CA2088293A1 - Cell line and assay to determine biological activity of retinoids - Google Patents
Cell line and assay to determine biological activity of retinoidsInfo
- Publication number
- CA2088293A1 CA2088293A1 CA 2088293 CA2088293A CA2088293A1 CA 2088293 A1 CA2088293 A1 CA 2088293A1 CA 2088293 CA2088293 CA 2088293 CA 2088293 A CA2088293 A CA 2088293A CA 2088293 A1 CA2088293 A1 CA 2088293A1
- Authority
- CA
- Canada
- Prior art keywords
- cell line
- beta
- rar
- retinoid
- transfected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
Abstract
ABSTRACT OF THE INVENTION
The present invention relates to an assay and cell lines for determining the biological effects of retinoids. The cell line is derived from a human lung tumour line which is tumorigenic in nude mice and expressed undetectable levels of the .beta. receptor of retinoic acid by introducing in a stable fashion the DNA coding for this receptor. The novel line is non-tumorigenic and grows in culture at a rate which is reduced in the presence of 10-7 M retinoic acid. This line is useful in determining the biological effects of novel retinoids which act in particular by activating RAR.beta..
The present invention relates to an assay and cell lines for determining the biological effects of retinoids. The cell line is derived from a human lung tumour line which is tumorigenic in nude mice and expressed undetectable levels of the .beta. receptor of retinoic acid by introducing in a stable fashion the DNA coding for this receptor. The novel line is non-tumorigenic and grows in culture at a rate which is reduced in the presence of 10-7 M retinoic acid. This line is useful in determining the biological effects of novel retinoids which act in particular by activating RAR.beta..
Description
~08~293 :
.
CELL LINE AND ASSAY TO DETERMINE BIOhOGICAL
ACTIVITY OF RETINOIDS
~ .
~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 - 2 - 20 88~g3 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 ~ ` ~ ;; ' ` L
~` ~ 3 ~ ~08~293 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 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 2~293 ~
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; ~`
'~8~293 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:
.. . .
~8~293 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.
~1~8~293 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 ~. . ~ . . :, ~: :
, : ,. . ,~ -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 2~88293 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.
.,' '. . , . ! ' . . ~ ` ., `: " `, ` `, , .. :,, ~ :
2~8~293 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.
~: ,. , - .: . : . : ,. . ..
~88293 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.
.
CELL LINE AND ASSAY TO DETERMINE BIOhOGICAL
ACTIVITY OF RETINOIDS
~ .
~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 - 2 - 20 88~g3 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 ~ ` ~ ;; ' ` L
~` ~ 3 ~ ~08~293 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 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 2~293 ~
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; ~`
'~8~293 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:
.. . .
~8~293 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.
~1~8~293 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 ~. . ~ . . :, ~: :
, : ,. . ,~ -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 2~88293 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.
.,' '. . , . ! ' . . ~ ` ., `: " `, ` `, , .. :,, ~ :
2~8~293 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.
~: ,. , - .: . : . : ,. . ..
~88293 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.
Claims (8)
1. A method of determining the retinoic acid (RA)-like biological activity of a retinoid which substantially activates the human RAR.beta. comprising:
a) transfecting a parental cell line being at least tumor-derived or tumorigenic with sequences encoding said RAR.beta., said parental cell line expressing a substantially low level of RAR.beta.;
b) isolating the transfected cell line of step a) expressing the transfected RAR.beta. 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;
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.
a) transfecting a parental cell line being at least tumor-derived or tumorigenic with sequences encoding said RAR.beta., said parental cell line expressing a substantially low level of RAR.beta.;
b) isolating the transfected cell line of step a) expressing the transfected RAR.beta. 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;
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.
2. A method according to claim 1, wherein said transfected cell line is the cell line R.beta.c24 or R.beta.c64.
3. A tumor-derived or tumorigenic modified cell line which substantially expresses RAR.beta. and has a suppressed tumorigenicity once modified.
4. A cell line according to claim 3, wherein said cell line is a transfected cell line.
5. A cell line according to claim 4, wherein said cell line is obtained according to the method of claim 1.
6. A cell line according to claim 3, wherein said cell line is the cell line R.beta.c24 or R.beta.c64.
7. A cell line according to claim 3, wherein said cell line is used for testing a retinoid for RA-like biological activity.
8. Use of a cell line according to claim 3 for testing a retinoid for RA-like biological activity.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2088293 CA2088293A1 (en) | 1993-01-28 | 1993-01-28 | Cell line and assay to determine biological activity of retinoids |
| PCT/CA1994/000032 WO1994017407A1 (en) | 1993-01-28 | 1994-01-20 | Cell line and assay to determine biological activity of retinoids |
| AU58770/94A AU5877094A (en) | 1993-01-28 | 1994-01-21 | Cell line and assay to determine biological activity of retinoids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2088293 CA2088293A1 (en) | 1993-01-28 | 1993-01-28 | Cell line and assay to determine biological activity of retinoids |
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| AU (1) | AU5877094A (en) |
| CA (1) | CA2088293A1 (en) |
| WO (1) | WO1994017407A1 (en) |
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| CA2020198A1 (en) * | 1989-06-30 | 1990-12-31 | Pierre Chambon | Human and mouse retinoic acid receptors and genes encoding for them |
| AU4375493A (en) * | 1992-05-15 | 1993-12-13 | Ludwig Institute For Cancer Research | Isolated protein receptors, antibodies which bind thereto, nucleic acid sequence coding therefor, and uses thereof |
-
1993
- 1993-01-28 CA CA 2088293 patent/CA2088293A1/en not_active Abandoned
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1994
- 1994-01-20 WO PCT/CA1994/000032 patent/WO1994017407A1/en active Application Filing
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| WO1994017407A1 (en) | 1994-08-04 |
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