and cultured
according to either LS180 or LS174T protocols. The scraped
cell cultures were analyzed at 3 early passage levels (Chart
1C). A variety of chromosome complements was observed, but
the translocation was not seen during early subcultivation. In
the early subcultures processed by mechanical scraping, all
cells with 47 chromosomes possessed an extra D group chro
mosome, which was often an extra No. 15 chromosome. At TC
4, cells dissociated by either trypsin treatment or scraping
were compared. The 47,XX + D line was present using both
culture methods, but the proportion was diminished in the
enzyme-treated cultures. LS tumor cells possessing a 45-chromosome complement were characterized by random losses at
early passages, but some cells with the 45,X karyotype were
observed.
Giemsa-banded chromosomes were evaluated in 40 normal
cultured peripheral blood leukocytes and 60 NB(LS174T) cells
at TC 14.8. Analysis of the karyotypes from the leukocyte
culture revealed a 46,XX chromosome complement with no
apparent structural abnormalities (Chart 1D). The modal kary
otype of NB(LS174T) cells was 46,XX; cells containing less
than 46 chromosomes exhibited random losses. At TC 14.8,
the culture contained mainly fibroblastic cells with some epi
thelial elements. One cell was observed with a 47.XX + 8 chro
mosome complement. Another cell (46,XXq —¿)
was observed
with a deleted long arm of one X chromosome reminiscent of
the deleted X chromosome observed in LS180 cells carrying
43
44
45
46
47
4N
Number of Chromoiomei
Table 2
Allozyme phenotypes of enzyme loci in cell lines and HeLa control
Allozyme phenotypes of cell lines
Enzyme lo
cusAcPACÓN,ADArbcEsDGePDHa-glucGOTmGLYIPep
LS0.440.990.810.810.990.940.960.360.990.980.980.900.560.
-APep
-CPep
-D6PGDPGM,PGMiME„3
Frequency ofHeLaab111A112111A1112the
eachpolymorphic
LS174TLS174Tab1,i}A122,
LS180.LS180ab111B112111A122NBO-S17-4T)ab111B2A122r-re-quen
NBÕLS174T) phenotypes of
onpaper
enzymetheHopkinson
locus in Caucasian populations based
from
of Harris anddata
(9).
the translocation
described previously. The B-group chromo
somes appeared to be normal (Fig. 2).
Isozyme Analysis. The allozyme phenotypes for the 15
enzyme loci of the 3 cell lines and HeLa are shown in Table 2.
The phenotype at every locus for any one cell line represents
the genetic signature of that cell line (22). Each of the 3 LS cell
lines differed from HeLa at the G6PDH, PGM3 and MEm loci.
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1445
L. P. Rutzky et al.
These data indicate that LS174T, LS180, and NB(LS174T)
cells are not HeLa contaminated.
LS174T, LS180, and
NB(LS1 74T) cells have identical genetic signatures, a result
consistent with their derivation from a single individual. Since
the frequency of that genetic signature in the Caucasian pop
ulation (calculated as the product of the frequencies of their
phenotype at each locus) is low (2.6 x 10~4), the probability
of one of these cell lines being a contaminant of independent
origin is negligible (the square of the frequency of the genetic
signature in the population or 6.8 x 10~8). This leads to the
conclusion that LS174T, LS180, and NB(LS174T) are all de
rived from the same individual, and any differences observed
between them cannot be accounted for by contamination with
another cell line. Stable isozyme patterns were observed at
passage levels 6, 25, 45, 49, 70, and 73 for LS174T cells and
at passage levels 12 and 18 for LS180 cells.
DISCUSSION
This report describes the karyotypic evolution of 2 human
colon adenocarcinoma cell lines, LS180 and LS174T, derived
from the same tumor specimen 5 years ago. The LS180 culture
has been mechanically scraped and has not been exposed to
exogenous proteolytic enzymes (37). Initially, some of the
primary cultures were either trypsinized to produce the LS174T
cell line or scraped to produce the LS180 line. Most of the
scraped cells grow as multicellular clusters or spheroids that
float in the medium until they become heavy enough to adhere
to the flask surface. The cell lines have been cultured sepa
rately and characterized. LS180 and LS1 74T cells had modal
chromosome complements of 45,X. LS180 developed a translocation after the fifth passage. Early subcultures of LS180 and
LS174T were composed of cells with chromosome comple
ments 46,XX, 45,X, and 47.XX + D (Chart 1, A and B). The
karyotype of the original tumor was not determined. The early
disappearance of the 46,XX line in both cultures may have
correlated with the paucity of fibroblastic cells. The 45,X stemline was selectively preserved during serial cultivation in both
LS180 and LS174T cell lines (Chart 1, A and B) (see Ref. 37).
These data indicate that the 45,X karyotype possessed a
growth advantage under the conditions used. The 47,XX + D
line was lost during early subculture (Chart 1, B and C),
suggesting that it may have been sensitive to in vitro propa
gation and to trypsin exposure (Chart 1C). Trypsin enters cells,
and it may interfere with cell processes (12). The patient's
peripheral blood leukocytes and cultured normal bowel cells
exhibited a normal 46,XX karyotype in 83 and 70% of the cells
examined, respectively (Chart 1D).
