Title: Immortalized human keratinocyte cell line
Abstract: A spontaneously immortalized human keratinocyte cell line is disclosed. In a preferred embodiment, this cell line is ATCC 12191. In another embodiment of the invention, a method of assaying the effect of a test tumor cell modulation agent is disclosed. The method comprises the steps of obtaining a human stratified squamous epithelial cell culture, wherein the culture comprises human malignant squamous epithelial cells and spontaneously immortalized human keratinocytes, wherein the culture forms a reconstituted epidermis. One then treats the epidermis with a test tumor cell modulation agent and evaluates the growth of the malignant cells within the epidermis.
Patent Number: 6,884,595 Issued on 04/26/2005 to Allen-Hoffmann, et al.
| Inventors:
|
Allen-Hoffmann; B. Lynn (Madison, WI);
Schlosser; Sandra J. (Oregon, WI)
|
| Assignee:
|
Wisconsin Alumni Research Foundation (Madison, WI)
|
| Appl. No.:
|
299938 |
| Filed:
|
November 19, 2002 |
| Current U.S. Class: |
435/7.21; 435/29; 435/371 |
| Intern'l Class: |
G01N 033//56.7; C12Q 001//02; C12N 005//08 |
| Field of Search: |
435/721,347,371,373,402,408,29
|
References Cited [Referenced By]
U.S. Patent Documents
| 5491084 | Feb., 1996 | Chalfie et al.
| |
| 5989837 | Nov., 1999 | Allen-Hoffmann et al.
| |
| 6214567 | Apr., 2001 | Allen-Hoffmann et al.
| |
| 6514711 | Feb., 2003 | Allen-Hoffmann.
| |
Other References
L. Allen-Hoffmann, et al., Proc. Nat'l Acad. Sci. USA 81:7802-7806, 1984.
B.L. Allen-Hoffmann, et al., "Use of RHEK-1 Immortalized Human Keratinocytes
for Detection of Induced Mutation at the Hypoxanthine-guanine Phosphoriboxyltransferase
Locus," Inter. J. Oncol. 3:619-625 1993.
H.P. Baden, et al., "Isolation and Characterization of a Spontaneously Arising
Long-lived Line of Human Keratinocytes (NM1)," In Vitro Cell Dev. Biol. 23(3):205-213, 1987.
P. Boukamp, et al., "Cell Keratinization in a Spontaneously Immortalized Diploid
Human Keratinocyte Cell Line," J. Cell Biol. 106:761-771, 1988.
P. Boukamp, et al., "Normal Keratinization in a Spontaneously Immortalized Aneuploid
Human Keratinocyte Cell Line," J. Cell Biol. 106:761-771, 1988.
J.A. Garlick and L.B. Tachman, "Fate of Human Keratinocytes during Reepithelization
in an Organotypic Culture Model," Lab. Invest. 70(6):916-924, 1994.
J.A. Garlick, et al., "Re-epithelialization of Human Oral Keratinocytes In Vitro,"
J. Dent. Res. 75(3):912-918, 1996.
J.A. Garlick, et al., "Skin Substitutes-Tissue Models for Cancer Biology," Bioengineering
of Skin Substitutes, Sep. 18-19, Boston, MA, 1997.
C. Gilles, et al. "Immortalization of Human Cervical Keratinocytes by Human Papillomavirus
Type 33," Int. J. Cancer 53:872-879, 1993.
M.A. Pickart, et al., "Development of an Organotypic Culture System for the Study
of Human Squamous Cell Carcinoma," Proc. Am. Assoc. Canc. Res. 40:192, 1999 (Abstract).
R.H. Rice, et al., "Elevation of Cell Cycle Control Proteins During Spontaneous
Immortalization of Human Keratinocytes," Mol. Biol. Cell 4:185-194, 1993.
D. Strickland, "Organogenesis gets Approval from FDA from Graftpatch: Stock Jumps
7 Percent," Bioworld Today 8(154):1-4, 1997.
|
Primary Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Quarles & Brady LLP
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with United States government support awarded by the
following agency: NIH Grant No. AR40284. The United States has certain rights in
this invention.
Parent Case Text
This application claims priority to and is a continuation of Ser. No. 09/769,124,
filed Jan. 24, 2001, and issued as U.S. Pat. No. 6,485,724, which is a continuation
of Ser. No. 09/277,295, filed Mar. 26, 1999, issued as U.S. Pat. No. 6,214,567,
which was a continuation of Ser. No. 09/114,557, filed Jul. 13, 1998, issued as
U.S. Pat. No. 5,989,837.
Claims
1. A method for screening compounds comprising:
1) providing:
a) a culture comprising a human keratinocyte cell line, wherein said cell line
i) is immortalized; ii) is nontumorigenic; iii) forms cornified envelopes when
induced to differentiate; iv) undergoes normal squamous differentiation in organotypic
culture; and v) maintains cell type-specific growth requirements and wherein the
cell line originated with ATCC CRL 12191; and
b) a test compound;
2) treating said host cells with said one or more test compounds; and
3) monitoring said culture.
2. The method of claim 1, wherein said culture is an organotypic culture.
3. The method of claim 2, wherein said culture is a monolayer culture.
4. The method of claim 1, wherein said cell type-specific growth requirements
include requirements selected from the group consisting of 1) exhibition of morphological
characteristics of normal human keratinocytes when cultured in standard keratinocyte
growth medium in the presence of mitomycin C-treated 3T3 feeder cells; 2) dependence
on epidermal growth factor for growth; and 3) inhibition of growth by transforming
growth factor β1.
5. The method of claim 1, wherein said cell line comprises an exogenous gene.
6. The method of claim 5, wherein said exogenous gene is green fluorescent protein.
7. The method of claim 1, wherein said compounds are selected from the group
consisting of cytostatic compounds, therapeutic compounds, and cosmetic compounds.
8. The method of claim 1, wherein said culture comprises ATCC CRL-12191 cells.
9. A method for screening compounds comprising:
1) providing:
a) a culture comprising a human keratinocyte cell line, wherein the cell line
originated with ATCC CRL 12191 wherein said cell line i) is immortalized; ii) is
nontumorigenic; iii) forms cornified envelopes when induced to differentiate; iv)
undergoes normal squamous differentiation in organotypic culture; and v) maintains
cell type-specific growth requirements, wherein said cell type-specific growth
requirements include requirements selected from the group consisting of 1) exhibition
of morphological characteristics of normal human keratinocytes when cultured in
standard keratinocyte growth medium in the presence of mitomycin C-treated 3T3
feeder cells; 2) dependence on epidermal growth factor for growth; and 3) inhibition
of growth by transforming growth factor β1; and
b) a test compound;
2) treating said host cells with said one or more test compounds; and
3) monitoring said culture.
10. The method of claim 9, wherein said culture is an organotypic culture.
11. The method of claim 9, wherein said culture is a monolayer culture.
12. The method of claim 9, wherein said cell line comprises an exogenous gene.
13. The method of claim 12, wherein said exogenous gene is green fluorescent protein.
14. The method of claim 9, wherein said compounds are selected from the group
consisting of cytostatic compounds, therapeutic compounds, and cosmetic compounds.
