Note: Descriptions are shown in the official language in which they were submitted.
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CELL LINE AND ASSAY TO DETERMINE BIOhOGICAL
ACTIVITY OF RETINOIDS
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~CKGROUND OF T~E INVENTION
(a~ Field of the Invention
The invention relates to a method of
determining which synthetic analogues of retinoic acid
may have useful biological activity, particularly
through activation of the ~-receptor of retinoic acid.
~b) Description of Prior Art
Retinoic acid (RA) and related compounds,
collectively known as retinoids, plays a variety of
very important roles in the development and the
differentiation in humans and other mammals. The
growth, differentiation and homeostasis of the tissues
which make up the epithelial layer of cells which
serves as a boundary between the individual and the
environment, such as skin, bronchial tubes, stomach
lining among many others, are known to be in may cases
controlled by retinoic acid. For example, a lack of
retinoic acid, or its metabolic precursor vitamin A, in
the diet tends to trigger the proliferation of cells of
a certain characteristic type, called squamous, in
bronchial epithelium. It is of particular interest
that this cellular proliferation is thought to
represent one of the first steps in the development of
epidermoid cancer of the lung.
Retinoids are in most respects inactive as free
molecules, but they can exert their biological activity
after binding in a specific fashion to any of a number
of different protein molecules called RA receptors.
Several classes of RA receptors are present in cells.
Those which are most important in mediating the effect
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of RA are nuclear receptors, which are of two classes,
RAR's and RXR's. The natural ligand for these receptors
are respectively, all-~ons RA and 9-cis RA. Each class is
made up of three different receptors, called a, ~, and ~
5 ~, each coded for by different genes. The RNA - ;
transcribed from most of these genes can be processed
("spliced") in various ways, giving rise to isoforms of
the various RA receptor proteins. For example, RAR~
exists as four isoforms, 1, 2, 3 and 4, in the mouse;
the only human isoform of RAR~ described to date is
analogous to the murine RAR~2.
Each of the nuclear RA receptor proteins has a
region ("domain") which binds RA (called the E domain),
and a DNA-binding domain (C domain). Once activated by
binding RA in the E domain, the C domain of the
receptor interacts with a specific nucleotide sequence,
or RA response element, and is then capable of
stimulating the rate of transcription of the genes
which have the RA response element in the adjacent
~promoter) DNA sequence.
As described above, RA is important for growth
control, and it is also important as an agent of cancer
prevention. Epidemiological studies have clearly
established an inverse correlation between vitamin A
dietary intake and the risk of a variety of cancers of
epithelial tissues, the best studied being lung cancer
among smokers. This implication of RA as a cancer
suppressing agent has prompted many attempts to develop
retinoid-based chemoprevention therapies. Some
successful examples exist, such as prevention of second
primary tumours in patients with squamous cell
carcinomas of the head and neck.
The potential applications of RA therapy are
very great. However, the high doses necessary for a
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pharmacological effect bring on a number of undesirable
side effects, such as systemic toxicity and
teratogenicity. These effects are sufficiently serious
as to hamper the full exploitation of this therapy.
For this reason much effort has been invested in the
synthesis of analogues of RA (retinoids) in order to
develop new agents which have the beneficial activity
of RA with reduced side effects.
An important step in the evaluation of the
potential usefulness of a given retinoid is an in vitro
test of biological activity. A variety of assays are
currently in use, all of which are based upon a known
effect of RA on cultured cells or on the specific RA
receptors.
Prior assays of bioloaical activity of retinoids
Systems where RA induces di.fferenhahon.
Several cell lines exist which will grow in the
~bsence of RA but upon addition of this compound will
differentiate, that is, undergo a predictable set of
changes in characteristics. For example, the line
HL60, a hematopoietic cell line derived from a patient
with promyelogenous leukemia, will differentiate into
granulocytic cells in the presence of 10-6 M RA. Many
investigators have used this system to quantitatively
assay the biological activity of synthetic retinoids in
the expectation that the ability to trigger
differentiation correlates with in vivo biological
activity. Similarly, in another cell type, cultured
human keratinocytes, the production of new cellular
structures (cross-linked envelopes) or appearance or
disappearance of certain proteins called keratins upon
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RA treatment has been used to determine retinoid
activity.
The shortcoming of these assays is that the
cells used express RARa, ~ and/or y, to various
extents, and it is impossible to distinguish which
receptor is being activated by a given retinoid. This
is important, because from the teachings of Lehman et
al. (Cancer Res. 51, 4804 (1991)) and Apfel et al.
(Proc. Natl. Acad. Sci. USA 89, 7129 (1992)) it is
known that retinoids have different activities on the
different RAR receptors. It is useful to be able to
make these distinctions, since different RAR's may be
responsible for the different biological effects,
undesirable and desirable, of RA.
Sys~ems where ~A acfivahon of in~ividual RAR or RX~ receptors is measured
The systems, as described by Apfel et al.
(Proc. Natl. Acad. Sci. USA 89, 7129 (1992)), Lehman et
al. (Cancer Res. 51, 4804 (1991)) and Nagpal et al.
