Immortalized human bronchial epitherial mesothelial cell lines

Immortalized human bronchial epithelial and human mesothelial cell lines have been obtained. Various uses of these cell lines have been described.

BACKGROUND OF THE INVENTION 
The present invention is related to immortalized celllines. More 
particularly, the present invention is related to immortalized human 
bronchial epithelial and human mesothelial cell lines or cell lines 
derived therefrom. 
Lung cancer is one of the more common forms of cancer and the cell type in 
which the majority of these cancers arise is the bronchial epithelial 
cells. Mesothelial cells are a less common, but important, site of origin 
of lung cancer. Both, normal human bronchial epithelial cells and normal 
human mesothelial cells could be cultured in vitro, but only for a limited 
period of time before cellular replication ceases. When transformed by 
transfection of the viral Harvey ras oncogene (Yoakum, et al., Science 
227:1174, 1985), human bronchial epithelial cells replicate for longer 
periods of time, but these cells are tumorigenic, grow in serum-containing 
media as do carcinoma cell lines, and have been constructed to contain an 
oncogene closely related to oncogenes sometimes found in human carcinomas. 
Similarly, human bronchial carcinoma cell lines and mesothelioma cell 
lines are tumorigenic Clearly, such tumorigenic cell lines are 
undesirable, inter alia. for carcinogenic studies. 
SUMMARY OF INVENTION 
It is, therefore, an object of the present invention to provide 
non-tumorigenic human cell lines of bronchial epithelial and of 
mesothelial cell origin with unlimited proliferative potential and capable 
of growing in the same serum-free media as their normal counterpart cells, 
and which do not contain an oncogene found in naturally occurring tumors. 
Other objects and advantages of the present invention would become apparent 
from the Detailed Description of Invention. 
DETAILED DESCRIPTION OF INVENTION 
The above and other objects and advantages of the present invention are 
achieved by non-tumorigenic, human bronchial epithelial cell lines 
continually growing when cultured in vitro in suitable growth medium. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are now described. All publications mentioned hereunder are 
incorporated herein by reference. 
The term "immortalized" as used herein means that the cell line grows 
continually without senescence when cultured in vitro in a suitable growth 
medium. 
General Method for Construction of Cell Lines 
Normal human bronchial epithelial (NHBE) cells were cultured from explants 
of necropsy tracheobronchial specimens from noncancerous individuals as 
described by Lechner, et al.,: J. Tissue Culture Methods 9:43-48, 1985. 
Normal human mesothelial (NHM) cells were cultured from pleural effusions 
or ascites fluids as described by Lechner et al., (Proc. Natl. Acad. Sci. 
U.S.A. 82:3884-3888, 1985). The cells were infected with SV40 virus or 
with adenovirus-12 SV40 hybrid virus, or transfected with a recombinant 
plasmid containing the Rous sarcoma virus long terminal repeat and the 
ori-SV40 early region by strontium phosphate coprecipitation (Brash, et 
al.: Molec. Cell. Biol., 1987). Colonies of cells transformed by each of 
these three methods were easily recognizable morphologically using phase 
contrast microscopy and were individually trypsinized and serially 
passaged. In all cases the lifespan of these cultures was extended 
compared to NHBE or NHM; most of the cultures underwent a prolonged period 
of senescence referred to as "crisis". With continued culture, in some 
cases colonies of cells which had escaped senescence arose; such surviving 
colonies were subsequently passaged for extended period of time and showed 
unlimited growth potential. Like NHBE cells, but unlike bronchial 
carcinoma cells, some of the cell lines thus derived retained the capacity 
to undergo squamous differentiation in response to serum exposure. 
Injection of these cells into irradiated athymic nude mice did not result 
in formation of tumors after periods of up to nine months. Furthermore, 
these cell lines were found to be suitable recipients for transfection of 
additional oncogenes and useful for testing the cytotoxicity potential of 
chemical and physical agents, the growth inhibition or promoting 
capability of biological agents, and squamous differentiating potential of 
chemical and biological agents.

