[CANCERRESEARCH38, 3033-3043,September1978)
0008-5472/78/0038-0000$02.00
Cultural, Morphological, Cell Membrane, Enzymatic, and Neoplastic
Properties of Cell Lines Derived from a Hodgkin's Disease Lymph
Node1
Audrey N. Roberts,2 Kirby L. Smith, Barry L. Dowell,3 and Andrea K. Hubbard
Department of Microbiology, university of Tennessee Center for the Health Sciences, Memphis 38163 (A. N. R., K. L. S., B. L. 0., A. K. H.J, and Department
of Hematology-Oncology,St. JudeChildren'sResearchHospital,Memphis,Tennessee38101(K. L. S.!
ABSTRACT
A neoplasticcell line (designated HuT,,) has been es
tablished in continuousculture from an involved lymph
node of a patientwith Stage IIA Hodgkin'sdisease of the
mixedcellularftytype. The HuT1,line hasbeen morpholog
ically heterogeneous,consistingof mononucleatelymph
old-like cells, polygonalepithelloidcells, and mono-, bi-,
and muftinucleategiant cells. Four clones inftiatedfrom
Isolatedbinucleategiant cells of the HuT,, line also have
been successfully
established
as continuous
cell lines.
The cloned lines have been morphologicallydistinctand
more homogeneous, although typical giant cells have
consistentlyappearedthroughoutthe long-termcultureof
each. The HuT1 lines have grown as monolayers in
McCoy's Medium 5A supplementedwith 10% fetal calf
serum, with generation times of 12 to 14 hr and high
saturation densities. Cytogenetic studies showed that
early and later passages of HuT,1cells were aneuplold,
and all cell lines were successfullyheterotransplantedin
the hamstercheek pouch.Repeatedindirectimmunofluo
rescence examinations
have shown each cell line to be
negative for Epstein-Barrvirus nuclear antigen. Indirect
lmmunofluorescence
tests In which monospecific
immu
noglobulinswere used revealed positivemembranereac
tions for the y (heavy)-chaIn and K (Iight)-chain of human
malignant cell of Hodgkin's disease have been hampered
by failure ho establish in vitro monotypic cell lines derived
from the characteristic neoplashic cells. Numerous investi
gators have explored the cytologic, histochemical, and
immunological properties of the large mononuclear cell
population and the binucleate or mulhinucleate Reed-Stern
berg cells of Hodgkin's disease with the use of fresh biopsy
specimens or cells cultured from involved tissues for limited
periods. The heterogeneity of these preparations has re
suIted in a diversity of suggestions regarding the cell of
origin, including the reticulum cell or hishiocyte (1, 24, 66),
transformed lymphocytes (40, 47, 60), T-lymphocytes (4, 13,
47), and B-lymphocytes (2, 5, 12, 19, 20, 29, 34, 63). Many
short-term cultures have displayed cells of mixed morphol
ogy which usually could not be serially passed (15, 52, 62,
65). Other long-term cultures (27, 51, 58) have not been
clearly distinguished from lymphoblashoid B-cell lines nat
urally infected with EBV4 (46).
Recently, Kaplan and Gartner (30, 31) have initiated
cultures of mono-, bi-, and mulhinucleale giant cells from
involved spleens of Hodgkin's patients. Although perma
nent cell lines have not yet been established from these
slow-growing cultures, they have been confirmed as neo
plastic by demonstration of aneuploidy and heherotrans
plantability. Tests on several of these have revealed prop
erties characteristic of macrophages, including phagocyto
sis formation of IgGEA and IgMEAC rosettes, and synthesis
and secretion of lysozyme (31). Long et a!. (38, 39, 69) have
successfully established continuous monolayer and sus
pension cell lines from Hodgkin's splenic tumor nodules.
immunoglobulinG in approximately20% of viable cells in
each line; however,direct Immunofluoreecencewith anti
humanimmunoglobulinG F(ab')@
reagentfailedto confirm
these reactions. Rosefte tests for B- and T-Iymphocyte
and macrophage membrane receptors yielded negative Bi- and muihinucleate giant cells have been prominent in
results. All cell lines were stronglyphagocyticfor latex each cell line. Cyhogenetic studies on many of these have
particles and neutral red dye. Cytochemical stains of the
demonstrated
aneuploid karyotypes (39), while 4 of the
monolayersrevealed abundantesterase, fluoride-resist monolayer cultures have been heterotransplantable when
ant nonspecificesterase, acid phosphatase,and leucine tested in nude mice (69). Although distinct differences in
amlnopeptldase
activities,
while lysozyme assays were
negative. Although some properties of the HuT,1 lines
have suggesteda macrophagederivation,an undifferen
tiated lymphoidcell originof the Hodgkin'sneoplasticcell
remainsa possibllfty.
INTRODUCTION
Attempts ho identify and determine the origin of the
membrane receptors have been shown between the cells
cultured in suspension and monolayers, some of the prop
erties uniformly demonstrated by both have suggested a
macrophagederivation (39). However,the cells have lacked
certain characteristic macrophage functions, such as
IgGEA rosette formation and phagocytosis. Thus, the cell
cultures established from Hodgkin's tissues have not dem
onstrahedconsistent properties.
4 The
I These
studies
were
supported
in
part
by
funds
from
the
Mid-South
Cancer Research Foundation, Memphis, Tenn.
2 To whom
requests
3 Present
address:
for reprints
Department
should
be addressed.
of Microbiology
and
University Medical Center, Durham, N. C. 27710.
Received January 30, 1978; accepted June 12, 1978.
SEPTEMBER
1978
Immunology,
Duke
abbreviations
used
are:
EBV,
Epstein-Barr
virus;
IgGEA,
lgG
anti
body-sheepRBC complexes; IgMEAC,1gMantibody-sheepRBC-comple
ment complexes; FCS, fetal calf serum; MEM, minimal essential medium;
EBNA, Epstein-Barr virus nuclear antigen; SRBC, sheep RBC; E-rosette,
erythrocyte (sheep RBC)-forming rosette; IgMEA, 1gM antIbody-sheep RBC
complexes; PBS. phosphate-buffered saline (0.15 N NaCl-0.0028 N mono
basic sodium phosphate-0.0072 u dibasic sodium phosphate).
