Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1298220
1 IM~ORTALIZED RUMAN CELL LINES
2 BAC~GROUND OF THE INVENTION
3 The present invention is related to immortalized
4 cell lines. More particularly, the present invention is
related to immortalized human bronchial epithelial and
6 human mesothelial cell lines or eell lines derived
7 therefrom.
8 Lung eancer is one of the more common forms of
9 eaneer and the eell type in which the ma~ority of these
eaneers arise is the bronchial epithelial eells.
11 Meaothelial eells are a less eommon, but important, site
12 of origin of lung eaneer. Both, normal human bronehlal
13 epithelial eells and normal human mesothelial eells eould
14 be eultured in vitro, but only for a limited period of
time before eellular replication eeases. When
16 transformed by transfeetion of the vlral Harvey ras
17 oncogene (Yoakum, et al., Science, 227:1174, 1985), human
18 bronchial epithelial cells replicate for longer periods
l9 of time, but these cells are tumorigenie, grow in
serum-eontaining media as do earcinoma cell lines~ and
21 have been constructed to contain an oncogene closely
22 related to oncogenes sometimes found in human
23 carcinomes. Similarly, human bronchial carcinoma cell
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1 lines and mesothelioma cell lines are tumorigenic.
2 Clearly, such tumorigenic cell lines are undesi~able,
3 inter alia, for carcinogenic studies.
4 SUMMA~Y OF INVENTION
It is, therefore, an object o~ the present
6 invention ~o provide non-tumorigenic human cell lines of
7 bxonchial epithelial and of mesothelial cell origin with
8 unlimited proliferative potential and capable of growing
9 in the same serum-free media as their normal counterpart
cells, and which do not contain an oncogene found in
11 naturally occurring tumors.
12 The present invention provides a non-tumorigenic, human bronchial
13 epithelial or mesothelial cell line or derivative thereof growing without
14 senescence when cultured in vitro in growth medium, said cell lines being
selected from the group consisting of those having the identifying
16 characteristics of ATCC CRL 9608, 9609, 9442, 9443, 9444, 9482 and 9483.
17 In another aspect, the invention provides a kit for screening
18 carcinogenic or chemotherapeutic agent comprising a container containing
19 a non-tumorigenic, human bronchial epithelial or mesothelial cell line or
derivative thereof as described above growing without senescence when
21 cultured in vitro in growth medium.
22 Also disclosed is a method for testing carcinogenicity of an
23 agent, comprising culturing the cell line as descirbed above with an
24 agent suspected of being carcinogenic and determining formation of
abnormal cellular mass by said cell line, the formation of abnormal
26 cellular mass being indicative of carcinogenicity of said agent.
27 Finally, there is described a method for testing antineoplastic
28 activity of an agent, comprising culturing the cell line as described
29 above with a potential antineoplastic agent and determining growth of
said cell line, a lack of growth of said cell line being indicative of
31 antineoplastic potency of said agent.
.
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1 Other objects and advantages of the present
~ invention would become apparent from the Detailed
3 Description of Invention.
4 DETaILED DESC~IPTION OF INVENTION
~he above and other objects and advantages of the
~ present invention are achieved by non-tumorigenic, human
7 bronchial epithelial cell line continually growing when
8 cultured in vitro in suitable growth medium.
9 Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as
11 commonly understood by one of ordinary skill in the art
12 to which this invention belongs. Although any methods
13 and materials similar or equivalent to those described
14 herein can be used in the practice or testing of the
present invention, the preferred methods and materials
16 are now described.
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~29~3Z20
1 The terrn "immortalized" as used herein means that
2 the cell line grows continually without senescence when
3 cultured in vitro in a suitable growth medium.