EXAMPLE 1 
Development of BES-1A1.6 Cell Line 
Normal human bronchial epithelial (NHBE) cells were cultured from explants 
of autopsy specimens from noncancerous individuals as described by 
Lechner, et al.: J. Tissue Culture Methods 9: 43-48,1985. The cells were 
cultured in a serum-free medium, LHC-9, consisting of LHC basal nutrient 
medium with calcium 0.08 mM, L-glutamine 1 mM, trace elements, gentamicin 
50 .mu.g/ml, insulin 5 .mu.g/ml, transferrin 10 .mu.g/ml, hydrocortisone 
200 nM, epidermal growth factor 5 ng/ml, phosphoethanolamine 0.5 .mu.M, 
ethanolamine 0.5 .mu.M, epinephrine 0.5 .mu.g/ml, retinoic acid 0.33 nM, 
triiodothyronine 10 nM, and bovine pituitary extract (Lechner, et al., 
supra). In the initial stages of the development of this cell line LHC-8 
medium which contains the ingredients listed for LHC-9, with the exception 
of epinephrine and retinoic acid, was used. 
NHBE cells were harvested by trypsinization and seeded in 10 ml growth 
medium into 100 mm culture dishes (Lux, Miles Scientific, Naperville, 
Ill.) whose growth surfaces had been coated with a solution of bovine 
serum albumin, fibronectin and collagen (Lechner, et al. supra). 
SV40 virus was prepared in CV-1 cells as described by Su, et al.: J. Virol. 
28: 53-65, 1978. NHBE cells were exposed at 37.degree. C. for 90 min. at a 
multiplicity of infection of approximately 1. The cells were subcultured 
twice in LHC-8 medium, and exposed to 1% fetal calf serum (FCS) in LHC-8 
medium for 47 days. Sixty-one days after infection three colonies of 
transformed cells were individually subcultured by trypsinization. All 
subsequent culture of these cells was in serum-free LHC-8 medium. The cell 
strains thus derived were designated as BES-1A. Two of these strains 
(BES-1A1 and BES-1A2) were subcloned by limiting dilution. 
All of these clonal isolates continued to proliferate for about 18 weeks at 
which time the cultures senesced (i.e., entered culture "crisis"). After a 
further period of 11 weeks, proliferating cells appeared in a subcloned 
culture designated BES-1A1.6. From these cells a line was established 
which remains in culture more than one year from the time of the initial 
SV40 infection. These cells are non-tumorigenic. 
The BES-1A1.6 line has the special property of being resistant to the 
squamous differentiation-inducing effects of serum. Whereas NHBE cells are 
able to be induced to undergo squamous differentiation when exposed to 
serum, bronchial carcinomas are resistant to this effect (Lechner et al., 
Cancer Res. 43:5915-5921, 1983). TBE cells lines (human bronchial 
epithelial cells transformed by the v-Ha-ras oncogene are tumorigenic and 
are resistant to this effect of serum. The non-tumorigenic BES-1A1.6 cell 
line is, therefore, intermediate between normal and fully malignant 
bronchial epithelial cells in this respect. 
EXAMPLE 2 
Development of the BEAS-2B Cell Line 
NHBE cells were cultured from explants of autopsy specimens from 
noncancerous individuals as described by Lechner, et al., supra. The cells 
were cultured in a serum-free medium, LHC-9, harvested by trypsinization 
and seeded in 10 ml growth medium into 100 mm culture dishes (Lux, Miles 
Scientific, Naperville, Ill.) whose growth surfaces had been coated with a 
solution of bovine serum albumin, fibronectin and collagen (Lechner, et 
al., supra). 
Adenovirus 12-SV40 (Ad12SV40) hybrid virus (Schell, et al. Proc. Natl. 
Acad. Sci. U.S.A. 55:81-88, 1966) was grown in Vero cells as described by 
Rhim, et al.: Proc. Natl. Sci, U.S.A. 78: 313-317, 1981. NHBE cells were 
exposed to the virus at 37.degree. C. for four hours at a multiplicity of 
infection of approximately 100. When the cultures reached confluenoe, each 
dish was subcultured into two 75 cm.sup.2 flasks, the cells were allowed 
to reach confluence again and then were re-fed twice weekly until 
transformed colonies appeared and the normal cells senesced. Senescence of 
the normal cells was accelerated by exposing the cultures to 1% FCS in 
LHC-9 for 28 days (Lechner, et al.: Differentiation 25: 229-237, 1984); 
all subsequent culture of these cells was in serum-free LHC-9 medium. 