3033
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A. N. Roberts et al.
The controversy surrounding the identity of the Hodgkin's
tumor cell can be resolved only by accumulating further
data from additional established cell lines. In this report we
describe experiments undertaken to characterize the prop
erties of a continuous cell line (designated HuT,, and
initiated by Dr. K. L. Smith, St. Jude Children's Research
Hospital) initiated from an involved lymph node of a patient
with Hodgkin's disease of the mixed cellularity type. The
properties of 4 continuous cloned cell lines have been
compared with those of the parent HuT1, line. Each clone
was established from a binucleate, Reed-Sternberg-like cell
isolated from HuT11cultures during early serial passage.
Our investigations described herein include cultural char
adheristics, morphology, heterotransplantation , immunoflu
orescence tests for membrane immunoglobulins, rosette
assays for lymphocyte and monocyte surface markers,
histochemistry, phagocytosis, and cytogenehic analyses.
MATERIALS AND METHODS
Tumor Donor. An enlarged cervical lymph node was
surgically excised from a 6-year-old female patient. Histo
logical examination of a portion of the node confirmed the
diagnosis of Hodgkin's disease of the mixed cellularity type.
The pathological stage of disease was recorded as lIA after
physical examination, chest X-rays, i.v. pyelograms, bone
marrow biopsy, and staging laparohomy. This patient was
diagnosed 6 months following the detection of Stage IVB
Hodgkin's disease of the same type in an 11-year-old male
sibling.
Cell Preparationand Culture.The lymphnodewas proc
essed for culture within 1 hr of excision. The tissue was
finely minced ; the fragments were washed in Hanks' bal
anced salt solution and then dissociated in 0.1%
trypsin:EDTA
(1:1)solution
by rapidagitation
for20 mm at
25°.The residual tissue fragments were allowed ho settle,
and the cells were washed and resuspended in McCoy's
Medium 5A (Microbiological Associates, Inc., Bethesda,
Md.) supplemented with 10% heat-inactivated FCS (Micro
biological Associates) and containing penicillin (50 unihs/
ml), shrephomycin (50 @tg/mI),and kanamycin (50 @g/ml);
(Kantrex; Bristol Laboratories, Syracuse, N. Y.). This cul
lure medium was routinely used for all experiments, unless
otherwise specified. The cell suspension was distributed
into 75-sq cm Falcon culture flasks (Falcon Plastics, Ox
nard, Calif.), 2 ho 3 x 10@cells/flask, and incubated in
stationary cultures at 37°.The resulting cell monolayers
were first passaged at 2- ho 6-week intervals. After 11
passages, more rapid cell growth occurred, allowing weekly
passages at transfer ratios of 1:6 ho i :12. This established
cell line has been designated HuT11 and currently is in
excess of 200 serial passages.
Cultures of Cloned Cell Lines. After approximately 20
passages, HuT,, cells from stock cultures were plated in 60x iS-mm Falcon plastic dishes containing 10 ml of culture
medium at a concentration of approximately 50 cells/dish.
After overnight culture in a humidified incubator at 37°
under 5% CO2, giant binucleate cells were isolated micro
scopicaily with a Teflon cylinder attached to the plate with
a coal of silicon grease. Fresh medium was added, and
incubation was continued for 3 to 5 days ho allow develop
3034
ment of the isolated clones. The clones then were dispersed
with 0.1% trypsin, and the cells were washed and subcul
lured in 75 sq cm Falcon flasks. Four cloned cell lines have
been established
and have been designatedHuT11-1
,-2,-5,
and -6. Each clone has been passaged at weekly intervals
at transfer ratios of 1:6 to i :i2 and currently is in excess of
150 serial passages.
Cell Counts and Viabilfty Determination. Cell counts
were performed at x400with a hemacytomeher, and viability
was determined by Irypan blue exclusion. An aliquol of the
cell suspension was mixed with an equal volume of 0.4%
Irypanblueand incubatedat25°for
i mm, and a minimum
of 500 cells was counted. The results were recorded as the
percentage of cells showing dye exclusion. All cell counts
reported are viable cell numbers.
Preparation of Cells for MorphologicalStudies. Cells
from Irypsinized monolayers were resuspended ho 5 x 10@
cells/mi, then planted in i6- x i50-mm culture tubes or
Leightontubescontaining
9-x 35-mm covergiasses,
2 mI/
tube, and incubated at 37°until the monolayers reached
50% confluency. The coverglasses were removed, rinsed in
0.oi M PBS, pH 7.2, and fixed for iO mm either in absolute
methanol for staining with May-GrUnwald-Giemsa or
Wright's Giemsa or in Carnoy's solution for staining with
methyl green-pyronin . Morphological observations were
made with a Zeiss standard bright-field fluorescence univer
sal microscope fitted with xi2.5 eyepieces, x400 and
xi000 apochromat oil immersion objectives, and an Ophi
var. Photomicrographs were made with the use of a Zeiss
camera attachment equipped with a Leica 35-mm camera
body and Kodak Plus-X Pan film.
Determinationof Cell Size. Aliquotsof diluted suspen
sion from trypsmnized monolayers were observed with he
macytometers to avoid cell compression. The diameter of
each cell was measured at x450 to the nearest i .45-sm unit
of an eyepiece reticle, previously calibrated with a stage
micrometer (American Optical Co. , Buffalo, N. Y.). The
diameters were recorded for a minimum of 300 cells from
each preparation.
Growth Rate Determinations. Cells were dispersed from
cultures in logarithmic growth with the use of 0.i% trypsin
in Hanks' balanced
salt solution,
washed,
resuspended
in
culture medium, and counted. The suspension was ad
jushed to a final concentration of 2 x 10@cells/mI and
seeded in 60-mm-diameter Falcon No. 3045 multiwell cul
lure dishes, 5 mI/well. The cultures were grown at 37°in a
humidified incubator under 5% CO2, and the medium was
changed at 24-hr intervals. Monolayers from 4 dishes were
counted at daily intervals through Day 9. The cells from
each dish were dispersed with Irypsin and removed with 3
medium washes and then pelleled by centrifugahion, resus
pended, and counted.