4 General Method for Construction of Cell Lines
Normal human bronchial epithelial (NHBE) cells
6 were cultuxed from explants of necropsy tracheobronchial
7 specimens from noncancerous individuals as described by
8 Lechner, et al., J. Tissue Culture Methods 9:43-48,
9 1985. Normal human mesothelial (NHM) cells were cultured
from pleural effusions or ascites fluids as described by
11 Lechner, et al, (Proc. Natl. Acad. Sci. USA 82:3884-3888,
12 1985). The cells were infected with SV40 virus or with
13 adenovirus-12 SV40 hybrid virus, or transfected with a
14 recombinant plasmid containing the Rous sarcoma virus
long terminal repeat and the ori-SV40 early region by
16 strontium phosphate coprecipitation (Brash, et al.,
17 Molec. Cell. Biol.~, 1987). Colonies of cells transformed
18 by each of these three methods were easily recognizable
19 morphologically using phase contrast microscopy and were
individually trypsinized and serially passaged. In all
21 cases the lifespan of these cultures was extended
22 compared to NHBE or NHM; most of the cultures underwent a
23 prolonged period of senescence referred to as "crisis".
24 With continued culture~ in some cases colonies of cells
which had escaped senescence arose; such surviving
26 colonies were subsequently passaged for extended period
27 of time and showed unlimited growth potential. Like NHBE
28 cells, but unlike bronchial carcinoma cells, some of the
29 cell lines thus derived retained the capacity to undergo
squamous differentiation in response to serum exposure.
31 Injection of these cells into irradiated athymic nude
32 mice did not result in formation of tumors after periods
33 of up to nine months. Furthermore, these cell lines were
1;~9~3Z~V
1 found to be suitable recipients for transfection of
2 additional oncogenes and useful for testing the
3 cytotoxicity potential of chemical and physical agents,
4 the growth inhibition or promoting capability of
biological agents, and squamous differentiating potential
6 of chemical and biological agents.
7 EXAMPLE 1
8 Development of BES-lAl.6 Cell Line
9 Normal human bronchial epithelial (NHBE) cells
were cultured from explants of autopsy specimens from
11 noncancerous individuals as described by Lechner, et al.,
12 J. Tissue Culture Methods 9: 43-48, 1985. The calls were
13 cultured in a serum-free medium, LHC-9, consisting of LHC
14 basal nutrient medium with calcium 0.08 mM, L-glutamine
lmM, trace elements, gentamicin 50 ~g/ml, insulin 5
16 ~g/ml, transferrin 10 ~g/ml, hydrocortisone 200 nM,
17 epidermal growth factor 5 ng/ml, phosphoethanolamine 0.5
18 ~M, ethanolamine 0.5 ~M, epinephrine 0.5 ~g/ml, retinoic
19 acid 0.33 nM, trilodothyronine 10 nM, and bovine
pltuitary extract (Lechner, et al., supra). In the
21 initial stages of the development of this cell llne LHC-8
22 medium which contains the ingredients listed for LHC-9,
23 with the exception of epinephrine and retinoic acid, was
24 used.
NHBE cells were harvested by trypsinization and
26 seeded in 10 ml growth medium into 100 mm culture dishes
27 (Lux, Miles Scientific, Naperville, IL) whose growth
28 surfaces had been coated with a solution of bovine serum
29 albumin, fibronectin and collagen (Lechner, et al.,
supra).
31 SV40 virus was prepared in CV-l cells as described
32 by Su, et al., J. Virol. 28: 53-65, 1978. NHBE cells
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1 were exposed at 37C for 90 min. at a multiplicity of
2 infection of approximately 1. The cells were subcultured
3 twice in LHC-8 medium, and exposed to 1% fetal calf serum
4 (FCS) in LHC-8 medium for 47 days. Sixty-one day~ after
infection three colonies of transformed cells were
6 individually subcultured by trypsinization. All
7 subsequent culture of these cells was in serum-free LHC-8
8 medium. The cell strains thus derived were designated as
9 BES-lA. Two of these strains (BES-lAl and BES-lA2) were
subcloned by limiting dilution.
11 All of these clonal isolates continued to
12 proliferate for about 18 weeks at which time the cultures
13 senesced (i.e., entered culture "crisis"). After a
14 further period of ll weeks, proliferating cells appeared
ln a subcloned culture designated BES-lAl.6. From these
16 cells a line was established which remains in culture
17 more than one year from the time of the initial SV40
18 infection. These cells are non-tumorigenic.