15. The method of claim 9, wherein said culture comprises ATCC CRL-12191 cells.
16. A method for screening compounds comprising:
1) providing:
a) a culture comprising a human keratinocyte cell line, wherein the cell line
originated with ATCC CRL 12191, wherein said cell line i) is immortalized; ii)
is nontumorigenic; iii) forms cornified envelopes when induced to differentiate;
iv) undergoes normal squamous differentiation in organotypic culture; and v) maintains
cell type-specific growth requirements, wherein said cell type-specific growth
requirements include requirements selected from the group consisting of exhibition
of morphological characteristics of normal human keratinocytes when cultured in
standard keratinocyte growth medium in the presence of mitomycin C-treated 3T3
feeder cells; dependence on epidermal growth factor for growth; and inhibition
of growth by transforming growth factor β1; and vi) and comprises an exogenous
gene; and
b) a test compound;
2) treating said host cells with said one or more test compounds; and
3) monitoring said culture.
17. The method of claim 16, wherein said culture is an organotypic culture.
18. The method of claim 16, wherein said culture is a monolayer culture.
19. The method of claim 18, wherein said exogenous gene is green fluorescent protein.
20. The method of claim 16, wherein said compounds are selected from the group
consisting of cytostatic compounds, therapeutic compounds, and cosmetic compounds.
21. The method of claim 16, wherein said culture comprises ATCC CRL-12191 cells.
Description
CROSS-REFERENCE TO RELATED APPLICATION
Not applicable.
BACKGROUND OF THE INVENTION
Human Keratinocytes
Human keratinocytes isolated from stratified squamous epithelia can be readily
cultivated in vitro (reviewed in Leigh, et al., 1994). Cultivated keratinocytes
replicate readily during early passage and can generate large numbers of cells
which exhibit certain features of squamous differentiation in vivo. When cultured
normal human keratinocytes are transplanted onto mice, epidermal tissue architecture
is regenerated over time in an orderly fashion (Breitkreutz, et al., 1997). The
ease of cultivation and transplantation of human keratinocytes coupled with the
accessibility of skin for the grafting procedure and subsequent monitoring, make
this somatic cell type attractive for therapeutic gene delivery. However, due to
the initiation of terminal differentiation, transgene expression in keratinocytes
is consistently lost regardless of the gene expression strategy used. Several reports
have shown that genetically engineered human keratinocytes can recapitulate full
thickness epidermis, thus demonstrating that cells with stem cell-like properties
were present in the transplanted population of cells (Choate and Khavari, 1997;
Choate, et al., 1996; Gerrard, et al., 1993; Garlick, et al., 1991; Greenhalgh,
et al., 1994; Vogel, 1993, Fenjves, 1994).
In Vitro Tissue Culture Assays Utilizing Human Cells Derived from Stratified
Squamous Epithelia
In vitro assays using monolayer cultures of adherent cells which maintain the
normal in vivo tissue context do not exist for human tissues. Animal models do
have the capacity to mimic some of the processes involved in the response of human
tissue therapies. However, animal systems lend themselves only to qualitative and
subjective scoring of tumor repopulation. Historically, simple in vitro growth
assays have used monolayer cultures of rodent or human cell lines on plastic tissue
culture dishes. Colony size or cell number are assessed in order to estimate the
extent of survival and repopulation of cancer cells following radiation treatment.
A manor drawback of this approach is that it does not account for any adhesive
or paracrine growth factor signals within the tumor cell environment. For this
reason, studies on the growth of tumor cells in the absence of normal surrounding
tissue may not accurately reflect the in vivo growth characteristics of tumor cells.
For example, human head and neck (H&N) tumors are diagnosed in 43,000 patients
in the United States every year and in over 750,000 patients worldwide. Although
tumor recurrence near the site of the primary tumor is the predominant cause of
treatment failure and death for these patients, little is know about the molecular
events contributing to tumor regrowth following treatment. Clinical and radiobiological
evidence suggests that tumor proliferation rates may actually increase following
wounding due to radiation exposure (Hall, E. J., 1988; Petereit, D. G., et al.,
1995). It has been suggested that the wound environment provides potent tumor growth
signals (Haddow, A., 1972). For example, extracellular matrix (ECM) glycoproteins
present in the wound bed provide and/or sequester potent growth stimuli required
for normal tissue regeneration. It is clear from these observations that the tissue
context in which a tumor initially develops and/or regrows following failed cancer
treatment may have significant impact on tumor growth.
Needed in the art of cell biology is a spontaneously immortalized human keratinocyte
cell line with near normal chromosomal complement and a method for using this immortalized
cell line in an in vitro tissue assay.
SUMMARY OF THE INVENTION
In one embodiment, the present invention is an immortalized human keratinocyte
cell line, wherein the cell line comprises a normal chromosomal complement of 46
with the exception of an extra isochromosome on the long arm of chromosome 8. In
one particularly advantageous embodiment, the cell line is ATCC CRL 12191.
In another embodiment, the present invention is a transgenic immortalized human
keratinocyte cell line transfected with a heterologous gene. This gene may be a
marker gene, most preferably green fluorescent protein (GFP).
The present invention is also a method of assaying the effect of a test tumor
cell therapeutic agent by obtaining a human stratified squamous epithelial cell
culture comprising malignant squamous epithelial cells, preferably human squamous
carcinoma cells (SCC), and spontaneously immortalized human keratinocytes. This
culture is then formed into a reconstructed epidermis. One may then treat the epidermis
with a test tumor cell therapeutic agent and evaluate the growth of the SCC within
the epidermis.
It is an advantage of the present invention that an immortalized keratinocyte
cell line is provided.
Other objects, features and advantages of the present invention will become
apparent after review of the specification, claims and drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is a chromosomal analysis of BC-1-Ep/SL cells. Karyotypic analysis was
performed on BC-1-Ep/SL cells at passage 31. The cells contained 47 chromosomes
due to an extra isochromosome of the long arm of chromosome 8. The extra chromosome,
i(8q), is not seen in the parental keratinocytes (BC-1-Ep passage 3) which exhibited
a normal male karyotype.
FIG. 2 demonstrates the requirement for epidermal growth factor (EGF) for serial
passage of EC-1-Ep/SL cells. BC-1-Ep/SL cells were serially passaged in standard
media +/-10 ng/ml EGF. The cells survived without EGF but grew poorly.
FIG. 3 demonstrates that transforming growth factor-β-1 (TGF-β1)
inhibits growth of BC-1-Ep/SL keratinocytes. The parent cells, BC-1-Ep (6°)
and BC-1-Ep/SL (28°) were plated in standard media without EGF or a 3T3 feeder
layer. Cells were treated +/-5 ng/ml TGF-β1 when the cells were ˜20%
confluent in standard media without EGF. Cells were counted 3-5 days later. The
effect of TGF-β1 treatment is shown as a percentage of controls.
