DNA construct and in vitro test for detecting tumor promoters by

Details DNA construct and in vitro test for detecting tumor promoters by DNA construct and in vitro test for detecting tumor promoters by

1994-07-25

1997-06-17

Ketter, James

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

435 6, 4353201, C12Q 168, C12Q 170

Patent

active

056395965

DESCRIPTION:

BRIEF SUMMARY
The invention relates to a DNA construct and to a method of detecting tumor promoters with the use of this DNA construct.
Tumor promoters are substances which, in combination with doses of solitary carcinogens that are in themselves not tumorigenic, induce the development of tumors or carcinomas. Solitary carcinogens are substances which produce tumors when applied individually (in animal tests) in dependence on the dosage. However, the sole influence of tumor promoters does not produce cancer in the sense of their definition, but it enhances to a considerable degree the development of tumors in organs which have been initiated by the exogenous influence of a solitary carcinogen. Simple and quickly performed tests systems are already available to identify solitary carcinogens. However, in the past, the substance class of tumor promoters could be identified only by way of animal experiments, for example, on the skin of a mouse or the intestines of a rat.
A starting procedure for a test to search out tumor promoters described by H. zur-Hausen, G. W. Bornkamm, R. Schmidt, and E. Hecker, entitled "Tumor Initiators and Promoters in the Induction of Epstein-Barr Virus", in Proceedings of the National Academic Science [sic] (U.S.A.), Volume 76, pages 782-785 (1979), provides for a determination of the property of substances that induces the Epstein-Barr virus (EBV) by means of an immunofluorescence display of EBV-coded antigens (so-called early antigens). However, this technique requires personnel that are trained in virology and serology procedures. Moreover, this process is time consuming and, due to the subjective evaluation of immunofluorescence stained cells, not accurately standardizable.
The final proof of the tumor promoting property of a substance can be brought only by the initiation of tumors in an in-vivo animal experiment, for example, on the skin of a mouse. However, such in-vivo experiments are not suitable as search tests for testing a plurality of substances.
It is now an object of the invention to make available a DNA construct and an easily performed quantitative method of the above-mentioned type.
This is accomplished by claims 1 and 8. The remaining claims define advantageous features of the invention.
The invention will now be described in greater detail with reference to two embodiments thereof and FIGS. 1 to 5.
FIG. 1 is a schematic representation of the configuration of the DNA construct,
while FIGS. 2 and 3 are schematic representations of two DNA constructs with points of intersection and their precise positions.
FIG. 4 shows a Southern-blot analysis of various pHEBO-DR-CAT transfectants (A4, D2, D4), and
FIG. 5 depicts a CAT assay of the pHEBO-DR-CAT transfected Raji sublines D4, A4 and D2.
The base sequence (SEQ ID NOS: 1 and 2) and mapping of these two DNA constructs are given in Tables 1 and 2.
The invention is based on the observation that different classes of tumor promoters, such as diterpene esters, indole alkaloids or polyacetates, Teleocidin, transforming growth factor .beta. (TGF.beta.), induce a lytic or abortive virus cycle in lymphatic cells containing the Epstein-Barr virus (EBV). The EBV genome contains a large number of genes that are not expressed in the latent state and are induced only upon the induction of a lytic or abortive cycle. The control region of the particularly strongly induced DR gene responsible for this activation (DL or others are possible) was cloned by molecular biology methods ahead of the coding region of a so-called reporter gene. The vector (pHEBO) employed for this purpose carries the episomal replication origin of EBV and two resistance genes one of which permits the selection of transfected eukaryotic cells with hygromycin and the other the selection in procaryotes.. This vector is described by B. Sugden, K. Marsh and J. A. Yates as "[A] vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein virus" in Molecular and Cellular Biology, Volume 5, pages 410-413 (1985). The construct obtained in

REFERENCES:
Cancer Research Clinical Oncology (Suppl. Part 1), vol. 116, Springer International, New York, US, p. 99; A. Polack et al: Description of an EBV system detecting tumor promoters of the diterpene esters type with high . . . .
Biological Abstracts, vol. 86, 1988: G.D. Kutuzova et al Synthesis of active luciferase of the glow worm Luciola mingrelica and its stability in the oocytes of the frog Xenopus laevis. p. 410.
Biochemical Society Transactions, vol. 18, No. 3, Jun. 1990, pp. 459-460; G. Sala-Newby et al: Production of translatable firefly luciferase mRNA in vitro from cloned cDNA.
Journal of Virology, vol. 56, No. 3, 1985, pp. 987-995; G. Laux et al: Structure and evolution of two related transcription units of Epstein-barr virus carrying small tandem repeats.
Molecular and Cellular Biology; vol. 5, No. 2, Feb. 1985 pp. 410-413; b. Sugden et al: A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-bar virus.
Biochimica et Biophysica Acta, vol. 949, 1988, pp. 206-212; A. Jalanko et al: An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.
Chavrier et al., J. Virol., vol. 63, 1989, pp. 607-614.
de Wet et al., Mol. Cell. Biol., vol. 7, 1987, pp. 725-737.
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 1989, pp. 16.33-16.37 and 16.59, CSH Laboratory Press, NY.
Takada et al., International J. Cancer, vol. 33, 1984, pp. 491-496.
zur Hausen et al., Proc. Natl. Acad. Sci. (USA), vol. 76, No. 2, pp. 782-785, Feb. 1979, "Tumor initiators and promoters in the induction of Epstein-Barr Virus."

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