19 The BES-lA1.6 line has the special property of
being resistant to the squamous differentiation-inducing
21 effects of serum. Whereas NHBR cells are able to be
22 induced to undergo s~uamous differentiation when exposed
23 to serum, bronchlal carcinomas are resistant to this
24 effect (Lechner, et al, Cancer Res. 43:5915-5921, 1983).
TBE cells lines (human bronchial epithelial cells
26 transformed by the v-Ha-ras oncogene are tumorigenic and
27 are resistant to this effect of serum. The
28 non-tumorigenic LES-lA1.6 cell line is, therefore,
29 intermediate between normal and fully malignant bronchial
epithelial cells in this respect.
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1 EXAMPLE 2
2 Development of the BEAS-2B Cell Line
3 NHBE cells were cultured from explants of autopsy
4 specimens from noncancerous individuals as describe-d by
Lechner, et al., supra. The cells were cultured in a
6 serum-free medium, LHC-9, harvested by trypsinization and
7 seeded in 10 ml growth medium into 100 mm culture dishes
8 (Lux, Miles Scientific, Naperville, IL) whose growth
9 surfaces had been coated with a solution of bovine serum
albumin, fibronectin and collagen (Lechner, et al.,
11 supra).
12 Adenovirus 12-SV40 (Adl2SV40) hybrid virus
13 (Schell, et al., Proc. Natl. Acad. Sci. USA 55:81-88,
14 1966) was grown in Vero cells as described by Rhim, et
al., Proc. Natl. Sci, USA 78: 313-317, 1981. NHBE cells
16 were exposed to the virus at 37C for four hours at a
17 multiplicity of infection of approximately 100. When the
18 cultures reached confluence, each dish was subcultured
19 into two 75 cm2 flasks, the cells were allowed to reach
confluence again and then were re-fed twice weekly until
21 transformed colonies appeared and the normal cells
22 senesced. Senescence of the normal cells was accelerated
23 by exposing the cultures to 1% FCS in LHC-9 for 28 days
24 (Lechner, et al., Differentiation 25: 229-237, 1984); all
subseguent culture of these cells was in serum-free LHC-9
26 medium. Individual colonies were subcultured 41 days
27 after the viral infection and cell strains thus derived
28 from this experiment were designated BEAS-2.
29 One of the clonal cultures thus derived, BEAS-28,
has proliferated continuously for more than a year and
31 appears to be permanently established. Cells from this
32 cell line injected as passage 18 into athymic nude mice
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1 have not formed tumors after one year. This cell line
2 retains the ability to undergo squamous differentiation
3 in response to serum; of the cell lines developed BEAS-2B
4 was the most sensitive to this effect and is thus
particularly useful for studies of differentiation-
6 inducing agents. It is able to form an epithelium in
7 de-epithelialized rat tracheas implanted subcutaneously
8 in athymic nude mice and is thus particularly suitable
9 for in vivo studies, especially of chemical
carcinogenesis. In assays of invasiveness using matrigel
11 coated filters and Boyden chambers (Albini, et al.,
12 Cancer 47:3239, 1987) BEAS-2B cells were similar to NHBE
13 cells and 100 times less invasive than TBE-l cells.
14 EXAMPLE 3
Development of the BET-lA Cell Line
16 NHBE cells were cultured from explants of autopsy
17 specimens from noncancerous individuals as described by
18 Lechner, et al., 1985, ~E~ The cells were cultured in
19 a serum-free medium, LHC-9, harvested by trypsinization
and seeded in 10 ml growth medium into 100 mm culture
21 dishes (Lux, Miles Sclentific, Naperville, IL) whose
22 growth surfaces had been coated with a solution of bovine
23 serum albumin, fibronectin and collagen (Lechner, et al.,
24 1985, supra).
The cells were transfected with a plasmid, pRSV-T
26 (obtained from National Cancer Institute), which is an
27 SV40 ori- construct containing the SV40 early region
28 genes and the Rous sarcoma virus long terminal repeat
29 (LTR).
