Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN ESOPHAGEAL EPITHELI~L CELL LINES
FIELD OF THE I_NVENTION
The present invention relates to human epithelial
cells that originate from the esophagus and are immortal-
ized in culture, particularly to such cells geneticallytransformed with genes from tumor viruses, especially,
SV40 early region genes. The present invention also
relates to methods of using such cells for detection of
various types of agents, for instance, chemicals that can
cause cancer.
BACKGROUND OF THE INVENTION
Epidemiological evidence indicates that esophageal
cancer is associated with exposure to chemical carcinogens
in the environment and in the diet. Factors associated
with an increased risk of developing esophageal cancer
include tobacco, alcoholic beverages, and moldy foods, all
of which contain nitrosamines; and asbestos. As in most
forms of cancer, esophageal neoplasms probably are the
result of a sequence of genetic and phenotypic changes in
the esophageal epithelium.
For example, the phenotypic keratin patterns of
squamous cell carcinomas of the esophagus are consistently
different from those of the normal tissue (S.P. Banks-
Schlegal and C.C. Harris, Cancer Res. 1984, 44: 1153-
1157.) (M.P. Grace, K.H. Kim, L.D. True and Fuhs, 1985,
Cancer Res. 45: 841-846). Like epidermal keratinocytes,
the esophageal epithelial cell is able to form crosslinked
envelopes when it terminally differentiates (S.P. Banks-
Shlegal and C.C. Harris, Cancer Res. 1986, 250-258. This
; 30 funct~on is variably expressed in esophageal carcinomas
- (S.P. Banks-Shlegal and C.C. Harris, Cancer Res. 1984,
44:1153-1157 and carcinoma cell lines (S.P. Banks-Shlegal
and C.C. Harris Cancer Res. 1986, 250-25~). In addition,
a number of tumor associated antigens have been found in
esophageal neoplasms that are not present in normal cells,
including such antigens as human chorionic gonadotropin,
placental lactogen, ~-fetoprotein, carcinoembryonic
antigen, and nonspecific crossreacting antigen (C.L. Burg- -
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Kurland, D.M. Purnell, J.W. Combs, E.A. Hillman, C.C.
Harris and B.F. Trump, 1986, 46: 2936-2943).
To facilitate discovery of agents that represent
important causes of esophageal cancer in the human envi-
ronment, as well as development of preventive measures andtreatments, there is a need for cell culture systems
suitable for investigating chemical carcinogenesis in
esophageal epithelial cells. These cells are the precur-
sors of esophageal tumors, most of which are classified as
squamous cell carcinomas (S.F. Stinson and G. Reznik
Cancer of the Esophagus, Vol. 2., pp. 139-168, Boca Raton,
CRC Press, Inc. 1982). In investigations with the rat
esophagus, treatment of explanted tissue cultures with N-
nitrosobenzylmethylamine (NBMA) led to the establishment
of epithelial cell lines that, after prolonged subculture,
produced well-differentiated squamous cell carcinomas
following transplantation in vivo. (G.D. Stoner, M.S.
Babcock, G.A. Cothern, J.E. Klaunig, W.T. Gunning III, and
S.M. Knipe, 1982, Carcinogenesis 3: 629-634). There was a
20 positive correlation between transformation of these cells
and alterations in: (a) cytoskeletal microfilaments, (b)
response of the cells to extracellular calcium ion concen-
tration and serum-induced terminal differentiation (G.D.
Stoner, M.S. Babcock, M.M. McCorquodale, W.T. Gunning III,
25 R. Jamasbi, N. Budd, and B. Hukku, Comparative properties
of- untreated and N-nitrosobenzylmethylamine-transformed
rat esophageal epithelial cell lines (in press); and, (c)
the tendency of the cells to accumulate cholesterol
sulfate when grown in medium containing high calcium (J.E.
Rearick, G.D. Stoner, M.A. George, and A.M. Jetten, 1988,
~ancer Res. 48:5289-5295). However, comparisons between
NBMA transformed cells and normal cells were frequently
compromised by the marked tendency of rat esophageal
epithelial cells to undergo spontaneous neoplastic trans-
formation. Moreover, there are distinct histologicaldifferences between rat and human esophageal epithelium.
In view of these difficulties with rat cells,
there is a need to develop cultures of human esophageal
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WO9l/OS~62 PCT/US90/0~62
epithelial cells for transformation studies. Normal human
cells in general are known to be stable with respect to
neoplastic transformation in culture, and have never been
convincingly demonstrated to undergo spontaneous transfor-
mation. (G.H. Sack, Jr., 1981, In Vitro, 17: 1-19).