In the LS180 cell line, a translocation occurred from the long
arm of an X chromosome to the long arm of the No. 5 chro
mosome between passages 5 and 19.4 (Fig. 1). If the translo
cation occurred in a 46,XX line cell, subsequent loss of the
normal X chromosome may have produced the 45,X line bear
ing the translocation. It is interesting that one chromosome
complement (46,XXq-)
with a deleted X chromosome was
detected in the NB(LS174T) culture after 14 serial subcultivations (Fig. 2). In the LS180 cell line, the 45,X,t(Xq->5q) translocation was detected in all cells studied following 19.4 serial
subcultivations. The uniform occurrence of the translocation
suggests that it may be associated with the acquisition of
additional survival potential. While the significance of the dele
1446
tion and translocation is unknown, it is possible that an "unsta
ble" area existed on an X chromosome in the premalignant
tissue. During prolonged serial subcultivation the X chromo
some may have become unstable, undergone breakage and
rearrangement, and become visible in the NB(LS174T) and
LS180 cultures. Fragile sites have been demonstrated on the
X chromosome of cultured human lymphocytes (36). Alterna
tively, the translocation may have occurred in a 45,X cell which
was selectively preserved during serial subcultivation. Cells
preserved in liquid nitrogen from a primary culture selectively
retained the 45,X karyotype during early subculture, although
the translocation and other marker chromosomes were not
observed during 6 subcultivations.
Analysis of fresh cells or short-term cultures of human colon
neoplasms revealed that primary tumors were characterized by
modal chromosome numbers less than 50; metastatic tumors
had modal chromosome numbers greater than 60 (17). The LS
tumor lines were established from a primary lesion; the near
diploid karyotype of LS174T and LS180 substantiates the
observations of Lubs (1 7). Many cultured human colon tumors
have near diploid chromosome complements (7, 8, 16, 28, 39),
but other lines possessed heteroploid (16, 21, 26, 32) or
variable modes (8). LS174T and LS180 tumor cells were
characterized by a stable hypodiploid chromosome comple
ment that was observed during this study. Karyotype stability
has been described in several additional cultured human tumor
cells (1, 18, 19, 27). Although the cytogenetic relationship
between the original tumor and established cell cultures is
often unknown, the presence of karyotype stability is an im
portant prerequisite for providing uniform materials in longterm studies. Establishment of human tumor cultures from
pleural effusions permitted the comparison of the tumor and
culture karyotypes and identified the presence of HeLa-like
marker chromosomes in the fresh and cultured specimens (24).
Genetic signature analysis confirmed the common origin of
LS180, LS174T and NB(LS174T) cell lines, suggesting that
differences in extractable cell-surface components (14), carcinoembryonic antigen production (37), culture morphology,
and population-doubling time (38) observed among them were
not due to contamination with other cells. Stable isozyme
patterns observed in LS1 74T and LS180 cells were consistent
with isozyme patterns reported previously (2).
Serial karyotype and genetic signature analysis of LS174T
and LS180 cells revealed the development of stable chromo
some patterns within the subculture range used during previous
studies (14, 15, 29, 30, 31, 37, 38). This report emphasizes
the importance of continuous detailed monitoring of established
cell lines for retention of innate properties with increased in
vitro age. Compilation of longitudinal characterization data is
useful in evaluation of established human tumor cell cultures
as relevant cancer models. Genetic signature and karyotype
analysis combined to produce an effective method to charac
terize the origin of the cell lines. These lines may help to identify
the role of critical chromosomes or chromosomal segments in
regulating malignant growth. Cell fusion studies between malig
nant and nonmalignant cells (10, 35) indicated that the pres
ence of chromosomes from the nonmalignant donor cells
tended to suppress growth and malignancy in the hybrids.
Chromosome balance is critical to the production of tumors by
transformed cells (4, 5). The availability of 2 established,
closely related human colon tumor cell lines from the same
CANCER
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Karyotype and Genetic Signature of Human Colon Tumor Cells
specimen, differing cytogenetically in a translocation, provides
a useful resource for studying X and No. 5 chromosome gene
function in human colon cancer cell biology.
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21. Noguchi, P., Wallace. R., Johnson, J., Earley. E. M., O'Brien, S.. Ferrone.
ACKNOWLEDGMENTS
S., Pellegrino, M. A., Mustien, J., Needy. C.. Browne. W., and Petricciani. J.
Characterization of WiDr: a human colon carcinoma cell line. In Vitro (Rockville), 75 401-408. 1979.
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We thank Miriam Calenott and Billie White for their excellent technical assist
ance.
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1447
L. P. Rutzky et al.
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45. X.t( Xa.5a)
Fig. 1. Karyotype of G-banded LS180 chromosomes observed at TC 19.4.
Arrows, translocation from the long arm of the X to the long arm of the No. 5
chromosome.
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Fig. 2. Karyotype from a G-banded NB(LS174T) cell. Arrow, deleted X chro
mosome reminiscent of the LS180 X chromosome following translocation.
1448
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1980 American Association for Cancer Research.
Longitudinal Karyotype and Genetic Signature Analysis of
Cultured Human Colon Adenocarcinoma Cell Lines LS180 and
LS174T
Lynne P. Rutzky, Celia I. Kaye, Michael J. Siciliano, et al.
Cancer Res 1980;40:1443-1448.
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