FIG. 4 demonstrates growth factor requirements of BC-1-Ep/SL cells. BC-1-Ep/SL
cells at passage 31 were grown in 2.5% FCS+3F12:1DME+10 ng/ml EGF supplemented
with no additional growth factors (˜GF), 0.4 μg/ml hydrocortisone (HC),
8.4 ng/ml cholera toxin (CT), 24 μg/ml adenine (Ade), 5 μg/ml insulin
(Ins), or all growth factors (+GF). Cell growth increased in the presence of each
growth factor alone, however optimal growth was achieved in the presence of all
growth factors.
FIG. 5 shows that increased cell confluence reduces transient transfection efficiency
in BC-1-Ep/SL cells. BC-1-Ep/SL cell were transfected with the GFP containing plasmid
pGreenLantern (Gibco) and pcDNA3 neo (Invitrogen) using GeneFECTOR (VennNova Inc.).
A range of 15-20 μg of linearized pGreenLantern and 5-6.7 μg of pcDNA3neo
was used. Cell numbers were obtained by trypsinization and counting of keratinocytes
using a hemacytometer. Low density was defined as 6-7.5×10
5 cells/100
mm dish. High density was defined as 3-4×10
6 cells/100 mm dish.
Transfection efficiency was obtained using fluorescence activated cell sorting
(FACS). Each bar of the graph represents a single experiment.
FIG. 6 is a cross-sectional schematic of skin and organotypic SCC13y
GFP+/BC-1-Ep/SL
culture. FIG. 6A is a schematic of the multiple layers present in skin. FIG. 6B
is a schematic of a tumor/normal tissue model consisting primarily of three components
as shown. The base layer is a dermal-equivalent consisting of collagen and fibroblasts.
Above this layer, is the reconstructed epidermis formed by differentiating BC1-Ep/SL
epithelial cells (shaded). Within this epidermal equivalent is an SCC13 y
GFP+
tumor foci (not shaded).
BRIEF DESCRIPTION OF THE INVENTION
A. Immortalized Human Keratinocyte Cell Line
Human keratinocytes with stem cell-like properties are the optimal target for
stable transfection of exogenous genes. Stable transfectants are produced when
exogenous DNA is introduced into a cell and integrates into the host chromosomes.
Subsequent daughter cells express the gene product of the transgene and are stable
if the expression is propagated indefinitely throughout subsequent generations.
For gene therapy applications, it is essential to use stem cell-like keratinocytes
with the ability to regenerate full thickness epidermis for multiple cycles of
tissue turnover while maintaining expression of the transduced gene of interest.
In one embodiment, the present invention is a spontaneously immortalized human
keratinocyte cell line, BC-1-Ep/SL, which maintains cell type-specific growth requirements,
expresses differentiation markers, and can be stably transfected. BC-1-Ep/SL was
deposited on Sep. 20, 1996 with the American Type Culture Collection at 10801,
University Boulevard, Manassas, Va. 20110-2209, USA, under Accession No. CRL-12191
under the terms and conditions of the Budapest Treaty.
BC-1-Ep/SL keratinocytes are not tumorigenic and undergo squamous differentiation
in organotypic culture. Organotypic cultures are cultures in which keratinocytes
are grown on a substrate that resembles the dermis and are exposed to the air-medium
interface (Leigh ant Watt 1994).
We envision that this human keratinocyte cell line will serve as a source of
long-lived
epidermal progenitor cells capable of supporting high efficiency and long-term
epidermal transgene expression. BC-1-Ep/SL keratinocytes represent an important
new cellular reagent for the study of growth and differentiation in stratified
squamous epithelia.
In another embodiment, the present invention is a immortalized human keratinocyte
cell line, preferably BC-1-Ep/SL, preferably ATCC CRL-12191, comprises at least
one transgene. The Examples below demonstrate a preferred method of creating a
transgenic cell line. Many other methods would be apparent to one of skill in the
art of molecular biology. We have developed a method whereby the transfected BC-1-Ep/SL
keratinocytes are identified based on the expression of GFP and are selected based
on morphology and degree of GFP expression, and as such preserve the highest degree
of the cells pre-transfection, natural state/qualities. This technique avoids the
potential character-altering pressures due to selection based on a dominant selectable
marker with standard chemical agents such as F418, methotrexate, hygromycin-B,
aminopterin, mycophenolic acid, and zeocyn.
We envision that one would wish to use the immortalized human keratinocyte cell
line in a variety of ways such as formation of an organotypic culture, with monolayer
cell culture, a human tissue-engineered product appropriate for short- and/or long-term
grafting to humans or research animals, and as a biofilm component, e.g., as part
of a machine which produces gene products to continually be administered to a patient intravenously.
B. Use of BC-1-Ep/SL as a Model Organotypic System
As described above, the cell line of the present invention has the substantial
advantage of reproducing the tissue architecture of normal human stratified squamous
epithelia. This model is suitable as an assay of human tumor repopulation. By entirely
reconstructing malignant human epithelial cell growth within a tissue-like environment,
tumor cell growth characteristics can be monitored in a physiologically relevant context.
In addition, quantitative endpoints can be monitored because each individual
tumor
cell could be genetically engineered to express a marker protein, such as green
fluorescent protein (GFP). (The Examples below demonstrate a preferred method of
transfecting BC-1-Ep/SL with a marker gene.) This model system represents a technological
advancement for the screening of drugs or agents used in the treatment of cancers
of stratified squamous epithelia. Illustrative cancers include cancers of the head
and neck, skin, oral mucosa, cervix, and trachea.
Therefore the present invention is a human stratified squamous epithelial
model system designed to measure the rate of tumor cell growth and repopulation
(see FIG. 6 in Examples for schematic).
In one embodiment, the model system consists of an organotypic co-culture of
genetically
marked human squamous cell carcinoma (SCC) cells and unmarked spontaneously immortalized
human keratinocytes, BC-1EP/SL. The BC-1-EP/SL keratinocyte line is a critical
component in the model system because the cells provide a reproducible environment
context in which to compare the growth characteristics of SCC cells. The BC-1-EP/SL
immortalized cell line is highly advantageous because it maintains all the characteristics
of a normal keratinocyte, i.e., terminal differentiation, apoptosis, and non-tumorigenic
characteristics remain.
The Examples below disclose a preferred method of creating a reconstructed epidermis.
In brief, BC-1-EP/SL and SCC cells are seeded onto a base of collagen containing
normal human fibroblasts. The co-culture is seeded at various dilutions of marked
SCCs in a standard number of BC-1-EP/SL keratinocytes.
The in vitro tumor/normal tissue model of the present invention will also aid
in experimentation of the molecular mechanisms responsible for tumor progression
and repopulation. This same model may also be used to identify potential cytostatic
agents which can slow tumor regrowth and chemopreventive agents with specific tumor
selectivity. For example, the reconstituted organotypic cultures can be treated
with a variety of tumor cell modulation agents, such as physical (e.g., x-irradiation)
or chemical/biological agents (i.e., chemotherapeutic agents, cytostatic drugs),
and the cultures monitored, preferably using the GFP labelling, to quantify the
extent or lack of SCC cell (re)growth. In this way, the BC-1-EP/SL model system
will contribute to the identification and development of antiproliferative agents
for cancer patients receiving curative therapies for cancers of the head and neck,
skin, oral mucosa, cervix, trachea, and other epithelia or novel, new anticancer
strategies, in general.