The same protocol was used for growth rate studies in
each of the following: McCoy's medium supplemented with
0.1 , 0.5, 1.0, 5.0, 10, and 20% FCS, 10% newborn calf
serum (Microbiological Associates), and 10% human serum
(Flow Laboratories, Rockville, Md.); Dulbecco's MEM, Ea
gles's MEM, and Roswell Park Memorial Institute Medium
1640 (Microbiological
Associates), each supplemented with
10% FCS. All sera were heal inactivated (56°,30 mm) before
use.
CANCER RESEARCHVOL. 38
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Properties of Hodgkin's Cell Lines
Heterotranspiantation.The tumorigenic properties of
each cell line were assayed in young female Syrian ham
sters (17). Two- to 4-day monolayers were dispersed with
0.1% hrypsin, and the cell suspensions were washed in
serum-free Dulbecco's MEM. An aliquot of each suspension
was counted , and the cells were diluted to the desired
concentration in 0.1 ml. The hamsters were lightly anesthe
tized with ether and the cell inoculum was injected s.c. into
the cheek pouch. The presence of humor development and
the size of humor growth were recorded at weekly intervals,
until
thehumors became necrotic.
For transplantation primary or transplanted humors were
excised after 2 weeks of growth and washed with serum
free Dulbecco's MEM. Each humor was trimmed of excess
epihhelial tissue and finely minced. The tissue fragments
were allowed to settle, and the cell suspension then was
passed through sterile gauze ho remove residual fragments.
The cells were washed twice in medium, resuspended ho
the desired concentration, and inoculated as described
above. For histological examination slices of primary and
transplanted tumors were fixed in neutral formalin, sec
honed, and stained with hemahoxylin and eosin. Touch
preparations from freshly sliced tumors were fixed and
stained with hemaloxylin and eosin or methyl green-py
ronin.
resuspended in PBS, as described above. The final mono
nuclear suspensions usually contained about 90% lympho
cytes, and viability was greater than 98%.
indirect immunofluorescence was used to detect surface
immunoglobulin on the viable cell preparations. The mono
specific intermediate antisera included goat anti-human y,
a,
@,
8, €,K and A chains
(Cappel
Laboratories,
Cochran
yule, Pa.). The sera were heat inactivated for 30 mm at 56°
and ultracentrifuged (150,000 x g for 30 mm) before use.
Monospecificity of the sera was tested in immunoelectro
phoresis with normal human serum, human immunoglobu
lin reference sera (Kallestad Laboratories, Chaska, Minn.),
human K and A Bence Jones proteins (Behring Diagnostics,
Somerville, N. J.), and human lgD and IgE reference sera
(Behring Diagnostics). The viable cell assay used was mod
ified from a method described previously (44). Briefly, in
duplicate, 0.2 ml of cell suspension was mixed with 0.2 ml
of antiserum (1:2) and incubated at 4°for 60 mm. The cells
then were suspended to 10 ml in PBS, pelleted by centrifu
gahion, and washed 3 times with 10 ml of PBS ho minimize
the possibility of soluble immune complex formation. The
cell pellets were resuspended ho the original 0.2-mI vol
umes, mixed with 0.2 ml of fluorescein-conjugated rabbit
IgG against goat lgG (1:4; Cappel Laboratories), incubated,
and washed as before. The final cell pellets were mounted
Tests for EBNA. The anticomplementindirectimmuno in phosphate-buffered glycerol, pH 8.0, and observed by
fluorescence test (55) was used to detect the presence of fluorescence microscopy. The percentage of positive cells
EBNA. Raji lymphoblashoid cells, a nonproducer cell line was determined by counting at least 500 cells in each
derived from Burkitt's lymphoma (53), served as the positive
preparation.
control. The Raji cell line was kindly provided by Dr. Robert
Additional controls were included ho rule out possible
Naegele (St. Jude Children's Hospital) and grown in station
nonspecific globulin binding due to Fc receptors and non
specificity due to membrane bound FCS components.
ary suspension cultures in McCoy's medium supplemented
with 10% FCS. Tests were conducted in duplicate on at These included: (a) the direct tests with undiluted fluores
least 2 passage levels of each HuT11cell line. Smears of the cein-conjugated goat IgG F(ab')2 against human lgG F(ab')2
(Cappel Laboratories); (b) the direct test with fluorescein
cell suspensions were prepared on 24- x 50-mm cover
glasses, air-dried , and fixed in acetone:methanol (1:1) at conjugated rabbit lgG against goat lgG; (C)the indirect test
—20°
for 3 mm. The positive serum used was obtained
with undiluted normal goal serum as the intermediate
through the courtesy of Dr. Gertrude Henle (Division of serum; (d) indirect tests with goal anti-bovine serum and
Virology, The Children's Hospital of Philadelphia, Philadel
goat anti-bovine lgG (1:2; Miles Laboratories, Elkhart, Ind.)
phia, Pa.). The procedures for incubation with antiserum
as the intermediate globulins; and (e) blocking tests per
have been described (55). Observations were made with a formed by incubation with rabbit anti-bovine serum or
Zeiss fluorescent illuminator, dark-field condenser, BG 12 rabbit anti-bovine lgG (1:2; Cappel Laboratories) prior to
exciter filler, and No. 53 and 44 barrier filters.
indirect tests with the monospecific heavy- and light-chain
ImmunofluorescenceStudies for Surface Immunoglob antisera. Viabilities of the final cell preparations usually
ulins. Tests were conducted with passages 39, 87, and 124 were greater than 90%.
of the HuT,, cell line and passages 44 and 80 of each of the
Lymphocyteand MonocyteMembraneMarkers. Rosette
clones. Normal human peripheral blood lymphocytes and tests with the use of freshly collected SRBC in Alsever's
Raji cells were used as positive and negative controls,
solution were performed with passages 39, 80, and 132 of
respectively. Four- to 6-day monolayers of HuT,1 cells were HuT,, cells and passages 42 and 85 of each of the clones.
dislodged from the surface with a rubber policeman and Normal human peripheral blood lymphocytes and Raji cells
easily dispersed into a single-cell suspension by rapid, were used as positive and negative controls, respectively.
gentle pipetting. Approximately 3 x 10@cells were washed The cell preparation and washing procedures were as
3 limes with 50 ml of 0.01 M PBS, pH 7.6, at 4°.The cells described in the fluorescence studies, with the use of
were resuspended in buffer ho 1 x 10@cells/mI. Viability
modified barbital buffer, pH 7.4 (26). The tests for E-rosette
usually was greater than 98%. Raji cells were collected
and IgGEA, IgMEA, and IgMEAC rosette formation were
directly from suspension cultures and prepared in the same conducted essentially as described by Hunter et al. (26).
manner.