(Cell 70, 1007 (1992)) involve the construction of
plasmids allowing expression of specific RAR or RXR
receptors, cotransfection into cultured mammalian cells
of these plasmids with a plasmid carrying a gene
(reporter gene) coding for a meâsurable protein such âS
~ galactosidase with a promoter which includes a RA
response element sequence. In this way the activity of
the RA receptor being studied is measured by assaying
the product of the reporter gene. The abllity of
synthetic retinoids to activate the individual RA
receptor proteins can be determined and compared to the
activity of RA itself.
The shortcoming with this approach is that the
assay measures only the first step in the signalling
cascade of RA, and does not measure a biological
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function. It is useful to be able to assess what effect
a given retinoid will have on some biologically
important properties such as growth rate,
differentiation or tumorgenicity.
S~MMARY OF THE INVENTION
One aim of the present invention is to provide
with a tumor-derived or tumorgenic modified cell line
which substantially expresses RAR~ and has a suppressed ~ -
tumorgenicity once modified, said cell line is used for
testing a retinoid for RA-like biological activity.
Another aim of the present invention is to
provide for an assay for the in vitro testing of a
retinoid for RA-like biological activity using the
tumor-derived or tumorgenic modified cell line of the
present invention.
In accordance with the present invention there
is provided a method of determining the retinoic acid
(RA)-like biological activity of a retinoid which
substantially activates the human RAR~ comprising:
a) transfecting a parental cell line being at
least tumor-derived or tumorigenic with sequences
encoding said RAR~, said parental cell line expressing
a substantially low level of RAR~;
b) isolating the transfected cell line of step a)
expressing the transfected RAR~ sequences at a
sufficient level to substantially inhibit
tumorigenicity;
c) determining the growth rates of the isolated
transfected cell line of step b) and the parental cell
line of step a) in cell culture, with the addition of
RA at various concentrations and in the absence of
added RA; ~`
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d) determining the growth rates of said lines of
step c) with the addition of a retinoid at various
concentrations;
e) comparing the effect of said retinoid on cell
growth with the effect of RA.
The present invention provides a cell line
which is derived from the tumorigenic RAR~-deficient
cell line CALU-l and which carries, as a permanent
component of its genetic makeup, a sequence of DNA
which codes for the isoform of RAR~ which is analogous
to murine RAR~2. This cell line is non-tumorigenic,
and in the presenc eof 10-7 M RA displays the novel
characteristic of a substantial and reproducible
reduction in growth rate compared to that in the same
medium without RA. The parental CALU-l cell line, in
contrast, exhibits a slightly higher growth rate when
the medium is so supplemented, and serves as a control
to monitor retinoid toxicity. In carying out the
invention, cell suspensions of the two cell lines are
prepared and known numbers of cells are seeded in
several petri dishes. The growth medium of some dishes
is supplemented with the desired concentration of
either RA or the retinoid to be tested. Cell growth is
determined by counting cells at regular time intervals
and the growth curves are compared to determine the
effect of the tested retinoid on cell growth of the two
lines relative to the effect of RA.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the nature of
the invention, reference will now be made to the
accompanying drawings, showing by way of illustration a
preferred embodiment thereof, and wherein:
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Fig. 1 is a representation of the sequence
which was transfected into the CALU-l cells line to
generate the lines R~cl9, R~c24, R~c57 and R~c64; and : ~
Fig. 2 is a representation of RNase protection ~::
assays performed on the liens CALU-l and the
t.ransfectants R~c24, R~c57 and R~c64.
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DETAILED DESCRIPTION OF T~E INVENTION
The test system in accordance with the present
invention, as exemplified herein, involves the creation
of a novel cell line derived from a tumorigenic cell
line, said tumorigenic line being characterized by a
lack of detectable RAR~ messenger RNA. This novel cell
line is constructed by transfecting cDNA sequences, as
described in Figure 1, which encode genetic information
specifying the synthesis of the human analogLe of
murine RAR~2, with the SV40 late promoter sequences
inserted upstream of said cDNA sequences in order to
allow a high level of transcription of the cDNA
sequences in the transfected cell. In order to isolate
the rare cell which was successfully transfected, the
said cDNA sequences were cotransfected with a
dominantly acting selectable marker, as described in
Example 1, in a manner similar to the teachings of
Stambrook and Tischfield (US Patent 4,792,520). Further
characterization is performed, as described in Example
2 below, to demonstrate that the transfected sequence
is indeed expressed, and to test the degree of
tumorigenicity by injection into nude (athymic) mice.