Therefore, normal human esophageal epithelial cells appear
to offer an advantageous system, for instance, for detec-
tion of substances with carcinogenic potential for the
human esophaqus.
On the other hand, human esophageal epithelial
cells are known to have a limited lifespan and replicative
capacity in culture (C.C. Harris, 1987, Cancer Res. 47:1-
lO; K. Sasajima, J.C. Willey, S.P. Banks-Schlegel, and
C.C. Harris, 1987, J. Nat. Can. Inst. 78:419-423), which
severely limits the number of cells that can be produced
from a single human tissue specimen. To provide such
cells in quantities and with the reproducible quality
needed for extensive screening of chemicals for carcinoge-
nicity or potential for protection against such carcino-
gens, for instance, the replicative potential of these
cells would need to be extended. Although human esophage-
al epithelial cells that are neoplastic have an unlimited
ability to replicate in culture, whether neoplastic b~
virtue of derivation from a cancer or by neoplastic
transformation with a tumor virus in culture, such trans-
formed cells are, of course, useless for applications
requiring normal cells to detect carcinogenic activities.
The neoplastic transformation of normal cells into
cells that can cause tumors (for example, upon injection
into rodents) is thought to result from multiple cellular
changes. Evidence supporting this concept comes from
studies which compared the neoplastic transformation of
normal cells and cell lines that were immortalized by
genetic transformation with viral and cellular oncogenes.
Unlike normal cells, such as those in primary cultures of
epithelial cells which are prepared directly from tissues,
immortalized cells have the ability to replicate in
culture for an indefinite number of generations.
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The results of the comparative studies indicate
that certain oncogenes induce immortalization in normal
cells but, by themselves, cannot induce frank neoplasia as
evidenced by tumorigenicity. However, an immortalization
gene can cooperate with a second class of oncogene to
induce tumorigenic potential. On the other hand, at least
some cells that have become immortalized without addition
of an exogenous immortalizing oncogene, nevertheless
require only the second type of oncogene for complete
neoplastic transformation into tumor forming cells.
It is known that normal human epithelial cells
have a limited lifespan in culture tC.C. Harris, 1987,
Cancer Res. 47:1-10), even when optimal serum-free media
are used (J.F. Lechner, and M.A. LaVeck, 1985, Meth. 9:43-
48). It is also known that certain human cells infectedwith the SV40 tumor virus have extended lifespans in
culture (V. Defendi, P. Naimski and M.L. Steinberg, 1982,
J. Cell Physiol. Suppl., 2:131-140). However, in many
cases, SV40-infected cells undergo a condition called
crisis during which the rate of proliferation markedly
; decreases or ceases entirely (G.H. Sack, Jr., 1981, In
Vitro, 17:1-19). Following crisis, a culture of any given
type of human cells infected with SV40 may eventually die;
or, in some cases, a few individual cells will eventually
replicate sufficiently to produce a visible colony of
descendants. This phenomenon of crisis is poorly under-
stood and may be equivalent to senescence in non-
transfected cells, in which the cells appear to "age" and
lose the ability to replicate. Crisis is not a universal
phenomenon, however, since there are reports that SV40-
transformed amnion epithelial cell lines (E. Gaffney, J.
Fogh, L. Ramos, J.D. Loveless, H. Fogh, and A.M. Dowling,
1970, Cancer Res. 30:1668-1676), and SV40-transformed
foreskin keratinocytes (M.L. Steinberg and V. Defendi,
1979, Proc. Nat. Acad. Sci. USA, 76:801-805), became
immortalized without the intervention of a crisis.
Thus, the art of immortalizing human cells is
highly unpredictable, not only with respect to the course
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of the process in those cases that are successful, but
also in the matter of achieving eventual success with any
given new tissue source.
It has been reported (S.E. Chang, 1986, Biophys.
Acta, 823:161-194, 1986; P. Kahn, W.C. Topp, and S~Io
Shin, 1983, Virology, 126:348-360; Y. Ohnuki, J.F.
Lechner, S.E. Bates, L.W. Jones and M.E. ~aighn, 1982;
Cell Genet., 33:170-178; B.J. Christian, L.J. Loretz, T.D.