The SRBC used in E-rosette tests were pretreahed with
For preparation of normal human lymphocytes, 70 ml of neuraminidase (2.5 units/mI; Calbiochem, La Jolla, Calif.)
venous blood containing EDTA were subjected to Ficoll
for 30 mm at 37°,washed 3 times in barbital buffer, and
Hypaque centrifugation (6), and the mononuclear cells, diluted ho a 0.5% suspension. The lgG and 1gM antisera ho
collected from the gradient interphase, were washed and SRBC were obtained from Cordis Laboratories, Miami, Fla.
SEPTEMBER1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
3035
A. N. Roberts et al.
The CS-deficient complement used to prepare IgMEAC was
collected from retired breeder AKR mice (The Jackson
Laboratory, Bar Harbor, Maine) and shored at —70°.
All
rosette tests were performed in triplicate, with incubation
at 5°and 25°for 1 to 2 hr.
(Fig. 1). The smaller cells (8 to 14 .tm) were round and
lymphoid-like or spindle-shaped with a single nucleus pos
sessing 1 or more prominent nucleoli. Mononucleate polyg
onal cells (12 to 18 @m)
and giant cells (30 to 48 @m)
with 1
to 8 nuclei also were prevalent. Throughout all passage
Phagocytosls.Latex beads, 1.01 @m
in diameter (Dow levels the cultures have consistently demonstrated numer
ous binucleate giant cells that resemble the typical Reed
Diagnostics, Indianapolis, md.), were washed in culture
Shernbergcell(Fig.
2).
medium, resuspended to 5 x 10@particles/mI, and then
Each of the 4 cloned cell lines, initiated from a single
added ho 50% confluent monolayers of HuT11 cells (18).
binucleahe Reed-Shernberg-like cell of the HuT11line, dem
After various intervals of incubation at 37°,the monolayers
onstrated a more homogeneous cell morphology. The pre
were washed 5 times with 0.01 M PBS, pH 7.2, then
dominant cells of clone HuT,1-1 were polygonal, possessing
dispersed with hrypsin, and prepared as smears on slides.
a single nucleus with 1 or more prominent nucleoli (Fig. 3).
The smears were fixed for 5 mm in absolute methanol,
stained with eosin, and counted microscopically for cells This clone also contained a few lymphoid-like cells and
with membrane-bound and phagocytosed particles. The sparsely distributed bi- and multinucleale giant cells. The
majority of cells in clone HuT11-2were lymphoid-like, some
smears then were immersed overnight in xylene ho solubil
ize exhracellular particles and recounted for cells with
with eccentric nuclei (Fig. 4). These cultures also consist
internalized particles. In addition, 50% confluent coverglass
ently demonstrated a few mononucleate and mulhinucleahe
monolayers of HuT11 cells were incubated at 37°with a giant cells that frequently contained inhracytoplasmic vacu
0.01% neutral red solution and then examined microscopi
oles. Clone HuT11-5consisted of spindle-shaped and polyg
cally as wet-mount preparations for dye incorporation.
onal cells with a clustered pattern of growth (Fig. 5). When
Enzyme Histochemistryand LysozymeAssays. Mono treated with trypsin, the monolayers tended to disperse in
layers of the HuT,1 and cloned cell lines at various passage
small aggregates. These cells also showed a greater varia
levels were dispersed with Versene and prepared as smears
lion in size than did cells of the other cloned lines. Cells
that morphologically resembled macrophages predomi
on slides for enzyme histochemistry, as described previ
ously (42, 43). Buffy coal leukocytes from normal human
nated in clone HuT11-6,most of which were mononucleate
with prominent nucleoli and numerous cytoplasmic vacu
peripheral blood were used as controls. Assays were con
oles (Fig. 6). Mono- and binucleate giant cells were more
ducted for acid phosphatase (3, 64), alkaline phosphatase
prevalent than in the other cloned cell lines. Features in
with the use of naphthol AS-TA phosphate (7, 64), nonspe
common with all cell lines have included hyperpyroninophi
cific esherases with the use of the substrates a-naphlhol
acetate and naphthol AS acetate (21, 59, 64), fluoride
ha and frequent observation of mitohic figures in binucleate
cells.
resistant esherase (36), leucine aminopeptidase (8, 64), and
Growth Rate. The HuT,1 cell line demonstrated a rapid
peroxidase (49). The lysoplahe technique (48) was used to
population-doubling time of 12.3 ±0.6 hr during the first 4
assay for the presence of lysozyme in medium from 4-day
days of logarithmic growth. The cloned lines had compara
HuT11cell monolayers and in each of 3 extracts prepared
ble growth rates, ranging from 12.1 ±1.7 (clone HuT,,-2) to
from approximately 1 x 10@cells. The extraction proce
13.6 ±0.8 (clone HuT11-6)hr. Comparisons of the growth
dures were: (a) incubation for 5 mm at 4°in 0.3 ml of 0.5%
rates of early serial passages (passages 32 to 34) with those
or 1.0% Nonidet P-40 (Particle Data Laboratories, Ltd.,
Elmhursh, Ill.); (b) 6 rapid freeze-thaw cycles in 0.3 ml of
of long-term cultures (passages 67 to 69 of the clones and
passages 92 and 155 of the HuT1, line) revealed no signifi
0.15 M phosphate buffer, pH 6.3; and (C) sonic disruption
for three 30-sec cycles (Model 185 sonicalor; Heal-Systems
cant differences. The monolayers grew to high saturation
densities, ranging from 67 x 10@cells/sq cm (clone HuT,1Ultrasonics, Inc. , Plainview, N. Y.) in 0.3 ml of buffer. Cell
debris was removed from all samples by centrifugation at 6) to 74 x 10@cells/sq cm (clone HuT,,-2), after 9 days in
culture. The high densities were due to multilayered cell
10,000 x g for 10 mm at 4°.All extracts were assayed
growth, with loosely attached clusters of rounded cells
immediately after preparation. Egg while lysozyme was
used as a standard.
prevalent in all of the cultures. Trypan blue exclusion tests
revealed that over 98% of the clustered cells were viable.