In carrying out the invention, the novel cell
lines, with readily detectable levels of RAR~2 mRNA and
with greatly reduced tumorigenicity, were then
subjected to the assay described in Example 3, for
testing the effect of 10-7 to 10-9 M RA on the growth
rate of the cell. As a control, the untransfected
parental line, CALU-l, was subjected to the same assay,
in the presence or absence of the same concentrations
of RA. The growth rate was determined from a graph in
which the results of cell counts were plotted on semi-
logarithmic graph paper and the results of several
repeat experiments were averaged. As described in
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Example 3, all the transfected cell lines with high
levels of RAR~2 mRNA (R~cl9, R~c24, R~c64) exhibited an
increased cell doubling time, by 20-60%, in 10-7 M RA,
whereas no increase at all was seen with a control
txansfectant which only carried the dominant selectable
marker (Neoc30) or the parental CALU-l line. The
transfectant R~c57 which expressed only about 25% of
the level of RAR~ mRNA detected in R~c24, and did not
have greatly reduced tumorigenicity, exhibited on
average a very small increase in cell doubling time,
but it was not sufficient to be statistically
significant.
In order to disclose more clearly the nature of
the present invention, the following illustrative
Examples are given.
~xample 1
Transfection of a tumorigenic cell line not
expressing RAR~ with DNA sequences allowing the
creation of a novel RAR~2-expressing lines. CALU-l
(available from the American Type Culture Collection,
Rockville, MD), a line known to be tumorigenic and
shown previously by Houle, Leduc and Bradley (Genes
Chromosomes and Cancer 3,358, 1991) to have
undetectable levels of RAR~ mRNA was transfected with
the plasmid described in Figure 1. Cells were
trypsinized, and 10-7-10-8 were washed in phosphate
buffered saline (PBS), centrifuged, and washed twice
with 5 ml of Opti-MEM medium (Gibco BRL). After
centrifugation, the cells were resuspended in the
transfection medium of 1.4 ml of Opti-MEM' medium, 300
~g of Lipofectin (Gibco BRL), 40 ~g of pAG60 DNA
(described by Colbère-Garapin et al., J. Molecular
Biology 150, 1981) linearized by ClaI and where
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appropriate, 40 ~g of pBH-4 ~the filled in 1.7 kb
BamHI-HindIII fragment of pBLhRAR~ (provided by Magnus
Pfahl) cloned in the SmaI site of pSVL from Pharmacia)
'inearized by PstI. the mix was incubated at 37 for up
to 20 minutes, diluted into 80 volumes of _medium
supplemented with 10% fetal calf serum and plated in
two 150 mm dishes, to which G418 (Gibco BRL, effective
concentration 400 ~g/ml) was added after 72 hours.
Resistant clones, arising at a frequency of _10-6 were
picked 3 to 4 weeks later. RNA was extracted and
analysed by RNase protection.
~xample 2
Characterization of transfected cell lines with
respect to RAR~ expression and tumorigenicity. the
expression of RARB in CALU-l cells and their
transfected derivatives described in example 1 were
determined using the technique of RNase protection, as
taught by, among others, Houle et al., Gene Chromosomes
and Cancer 3, 358, 1991. Using a control ~actin probe,
relative levels of RAR~ were estimated in the
transfected lines by measuring the relative intensities
of the protected bands and normalizing for the
intensity of the ~actin bands (see Figure 2). These
were found to be: CALU-l, <0.03; RNeoc30, ~0.03;
R~cl9, approximately 0.5; R~c24, approximately 0.5;
R~c57, approximately 0.13; and R~c64, approximately
1Ø Tumorigenicity of each line was determined by
injection of 5X106 cells into each of several nude
mice, according to standard practice. The results of
latency and growth rates of the tumours are summarized
in Table 1.
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Table l
Tumorigenicity of the CALU-l cell line and its
derivatives
Cell line RAR~ Number tumorsl Mean latency Mean doubling
expressionnumber injections (weeks~ time in weeks
(range)
CALU-1 - 14/15 8.6 1.5(1-2.3)
RNEOC30 - 6/6 6.5 ~1.2(1.03-1.5)
R~c19 ++ 7/12 19 5.6(3.5-7.0)
R~c24 ++ 6/s 16 4.1 (2.~7.0)
R~c57 + 2/4 13 2.7(2.~3.5)
R~c~ +++ ol4 ~16
Example 3
Cells of the lines CALU-l, the negative control
transfectant Neoc30 and RAR~-expressing transfectants
described in Examples l and 2 were added at 5x104
: :::
l0 cells/dish and grown in a-medium with or without ~ :
supplementing with 10-7M RA. At one or two day . ~
intervals dishes were trypsinized and cells were ~ .
counted Doubling time was calculated and the results
are summarized in Table 2.
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Table 2
Growth rates of CALU-l and transfected derivatives in
the presence or absence of RA
Cell line RAR~ Doublingtime, days
expression Without RA With RA
CALU-1 - 1.25 1.20
c30 - 1.4 1.25
c19 + 1.35 1.70
c24 + 1.28 1.81
c57 + 1.53 1.55
c64 + 1.75 2.85
These cell lines of the present invention are
readily available from Mr. Edward Bradley's laboratory
at Institut du Cancer de Montréal, 1560 Sherbrooke
East, Montreal, Quebec H2L 4Ml.
While the invention has been described with
particular reference to the illustrated embodiment, it
will be understood that numerous modifications thereto
will appear to those skilled in the art. Accordingly,
the above description and accompanying drawings should
be taken as illustrative of the invention and not in a
limiting sense.