Tumor cell growth may be most conveniently monitored in the following manner:
The total number of tumor cells and ratio to the number of BC-1-Ep/SL cells can
be most conveniently monitored using flow cytometry techniques based on GFP fluorescence.
Tumor cell volume and localization is most conveniently monitored by confocal microscopy.
Cell-cell interactions are most conveniently monitored in histologic sections using
immunostaining and in situ hybridization techniques.
Introduction of GFP into mammalian cells is increasingly utilized for
in vitro and animal model systems. To date, there are no reports describing altered
mammalian cell growth characteristics due to GFP expression. The fact that these
models behave as expected suggests that GFP expression is innocuous and thus an
ideal marker protein. Observations in this study have also confirmed that introduction
of GFP into the human squamous cell carcinoma cell line, SCC13y, has no effect
on relevant biologic endpoints. The results observed in these experiments support
previous studies which suggest GFP expression does not interfere with growth and
differentiation characteristics of the cell types studied. Stability of GFP expression
is another variable which is critical to the success of models which utilized GFP
as a marker over time. Our experimental observations suggest that GFP expression
is stable in at least one malignant cell line, SCC13y. Other methods of genetic
marking, e.g., enzymatic activity and/or culture devices are being considered to
allow repeated assay of the same culture sample.
The co-culture of abnormal and normal human keratinocytes has recently been reported
by A. Javaherian and co-workers (Javaherian, A., et al., 1998). In this study,
keratinocyte cells (HaCat) which have been immortalized using genetic engineering
are grown in organotypic culture. Though immortal, these cells are not tumorigenic,
i.e., able to form malignant tumors in nude mice, and thus are not representative
of malignant cells in general. Since the SCC13y
GFP+ cell line is derived
from an actual patient tumor and is tumorigenic, it may be more appropriate for
studies aimed at eradication of malignant tumors.
The organotypic co-culture model may be useful for at least three critical problems
faced by the pharmaceutical and biotechnology industries: (1) how to screen for
novel cytostatic inhibitors of tumor repopulation, (2) how to determine patient-specific
responses to chemotherapy or radiotherapy prior to treatment, and (3) how to develop
novel, biologic therapeutic agents. Unlike traditional cytotoxic agents which target
the tumor cell directly, cytostatic inhibitors of tumor growth may target the individual
tumor or its microenvironment. This difference arises predominantly because the
primary aim of a cytotoxic agent is to kill the tumor cell; whereas, cytostatic
agents aim to slow or halt tumor proliferative expansion but do not necessarily
kill it. In theory, this can be achieved by direct interference with cellular proliferation
(e.g., cell cycle inhibitors) or by indirect alterations in the surrounding normal
tissue which make it less supportive of tumor growth (e.g., inhibition of wound
regenerative signals).
Novel antitumor strategies may be identified using organotypic tumor/normal
tissue co-culture because in vitro function of tumor and normal tissue responses
can be assayed directly in reconstructed human tissues. Current in vitro assays
based on the growth of monolayer cultures of human tumor cells fail in this regard
because they do not require normal tissue function nor do they account for the
influence of the microenvironment of the malignant cells. Understanding the tumor
microenvironment may be of particular importance an identification of chemopreventive
agents which focus on preventing the initiation of a tumor when it has yet to develop
aggressive malignant characteristics. It may also be important for prevention of
regrowth of malignant cells which survive conventional cytotoxic or surgical therapies.
Additionally, cytostatic agents with organotypic tumor/normal tissue co-culture,
may also be useful for concurrent use for clinical radiotherapy. In this case,
the capacity of cytostatic agents to slow the tumor growth during treatment may
be critical in eliminating the tumor and may attenuate the complications produced
by inflammation which accompanies conventional therapies.
Monitoring tumor regrowth in an organotypic tumor/normal tissue co-culture
model may also be useful for providing pretreatment patient-specific information
regarding therapy responsiveness and therapeutic ratio. Current assays have demonstrated
only a weak correlation between in vitro growth of human tumors in monolayer culture
and actual patient tumor response. Stronger correlations could be achieved by including
the normal tissue context within in vitro models.
Other applications may provide additional prognostic and/or biologic materials
with potential to directly impact patient treatment. For instance, cells from tumor
biopsies could be co-cultured with GFP-labelled BC-1-EP/SL cells, defined herein
as "inverse culture." An inverse culture model would provide a reproducible culturing
environment which closely mimics the in situ tumor microenvironment and would allow
assays of individual patient's tumor cells. Specific treatments could then be tested
with the biopsied tumor sample in culture before actual patient treatment commences.
In this way, treatments may be individually tailored to each patient based on test responses.
An important property of an organotypic tumor/normal tissue "inverse" co-culture
model is that it may closely mimic the tumor microenvironment specific to that
patient. This may be highly advantageous for development of some novel forms of
biologic treatment such as immune therapy. The goal of immune therapy is to prime
the patients own immune effector cells, e.g., B cells, helper T cells, cytolytic
T cells (CTLs), or natural killer cells (NK) in vitro, and then return these activated
cells to the patient in order to target and eradicate the tumor. Current in vitro
cultures may not be as efficient as organotypic cultures for the priming of immune
effector cells. A potential reason for lack of activation of the immune effector
cells is inefficient and/or unsuccessful presentation of the appropriate tumor
specific antigens (TSAs). An organotypic tumor/normal tissue "inverse" co-culture
model may eliminate this problem. Because it accurately reconstructs the in situ
tumor microenvironment, TSAs may be more likely to be expressed in organotypic
culture; and, thus, may have a higher chance of being presented to the immune effector cells.
B. Other Embodiments
1. Use as a universal donor epidermal cell type in living tissue products for
the repair and/or support of appropriate epithelial tissues. An example of an application
is venous leg ulcers, which affect about 1 million people in the United States
and 3 million worldwide, and other ulcer conditions such as diabetic ulcers and
pressure ulcers (bedsores), which affect approximately 10 million people worldwide.
The BC-1-Ep/SL keratinocyte cell line could also be used in a wide range of clinical
applications, for example, acute burn coverage, dermatological surgery wounds,
donor site wounds (for coverage after skin is harvested to be used elsewhere).
2. Use includes the development and testing of agents used in the formulation
of therapeutic, cosmeceutical, and cosmetic skin products applied in cream, lotion,
liquid, and spray forms. The cells provide a consistent source of human keratinocytes
to test toxicity, potency, and efficacy of such agents. Cells could be used in
monolayer culture for these assays or in the organotypic cultures. Organotypic
cultures could be used to develop and assay effects of agents on tissue architecture,
differentiation, cell replication and growth, barrier function, and tissue strength.
