Substances causing differentiation

Abstract

A DNA construct is described which contains a fusion gene under the control of a promoter. The fusion gene comprises at least one resistance gene and at least one reporter gene and is slightly toxic to a host cell transfected with that DNA construct. That DNA construct can be encoded on a plasmid or a virus. Further, a method is described for using the DNA construct to identify substances that may cause a differentiation in eukaryotic cells.

Description

CROSS REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 431,705, filed May 8, 2003, which is continuation of International Patent Application PCT / EP01 / 12660 filed on Oct. 31, 2001, designating the US, and published in German, which claims priority of German patent application DE 100 56 059.8 filed on Nov. 11, 2000, all which are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a method for identifying substances causing differentiation in eukaryotic cells, to DNA constructs, plasmids, viruses and cell lines used in said method, and to a method of preparing a pharmaceutical composition. [0004] 2. Description of the Related Art [0005] Differentiation of cells from stem cells is a general biological phenomenon during embryonic development, but also plays a very large part in regeneration processes in the adult organism (e.g....

AI Technical Summary

Benefits of technology

[0019] In view of the above, it is an object of the present invention to provide a method of the type mentioned at the beginning, which can be used to identify in a rapid, simple and reliable manner substances causing differentiation, and auxiliary substances which can be used in said method.

[0031] Up until now, no cell line has been described or known in which substances causing differentiation can be detected using an expression unit consisting of CMV promoter and hygromycin-GFP fusion. The system is very reliable, with the presence of hygromycin during routine cultivation of the cell line preventing the loss of said expression unit. The cell line has the intrinsic capability of virtually completely downregulating the CMV promoter only a few days after removing hygromycin. The promoter is upregulated again when adding substances causing differentiation.

[0032] The advantage compared to the known system is, inter alia, the use of a fusion gene which combines a plurality of proper-ties: (I) positive selectability by the hygromycin gene, i.e. the expression unit is retained in a stable manner when adding the antibiotic; (II) negative selectability, i.e. in the absence of hygromycin B the toxic effect of the GFP gene selects for cells in which the promoter is switched off; (III) identifiability due to intrinsic fluorescence of GFP. These properties render the system very stable and reliable.

[0039] The cell line U87-HGFP, which is still a further object of the invention, has a particular advantage in that the promoter can be switched off within a short time and reliably. Furthermore, the method can be automated, making it possible to screen many substances in a short time.

[0040] The cell line has the further advantage of being a tumor cell line so that the substances are searched for in a cancer cell which is thus not only a model system but also, at the same time, a test system. Another advantage is the fact that these cells differentiate with addition of the appropriate substances, and this can be seen due to the change in morphology.

Problems solved by technology

As in all important biological processes, disruptions may cause chronic diseases or may be lethal.

Despite great improvements in the methods for early diagnosis and therapy of tumor diseases, mortality is still very high and affected patients suffer immensely.

Thus, the largest class of cytostatics leads to damage of cellular DNA.

A disruption of this equilibrium is often present in tumor cells.

While for some classes of cytostatics there are already very good assay systems which also can be used to identify novel compounds, there is, however, a lack of methods for identifying, as mentioned above, such substances capable of causing differentiation.

Both tests have comparable sensitivities, but have the fundamental disadvantage that genotoxic action of substances can vary in bacteria and higher organisms.

The previously known assay systems for identifying DNA-damaging agents, however, are not suited to identify substances causing differentiation, since the mechanism is completely different.

The reporter cell line described by Biard et al. has the decisive disadvantage of being an inducible system.

Since there are, after 1992, no further publications regarding this cell line, it is neither known whether this cell line is stable nor whether it is suitable for detecting other differentiation processes as well.

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