Oberley and A. Reznikoff, 1987, Cancer Res., 47:6066-
6443), that human and other primate cells immortalized by
SV40 virus are nontumorigenic in certain mice which are
athymic, having an immune system disorder that allows
transplantation of tissues from other species without
rejection. (Since these creatures also happen to be
hairless due to a genetic defect, they are generally known
as "nude" mice.) Therefore, infection with SV40 appears
to offer a means for extending the replicative potential
of at least some types of cultured human epithelial cells.
A problem with the use of infectious SV40 virus to
immortalize cells is that, although the infection with
SV40 is not lethal and tends to be self-limiting, the
potential for resurgence of more active viral replication
persists. Such replication may alter the physiology of
the cells and thus render them unreliable for long term
culture and chemical testing purposes. Accordingly, the
present inventors have been involved in research efforts
to extend the replicative potential of various human
epithelial cells without introducing genes allowing
complete viral synthesis. Within the past year they have
demonstrated that genetic transformation with a plasmid
containing only certain SV40 genes, namely the so-called
early region genes, leads to immortalization of human
cells derived from tissues other than esophageal epitheli-
um. These include: epithelial cells from the bronchus
(R.R. Reddel, et al., 1988, Cancer Res., 48.1904-1909; see
also U.S. Patent Application Ser. No. 07/114,508 filed
October 30, 1987) mesothelium (Y. Ke, et al., 1989, J.
Path~l. 134:979-991; see also U.S. Patent Application Ser.
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No. 07/114,508), and neonatal prostate (M.E. Kaighn, et
al., 1989, Cancer Res. 49:3050-3056).
The plasmid used to immortalize these cells, which
is called pRSV-T, is known in the art (D.E. Brash, R.R.
Reddel, M. Quanrad, K. Yang, M.P. Farrell, and C.C.
Harris, 19~7, Mol. Cell. siol., 7:2031-2034). It also
comprises certain genetic elements from another tumor
virus, the Rous sarcoma virus. (Since this plasmid
comprises viral qenes, the process of genetic transforma-
tion in this instance is referred to as "transfection", aterm of the art that is a hybrid of the words "transforma-
tion" and "infection". Even though the viral genes of the
; plasmid are insufficient to produce infectious virus, the
use of the term transfection here i~ convenient for
distinguishing the process from that of neoplastic trans-
formation.
Further, the present inventors have shown that a
transfection procedure using strontium phosphate (D.E.
Brash, et al., 1987, supra) has yielded stable trans-
fectants (i.e., transfected cells) in the several humanepithelial cell types cotransfected with the plasmid pRSV-
T (as cited above), and that this strontium procedure is
more effective than the traditional calcium phosphate
method for insertion of DNA into epithelial cells that are
sensitive to high levels of calcium.
. SUMMARY OF THE INVENTION
The present invention contemplates the application
of methods of recombinant DNA technology to fulfill the
above needs for esophageal cell lines for chemical testing
and other purposes. More specifically, it is an object of
the present invention to provide a line of human esophage-
al epithelial cells or a derivative thereof having a
replicative capacity in cell culture that is enhanced
compared to normal cells, and is unable to produce tumors.
Further, it is an object of the present invention to
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provide a cell line of this type which replicates continu-
ously in cell culture.
As described in the following Examples, the normal
human esophageal epithelial cells can be made to grow
continuously by transfecting normal ~sophageal epithelial
cells with the T antigen gene of SV40 virus. The plasmid
containing the SV40 early region genes further comprises a
genetic element derived from another tumor virus, the long
terminal repeat (LTR) or Rous sarcoma virus, which serves
to stimulate transcription of the SV40 DNA sequences.
Other constructs can be used for the same purpose, accord-
ing to the methods well known in the art of genetic
engineering.
Transfection or infection can be accomplished by
use of a virus or a plasmid obtaining the T antigen gene
of the SV40 virus. Either transfection or infection may
lead to transformation of the cell line. Other transfor-
mation vectors may be useful, such as papilloma virus or
Epstein Barr virus. Techniques for making continuous
human cell lines are described in the following referenc-
es: Grahm, F.L., Smiley J., Russell, W.C. and Nairn, R.