Individual colonies were subcultured 41 days after the viral infection and 
cell strains thus derived from this experiment were designated BEAS-2. 
One of the clonal cultures thus derived, BEAS-2B, has proliferated 
continuously for more than a year and appears to be permanently 
established. Cells from this cell line injected as passage 18 into athymic 
nude mice have not formed tumors after one year. This cell line retains 
the ability to undergo squamous differentiation in response to serum; of 
the cell lines developed BEAS-2B was the most sensitive to this effect and 
is thus particularly useful for studies of differentiation-inducing 
agents. It is able to form an epithelium in de-epithelialized rat tracheas 
implanted subcutaneously in athymic nude mice and is thus particularly 
suitable for in vivo studies, especially of chemical carcinogenesis. In 
assays of invasiveness using matrigel coated filters and Boyden chambers 
(Albini et al., Cancer 47:3239, 1987) BEAS-2B cells were similar to NHBE 
cells and 100 times less invasive than TBE-1 cells. 
EXAMPLE 3 
Development of the BET-b 1A Cell Line 
NHBE cells were cultured from explants of autopsy specimens from 
noncancerous individuals as described by Lechner, et al., 1985, supra. The 
cells were cultured in a serum-free medium, LHC-9, harvested by 
trypsinization and seeded in 10 ml growth medium into 100 mm culture 
dishes (Lux, Miles Scientific, Naperville, Ill.) whose growth surfaces had 
been coated with a solution of bovine serum albumin, fibronectin and 
collagen (Lechner, et al., 1985, supra). 
The cells were transfected with a plasmid, pRSV-T (obtained from National 
Cancer Institute), which is an SV40 ori- construct containing the SV40 
early region genes and the Rous sarcoma virus long terminal repeat (LTR). 
Transfection was by DNA strontium phosphate coprecipitation as described by 
Brash, et al., Molec. Cell Biol. 7: 2031-2034, 1987. 5.times.10.sup.5 NHBE 
cells plated in 100 mm dishes were transfected with 10 .mu.g DNA 
precipitated at pH 7.8. The cells were exposed to the precipitate for 4 hr 
before glycerol shock (Brash, et al., supra). Three days after 
transfection the cells were passaged; thereafter the cell culture medium 
was changed twice weekly until subculturing of transformed colonies. Upon 
confluence the cells were passaged a second time, and senescence of normal 
cells was hastened by exposure to LHC-9 medium with 1% FCS for 46 days. 
One colony only was subcultured at day 61 after transfection. All 
subsequent culture of these cells was in serum-free LHC-9 medium, and the 
cell strain thus obtained was designated BET1A. 
These cells continued to proliferate for about 16 weeks at which time the 
culture senesced (i.e., entered "crisis"). After a further 13 weeks, 
colonies of dividing cells appeared from which a cell line has become 
established; BET1A cells have been in culture for more than a year from 
the time of initial transfection. These cells are non-tumorigenic, and 
retain the ability to undergo squamous differentiation in response to 
serum. 
EXAMPLE 4 
Development of the BET-2A Cell Line 
NHBE cells were cultured from explants of autopsy specimens from 
noncancerous individuals as described previously herein above. The cells 
were cultured in a serum-free medium, LHC-9, harvested by trypsinization 
and seeded in 10 ml growth medium into 100 mm culture dishes (Lux, Miles 
Scientific, Naperville, Ill.) whose growth surfaces had been coated with a 
solution of bovine serum albumin, fibronectin and collagen (Lechner, et 
al., 1985, supra). 
The cells were transfected with a plasmid, pRSV-T, (obtained from National 
Cancer Institute) which is an SV40 ori- construct containing the SV40 
early region genes and the Rous sarcoma virus long terminal repeat (LTR). 