3. Use includes recipient cell for the cultivation of biological agents, such
as human papilloma viruses, so that vaccines against viruses can be produced. The
cell line could also be used to develop and test antiviral drugs and agents. Over
60 different types of human papilloma viruses produce warts in humans, including
genital warts and viral lesions that are tightly associated with the development
of cervical cancer in women and penile cancer in men. Human papillomaviruses are
small double-stranded DNA viruses which are widespread in the human population.
They are strictly epitheliotropic and infect only cutaneous and mucosal skin from
a range of anatomic sites. Human papillomaviruses replicate only in human epithelial
cells which are undergoing differentiation.
EXAMPLES
1. Normal Growth and Differentiation in a Spontaneously Immortalized Near-Diploid
Human Keratinocyte Cell Line, BC-1-Ep/SL
A. Materials and Methods
Cell Culture
Normal keratinocytes (BC-1-Ep) were isolated from newborn human foreskin.
Keratinocyte cultures were established by plating aliquots of a single cell suspension
in the presence of mitomycin C-treated Swiss mouse 3T3 fibroblasts as described
by Allen-Hoffmann and Rheinwald (1984). The standard keratinocyte culture medium
was composed of a mixture of Ham's F12:Dulbecco's modified Eagle's medium (DME),
(3:1, 0.66 mM calcium, supplemented with 2.5% fetal calf serum (FCS), 0.4 μg/ml
hydrocortisone (HC), 8.4 ng/ml cholera toxin (CT), 5 μg/ml insulin (Ins),
24 μg/ml adenine (Ade), 10 ng/ml epidermal growth factor (EGF), 100 units
penicillin and 100 μg/ml streptomycin (P/S). The cells were passaged at weekly
intervals at 3×10
5 cells on a 100 mm
2 tissue culture
dish with feeders. Transformed cells, BC-1-Ep/SL (spontaneous line), appeared at
passage 16. BC-1-Ep/SL cells at passage 55 tested negative for mycoplasma (Wisconsin
State Laboratory of Hygiene, Madison, Wis.). Recombinant human EGF obtained from
R+D Systems. Transforming growth factor beta (TGF-(1) was purified from human platelets.
Chromosomal Analysis
Cells in log phase growth were arrested in metaphase with 50 ng/ml colcemid,
then trypsinized and pipetted from the flask for centrifugation. After removal
of the media and trypsin, the cells were suspended in a hypotonic 75 mM KCl solution
for 20 minutes, fixed with 3:1 methanol/acetic acid three times and dropped onto
glass slides. Slides were aged two weeks, lightly trypsinized and stained with
Giemsa (Seabright, 1971). In each sample, the chromosomal identities and abberations
were determined in well-spread G-banded metaphases by photographic analysis and
the cutting of at least two karyotypes for band to band comparison of chromosomal homologs.
DNA Fingerprinting
DNA was isolated from keratinocytes using Qiagen QIAamp Blood Kit (Qiagen, Inc.,
Santa Clarita, Calif.). DNA fingerprint analysis used the GenePrint (Fluorescent
STR System according to protocols recommended by manufacturer. The twelve primer
pairs are divided into three quadriplexes (CTTv, FFFL, and GammaSTR). Each quadriplex
was amplified in separate reactions using 25 ng of DNA as template. Amplification
was preformed in a Perkin-Elmer 9700 thermal cycler (Perkin-Elmer, Corp., Norwalk,
Conn.). PCR products were electrophoresed on 42 cm×33 cm×0.4 mm polyacrylamide
gels in a BRL sequencing apparatus (Life Technologies, Inc., Gaithersburg, Md.).
Gels were then scanned on a Hitachi FMBIO(II Fluorescent Scanner.
Growth of BC-1-Ep/SL Cells in Athymic Mice
BC-1-Ep/SL cells were injected into nude athymic mice to determine if
they could form tumors. A suspension of 5×10
6 cells in 100 μl
Ham's F12 was infected subcutaneously into the flanks of six nude mice. As a negative
control, the parental BC-1-Ep 6(cells were injected at 3×10
6 cells/100
μl F12. As a positive control, SCC4 cells were injected at 3×10
6/100
μl F12. Mice were weighed and tumors measured 26 days later.
Suspension in Semi-solid Media
For suspension studies, preconfluent cultures were removed from culture dishes
with 0.5 mM EDTA, 0.1% trypsin and washed in serum-containing medium to inactivate
any residual trypsin. After a short centrifugation (440×g for 3 minutes),
cells were resuspended at 1×10
6 cells/ml in 3 parts Ham's F-12
plus 1 part DME made semi-solid with 1.68% methylcellulose (4,000 centipoises,
Fisher Scientific, Fairlawn, N.J.) as described in Sadek and Allen-Hoffmann, 1994
(Sadek, C. M. and B. L. Allen-Hoffmann, 1994). Cells were recovered from suspension
by repeated dilution with serum-free medium and centrifugation (440×g for
10 minutes). Following one rinse with phosphate-buffered saline (0.137 M NaCl;
2.7 mM KCl; 8.1 mM Na
2HPO
4; 1.4 mM KH
2PO
4;
pH 7.2) (PBS), cells were either resuspended in PBS (pH 7.2) to assay for CE formation
or lysed in SLS buffer (50 mM Tris; 10 mM EDTA, pH 8.0; 0.5% (w/v) sodium lauroyl
sarcosine) to determine DNA fragmentation. Control controls consisted of adherent
keratinocytes treated for similar times in 3 parts Ham's F-12 plus 1 part DME.
Northern Analysis
Cells were grown in standard keratinocyte culture medium on a 3T3 feeder layer.
The feeder layer was removed 24 hours prior to RNA isolation with 0.02% EDTA in
PBS. Poly A
+RNA was isolated from logarithmically growing cells as previously
described (Sadek and Allen-Hoffmann, 1994). Poly A
+RNA was electrophoresed
in a 1.2% agarose gel containing formaldehyde and electroblotted to a Zeta-probe
membrane (Bio-Rad Laboratories, Richmond, Calif.). The membrane was prehybridized
and then hybridized in the presence of a random primer [
32p]-dCTP-labeled
cDNA probe as recommended by the supplier. The cDNA probes used for detection include
rat glyceraldehyde-3-phosphate dehydrogenase, pGPDN5 (Fort, et al., 1985), monkey
TGF-β1 (Sharples, et al., 1987), EGF receptor (Xu, et a., 1984), mouse keratin
14 (gift from Dennis Roop), TGF-α (Kudlow, et al., 1988), and a 830-bp 5′
fragment of human c-myc (Miyamoto, et al., 1989).
Cornified Envelope (CE) Formation
Keratinocytes were removed from culture plates and recovered from suspension
as described previously. Cells from each treatment were counted and resuspended
in triplicate at 10
6 cells/ml in PBS (pH 7.2) containing 1% SDS and
20 mM dithiothreitol. Samples were boiled for 5 minutes in a waterbath and cooled
to room temperature. DNase (0.5 μg/ml) was added and CEs counted using a
hemacytometer. CE formation was calculated as a percentage of input cells.