Characteristics of a human cell line transformed by DNA
from human adenovirus type 5. J. Gen. Virol., 36: 59-72,
1977; Zur Hausen, H. Oncogenic herpes viruses In: J~
25 Tooze (ed.), DNA tumor viruses, Rev. ~d. 2, pp 747-798.
; Cold Spring Harbor, New York, Cold Spring Press, 1981;
DiPaolo, J.A. Pirisi, I., Popeseu, N., Yasumotoj S.,
- Poniger, J. Progressive changes induced in human and mouse
; cells by human Papillomavirus Type-16 DNA. Cancer Cells
30 5:253-257, 1987.
Further, it is an object of this invention to
provide a method for testing carcinogenicity of an agent,
comprising culturing the cells line of this invention with
an agent suspected of being carcinogenic; and determining
formation of an abnormal cellular mass (i.e., a trans-
formed "focus"~ by the cell line. The formation of a
focus or foci is indicative of carcinogenicity of an
; agent.
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Further, it is an object of the present invention
to provide a method for testing antineoplastic activity of
an agent, comprising culturing a cell line of this inven-
tion with a potential anti~neoplastic agent; and determin-
ing growth of the cell line. A lack of growth of the cellline is indicative of antineoplastic potency of an agent,
particularly for esophageal epithelial cells.
The present invention may be understood more
readily by reference to the following detailed description
of specific embodiments and the Example included therein.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Unless defined otherwise, all technical and
scientific terms used herein have the same meanings
commonly understood by one of ordinary skill in the art to
which this invention belongs. Although any methods and
materials similar or equivalent to this described herein
can be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
ABBREVIATIONS
The abbreviations used are: HE, human esophagus;
NHE, normal human esophagus; HBS, Hepes buffered saline;
BPE, bovine pituitary extract; SV40, Simian virus 40; CFE,
colony forming efficiency; PD, population doublings; EM,
electron microscopy.
DESCRIPTION
The term "immortalized" as used herein means that
the cell line grows continually without senescence when
cultured in a suitable growth medium.
The term "continuous cell line" as used herein
means that the cell line grows continually without senes-
cence when cultured in a suitable growth medium.
The "derivatives" of the esophageal cell line(s)
; of the present invention include cells which have been
further genetically altered by adding, for example, genes
for drug metabolizing enzymes, other oncogenes, anti-
oxidant genes, or other genes, thereby creating a continu-
ous derivative of the cell line.
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In one aspect, the present invention relates to a
line of human esophageal epithelial cells or a derivative
thereof having a replicative capacity in cell culture that
is enhanced compared to normal cells and being unable to
produce tumors. The general method for obtaining immor-
talized human esophageal epithelial cells is as follows.
Nor~al human esophageal (NHE) cells were obtained
from explant outgrowths of autopsy specimens from a
noncancerous male. Dispersed cell suspensions were plated
at 3-5 x 105 cells/dish and transfected with 10 ~g of
plasmid pRSV-T coprecipitated with strontium phosphate.
After 4 hrs, the cells were shocked with glycerol, as
described in Example 1 below. After the appearance of
foci of transformed cells, control and transfected cul-
tures were subcultured (2.5 x 105/100-mm dish). Control
strains could be subcultured for no more than 20 PDs,
after which they senesced.
pRSV-T-transfected cells (e.g., lines designated
HE-451, HE-457, see below) grew exponentially for approxi-
mately 50 PDs, after which they went into crisis. During
this crisis period which lasted for several months, the
majority of cells in both strains senesced. Recovery from
; this state depended upon their continued maintenance at
the highest possible density. Eventually, as described in
the Example below, one separate immortalized cell line,
designated HET-lA developed, from the HE-457 cultures.
A deposit of the cell lines of the present inven-
tion has been made at the American Type Culture Collec-
tion, 12301 Parklawn Drive, Rockville, Maryland, 20852,
U.S.A., on August 25, 1989, under accession number CRL-
10209 (HET-1A), in accordance with the Budapest Treaty.
The deposit shall be viably maintained, replaced if it
becomes non-viable, for a period of 30 years from the date
of the deposit, or for 5 years from the last date of
request for a sample of the deposit, whichever is longer,
and made available to the public without restriction in
accordancç with the provisions of
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the law. The Commissioner of Patents and Trademarks, upon
request, shall have access to the deposit.
The pRSV-T transfected cell lines expressed T-
antigen by immunoperoxidase nuclear staining. This is
consistent with the Southern blotting data indicating that
all lines contain integrated SV40 early region DNA. All
transfected lines were confirmed as epithelial by virtue
of their positive reaction with antibodies to keratin and
the presence of desmosomal junctions and cytoplasmic
microfilaments in EM preparations.