Cytogenetlc Analysis. Chromosome preparations were
On transfer to new culture dishes, these cells resumed the
made from monolayers of HuT1, cells in logarithmic growth.
The monolayers were incubated with colchicine (0.05 @g/ typical monolayer pattern of growth.
Effects of Serum and Medium on Cell Growth. With
ml) for 2 hr at 37°,dispersed with trypsin, and then swelled
medium changes at 24-hr intervals, growth rates of the
with 0.075 M KCI for 30 mm at room temperature. The cells
HuT1, cell monolayers were comparable in McCoy's Medium
were fixed with 3 changes of freshly prepared
5A supplemented with 5, 10, and 20% FCS. Logarithmic
methanol:glacial acetic acid (3:1) for a total of 30 mm and
growth occurred during the first 96 hr, with generation
stained with Giemsa (45).
times ranging from 12 to 13 hr. A reduced rate of growth
was observed in 0.1 , 0.5, and 1.0% FCS, with generation
RESULTS
times of 34, 29, and 26 hr, respectively. When the cells were
Cultural and MorphologicalProperties.The HuT,, cell cultured for 7 days without a medium change, 5% FCS was
required to support continued growth. In medium contain
line grew as a dense, loosely adherent monolayer of mono-,
ing 10% human serum or newborn calf serum, the monolay
bi-, and mulhinucleate cells of heterogeneous morphology
3036
CANCER
RESEARCH
VOL. 38
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Properties of Hodgkin's Cell Lines
ers detached from the culture surface and over 90% of the
cells were nonviable after 48 hr.
A comparison of the 9-day growth of HuT,1 cells in
different culture media supplemented with 10% FCS
showed that optimal growth occurred in McCoy's Medium
5A. These
cultures
reached
confluency
by 4 days and
demonstrated a 12-hr generation time. The cells continued
to grow at a reduced rate between 5 and 9 days, with an
increase in generation time to 26 hr. Roswell Park Memorial
Institute Medium 1640 supported a similar rate of cell
growthduringthefirst
3 days,whilegrowthwas reducedin
Dulbecco's MEM and minimal in Eagle's MEM.
Heterotransplantationof the HuT,, Cell Lines. HuT,1
cells (passage 93) were injected s.c. into hamster cheek
pouches at various concentrations to determine the cell
dose required for tumor induction. An inoculum of 1 x 10@
cells produced 100% tumor formation, while a minimum
dose of 100 cells was required for tumor induction. The
vascularized and consisted of malignant cells sparsely infil
trated by polymorphonuclear leukocytes, presumably of
host origin. The tumor cell morphology resembled a malig
nant histiocytic
lymphoma,
and mihoses were numerous.
Giant binucleale Reed-Slernberg-Iike cells were sparsely
distributed throughout the mononuclear humor cell masses.
ImmunofluorescenceTests for EBNA. Four serial pas
sages of the HuT,, cell line (through passage 78) and 2
passages of each clone have been tested in duplicate for
EBNA. All observations have been negative. In each test
over 95% of the Raji control cells were strongly positive for
nuclearfluorescence.
Studies for Lymphocyte and Monocyte Membrane
Markers. As a marker for B-lymphocytes,membraneim
munofluorescence tests for human immunoglobulins were
performed with viable cells from various passages of the
HuT,, cell line. The results consistently demonstrated ap
proximately 20% of cells positive for -yand K when reacted
50% tumor-producing
dose was calculated to be 5 x 102 with the monospecific goal antisera (Table 1). The cell
cells (54). Tests with earlier serial passages (passages 27 cultures and normal lymphocyte preparations contained a
and 45) of HuT,, cells yielded comparable data. The tumors
low percentage (average, 4%) of cells that nonspecifically
induced by HuT,, cells were transplanted through 3 sequen
bound globulin from normal goat serum, suggesting that
hal animal transfers. Inocula of 1 x 10@through 1 x 106 these cells possessed Fc receptors. Similar studies on
cells produced 100% tumor formation. After the third trans
passages 44 and 80 of each cloned line yielded comparable
fer a minimum of 100 cells still was required for tumor
results. The data supported the conclusion that a consistent
percentage of cells in each cell line showed monoclonal
induction. Thus, subpassage of the tumor cells produced
no apparent enhancement of humorigen icily.
synthesis of lgG. However, direct immunofluorescence with
goat F(ab')2
reagentdetectedonly0.1ho0.2% of cellswith
Passages 32 and 85 of each of the 4 cloned lines induced
100% tumor formation in hamsters within 2 weeks after this B-cell marker, indicating that most of the positive
injection with an inoculum of 1 x 10@cells. Cell dose-tumor
membrane reactions were nonspecific (67). The predomi
induction analyses were not performed with the clones.
nant morphological cell types showing positive reactions
In animals given injections of 10@to 10@HuT,, cells, with F(ab')2 reagent were mononucleale, ranging in size
individual tumors grew to a volume of 3000 cu mm within 4 from 8 to 20 @m.