Transfection was by DNA strontium phosphate coprecipitation as described 
previously (Brash, et al., supra). 5.times.10.sup.5 NHBE cells plated in 
100 mm dishes were transfected with 10 .mu.g DNA precipitated at pH 7.8. 
The cells were exposed to the precipitate for 4 hr before glycerol shock 
(Brash et al. supra). Three days after transfection the cells were 
passaged. Thereafter, the cell culture medium was changed twice weekly. 
Three transformed colonies were subcultured individually at 28 days 
following transfection, and the clonal cell strains thus derived continued 
to proliferate in culture for 11 weeks after which time the cultures 
senesced (i.e. entered "crisis"). After a further 36 weeks, colonies of 
dividing cells appeared in culture BET-2A from which a cell line has 
become established; BET-2A cells have been in culture for more than a year 
from the time of initial transfection. The BET-2A cell line, like the 
BES-1A1.6 cell line, appears to be resistant to the squamous 
differentiation-inducing effects of serum. Whereas NHBE cells are able to 
be induced to undergo squamous differentiation when exposed to serum, 
bronchial carcinomas are resistant to this effect (Lechner et al., Cancer 
Res. 43:5915-5921, 1983). TBE cell lines (human bronchial epithelial cells 
transformed by the v-Ha-ras oncogene) are tumorigenic and are resistant to 
this effect of serum. The non-tumorigenic BET-2A cell line is, therefore, 
intermediate between normal and fully malignant bronchial epithelial cells 
in this respect. 
EXAMPLE 5 
Development of MeT-5A Cell Line 
Human mesothelial cells were cultured as described by Lechner, et al.: 
Proc. Natl. Acad. Sci. U.S.A. 82: 3884-3888, 1985, and were transformed at 
a frequency of 2.times.10.sup.-4 by transfection using strontium phosphate 
coprecipitation (Brash, et al., supra) of a recombinant plasmid, pRSV-T, 
containing the SV40 virus early region. Colonies of cells transformed by 
the plasmid, pRSV-T, were isolated and propagated by serial passaging for 
periods of up to 140 days and 60-70 population doublings from the time of 
transfection, before cellular senescence occurred. This contrasts with the 
usual culture lifespan of normal mesothelial cells of 30 days and 15 
population doublings. Colonies of dividing cells arose from one such 
senescent culture, and from these colonies an immortalized cell line, 
MeT-5A, has been established by continued passaging. This cell line is 
non-tumorigenic. Although it has been maintained routinely in the 
serum-containing LHC-MM medium, it also grows well in a serum-free medium. 
EXAMPLE 6 
Development of BBM Cell Line 
BEAS-2B cells were transfected via strontium phosphate co-precipitation 
(Brash, D. E. et al., Molec. Cell Biol. 7:2031-2034, 1987) with a 
recombinant plasmid, B-myc/pSV2neo, which had been constructed by ligating 
a BamH1/EcoR1 fragment of the c-myc gene from the Burkitt's lymphoma cell 
line CA46 (Showe et al., Mol. Cell Biol. 5:501-509, 1985) to a BamH1/EcoR1 
fragment of the pSV2neo vector (Southern et al., Mol. Appl. Genet. 
1:327-341, 1982). BEAS-2B cells so transfected were selected in LCH-9 
medium with G418 (Geneticin), and colonies resistant to G418 were isolated 
individually and subcultured. The cell line arising from one such colony 
has been designated BBM. 
EXAMPLE 7 
Development of BZR Cell Line 
This cell line has been derived by infecting the BEAS-2B cell line with a 
recombinant containing the viral Harvey-ras (v-Ha-ras) oncogene. The cell 
line so derived is highly tumorigenic in athymic nude mice. 