The chromosomal alterations in the transfected
human esophageal epithelial cells are similar to those
previously reported after infection with SV40 virus (M.E.
Kaighn, K.S. Narayan, Y. Ohnuki, L.W. Jones, and JoF~
Lechner, 1980, Carcinogenesis, 1:635-645). HET-1A, a
hypodiploid line, has a chromosome complement similar to
that of pRSV-T immortalized human bronchial epithelial
cells (R.R. Reddel, et al., 1988, supra) and prostatic
epithelial cells (M.E. Kaighn et al. 1989, supra). The
chromosome number and structural aberrations of HET-lA
cells will probably increase with continued subculture,
` since this has occurred with pRSV-T immortalized bronchial
and prostatic epithelial cell lines.
None of the pRSV-T transfected cells tested,
including the HET-1A cell line, were tumorigenic in
athymic nude mice. In addition, they did not induce
transient nodules of carcinoma-like cells at the injection
` site as had been observed with carcinogen-
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treated rat esophageal epithelial cells and human neonatal
prostatic epithelial cells (M.E. Kaighn, R.R. Reddel, JoF~
Lechner, D.M. Peehl, R.F. Camalier, D.E. Brash, U. Saffio-
tti and C.C. Harris, 1989, Cancer Res. 49:3050-30563.
Immortalized HET-1A cells may represent an intermediate
between normal and neoplastic in which altered growth
control is offset by the capability of undergoing differ-
entiation when injected into mice ~M.E. Kaighn, et al~,
1989, supra; E.J. Stanbridge, C~J~ Der, C-J~ Doersen, R.Y.
Nishimi, D.M. Peehl, B.E. Weissman and J.E. Wilkinson
1982, Science, 215:252-259).
Since both calcium and fetal bovine serum are
known to have profound effects on growth and differentia-
tion of epithelial cells, the effects of these factors
were assessed by clonal titration experiments, as de-
scribed in the Example. A dose-dependent stimulation of
CFE was observed with maximal CFE at 0.3 mM Ca+~ in both
HET-1A and HET-2A cell lines. In contrast, fetal bovine
serum inhibited the CFE at all concentrations tested.
Half maximal inhibition was seen at 1% (HET-1A) and 3%
(HET-2A) serum. There was no significant effect of either
Ca~+ or serum on the clonal growth rate (PDs/day) of HET-
1A or HET-2A cell lines at all concentrations tested. -
UTILITY OF THE CELL LINES
Identification of potential carcinoqens, tumor
promoters and antagonists thereof. These cells are useful
for screening chemicals or other agents in the human
environment for the potential to neoplastically transform
the cells. Putative carcinogens or tumor promoters may be
added to the growth medium of the cells and the state of
transformation of the cells as a function of time and dose
of exposure may be ascertained using anchorage indepen-
dence growth, matrix invasion, cells to cell communication
assays, and/or nude mice tumorigenicity assays. Anti-
carcinogenic or anti-promoting agents may be added to the
medium with carcinogens or promoters and the state of
transformation as a function of time may be ascertained as
for carcinogens or promoters alone. Such screening could
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be used to identify specific esophageal toxins in the diet
or in other substances and to identify dietary or other
compounds that could prevent chemically-induced esophageal
cancer.
Identificatlon of potential chemotherapeutic
dru~s. These cells are also useful, either before or
after further alteration by other oncogenes or carcino-
gens, for screening chemicals particularly suitable for
the treatment of esophageal cancer and related diseases,
by growing them in culture medium containing the chemical
to be tested and then, after a suitable period of expo-
sure, determining whether and to what extent cytotoxicity
has occurred, e.g., by trypan blue exclusion assay or
related assays (Paterson, Methods Enzymol., 58:141, 1979),
or by growth assays such as colony forming efficiency as
described in the examples, herein, all of which are
standard techniques well known in the art.
Identification of anti-eso~haqeal cancer druas
which act by inducing terminal cell differentiation.
Chemical and biological substances are screened for their
ability to induce terminal differentiation by adding them
to the growth medium of these esophageal cells and then
after a suitable period of time, determining whether a
complex of changes occurs, including for example, the
phenotypic keratin patterns of the normal esophagus (S.P.
Banks-Schlegel and C.C. Harris, 1984, Cancer Res. 44:1153-
1157; M.P. Grace, K.H. Kim, L.D. True and E. Fuchs, 1985,
Cancer Res. 45:841-846). Induction of terminal differen-
tiation may be an effective way of controlling the growth
of cancer.