Analysis of Nucleosomal DNA Fragmentation
DNA was isolated and labeled as previously described (Sachsenmeier and Allen-Hoffmann,
1996). Briefly, 2.5×10
6 cells were lysed in 500 μl of 50
mM Tris, 10 mM EDTA pH 8.0 and 0.5% (w/v) sodium lauroyl sarcosine. The lysate
was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, v:v:v) and ethanol
precipitated. The DNA was dissolved in 20 μl TE buffer, pH 8.0, and quantitated
by absorption at 260 nm. Intact and fragmented DNA was 3′ end-labeled with
[α
32P]-ddATP using terminal dideoxynucleotidylexotransferase as
described by Tilly and Hsueh (1993). One half of each labeled sample was loaded
onto a 1.5% agarose gel and electrophoresed. Gels were dried with heat using an
SE 1200 Easy Breeze (Hoefer Scientific, San Francisco, Calif.) and exposed to Kodak
Biomax MR film.
Formation of Organotypic Cultures
Organotypic cultures were grown as previously described (N. Parenteau,
1994). A collagen raft was formed by mixing normal human neonatal fibroblasts,
CI-1-F, with Type I collagen in 10% FCS+F12+penicillin/streptomycin. Rafts were
allowed to contract for 5 days. The parent cells, BC-1-Ep (5°) and the BC-1-Ep/SL
(38°) cells were plated on the rafts at 3.5×10
5 cells in 50
μl 0.2% FCS+3F12:1DME+HC+Ade+Ins+CT+P/S containing 1.88 mM calcium. Cells
were allowed to attach 2 hours before adding an additional 13 mls of media (Day
0). On Day 1 and 2 cells were refed. On Day 4, cells were lifted to the air interface
with cotton pads and switched to cornification medium (2% FCS+3F12:1DME+HC+Ade+Ins+CT+P/S
containing 1.88 mM calcium). Cells were fed cornification medium every three days.
On Day 15 rafts were fixed with freshly made modified Karnovsky's fixative consisting
of 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4,
at room temperature for 3 hours. Before removing the culture media, fixative was
gently added to the cells on top of the raft to prevent cornified layers from floating
away. Subsequently, the culture media was aspirated and the culture wells filled
with fixative. The raft was cut in half with one half processed for light microscopy
and the other half for electron microscopy.
Tissue Sectioning
Fixed rafts were embedded in paraffin, sectioned and stained with hematoxylin
and eosin by Surgical Pathology, University Hospital, Madison, Wis. Stained sections
were viewed and photographed using an Olympus IX-70 microscope equipped with a
35 mm camera.
Electron Microscopy
Fixed cultures were washed 3 times with 0.1M cacodylate buffer, pH 7.4. Under
a dissecting microscope, a scalpel was used to detach the polyester mesh supporting
the raft culture from the plastic insert. The raft was cut with a scalpel into
approximately 2 mm×4 mm pieces which were stored overnight in buffer. Following
postfixation with 1% osmium tetroxide at 4° C., the keratinocytes were washed
4 times, 15 minutes each, with 0.1 M maleic acid, pH 6.5, before en bloc staining
with 2% aqueous uranyl acetate for 1 hour. After washing with distilled water,
keratinocytes were dehydrated with increasing concentrations of ethanol, 100% propylene
oxide, and infiltrated with 1:1 propylene oxide:eponate overnight. Rafts were embedded
in fresh Eponate in flat embedding molds and oriented so they could be sectioned
perpendicularly on a Reichert Ultracut E3 ultramicrotome equipped with diamond
knife. Thin sections were stained with lead citrate and examined in a Hitachi H-7000
electron microscope (Hitachi, San Jose, Calif.) operated a: 75 kV.
Transfection Cell Culture
For transfection experiments, BC-1-Ep/SL cells were plated at a density of 3×10
5
cells onto mitomycin C-treated Swiss mouse 3T3 fibroblasts in 100 mm dishes.
Cells were given 48 hours to adhere at which time the 3T3 layer was removed with
0.5 mM EDTA. Cells were rinsed twice with DME and serum-containing media was added.
Cells were transfected 24 hours later.
Plasmid DNA
Plasmid DNA was prepared with the Endotoxin Free Maxiprep Kit (Qiagen). pGreenLantern
was linearized using XmnI. pcDNA3 neo and pTracer-SV40 (Invitrogen) were linerarized
using BglII (Promega). Expression of green fluorescent protein (GFP) is driven
by a constitutively active CMV promoter in both pGreenLantern and pTracer-SV40 plasmids.
Determination of Optimal Conditions for Transfection of BC-1-Ep/SL Cells
BC-1-Ep/SL cells passage 30°-40° were transfected using the
polycationic lipid GeneFECTOR (VennNova). The transfection mix is made by adding
20-33 μg linearized plasmid DNA to 500 μl sterile milli-Q water for
each 100 mm dish. Different amounts of GeneFECTOR were added depending on the ratio
of total DNA to GeneFECTOR which varied from 1:2 to 1:4. The transfection mix was
swirled gently and incubated for 15 minutes at room temperature. Media was removed
from the BC-1-Ep/SL cells, plates were rinsed twice with DME and refed with 5 mls
of DME. The transfection mix was added to each plate in a drop-wise fashion and
cells were incubated for 5 hours at 37° C. under 5% CO
2. The medium
was removed and cells were rinsed twice with DME and refed with serum-containing
media. The cells were viewed 24 hours post-transfection with an IX-70 inverted
fluorescent microscope (Olympus) equipped with a GFP short band pass filter to
check for successful transfection before analysis or flow activated cell sorting (FACS).
Optimal Transient Transfection Efficiency
BC-1-Ep/SL cells passage 30°-40° were transfected using the
polycationic lipid GeneFECTOR (VennNova). The transfection mix was made by adding
15 μg linearized pGreenLantern and 5 μg pcDNA3neo (20 μg total
DNA) to 500 μl sterile milliQ water for each 100 mm plate of cells. Genefector
is then added at a quantity of three times that of the total DNA (1:3 ratio of
DNA to GeneFECTOR). The transfection mix was swirled gently and incubated for 15
minutes at room temperature. Media was removed from the BC-1-Ep/SL cells, plates
were rinsed twice with DME and refed with 5 mls of DME. The transfection mix was
added to each plate in a dropwise fashion and cells were incubated for 5 hours
at 37° C. under 5% CO
2. The medium was removed and cells were rinsed
twice with DME and refed with keratinocyte media. The cells were viewed as described previously.
Flow Cytometry
Twenty-four hours post-transfection, BC-1-Ep/SL cells were removed from
culture with 0.5 mM EDTA and 0.1% trypsin. After short centrifugation (440×g
for 5 minutes), cells were resuspended in serum-containing medium at 2×10
6
cells/ml. 500 μl of this cell suspension was filtered through 42 μm
mesh (Tetko, Inc.) and stained with 5 μg/ml propidium iodide (PI) immediately
prior to analysis. Transfected BC-1-Ep/SL cells were analyzed on either a FACScan
or FACSCalibur benchtop flow cytometer (both from Becton Dickinson) equipped with
a laser tuned to 488 nm. Ten thousand events were acquired and analyzed using CellQuest
software (Becton Dickinson) and analysis was restricted to live events only, based
on PI staining. Cell viability and transient transfection efficiency data were obtained.