The details of the construction are as follows. Zip-neo-v-Ha-ras 
recombinant retrovirus was constructed by recombining the pZipNeoSV(X) 
retrovirus (Cepko et al., Cell 37:1053-1062, 1984) at its unique Bam H1 
restriction enzyme site with a Bam H1 -linkered 1.3 Kb fragment of the H1 
clone (Ellis et al., J. Virol. 6;408-420, 1980) containing the v-Ha-ras 
oncogene. Recombinant DNA molecules containing the v-Ha-ras DNA in sense 
orientation with respect to the pZipNeoSV(X) retrovirus 5' long terminal 
repeat, were identified by standard DNA manipulation techniques and were 
used to transfect the psi.sup.2 packaging mutant cell line (Mann et al., 
Cell 33:153-159, 1983). Supernatants from these cells were shown to 
contain infectious retrovirus, and were used to infect the amphotrophic 
packaging mutant cell line, psiAM (Cone et al., Proc. Natl. Acad. Sci. 
U.S.A. 81:6349-6353 1984). Supernants from this cell line were titered and 
used to infect BEAS-2B cells. 
Following infection of BEAS-2B cells with this virus, G418 resistant cells 
were selected (Southern et al., J. Mol. Appl. Genet. 1:327-341, 1982) and 
serially subcultured; the cell line so derived was designated BZR. This 
cell line is highly tumorigenic, forming tumors with a latency period of 2 
weeks in 12/15 athymic nude mice each injected with 5.times.10.sup.6 cells 
subcutaneously. 
A deposit of the cell lines of the present invention has been made at the 
ATCC, Rockville, Md., on June 12, 1987 and July 14, 1987 under the 
accession numbers CRL 9608, 9609, 9442, 9443, 9444, 9482 and 9483, 
corresponding to cell lines BES-1A1.6, BEAS-2B, BET1A, BET-2A, MeT-5A, BBM 
and BZR, respectively. The deposits shall be viably maintained, replacing 
if it became 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 accordance with the provisions of the law. The Commissioner 
of Patents and Trademarks, upon request, shall have access to the deposit. 
UTILITY OF CELL LINES 
(1) Identification of potential chemotherapeutic drugs: These cells are 
useful for screening chemicals suitable for the treatment of cancer and 
related diseases, by growing them in vitro in medium containing the 
chemical to be tested and then, after a suitable period of exposure, 
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 
(MacDonald et al., Exp. Cell. Res. 50: 417, 1968), all of which are 
standard techniques well known in the art. 
(2) Studies of the control of squamous differentiation, and identification 
of chemical and biological agents which induce squamous differentiation: 
This is accomplished by assays previously described for normal human 
bronchial epithelial cells (Masui, Proc. Natl. Acad. Sci. U.S.A.. 83:2438, 
1986). As noted in the cell line specification, some retain ability to 
undergo squamous differentiation in response to serum. Induction of 
terminal differentiation may be an effective way of controlling the growth 
of cancer. Chemical and biological substances are screened for their 
ability to induce differentiation by adding them to the growth medium of 
these cells and then after a suitable time interval determining whether a 
complex of changes including cessation of DNA synthesis and the appearance 
of squamous morphology has occured. The cells are also useful for studies 
of the biological mechanisms of squamous differentiation, and the 
existence of both serum-resistant and serum-sensitive cell lines enables 
comparisons and identification of genes of their protein products involved 
in the process of differentiation. 
(3) Studies of metabolism of caroinogens and other xenobiotics: Carcinogens 
and other xenobiotics may be added to the growth medium of these cells and 
then the appearance of metabolic products of these compounds may be 
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 metabolites with DNA is determined. 
(4) Studies of DNA mutagenesis: 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 Enzymol. 58:308, 1979). Similarly, 
cell-mediated DNA mutagenesis, by cocultivating the cells with cell types 
known or suspected to be capable of secreting mutagenic compounds (Hsu et 
al., Proc. Natl. Acad. Sci. U.S.A. 75:2003, 1978). 
(5) Studies of chromosome damaging agents: Substances known or suspected to 
cause chromosomal damage may be added to the culture medium of these cell 
lines, and then the extent of chromosomal damage may be measured by 
techniques such as measurement of the frequency of sister chromatid 
exchange (Latt et al., In: Tice, R. R. and Hollaender, A., Sister 
Chromatid Exchanges. New York: Plenum Press, pp. 11 ff., 1984). 