Transfection was by DNA strontium phosphate
31 coprecipitation as described by Brash, et al., Molec.
32 Cell. Biol. 7: 2031-2034, 1987. 5 x 105 NHBE cells
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1 plated in 100 mm dishes were transfected with 10 ug DNA
2 precipitated at pH 7.8. The cells were exposed to the
3 precipitate for 4 hr before glycerol shock (Brash, et
4 al., supra). Three days after transfection the cells
were passaged; thereafter the cell culture medium was
6 changed twice weekly until subculturing of transformed
7 colonies. Upon confluence the cells were passaged a
8 second time, and senescence of normal cells was hastened
9 b~-exposure to LHC-9 medium with-1~ FCS for 46 days. One
colony only was subcultured at day 61 after
11 transfection. All subseguent culture of these cells was
12 in serum-free LHC-9 medium, and the cell strain thus
13 obtained was designated BET-lA.
14 These cells continued to proliferate for about 16
weeks at which time the culture senesced (i.e., entered
16 "crisis"). After a further 13 weeks, colonies of
17 dividing cells appeared from which a cell line has become
18 established; BET-lA cells have been in culture for more
19 than a year from the time of initial transfection. These
cells are non-tumorigenic, and retain the ability to
21 undergo s~uamous differentiation in response to serum.
22 EXAMPLE 4
23 Development of the BET-2A Cell Line
24 NHBE cells were cultured from explants of autopsy
specimens from noncancerous individuals as described
26 previously herein above. The cells were cultured in a
27 serum-free medium, LHC-9, harvested by trypsinization and
28 seeded in 10 ml growth medium into 100 mm culture dishes
29 (Lux, Miles Scientific, Naperville, IL) whose growth
surfaces had been coated with a solution of bovine serum
31 albumin, fibronectin and collagen (Lechner, et al., 1985,
32 supra).
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1 The cells were transfected with a plasmid, pRSV-T,
2 (obtained from National Cancer Institute) which is an
3 SV40 ori- construct containing the SV40 early region
4 genes and the Rous sarcoma virus lony terminal repeat
~LTR).
6 Transfection was by DNA strontium phosphate
7 coprecipitation as described previously (Brash, et al.,
8 supra). 5 x 105 NHBE cells plated in 100 mm dishes were
9 transfected with 10 ~g DNA precipitated at pH 7.8. The
cells were exposed to the precipitate for 4 hr before
11 glycerol shock (Brash, et al., supr_). Three days after
12 transfection the cel~s were passaged. Thereafter, the
13 cell culture medium was changed twice weekly. Three
14 transformed colonies were subcultured individually at 28
days following transfection, and the clonal cell strains
16 thus derived continued to proliferate in culture for 11
17 weeks after which time the cultures senesced (i.e.
18 entered "crisis"). After a further 36 weeks, colonies of
19 dividing cells appeared in culture BET-2A from which a
cell line has become established; BET-2A cells have been
21 in culture for more than a year from the time of initial
22 transfection. The BET-2A cell line, like the BES-lA1.6
23 cell line, appears to be resistant to the squamous
24 differentiation-inducing effects of serum. Whereas NH~E
cells are able to be induced to undergo squamous
26 differentiation when exposed to serum, bronchial
27 carcinomas are resistant to this effect (Lechner, et al.,
28 Cancer Res., 43:5915-5921, 1983). TBE cell lines (human
29 bronchial epithelial cells transformed by the v-Ha-ras
oncogene) are tumorigenic and are resistant to this
31 effect of serum. The non-tumorigenic BET-2A cell line
32 is, therefore, intermediate between normal and fully
33 malignant bronchial epithelial cells in this respect.
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1 EXAMPLE 5
2 Development of MeT-5A Cell Line
3 Human mesothelial cells were cultured as described
4 by Lechner, et al., Proc. Natl. Acad. Sci. USA, 82:
3884-3888, 1985, and were transformed at a frequency of
6 2 x 10-4 by transfection using strontium phosphate
7 coprecipitation (Brash, et al., supra) of a recombinant
8 plasmid, pRSV-T, containing the SV40 virus early region.