;Studies on the metabolism of carcinoqens and other
xenobiotics. Carcinogens and other xenobiotics may be
added to the growth medium of these cells and the appear-
ance of metabolic products of these compounds may be
,~35 monitored by techniques such as thin layer chromatography
;~or high performance liquid chromatography and the like,
and the interaction of the compounds and/or their metabo-
lites with DNA is determined.
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St~dies of DNA mutaqenesis. Substances known or
suspected to be mutagens may be added to the growth medium
of the cells and then mutations may be assayed, e.g., by
detection of the appearance of drug resistant mutant cell
colonies (Thompson, Methods En~ymol., 58:308, 1979).
Studies of chromosome damaainq agents. Substances
known or suspected to cause DNA or chromosomal damage may
be added to the culture medium of these cells lines, and
then the extent of chromosomal damage may be measured by
~0 techniques such as measurement of the frequency of sister
chromatic exchange (Latt et al., In: Tice, R.R. and
Hollander, A., Sister Chromatid Exchanges, New York:
Planum Press, pp. 11 ff., 1984), and DNA damage can be
determined by the measurement of unscheduled DNA synthesis
(Mirsalis, J.C., Banbury Report vol. 13, pp. 83-99, 1982).
Studies of maliqnant transformation bY additional
oncoqenes. The effects of viral agents and transferred
genes, including oncogenes and high molecular weight
genomic DNA from tumors, may be tested using standard
assays such as anchorage independent growth or tumor
- formation in athymic nude mice. Further, such cells
transformed by an additional oncogene can be used to
`~ screen for potential chemotherapeutic agents by the
techniques described above, especially those which may be
specific for cells transformed by the activation of
particular oncogenes or combination of oncogenes.
Studies of cellular responses to growth factors
and production of qrowth factors. These cells are partic-
ularly useful for identification and purification of
growth factors important for growth and differentiation of
human esophageal epithelial cells, since they grow in
serum-free media. Therefore, responses to added growth
factors or isolation or produced growth factors can be
readily accomplished without interference from serum and
its complexities.
It will be readily appreciated by one skilled in
the art that a kit for screening carcinogenic or antineo-
; plastic agents, for instance, or for any other usage
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described herein, is easily assembled, comprising contain-
er(s) containlng cells of the present invention. Other
components routinely found in such kits may also be
included with instructions for performing the test.
EXAMPLE
Development of the HET-lA cell line.
Cell Culture. Normal human esophageal (NHE) cells were
obtained from outgrowths of autopsy tissue from noncancer-
ous individuals as previousl~ described (G.D. Stoner and
J. Klaunig, In: T.G. Pretlow III and T.P. Pretlow (eds.)
Cell Separation: Methods and Selected Applications, Vol.
2, pp. 8192. New York, Harcourt, srace and Jovanovich,
1983). The outgrowths were suspended with PET [ 1% polyvi-
nylpyrrolidone, 0.02% ethylenebis-(oxyethylenenitrilo)
;15 tetraacetic acid, 0.2% crystalline tryspin in HBS, pH 7.4]
at room temperature and subcultured into tissue culture
dishes or T flasks which had been coated with a mixture of
100 ~g/ml bovine serum albumin, 10 ~g/ml bovine fibro-
nectin (both from Calbiochem), and 20 ~g/ml type 1 colla-
gen (Vitrogen 100j Collagen Corp., Palo Alto, CA) to
promote cell attachment (J.F. Lechner and M.A. LaVec~,
1985, J. Tissue Culture Meth. 9:43-48).
A serum-free medium, LHC-9, purchased from
Biofluids, Inc., was used in the early phase of this
research tJ.F. Lechner and M.A. LaVeck, 1985, supra). It
was found later that a serum-free formulation developed
~7' for human epidermal keratinocytes (S.T. Boyce and R.G.
Ham, ~n: M. Webber and L. Sekely (eds.) In Vitro Models
for Cancer Research, Vol. 3, pp. 245-274. Boca Raton,
Florida, CRC Press, 1985) was more appropriate for growth
of these esophageal epithelial cells. This formulation,
consisting of modified MCDB 153 supplemented with growth
factors (KGM) is available from Clonetics Corporation, San
Diego, CA. The basal medium (cKBM) used in these studies
,35 and purchased frcm Clonetics Corporation was a customized
;modification of KBM without phenol red. Other components
omitted from cKBM were added just before use CaC12, FeSO4,
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ZnC12, trace element concentrate, thymidine, phospho-
ethanolamine, ethanolamine, glutamine and antibiotics).