Extensive control tests to detect nonspe
weeks, but necrosis frequently occurred in later stages of cific binding of rabbit lgG and FCS components gave
growth due to surface abrasion . From sequential measure
completely negative results (see “Materials
and Methods―
ments of tumor size in 3 animals, the average doubling rate fordetails).
of tumor volume was 2.2 ±0.6days.This rateremained
Specific tests for Fc membrane receptors were unreward
relatively constant through 20 days of observation. Tumor
ing, with a low, insignificant percentage of cells in each cell
regression was never observed. The humors were highly
line demonstrating IgGEA and IgMEA rosette formation
Table 1
cellsThe
Immunofluorescence demonstration of membrane immunoglobulin on viable HuT,,
of0.01 cell-antiserum reactions were incubated for 60 mm at 4°,followed by 3 washes in 10 ml
M PBS, pH 7.6.%
reactions@'Human
of cells showing positive
immunoglobulin specificity of intermediate goat
antiserumb
Serial
@
F(ab')@'HuT,
Cells tested
passage
39
0.187
0.2124
0.1Lymphocytes'@
0.3Rajicells
lgG
‘y
a
j@
20.2
20.4
0
0
0
0
21.0
3.3
0
0
0
11.7
0
(1 Each
number
represents
0
the
average
0
percentage
a
sc
0
0
0
0
19.6
20.8
0
0
0
1.5
0
0
20.2
10.1
0
2.7
0
0
determined
from
A
0
500
0
to
1000
cells
lgG;
all
0
counted
in
duplicatepreparations.
U
Reactions
were
revealed
with
fluorescein-conjugated
rabbit
anti-goat
cells
tested
showed an average 4% (range 3.7 to 4.2%) nonspecific reaction with normal goat serum that has
been subtracted from the recorded values.
C Direct
immunofluorescence
(I Separated
from normal
with
human
goat
lgG
peripheral
F(ab')2
blood
against
human
in Ficoll-Hypaque
lgG
F(ab')2.
gradients;
each percentage
was derived from counts of duplicate lymphocyte preparations from each of 3 normal donors.
SEPTEMBER1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
3037
A. N. Roberts et al.
(Table 2). Likewise, tests for IgMEAC rosette formation
were essentially negative, demonstrating the absence of
complement receptors. A consistent but low percentage of
cells of each line formed weakly adherent, spontaneous E
rosettes with SRBC. These reactions were not convincing
as a marker for T-lymphocyles. A high percentage of cells
of the cells had incorporated delectable levels of neutral
(Table 3). Clone 1, however, showed weaker enzyme reac
tivities, and all cell lines were negative for alkaline phospha
tase and peroxidase. The cells retained nonspecific ester
ase staining in the presence of 4 x 10_2M sodium fluoride,
as did the monocytes in control leukocyte preparations.
Lysoplale lysozyme assays of 4-day culture medium and
extracts prepared from HuT,, cells were negative.
Cytogenetic Analysis. Chromosome counts of 50 mitohic
figures on each of 30 melaphase preparations from passage
42 of the HuT,, cell line showed a variation of 37 ho 190
chromosomes. Of these 405 were hypodiploid with a mode
of 44 chromosomes, 900 were hyperdiploid with a mode of
red, which increased
54 chromosomes,
from
various
serial
passages
of all cell
lines
exhibited
phagocytosis in the presence of latex particles and neutral
red dye (Table 2). Maximum uptake of the particles was
observed after 2 hr of incubation at 37°,with an average of
14 particles/positive
cell. After 15 mm of incubation,
in intensilywith
continued
most
incubation.
The dye was localized in cytoplasmic vacuoles and perinu
clearareas.
183 were hypoletraploid
with 77 ho 91
chromosomes, and 12 were hyperochoploid with approxi
malely 190 chromosomes. Frequent milotic abnormalities
were observed, including Iripolar and quadripolar spindles,
lagging chromosomes, and chromosome bridges. Similar
ranges in aneuploid chromosome numbers and comparable
EnzymeProperties.Cylochemicalstainsof the HuT,, cell
lines revealed strongly positive reactions for nonspecific
esterase, acid phosphalase, and leucine aminopeptidase
2Membrane
Table
receptors and phagocytic properties of HuT,
cellsPellets
aftergentle of treated
SRBC and test cells (100:1) were incubated
at 5°and 25°for 1 to 2 hr;
wasdetermined
resuspension, the percentage of rosette-forming cells (surrounded by 3 or more SRBC)
by counting
a minimum
of 200 cells.
The
percentage
of phagocytic
cells
was
determined
particlesand from counts of 500 cells after incubation of each monolayer at 37°with latex
neutral red dye for 2 hr and 15 mln, respectively.Rosette-forming
(%)(‘Serial
cells (%)@)
Phagocytosis
NeutralCells
redHuT1,
tested
passage1
98HuT,-1
97HuT,,-2
93HuT,,-5
97HuT,-6
E
IgGEA
39
42
42
42
22
21
22
19
8
6
5
3
4
7
3
3
2
3
1
2
86
85
82
91
42
23
36
7
27
4
17
2
16
93
4
98
0
5
0
93
0
0
Leukocytesd
5Rajicells
(1 Tests
on
b Average
different
serial
percentage
passages
of
of positive
each
cells
cell
IgMEA
line
in triplicate
gave
IgMEAC
consistent
tests.
Cell
Latex
results.
viabilities
were
greater
than
98%
and comparable rosette formation occurred at 5°and 25°.
C Average
percentage
of
positive
cells
in
triplicate
tests
of
monolayers
incubated
with
5
x
10@
latex particles (1.01 @m)
and 0.01% neutral red dye.
(I Buffy
coat
leukocytes
collected
from
normal
human
peripheral
blood;
rosette
studies
used
mononuclear cells prepared by Ficoll-Hypaque gradient separation. Each percentage was derived
from counts of triplicate
cell preparations
from each of 3 normal donors.