9 Colonies of cells transformed by the plasmid, pRSV-T,
were isolated and propagated by serial passaging for
11 periods of up to 140 days and 60 - 70 population
12 doublings from the time of transfection, before cellular
13 senescence occurred. This contrasts with the usual
14 culture lifespan of normal mesothelial cells of 30 days
and 15 population doublings. Colonies of dividing cells
16 arose from one such senescent culture, and from these
17 colonies an immortalized cell line, MeT-5A, has been
18 established by continued passaging. This cell line is
19 non-tumorigenic. Although it has been maintained
routinely in the serum-containing LHC-MM medium, it also
21 grows well in a serum-free medium.
22 EXAMPLE 6
23 Development of BBM Cell Line:
24 BEAS-2B cells were transfected via strontium
phosphate co-precipitation (Brash, et al., Molec. Cell
26 Biol., 7:2031-2034, 1987) with a recombinant plasmid,
27 B-mys/pSV2neo, which had been constructed by ligating a
28 BanHl/EcoRl fragment of the c-myc gene from the Burkitt's
29 lymphoma cell line CA46 (Showe, et al., Mol. Cell Biol.,
5:501-509, 1985) to a BamH1/EcoR1 fragment of the pSV2neo
31 vector (Southern, et al., Mol. Appl.
~Z98;~2()
1 Genet., 1:327-341, 1982). BEAS-2B cells so transfected
2 were selected in LCH-9 medium with G418 (Geneticin), and
3 colonies resistant to G418 were isolated individually and
4 subcultured. The cell line arising from one such colony
has been designated BBM.
6 EXAMPLE 7
7 Development of BZR Cell Line:
8 This cell llne has been derived by infecting the
9 BEAS-2B cell line with a recombinant containing the viral
Harvey-ras (v-Ha-ras) oncogene. The cell line so derived
11 is highly tumorigenic in athymic nude mice.
12 The details of the construction are as follows.
13 Zip-neo-v-Ha-ras recombinant retrovirus was constructed
14 by recombining the pZipNeoSV(X) retrovirus (Cepko, et
al., Cell, 37:1053-1062, 1984) at its unique Bam Hl
16 restriction enzyme site with a Bam H1-linkered 1.3 Kb
17 fragment of the Hl clone (Ellis, et al., J. Virol.,
18 36:408-420, 1980) containing the v-~a-ras oncogene.
19 Recombinant DNA molecules containing the v-Ha-ras DNA in
sense orientation with respect to the pZipNeoSV(X)
21 retrovirus 5' long terminal repeat, were identified by
22 standard DNA manipulation techni~ues and were used to
23 transfect the psi2 packaging mutant cell line (Mann, et
24 al., Cell, 33:153-159, 1983). Supernatants from these
cells were shown to contain infectious retrovirus, and
26 were used to infect the amphotrophic packagaing mutant
27 cell line, psiAM (Cone, et al., Proc. Natl. Acad. Sci.
28 USA, 81:6349-6353, 1984). Supernants from this cell line
29 were titered and used to infect BEAS-2B cells.
Following infection of BEAS-2B cells with this
31 virus, G418 resistant cells were selected (Southern, et
32 al., J. Mol. Appln. Genet., 1:327-341, 1982) and
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1 serially subcultured; the cell line so derived was
2 designated ~ZR. This cell line is highly tumorigenic,
3 forming tumors with a latency period of 2 weeks in 12/15
4 athymic nude mice each inJected with 5 x 106 cells
subcutaneously.
6 A deposit of the cell lines of the present
7 invention has been made at the ATCC, Rockville, Maryland,
8 on June 12, 1987 and July 14, 1987 under the accession
9 numbers CRL 9608, 9609, 9442~ 9443, 9444, 9482 and 9483,
10` corresponding to cell lines BES-lAl-6, BEAS-2B, BET-lA,
11 BET-2A, MeT-5A, BBM and BZR, respectively. The deposits
12 shall be viably maintained, replacing if it became
13 non-viable, for a period of 30 years from the date of the
14 deposit, or for 5 years from the last date of request for
a sample of the deposit, whichever is longer, and made
16 available to the public without restriction in accordance
17 with the provisions of the law. The Commissioner of
18 Patents and Trademarks, upon request, shall have access
19 to the deposit.