The growth medium (cKGM-AE consisted of cK~M
supplemented with 5 ng/ml epidermal growth factor, 1.4 ~M
hydrocortisone, 0.1 mM ethanolamine and phosphoethanol-
amine, 5 ~g/ml insulin, 40 ~g/ml bovine pituitary extract
(BPE), 250 ~g/ml bovine serum albumin, and 0.5 ~g/ml
epinephrine. Antibiotics were added as needed (100
; units/ml penicillin G, 100 ~g/ml kana~.ycin, 50 ~g/ml
gentamicin).
Cultures were monitored for mycoplasma contamina-
tion by culture on anexic agar and by DNA fluorochrome
staining of an indicator culture (R. DelGiudice and H~Eo
Hopps, In: G.J. McGarrity, D.G. Murphy, and W.W. Nicols
(eds.), Mycoplasma Infection of Cell Cultures, pp. 57-69.
New York, Plenum Publishers, 1978). No contamination was
detected.
Transfection. Subcultures of NHE cells were used
for transfection. Cells were plated at 3-5 x 105/100-mm
coated dish and transfected the next day with 10 ~g of
plasmid DNA coprecipita~ed with strontium phosphate as
; previously described (D.E. Brash et al., 1987, supra).
The plasmid, pRSV-T, obtained from Dr. Bruce Howard, NCI,
is an ori construct (i.e., lacks the SV40 site for origin
of DNA replication) containing the SV40 early region genes
and the Rous sarcoma virus long terminal repeat (R.R.
Reddel, Y. Ke, B.I. Gerwin, M.G. McMenamin, J.F. Lechner,
RoT~ Su, D.E. Brash, J.B. Park, J.S. Rhim and C.C. Harris,
1988, Cancer Res., 48:1904-1909). Four hrs after trans-
fection, the cells were shocked with 15% glycerol in HBS,washed 3 times with LHC basal medium and incubated in LHC-
9 medium. After the appearance of f GCi of transformed
cells (3-4 weeks) the cells were subcultured (5 x 105/100-
mm dish). The cultures were fed 3 times per week with
fresh LHC-9 and transferred at weekly intervals at a
^ seeding density of 5 x 105 cells per 100-mm dish or T75
flask.
.
:
- 16 -
~ n this and the followin~ Example, the newly
developed strontium phosphate procedure for transfection
(D.E. Brash, et al., 1987, supra) was used, and yielded
stable transfectants with an efficiency of 2-4 x 10~5O
This fre~uency is 5 to 10-fold lower than that reported
for human cells from the bronchial epithelium (R.R.
Reddel, et al., 1988, supra) mesothelium (Y. Ke et al.,
1989, supra) and neonatal prostate (M.E. Kaighn et al,
1989, supra) transfected with the same plasmid. The
reason for the observed lower incidence of transfection in
HE cells is unknown.
The surviving cells of strain HE-457 formed two
discreet colonies in a single flask. One of these contin-
ued to grow following isolation; the other did not sur-
vive. At passage 14, this strain (HET-1A) was switched to
cKGM-AE medium. Its growth has accelerated, and it has
doubled at least 143 times thus far.
Characterization. Pre-crisis cultures and the
HET-1A cell line were characterized by immunohistochem-
istry. Keratin staining was intensely positive in pre-
crisis cells (HE-457), especially in closely apposed foci
of cells. Vimentin was also positive, although to à
lesser extent than keratin. Numerous atypical nuclear
features were observed including dysplasia and para-
nuclear clearing (koilocytosis). Keratin stained posi-
tively in all HET-1A cells. HET-1A cells stained hetero-
geneousiy for vimentin.
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- 17 -
The overall staining pattern of both pre- and post-crisis
cells was consistent with their epithelial origin.
Transmission electron microscopy also confirmed that all
pre- and post-crisis cells are of epithelial origin since
they contained tonofilaments and were jointed by desmosomal
junctions.
Clonal growth assays. ~esponse to growth factors and
inhibitors was assessed by a clonal growth assay (J.F.
