WO1994017407A1 - Lignees cellulaires et test pour determiner l'activite biologique des retinoides - Google Patents

Lignees cellulaires et test pour determiner l'activite biologique des retinoides Download PDF

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Publication number
WO1994017407A1
WO1994017407A1 PCT/CA1994/000032 CA9400032W WO9417407A1 WO 1994017407 A1 WO1994017407 A1 WO 1994017407A1 CA 9400032 W CA9400032 W CA 9400032W WO 9417407 A1 WO9417407 A1 WO 9417407A1
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WIPO (PCT)
Prior art keywords
cell line
rarβ
retinoid
transfected
biological activity
Prior art date
Application number
PCT/CA1994/000032
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English (en)
Inventor
Benoit Houle
Abdelmajid Belouchi
W. Edward C. Bradley
Original Assignee
Universite De Montreal
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Universite De Montreal filed Critical Universite De Montreal
Priority to AU58770/94A priority Critical patent/AU5877094A/en
Publication of WO1994017407A1 publication Critical patent/WO1994017407A1/fr

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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

Definitions

  • 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.
  • 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.
  • 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
  • Retinoids are in most respects inactive as free molecules, but they can exert their biological
  • RA receptors 30 activity after binding in a specific fashion to any of a number of different protein molecules called RA receptors.
  • RA receptors Several classes of RA receptors are present in cells. Those which are most important in mediating the effect of RA are nuclear receptors,
  • RAR's and RXR's which are of two classes, RAR's and RXR's.
  • the natural ligand for these receptors are respectively, all-trans RA and 9-cis RA.
  • Each class is made up of three different receptors, called ⁇ , ⁇ , and ⁇ , 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.
  • 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.
  • 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 tumors in patients with squamous cell carcinomas of the head and neck.
  • the line HL60 a hematopoietic cell line derived from a patient with promyelogenous leukemia, will differentiate into granulocyte 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.
  • the ability 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 signaling cascade of RA, and does not measure a biological 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.
  • 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.
  • 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; 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 parental cell line being at least tumor-derived or tumorigenic with
  • the present invention provides a cell line which is derived from the tumorigenic RAR ⁇ -deficient cell line CALU-1 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 presence of 10 " ? 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-1 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.
  • 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.
  • Fig. 1 is a representation of the sequence which was transfected into the CALU-1 cells line to generate the lines R ⁇ cl9, R ⁇ c24, R ⁇ c57 and R ⁇ c64; and Figs. 2A and 2B are a representation of RNase protection assays performed on the lines CALU-1 and the transfectants R ⁇ c24, R ⁇ c57 and R ⁇ c64.
  • the test system in accordance with the present invention 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 Fig. 1, which encode genetic information specifying the synthesis of the human analogue 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.
  • 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.
  • the novel cell lines 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.
  • the untransfected parental line, CALU-1 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.
  • Example 1 Transfection of a tumorigenic cell line not expressing RAR ⁇ with DNA sequences allowing the creation of a novel RAR ⁇ 2-expressing lines.
  • CALU-1 is readily available from the American Type Culture Collection under accession number HTB 54 (Rockville, MD), and is a cell 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 Fig. 1.
  • Opti-MEMTM medium phosphate buffered saline (PBS), centrifuged, and washed twice with 5 ml of Opti-MEMTM medium (Gibco BRL). After centrifugation, the cells were resuspended in the transfection medium of 1.4 ml of Opti-MEMTM 1 medium, 300 ⁇ g of LipofectinTM (Gibco BRL), 40 ⁇ g of pAG60 DNA (described by Colbere-Garapin et al., J.
  • Tumorigenicity of each line was determined by injection of 5x10 ⁇ cells into each of several nude mice, according to standard practice. The results of latency and growth rates of the tumors are summarized in Table 1.
  • the R ⁇ c64 cell line of the present invention has been deposited at the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, MD 20852 USA) on under accession number

Abstract

La présente invention concerne un test et des lignées cellulaires pour déterminer les effets biologiques des rétinoïdes. La lignée cellulaire est obtenue à partir d'une lignée tumorale humaine provenant des poumons qui est tumorigène chez les rats sans poils et elle exprime des niveaux indétectables du β-récepteur de l'acide rétinoïque lorsqu'on introduit d'une manière stable l'ADN codant pour ce récepteur. La nouvelle lignée est non tumorigène et elle se développe en milieu de culture à une vitesse qui est diminuée par la présence d'acide rétinoïque 10-7 M. Cette lignée est utile pour déterminer les effets biologiques de nouveaux rétinoïdes qui agissent en particulier en activant le RARβ.
PCT/CA1994/000032 1993-01-28 1994-01-20 Lignees cellulaires et test pour determiner l'activite biologique des retinoides WO1994017407A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU58770/94A AU5877094A (en) 1993-01-28 1994-01-21 Cell line and assay to determine biological activity of retinoids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA 2088293 CA2088293A1 (fr) 1993-01-28 1993-01-28 Lignee cellulaire et essai pour la determination de l'activite biologique des retinoides
CA2,088,293 1993-01-28

Publications (1)

Publication Number Publication Date
WO1994017407A1 true WO1994017407A1 (fr) 1994-08-04

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AU (1) AU5877094A (fr)
CA (1) CA2088293A1 (fr)
WO (1) WO1994017407A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009387A1 (fr) * 1994-09-21 1996-03-28 Worcester Foundation For Biomedical Research Traitement du cancer par expression du recepteur du facteur de differenciation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0411323A2 (fr) * 1989-06-30 1991-02-06 Institut National De La Sante Et De La Recherche Medicale (Inserm) Récepteurs de l'acide rétinoique chez l'homme et chez la souris et les gènes codant pour ces récepteurs
WO1993023538A1 (fr) * 1992-05-15 1993-11-25 Ludwig Institute For Cancer Research Recepteurs de proteines isolees, anticorps se fixant a ces proteines, sequence d'acides nucleiques codant pour ces recepteurs, et leur applications

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0411323A2 (fr) * 1989-06-30 1991-02-06 Institut National De La Sante Et De La Recherche Medicale (Inserm) Récepteurs de l'acide rétinoique chez l'homme et chez la souris et les gènes codant pour ces récepteurs
WO1993023538A1 (fr) * 1992-05-15 1993-11-25 Ludwig Institute For Cancer Research Recepteurs de proteines isolees, anticorps se fixant a ces proteines, sequence d'acides nucleiques codant pour ces recepteurs, et leur applications

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
B.HOULE ET AL.: "Tumor-suppressive effect of the retinoic acid receptor Beta in human epidermoid lung cancer cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA), vol. 90, no. 3, 1 February 1993 (1993-02-01), pages 985 - 989 *
C.APFEL ET AL.: "A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA), vol. 89, August 1992 (1992-08-01), pages 7129 - 7133 *
C.NASTRUZZI ET AL.: "New Synthetic Retinoids: Effects on Proliferation and Differentiation", ANTICANCER RESEARCH, vol. 9, no. 5, 1989, pages 1377 - 1384 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5789187A (en) * 1992-08-27 1998-08-04 Worcester Foundation For Experimental Biology Identification of differentiation factor receptors which inhibit the tumorigenicity of neuroblastoma cells in a ligand-independent manner
WO1996009387A1 (fr) * 1994-09-21 1996-03-28 Worcester Foundation For Biomedical Research Traitement du cancer par expression du recepteur du facteur de differenciation

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CA2088293A1 (fr) 1994-07-29
AU5877094A (en) 1994-08-15

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