UTILITY OF CELL LINES
21 (1) Identification of ~otential chemotherapeutic
22 drugs: These cells are useful for screening chemicals
23 suitable for the treatment of cancer and related
24 diseases, by growing them in vitro in medium containing
the chemical to be tested and then, after a suitable
26 period of exposure, determining whether and to what
27 extent cytotoxicity has occurred, e.g. by trypan blue
28 exclusion assay or related assays (Paterson, Methods
29 Enzymol., 58:141, 1979), or by growth assays such as
colony forming efficiency (MacDonald, et al., Exp. Cell.
31 Res., 50: 417, 1968), all of which are standard
32 techniques well known in the art.
lZ982;20
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1 (2) Studies of the control of squamous
2 differentiation, and identification of chemical and
3 biological agents which induce squamous differentiation:
4 This is accomplished by assays previously described for
normal human bronchial epithelial cells (Masui, Proc.
6 Natl. Acad. Sci. USA, 83:2438, 1986). As noted in the
7 cell line specification, some retain ability to undergo
8 sguamous differentiation in response to serum. Induction
9 of terminal differentiation may be an effective way of
controlli`ng the growth of cancer. Chemical and
11 biological substances are screened for their ability to
12 induce differentiation by adding them to the growth
13 medium of these cells and then after a suitable time
14 interval determining whether a complex oi changes
including cessation of DNA synthesis and the appearance
16 of squamous morphology has occured. The cells are also
17 useful for studies of the biological mechanisms of
18 squamous differentiation, and the existence of both
19 serum-resistant and serum-sensitive cell lines enables
comparisons and identification of genes of their protein
21 products involved in the process of differentiation.
22 (3) Studies of metabolism of carcinogens and
23 other xenobiotics: Carcinogens and other xenoblotics may
24 be added to the growth medium of these cells and then the
appearance of metabolic products of these compounds may
26 be monitored by techniques such as thin layer
27 chromatography or high performance liquid chromatography
28 and the like, and the interaction of the compounds and/or
29 their metabolltes wlth DNA ls determined.
(4) Studies _ of DNA mutagenesis: Substances
31 known or suspected to be mutagens may be added to the
32 growth medium of the cells and then mutations may be
33 assayed, e.g., by detection of the appearance of drug
34 resistant mutant cell colonies (Thompson, Methods
129~3~2()
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1 Enzymol., 58:308, 1979). Similarly, cell-mediated DNA
2 mutagenesis, by cocultivating the cells with cell types
3 known or suspected to be capable of secreting mutagenic
4 compounds (Hsu, et al., Proc. Natl. Acad. Sci. USA,
75:2003, 1978).
6 (5) Studies of chromosome damagin~ agents:
7 Substances known or suspected to cause chromosomal damage
8 may be added to the culture medium of these cell lines,
9 and then the extent of chromosomal damage may be measured
by techniques such as measurement of the frequency of
11 sister chromatid exchange (Latt, et al., In: Tice, R.R.
12 and Hollaender, A., Sister Chromatid Exchanges. New York:
13 Plenum Press, pp. 11 ff., 1984).
14 ~ (6) Studies of malignant transformation by
chemical, physical and viral agents, and transferred
16 genes including oncogenes and high molecular weight
17 genomic DNA from tumors, using standard assays such as
18 anchorage independent growth or tumor formation in
19 athymic nude mice. For example, a cloned cellular
oncogene from a human tumor has been transferred into the
21 BEAS-2B cell line; the cell line thus derived is BBM.
22 This cell line has been shown to be resistant to the
23 squamous differentiation inducing effects of serum. In a
24 second example, a cloned viral oncogene, v-Ha-ras, has
been introduced into the BEAS-2~ cell line; the cell line
26 thus derived is BZR. Thls cell line has been shown to be
27 able to form tumors in nude mice with a latency period of
28 two weeks, and is able to grow in an anchorage-
29 independent fashion in soft agar.