Cell Sorting
We used the following protocol to obtain stable GFP-expressing BC-1-Ep/SL cells.
Twenty-four hours post-transfection, cells were removed using 0.5 mM EDTA and 0.1%
trypsin. After short centrifugation (440×g for 5 minutes), cells were resuspended
in serum-containing medium at a density of 5-7×10
6 cells/ml. This
suspension was then filtered through 42 μm sterile mesh (Tetko, Inc.). Immediately
prior to sorting cells were stained with 5 μg/ml propidium iodide. Transfected
BC-1-Ep/SL cells were sorted on a FACStar Plus (Becton Dickinson) equipped with
a coherent argon laser tuned to 488 nm. Transfection efficiency data was obtained
with CellQuest software (Becton Dickinson). Cells were sorted at a rate of 2000/second
and samples were collected post-sort to check viability and GFP expression.
Colony Forming Efficiency
Colony forming efficiencies (CFE) were obtained by plating 1000 events onto
duplicate 60 mm plates in the presence of mitomycin C-treated Swiss mouse 3T3 fibroblasts.
After one week, plates were fixed for 10 minutes in 10% formalin, rinsed with tap
water, and stained overnight with methylene blue. Colonies were counted and divided
by the number of events plated to obtain the final CFE. Based on the CFE calculated,
the total number of colonies formed was obtained by multiplying the number of events
by the CFE. GFP-expressing colonies were counted 10-12 days post-sorting using
an IX-70 inverted fluorescent microscope with a GFP short band pass filter. The
number of GFP-expressing colonies was divided by the total number of colonies formed
to obtain a stable GFP expressing colony forming efficiency.
Isolation and Identification of Stable pGreenLantern Transfected BC-1-Ep/SL cells
BC-1-Ep/SL cells were transfected with pGreenLantern as described previously
and sorted based on fluorescence. Immediately following cell sorting, GFP-positive
PC-1-Ep/SL cells were reinitiated into culture. Cells were plated at low density
from 2-3×10
4 cells per 100 mm dish onto mitomycin C-treated Swis
mouse 3T3 fibroblasts. Cells were monitored every other day using an IX-70 inverted
fluorescent microscope (Olympus) with a GFP short band pass filter. Non-GFP expressing
colonies were removed by scraping and stable GFP expressing colonies were allowed
to expand. When GFP expressing colonies had grown to an estimated density of 1000
cells or more, they were isolated by ring cloning and replating onto 60 mm plates
with ring mitomycin C treated 3T3's and expanded.
Isolation and Identification of Stable pTracer-SV40 Transfected BC-1-Ep/SL cells
BC-1-Ep/SL cells were transfected using 20 μg of pTracer-SV40 and
a 1:4 ratio of DNA to GeneFECTOR (VennNova). Twenty-four hours post-transfection
cells were removed with 0.5 mM EDTA, 0.1% trypsin and resuspended at a density
of 2×10
6 cells/ml in serum containing medium. Three ×10
6
cells were replated onto two 100 mm plates with a mitomycin C-treated Swiss
mouse 3T3 fibroblasts. Forty-eight hours after passage, GFP-positive cells were
selected for 5 days with 250 μg/ml zeocin (Invitrogen). Stable GFP-expressing
BC-1-Ep/SL cells were purifiedusing sterile cell sorting and expanded as described previously.
Histological Analysis of Transfected Organotypic Cultures
GFP-expressing BC-1-Ep/SL cells at 43(passage were plated at a density
of 3×10
5 cells/collagen raft and grown in organotypic culture for
16 days. Raft cultures were fixed for at least one hour in 4% paraformaldehyde
before embedding in paraffin. Five μm sections were cut and alternate sections
were stained with hemotoxylin and eosin (H&E) by Surgical Pathology, UW-Madison.
Non-H&E sections were rehydrated, stained with 5 μg/ml Höechst dye (33258)
for 15 minutes, dehydrated and mounted using Cytoseal mounting media (Stephens
Scientific). Sections were viewed and photographed using an IX-70 inverted fluorescent
microscope (Olympus) equipped with a dual FITC-Höechst filter.
Analysis of GFP Expression by Confocal Microscopy
GFP-expressing BC-1-Ep/SL cells at 43° passage were plated at
a density of 3×10
5 cells/collagen raft and grown in organotypic
culture for 16 days. Raft cultures were fixed overnight in 4% paraformaldehyde-PBS
and rinsed for one hour in a 0.1 M glycine-PBS solution at 4°. Whole rafts
were mounted, coverslipped using Vectashield mounting media (Vector labs) and sealed
with rubber cement. GFP expression was analyzed using a confocal laser scanning
microscope (Nikon Diaphot 200) with excitation at 488 nm and detection at 500-530
nm bandpass filter. Images were taken at 10 μm intervals starting at the
upper cornified layer. The microscope is located in the W. M. Keck Neural Imaging
Laboratory, University of Wisconsin-Madison.
B. Results
Isolation of the BC-1-Ep/SL Cell Line
Cells were desegregated from a neonatal foreskin by trypsinization. Keratinocytes
were initiated into culture by plating an aliquot of the cell suspension onto a
mitomycin C-treated Swiss mouse 3T3 feeder layer in standard keratinocyte growth
medium containing 0.66 mM calcium. Fibroblasts were initiated into culture by plating
an aliquot of cell suspension onto a tissue culture plate containing Ham's F-12
medium supplemented with 10% fetal calf serum.
After approximately 9 days, primary cultures of keratinocytes designated strain
BC-1-Ep were cryopreserved and subcultured onto a feeder layer. Fibroblast cultures
were grown to near confluency and also cryopreserved. In early passages, the BC-1-Ep
cells exhibited no morphological or growth characteristics that were atypical for
cultured normal human keratinocytes. Cultivated BC-1-Ep cells exhibited stratification
as well as features of programmed cell death.
To determine replicative lifespan, the BC-1-Ep cells were serially cultivated
to senescence in standard keratinocyte growth medium at a density of 3×10
5
cells per 100 mm dish. By passage 15 most keratinocytes in the population
appeared senescent as judged by the presence of numerous abortive colonies which
exhibited large, flat cells.
However, at passage 16, keratinocytes exhibiting a small cell size were
evident. By passage 17, only the small sized keratinocytes were present in the
culture and no large, senescent keratinocytes were evident. The resulting population
of small keratinocytes that survived crisis appeared morphologically uniform and
produced colonies of keratinocytes exhibiting typical keratinocyte characteristics
including cell-cell adhesion and apparent squame production.
The keratinocytes that survived senescence were serially cultivated at a density
of 3×10
5 per 100 mm dish for 59 passages, demonstrating that the
cells had achieved immortality. The keratinocytes which emerged from the original
senescencing population are termed BC-1-Ep/spontaneous line (BC-1-Ep/SL).