(6) Studies of malignant transformation by chemical, physical and viral 
agents, and transferred genes including oncogenes and high molecular 
weight genomic DNA from tumors, using standard assays such as anchorage 
independent growth or tumor formation in athymic nude mice. For example, a 
cloned cellular oncogene from a human tumor has been transferred into the 
BEAS-2B cell line; the cell line thus derived is BBM. This cell line has 
been shown to be resistant to the squamous differentiation inducing 
effects of serum. In a second example, a cloned viral oncogene, v-Ha-ras, 
has been introduced into the BEAS-2B cell line; the cell line thus derived 
is BZR. This cell line has been shown to be able to form tumors in nude 
mice with a latency period of two weeks, and is able to grow in an 
anchorage-independent fashion in soft agar. 
(7) Use of cells altered by transfer of oncogenes as in paragraph 6 above 
to screen for potential chemotherapeutic agents (by the techniques 
described in paragraph 1 above) especially those which may be specific for 
cells transformed by the activation of particular oncogenes or combination 
of oncogenes. 
(8) Studies of cellular biochemistry, including changes in intracellular pH 
and calcium levels, as correlated with cell growth and action of exogenous 
agents including but not limited to those described in paragraphs 1 
through 7 above. To study intracellular pH and calcium levels, cells in 
suitable culture vessels are exposed to fluorescent indicator dyes and 
then fluorescence emissions are detected with a fluorescence 
spectrophotometer (Grynkiewicz et al., J. Biol. Chem. 260:3440-3450, 
1985). 
(9) Studies of cellular responses to growth factors and production of 
growth factors: Identification and purification of growth factors 
important for growth and differentiation of human bronchial epithelial 
cells. These cells are particularly useful for such an application since 
they grow in serum-free media. Therefore, responses to growth factors can 
be studied in precisely defined growth media and any factors produced by 
the cells may be identified and purified without the complication of the 
presence of serum. 
(10) Use of recombinant DNA expression vectors to produce proteins of 
interest. For example, the gene encoding a protein of therapeutic value 
may be recombined with controlling DNA segments (i.e. containing a 
promoter with or without an enhancer sequence), transferred into the cell 
(e.g., by strontium phosphate transfection) and then the protein produced 
may be harvested from the culture supernatant or a cellular extract by 
routine procedures well known in the art. 
(11) Studies of intracellular communication e.g., by dye scrape loading 
assays. To determine whether the cells growing in vitro have the ability 
to communicate via gap junctions, the cultures may be scraped, e.g., with 
a scalpel in the presence of a fluorescent dye in the growth medium. Cells 
at the edge of the wound are mechanically disrupted and therefore take up 
dye; whether intercellular communication has occurred may be ascertained 
by determining whether cells distant from the wound also contain dye. 
(12) Characterization of cell surface antigens: The cells are incubated 
with an antibody against the cell surface antigen of interest, and then 
reacted with a second antibody which is conjugated to a fluorescent dye. 
The cells are then evaluated using a fluorescence activated cell sorter to 
determine whether they are fluorescent and therefore possess the cell 
surface antigen. 
(13) hybrid studies for identification of tumor suppressor activity 
(Stanbridge et al., Science 215:252-259, 1982). To determine whether these 
cell lines contain tumor suppressor genes, they are fused to malignant 
tumor cells. The presence of tumor suppressor genes is indicated by loss 
of malignancy e.g., as detected by loss of ability to form tumors in 
athymic nude mice, in the hybrid cells. 
(14) Identification of novel genes, including transforming genes in 
naturally concurring cancers described in paragraph 6 above, growth factor 
genes as described in paragraph 9 above, tumor suppressor genes as 
described in paragraph 13 above, using standard molecular biological 
techniques (Davis et al., Methods in Molecular Biology, New York: 
Elsevier, 1986) and techniques such as cDNA subtraction cloning and the 
like. 
Of course, a kit for screening carcinogenic or antineoplastic agents and 
for any other usage as described herein supra, is easily assembled, 
comprising container(s) containing the cell line(s) of the present 
invention. Other components routinely found in such kits may also be 
included with instructions for performing the test. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and scope of 
the appended claims.