Table 3
Enzymeproperties of HuT1,cell monolayers
Histochemistry
studies
were performed
on at least 2 passage
levels of each HuT,, cell line, with the use of cell
smears prepared from Versene-d ispersed monolayers.
histochemistryHuT,HuT,-1HuT,-2HuT,-5HuT,-6Leukocytes@'Acidphosphatase
Enzyme
++(Ia-Naphthol
Alkalinephosphatase++b
+“a-Naphthol esterase+
+efluorideNaphtholacetate esterase + sodium+
+@‘Leucine AS
—+
acetate esterase+
+@‘Peroxidase—————++c
aminopeptidase+
a
Buffy
coat
leukocytes
—++++++++c
++
+±+
+++
+++
++
++
++
+++
++
++
++
++
++
++++
prepared
from
human
peripheral
blood;
replicate
smears
were
prepared
from
3
normal
donors.
b Positive
reactions
are
scored
C Polymorphonuclear
leukocytes
(1 Polymorphonuclear
leukocytes.
from
and
strong
(++)
to weak
(±) intensity
of stain
observed
in the
majority
of cells.
monocytes.
e Monocytes.
3038
CANCER RESEARCH VOL. 38
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Properties of Hodgkin's Cell Lines
mononucleate cells, similar to those described by Kaplan
and Gartner (31). Clones HuT,,-1 and -5 have demonstrated
a predominance of polygonal, epithelioid cells interspersed
with a few rounded and spindle-shaped cells and have more
closely resembled the Hodgkin's monolayer cell lines de
DISCUSSION
scribed by Long et al. (39). All of the morphological types
In this investigation properties of the HuT,, cell line, characteristic of the cloned lines have persisted in the
derived from an involved lymph node of a patient with Stage heterogeneous parent line, with no apparent exclusion or
selection of any cell types due ho long-term culture. Also,
IIA Hodgkin's disease, have satisfied the criteria of neopla
mono-, bi-, and mulhinucleate giant cells have consistently
sia. A consistent proportion of HuT1, cells morphologically
resembled the in vivo tumor cell population, and the cells appeared in the 5 cell lines, with more abundant distribution
have demonstrated a rapid growth rate through long-term
in the parent line and clone 5. Thus, the clonal investiga
continuous culture. Early and long-term serial passages of lions have revealed that progeny arising from an isolated,
binucleate giant cell may demonstrate considerable varia
HuT,, cells have shown consistent aneuploid karyolypes
and have been heterotransplantable, inducing rapid humor lion in cell morphology. These observations are suggestive
growth in the hamster cheek pouch with a minimum inocu
that the Hodgkin's neoplastic cells have characteristics of
lum of 100 cells. Repealed tests for EBNA have been more primitive, undifferentiated, stem-line cells. However,
negative, thus distinguishing the HuT,1 line from lympho
morphological variation of in vitro cultured cells is, at best,
a weak parameter for conclusive interpretation.
blastoid B-cell lines frequently initialed from human lymph
oid tissues and peripheral blood specimens (46).
Indirect immunofluorescence tests for EBNA on different
serial passages of the HuT,1 cell lines have been negative,
Four cloned cell lines have been established from iso
laled, binucleate, Reed-Slernberg-like cells of the HuT11 indicating the absence of EBV genome expression. The
line. To our knowledge, these are the first such cloned cell possible role of EBV in Hodgkin's disease has been ex
lines to be described. The capacity of the binucleate Hodg
plored by numerous investigators, due to the detection of
kin's cell for replication has long been a subject of contro
high EBV antibody liters in Hodgkin's disease patients (9,
versy. Cytological studies of tissues from Hodgkin's pa
22, 23, 25, 28, 33, 57). Interpretations from these serological
hients provided little evidence for milosis or DNA synthesis studies have implicated EBV as a causative agent of the
in binucleate or multinucleahe
Reed-Sternberg
cells (41,
disease (22). Others have accounted for the enhanced
50). It was concluded that these cells are “end
stage,―with
levels of anhi-EBV antibodies on the basis of compensating
humoral responses in patients with impaired cell-mediated
little or no capacity for replication. However, Boeckeretal.
(5) demonstrated
clonal proliferation of aneuploid, hypote
immunity (33, 35). Although there has been a distinct
traploid cells derived from Hodgkin's tissues in diffusion
association of high anhi-EBV liters with lymphocyte deple
chambers implanted i.p. into mice. More recently, observa
lion and sarcomahous types of Hodgkin's disease (28, 56),
lions of Inhaled thymidine incorporation into nuclear DNA Chan et al. (9) have demonstrated an increased cell-me
and of milotic figures in binucleate cells of neoplastic
dialed immune response to Epstein-Barr virion antigen in
Hodgkin's cultures have provided further evidence for their Hodgkin's disease patients. The ehiological significance of
mihohic capability (31). In our studies, the successful clonal elevated anti-EBV responses has been challenged, how
growth from isolated binucleate cells has confirmed their ever, by data from a recent 2-year serological case control
capacityforreplication.
study that failed ho reveal a relationship between antibody
The cultural characteristics of the HuT1, cloned lines have titers and disease prognosis (23). EBNA tests on the HuT,1
been very similar to those of the parent line. Each has cell lines, likewise, have failed to provide evidence for an
demonstrated a rapid growth rate, with a doubling time of etiological
roleof EBV in the mixed cellularity
type of
12 to 14 hr, thus producing monolayers of high saturation
disease.
Positive membrane reactions for lgG and K were revealed
density. Sustained cell growth has been accomplished only
in medium supplemented with FCS. Human serum and in approximately 20% of viable cells in each HuT,, line by
newborn calf serum obtained from commercial sources indirect immunofluorescence with heavy- and light-chain
failed to support growth or even maintain cell viability.
monospecific immunoglobulmns. Although tests for the
Although the cause for growth inhibition by these sera has other class-specific heavy-chain antigens and A light chain
not been explored, other investigators who have used were negative, as were numerous controls ho detect non
human serum or pleural fluid in culture media have encoun
specific globulin binding and FCS components, this per
tered difficulties in establishing permanent Hodgkin's cell centage of positive cells was not confirmed by direct im
lines(31).
munofluorescence with a specific IgG F(ab')2 reagent (67).