Lechner and M.E. Kaighn, 1979, J. Cell Physiol. 100:519-
529). Subconfluent cultures were suspended with PET, and
plated at 500-1000 cells/60 mm-coated dish containing 4 ml
of cKGM-AE from which the factor under consideration was
omitted. After overnight incubation, the medium was
removed and experimental medium was added. Plates were
fixed in 10% neutral buffered formalin and stained with
Giemsa after 6-8 days incubation. Both colony forming
efficiency (CFE) and clonal growth rate, (population
doublings/day (PD/d) were determined. Four replicate
dishes per variable were used for CFE assay, and at least
18 colony counts were averaged for determination of PD/day.
Chromosome and isozyme analyses. Chromosome studies
were performed by Dr. Ward D. Peterson, Children's Hospital
of Michigan, Detroit, MI, uslng standard methods.
Metaphases were stained with Giemsa and counted at low
power for ploidy determination. Exact counts on 30
metaphases were made on banded chromosomes, and at least 8
Karyotypes per cell line were prepared. Analyses of 8
isozymes were carried out using standard procedures.
The chromosomal profiles of SV40-T antigen
immortalized cell line, HET-1A showed that the line is
aneuploid and has its own complement of marker chromosomes.
HET-1A is hypodiploid with only about 5% of metaphases
examined in the hypotetraploid range.
;
SUBSTITUTE SHEET
- 18 -
DNA hybridization ~lot analysis. Cells were grown
to 80-85% confluence, trypsinized and collected by cen-
trifugation. DNA was isolated by SDS proteinase-K incuba-
tion followed by phenol/chloroform extraction and ethanol
precipitation. The purity of the DNA was assessed spec-
trophotometrically using the 260/280 nm ratio. DNAs (5 ~g
each) from cell strains, HE-457, HOC-517, and HB-56B, and
cell lines RE-149, BEAS-2B, and HET-lA were loaded into
individual wells of a S&S Slot Blot apparatus (Schleicher
and Schuell, Inc., Keene, New Hampshire). DNA was blotted
onto Hybond-N (A~ersham) nylon membrane. The samples were
probed with a nick-translated EcoRI-HindIII fragment of
- the plasmid, pRSV-T in 2x SSC-0.1% SDS at 65C for 16
hours. The membrane was washed to 0.2x SSC-0.1% SDS at
65C and autoradiographed at -75C. A sample (1 ~g) of
the plasmid DNA was used as a control.
The results of DNA blot hybridization analyses of
SV40-T antigen gene showed that cell line HET-1A, strain
HE-457, as well as positive control cell lines, HOC-517
and BEAS-2B (R.R. Reddel, et al., 1988, supra), were all
positive for pRSV-T plasmid DNA. HET-1A had slightly
fewer copies, although more than HE-457. Although cell
strain HOC-517 appears to have incorporated only a few
gene copies, longer autoradiographic exposures indicated
that it is definitely positive, and that the negative
controls, HB-56B and RE149, have no detectable copies of
the SV40-T gene.
Immunofluorescence and electron microscopy. Cells
were fixed with 3% buffered glutaraldehyde for transmis-
sion electron microscopy or in absolute ~ethanol forimmunofluorescence. Alternatively, cell suspensions were
fixed and attached to slides by cytocentrifugation. The
cells were stained with antibodies to cytokeratin and
vimentin by the immunoperoxidase technique (Y. Katoh, G.D.
Stoner, C.C. Harris, K.R. McIntire, T Hill, R. Anthony,
' ,
, .
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E. McDowell and B.F. Trump, 1979, J. Nat. Cancer Inst.
62:1177-1185), and with a monoclonal antibody to SV40
large T-antigen (Oncogene Science, Inc., Mineola, NY) by
immunofluorescence.
Tumorigenicity assay. HE-451 (passage 16), HE-457
(Passage 14), ~ET-1A (passage 10) cells were tested for
tumorigenic potential in athymic nude mice. Cells were
suspended with PET and injected s.c. into both the left
and right flanks of each animal. A total of 5 x 106 cells
in 0.1 ml of medium were injected into each flank, and at
least ten animals per cell line were used. Animals were
observed weekly for tumor development up to 12 months.
For purposes of completing the background descrip-
tion and present disclosure, each of the published arti-
cles, patents and patent applications heretofore identi-
fied in this specification are hereby incorporated by
reference into the specification.
The foregoing invention has been described in some
detail for purposes of clarity and understanding. It will
also be obvious that various combinations in form and
detail can be made without departing from the scope of the
invention~
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