(7) Use of cells altered by transfer of
31 oncogenes as in paragraph 6 above to screen for potential
32 chemotherapeutic agents (by the techniques described in
33 paragraph 1 above) especially those which may be specific
lZ~8ZZC~
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1 for cells transformed by the activation of particular
2 oncogenes or combination of oncogenes.
3 (8) Studies of cellular biochemistry, including
4 changes in intracellular pH and calcium levels, as
correlated with cell growth and action of exogenous
6 asents including but not limited to those described in
7 paragraphs- 1 through 7 above. To study intracellular pH
8 and calcium levels, cells in suitable culture vessels are
9 exposed to fluorescent indicator dyes and then
fluorescence emissions are detected with a fluorescence
11 spectrophotometer (Gryn~iewicz, et al., J. Biol. Chem.,
12 260:3~40-3450, 1985).
13 (9) Studies of cellular responses to growth
14 factors and production of growth factors: Identification
and purification of growth factors important for growth
16 and differentiation of human bronchial epithelial cells.
17 These cells are particularly useful for such an
18 application since they grow in serum-free media.
l9 Therefore, responses to growth factors can be studied in
precisely defined growth media and any factors produced
21 by the cells may be identified and pur$fied without the
22 complication of the presence of serum.
23 (10) Use of recomblnant DNA expression vectors to
24 produce proteins of lnterest. For example, the gene
encoding a protein of therapeutic value may be recombined
26 with controlling DNA segments (i.e. containing a promoter
27 with or without an enhancer sequence), transferred into
28 the cell (e.g., by strontium phosphate transfection) and
29 then the protein produced may be harvested from the
culture supernatant or a cellular extract by routine
31 procedures well known in the art.
32 (11) Studies of intracellular communication e.g.,
33 by dye scrape loading assays. To determine whether the
34 cells growing in vitro have the ability to communicate
12982~3
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1 via gap junctions, the cultures may be scraped, e.g.,
2 with a scalpel, in the presence of a fluorescent dye in
3 the growth medium. Cells at the edge of the wound are
4 mechanically disrupted and therefore take up dye; whether
intercellular communication has occurred may be
6 ascertained by determining whether cells distant from the
7 wound also contain dye.
8 (12) Characterization of cell surface antigens:
9 The cells are incubated with an antibody against the cell
surface antigen of interest, and then reacted with a
ll second antibody which is conjugated to a fluorescent
12 dye. The cells are then evaluated using a fluorescence
13 activated cell sorter to determine whether they are
14 fluorescent and therefore possess the cell surface
antigen.
16 (13) Cell-cell hybrid studies for identification
17 of tumor suppressor activity (Stanbridge, et al.,
18 Science, 215:252-259, 1982). To determine whether these
19 cell lines contain tumor suppressor genes, they are fused
to malignant tumor cells. The presence of tumor
21 suppressor genes is indicated by loss of malignancy e.g.,
22 as detected by loss of ability to form tumors in athymic
23 nude mice, in the hybrid cells.
24 (14) Identification of novel genes, including
transforming genes in naturally occurring cancers
26 dsscribed in paragraph 6 above, growth factor genes as
27 described in paragraph 9 above, tumor suppressor genes as
28 described in paragraph 13 above, using standard molecular
29 biological techniques (Davis, et al., Methods in
Molecular Biology, New York: Elsevier, 1986) and
31 techniques such as cDNA subtraction cloning and the like.
32 Of course, a kit for screening carcinogenic or
33 antineoplastic agents and for any other usage as
34 described herein supra, is easily assembled, comprising
1298~i~V
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1 container(s) containing the cell line(s) of the present
2 invention. Other components routinely found in such kits
3 may also be included with instructions for performing the
4 test.
It is understood that the examples and embodiments
6 described herein are for illustrative purposes only and
7 that various modifications or changes in light thereof
8 will be suggested to persons skilled in the art and are
9 to be included within the spirit and purview of this
application and scope of the appended claims.