Distinct differences have been observed in the morpho
Numerous immunofluorescence and immunohislochemical
logical characteristics of each cell line. Cells of the parent studies of Hodgkin's cells in fresh tissue specimens, cell
line have remained morphologically heterogeneous through suspensions, and short-term cultures from involved tissues
more than 200 serial passages, while each cloned line has have suggested that the giant tumor cells are derived from
been composed of a somewhat more homogeneous cell transformed B-lymphocytes (2, 5, 12, 19, 20, 29, 34, 63).
population. Rounded, lymphoid-like cells, 8 to 12 @min Pitfalls in these approaches, however, have included failure
diameter, have predominated in clone HuT11-2,while clone to identify nonspecific membrane-binding of test reagents
HuT11-6has been composed primarily of large (25 to 40 @tm) and hodistinguish cell-synthesized globulin from that which
chromosome abnormalities have been observed in meta
phase preparations of passages 24, 82, and 150 of this cell
line.
SEPTEMBER1978
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3039
A. N. Roberts et al.
is bound or internalized from external sources. In contrast,
studies by Kaplan et al. (30, 31) and Long et a!. (39) have
contributed mounting evidence against a B-cell origin of
the Hodgkin's neoplastic cell. The questions posed by our
immunofluorescence results remain to be resolved by fu
lure kinetic studies, possibly with mihogenic stimuli, ho
reveal de novo lgG synthesis, since the low percentage (0.1
ho 0.2%) of positive cell reactions with lgG F(ab')2 reagent,
although suggestive, cannot alone serve as convincing
evidence for the presence of B-lymphocytes.
The HuT1, cell lines lacked characteristic B-lymphocyte
and macrophage membrane markers, as shown by essen
hially negative rosette formation with IgGEA, IgMEA, and
IgMEAC. Other continuous Hodgkin's monolayer cell lines
also have failed ho demonstrate these markers (39), while
the shorter-term giant cell cultures of Kaplan and Gartner
(31) possessed Fc and complement receptors characteristic
of macrophages. Long et al. (39), however, have shown that
in vitro culture conditions may have a dramatic influence
on the expression of membrane markers, since suspension
cell lines that spontaneously arose from long-term negative
monolayers gave positive Fc and complement receptor
reactions. In contrast a high percentage of monolayer cells
demonstrated nonspecific E-rosette formation, while the
suspension cultures were negative. The HuT,, monolayers
also demonstrated a low percentage of weakly adherent E
rosettes. Although it has been demonstrated conclusively
that human T-Iymphocytes possess a receptor for SRBC
that can be isolated as a discrete molecular species (10),
many human cell lines unrelated to T-cells show nonspe
cific E-rosette formation after long-term culture in vitro (68).
These findings strongly emphasize that attempts to identify
cells of continuous cell lines through membrane receptor
functions should be approached with caution until the
sources of nonspecific reactivity have been revealed. In
addition, a profound effect of cell cycle kinetics on the
expression of membrane markers has recently been dem
onstrahed (32), indicating that cell synchronization may be
required for accurate
receptor
quantitahion.
Cylochemical stains of the HuT1, cell lines revealed abun
dant esterase, fluoride-resistant nonspecific eslerase, acid
phosphalase, and leucine aminopephidase activities, al
though clone HuT,,-1 showed somewhat weaker enzyme
reactivities. These enzymes are comparable with those
demonstrated in other continuous monolayer cell lines (39).
However, lysoplale lysozyme assays of HuT,1 cell extracts
and culture medium have been negative. Lysozyme secre
lion, a characteristic of macrophages, has been a consist
ent property of the short- and long-term Hodgkin's cell
cultures described by other investigators (31, 39). The HuT,,
cell lines demonstrated strong phagocytic activity for both
latex particles and neutral red dye. The giant monolayer
Hodgkin's cultures of Kaplan and Gartner (31) also have
been strongly phagocytic.
In conclusion, although some of the enzyme and phago
cytic properties
of the neoplashic HuT,, cell lines suggest a
derivation from the hishiocyhe-macrophage family of cells
(11, 14, 16, 61), other more definitive macrophage markers
such as Fc (37) and complement receptors and lysozyme
secretion have been lacking. Tests for characteristic B- and
3040
T-lymphocyte functions also have not been convincing,
although further kinetic studies will be necessary to clarify
the immunofluorescence results indicative of monoclonal
lgG synthesis. Since malignant transformation usually
leads ho some functional dedifferenhiahion of cells that may
be reflected by loss or gain of enzyme activities and mem
brane markers, we believe that our investigations have not
conclusively ruled out either a lymphocyte or macrophage
origin of the Hodgkin's neoplashic cell.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Robert Neagele, St. Jude Children's
Research Hospital, for his assistance with the EBNA tests. We also are
grateful to Dr. RameshwarSharmafor providing helpful commentson the
manuscript.
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3041
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A. N. Roberts et a!.
Fig. 1. Heterogeneous HuT,, cell monolayer (pas
sage 45) composed of polygonal, spindle-shaped, and
lymphoid-like cells, as well as numerous mono-, bi-,
and multinucleate giant cells. Giemsa, x 200.
Fig. 2. Typical binucleate Reed-Sternberg-like cell
of the HuT1,cell monolayer. Giemsa, x 1000.
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CANCER RESEARCH VOL. 38
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Spindle-shaped, rounded, and larger mononucleate polygonal cells of clone HuT,,-1 monolayer (passage 21). May-Grunwald-Giemsa. x 1000.
Monolayer of clone HuT-2 (passage 38) showing a predominance of rounded lymphoid-like cells. May-Grunwald-Giemsa, x 1000.
Clone HuT-5 monolayer (passage 38) composed of polygonal, rounded, and spindle-shaped cells. May-Grunwald-Giemsa, x 1000.
Mononucleate giant cells of clone HuT,,-6 (passage 46), showing the cytoplasmic vacuolization characteristic of this cell line. May-Grunwald
Giemsa,x 1000.
3043
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Cultural, Morphological, Cell Membrane, Enzymatic, and
Neoplastic Properties of Cell Lines Derived from a Hodgkin's
Disease Lymph Node
Audrey N. Roberts, Kirby L. Smith, Barry L. Dowell, et al.
Cancer Res 1978;38:3033-3043.
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