Cytogenetic Analysis and DNA Fingerprinting
Chromosomal analysis was conducted on the parental BC-1-Ep cells at passage
3 and BC-1-Ep/SL cells at passages 31 and 54. The parental BC-1-Ep cells have a
normal chromosomal complement of 46, XY. At passage 31 all BC-1-Ep/SL cells contained
47 chromosomes due to an extra isochromosome of the long arm of chromosome 8 (FIG.
1). No other gross chromosomal abnormalities or marker chromosomes were
detected. At passage 54 all cells contained the isochromosome 8, however, an additional
isochromosome of the long arm of chromosome 1 and a marker chromosome were present
in a small fraction of the population (Table 1 in Appendix 1). The BC-1-Ep/SL cells
have been screened for the presence of proviral HIV DNA sequences and found to
be negative. Regions of the HIV provirus were amplified enzymatically, hybridized
to radiolabelled HIV-1 specific DNA probes, and the amplified DNA separated by
size and visualized using agarose gel electrophoresis and autoradiography. Polymerase
chain reaction products were compared by size and specificity to known HIV-1 positive
and negative controls. The presence of HPV16, and 31 viral sequences were assessed
by Southern analysis and none were detected.
The DNA fingerprints for the BC-1-Ep/SL cell line and the BC-1-Ep keratinocytes
are identical at all twelve loci. The odds of the BC-1-Ep/SL cell line having the
parental BC-1-Ep DNA fingerprint by random chance is 4×10
-16. The
DNA fingerprints for ED-1-Ep, SCC4 and SCC13y are different from the BC-1-Ep pattern.
The data from our DNA fingerprint analysis of the BC-1-Ep/SL cell line proves it
arose from the parental BC-1-Ep cells. This data also shows that keratinocytes
isolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated to the BC-1-Ep
cells or each other. The BC-1-Ep/SL DNA fingerprint data provides an unequivocal
way to identify the BC-1-Ep/SL cell line.
BC-1-Ep/SL Keratinocytes are Not Tumorigenic in Athymic Nude Mice
To determine the tumorigenicity of the parental BC-1-Ep keratinocytes and the
immortal BC-1-Ep/SL keratinocyte cell line, cells were injected into the flanks
of athymic nude mice. The human squamous cell carcinoma cell line, SCC 4, was used
as a positive control for tumor production in nude mice. The injection of samples
was designed such that each animal received an injection of SCC 4 cells in one
flank and either the parental BC-1-Ep keratinocytes or the BC-1-Ep/SL cells in
the opposite flank. This injection strategy eliminated animal to animal variation
in tumor production and confirmed that the mice would support vigorous growth of
tumorigenic cells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor the
BC-1-Ep/SL keratinocytes (passage 35) produced tumors in nude mice. The results
of the tumorigenicity testing is shown in Table 2 in Appendix 1.
Growth Characteristics in vitro
BC-1-Ep/SL keratinocytes are nontumorigenic and exhibit morphological
characteristics of normal human keratinocytes when cultured in standard keratinocyte
growth medium in the presence of mitomycin C-treated 3T3 feeder cells. To further
evaluate the growth characteristics of the BC-1-Ep/SL cells, we examined the steady
state mRNA levels of known autocrine regulators of keratinocyte growth. Northern
analysis of mRNAs from the BC-1-Ep/SL cell line revealed that expression of autocrine
growth factors, such as transforming growth factor-α (TGF-α) and transforming
growth factor-β (TGF-β), as well as the levels of epidermal growth
factor receptor (EGFR) and c-myc, are similar, if not identical to the parental
BC-1-Ep keratinocytes.
We next determined which constituents of standard keratinocyte growth medium
are
required for optimal growth of BC-1-Ep/SL cells. Serial cultivation in the absence
of epidermal growth factor (EGF) resulted in a 60 to 90% reduction in cell number
at each passage, compared to EGF-containing control cultures (FIG. 1). The
dependence on EGF for growth of BC-1-Ep/SL cells appears to be a stable characteristic.
BC-1-Ep/SL cells at passage 50 continue to exhibit a dependence on EGF for optimal growth.
Another polypeptide growth factor that plays an important role in epidermal
homeostasis is transforming growth factor-β1 (TGF-β1). In vitro, TGF-β1
is an inhibitor of growth in cultured normal human keratinocytes (Pientenpol, et
al., 1990), however malignant transformation of keratinocytes often results in
attenuation of TGF-β1-induced growth inhibition (Bascom, et al., 1989; Parkinson,
et al., 1983; Pietenpol, et al., 1990; Rice, et al., 1992). Like the normal parental
BC-1-Ep keratinocytes, TGF-β1 inhibits the growth of the BC-1-Ep/SL cell
line (FIG. 3). The TGF-β1-induced growth inhibition is reversible
in both the parental and BC-1-Ep/SL keratinocytes.
To further characterize the requirements for optimal in vitro growth of the BC-1-Ep/SL
keratinocytes, cultures were cultivated in medium supplemented with 2.5% fetal
calf serum, EGF, and individual constituents of the standard growth medium. FIG.4
demonstrates that addition of insulin alone promotes a 15-fold increase in cell
number. However, addition of all constituents of standard keratinocyte growth medium
promotes a 30-fold increase in BC-1-Ep/SL cell number. These findings demonstrate
that the BC-1-Ep/SL cell line has maintained cell type-specific requirements for
growth in vitro.
Differentiation Characteristics in vitro
We next investigated whether BC-1-Ep/SL cells could undergo normal differentiation
in both surface culture and organotypic culture. We monitored a marker of squamous
differentiation, the formation of cornified envelopes (CE). In cultured human keratinocytes,
early stages of CE assembly result in the formation of an immature cornified envelope
composed of involucrin, cystatin-a and other proteins, which represent the innermost
third of the mature cornified envelope. We examined CE formation in the parental
cells and the BC-1-Ep/SL keratinocytes (Table 3 in Appendix 1). Less than two percent
of the keratinocytes from either the adherent parental cells or the BC-1-Ep/SL
cell line produce CE's. This finding is consistent with our previous studies demonstrating
that actively growing, subconfluent keratinocytes produce less than five percent
CE (Hines and Allen-Hoffmann, 1996). To determine whether the BC-1-Ep/SL cell line
is capable of producing CE's when induced to differentiate, the cells are removes
from surface culture and placed in suspension for 24 hours in medium made semi-solid
with methylcellulose. Many aspects of terminal differentiation, including differential
expression of keratins (Drozdoff and Pledger, 1993) and CE formation (Green, 1977)
can be triggered in vitro by loss of keratinocyte cell-cell and cell-substratum
adhesion. We found that the BC-1-Ep/SL keratinocytes produced as many and usually
more CE's, than the parental keratinocytes (Table 3 in Appendix 1). These findings
demonstrate that the BC-1-Ep/SL keratinocytes are not defective in their ability
to make this cell type-specific
