WO2011105968A2

WO2011105968A2 - A cell based system for rapid detection and quantification of genotoxicity

Info

Publication number: WO2011105968A2
Application number: PCT/SI2011/000010
Authority: WO
Inventors: Metka FILIPIČ; Tamara LAH TURNŠEK; Irena Hreljac; Maja ČEMAŽAR; Gregor SERŠA; Valerija ŽAGER; Urška KAMENŠEK
Original assignee: Nacionalni Inštitut Za Biologijo
Priority date: 2010-02-25
Filing date: 2011-02-22
Publication date: 2011-09-01
Also published as: WO2011105968A3; SI23294A

Abstract

The cell system for rapid detection and determination of genotoxicity is a biosensor cell system with metabolicaly competent human cells, stable transfected with reporter gene, which is operationally linked to the promoter of gene, which responds to DNA damage, and solves the problem of rapid and accurate detection and quantification of genotoxic substances and other samples. In the preferred embodiment of this invention metabolicaly competent human cells HepG2 cells stable transfected with reporter gene, preferred the gene that encodes the fluorescent protein DsRed2, and a promoter that responds to DNA damage, preferred the p21 gene promoter are used. The vector construct by which the HepG2 cells are transfected is of composed of coding sequence of nucleotides that encode for DsRed protein that is operatively linked in cis configuration to promoter region of p21 gene. The method for genotoxicity determination consists of the following steps: contact HepG2-p21-DsRed cells with tested chemical or sample, incubation at 37oC/ 5% CO2, measurement of the fluorescence of the expressed protein DsRed with a microtiter plate reader at 24 and 48 hour of exposure, determination of cells density by MTT assay, and calculation of the normalized values of induced fluorescence, which are qualitative and quantitative indicator of genotoxic activity of the tested chemical or sample.

Description

TITLE OF INVENTION: A CELL BASED SYSTEM FOR RAPID DETECTION AND QUANTIFICATION OF GENOTOXICITY

DESCRIPTION OF INVENTION

The present invention relates to a method for determining genotoxicity of an agent and a cell based system useful therefore. In particular, the invention describes a biosensor system for detecting DNA damage in metabolically competent cell cultures.

The problem that the current invention is solving is a method that enables fast and accurate measurement of genotoxicity with microtitre plate spectrofluorimeter. The biosensing system comprises metabolically active cells transfected with a construct comprising reporter gen coding for fluorescent DsRed2 protein that is operatively linked to promoter region of p21 gen, that specifically responds to DNA damage. Due to the reporter gene DsRed2 the product of which is out of the region of autofluorescence of cell components and cyclic organic chemicals the results are not prone to the errors due to the autofluorescence and thus no additional steps are required to determine the fraction of autofluorescence or the use of more demanding methods for fluorescence determination such as flow cytometry or fluorescent microscopy.

In general, for protecting the human health as well as for protecting the environment it is important to identify agents that may cause DNA damage. In many countries the law requires that data are provided that show that an agent is not genotoxic before it can be marketed. Various methods for detecting genotoxic agents are available but are all time and cost consuming. One well known standard test is the Ames test which requires a few days to complete and measures mutagenicity by using a bacterium strain having a defective gene which makes it unable to synthesize an essential amino acid. The bacterium is grown on a medium lacking this amino acid and an agent to be tested is added. If bacteria can grow, this is an indication that mutation has occurred and that the agent is mutagenic. Other tests are the in vitro micronucleus test and the mouse lymphoma assay (MLA), the former of which is an in vitro method for detection of chromosomal aberrations and the latter of which is also a test detecting mutations via defective allels. These known methods are unsatisfactory for rapid screening for different reasons. With these analytical tools testing can take many days and even weeks and large quantities of the candidate compound are necessary. Moreover, these methods are not feasible for high throughput screening. Genotoxic agents are compounds that can damage genomic DNA. "Damage" in this regard means that the function of the DNA is disrupted or hindered such that the DNA does not, does only partially or not correctly express a gene. The damage can occur in different ways by chemical modification of nucleotides that comprise the DNA or by breaking the phosphodiester bonds that connect the nucleotides or by disrupting associations between nucleic bases. As the function of the DNA is of fundamental significance the living organism has developed complex defensive mechanisms against DNA damage, namely cell cycle arrest, DNA damage repair and apoptosis, i.e. the controlled death of cells, which positively contribute to genomic stability. In bacteria, DNA damage or inhibition of its replication invokes a well characterized SOS-response with the induction of about 20 different genes (Walker, Ann. Rev. Biochem, 54, 425, 1985). An even larger number of genes are involved in cellular response to DNA damage in yeasts (Weinhart and Hartwell, Science, 421, 317, 1988; Rowley et al., Nature 356, 353, 1992) and in mammalian cells (Holbrook and Fornace, New Biologist 3, 825, 1991).

It is also known that it is possible to measure gene expression changes and reporter gene expression systems have been provided for this purpose. Expression of genes that are expressed as response to DNA damage has been used as marker for genotoxicity. Bacterial systems that are best known and explored in this regard are those using the expression of SOS response genes for identifying genotoxic effects (Ben-Israel and Ben-Israel, Appl. Env. Microbiol, 64, 4346, 1998, Quillardet et al. Biochemie, 64, 797, 1982; Ptitsyn et al., Appl. Env. Microbiolo., 63, 4377, 1997; Oda et al., Mutat. Res., 147, 219, 1985). Recently a further assay system has been developed using yeast Saccharomyces cerevisiae known as GreenScreen" (Cahill et al., Mutagenesis, 19, 105, 2004). In this GreenScreen test RAD54 promoter is fused to green fluorescent protein (GFP) and RAD54 is induced by DNA damage which results in production of GFP which again can be quantified. Another assay (Cat-Tox (L)) has been developed in mammalian cells in 1995 by Todd et al (Todd et al., Fundam. Appl. Toxicol, 28, 118, 1995) wherein transformed HepG2 cells have been transformed which consists of 14 recombinant cell lines, each containing a specific stress gene promoter or response element fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Four different DNA damage responsive genes are included in this system: growth arrest and DNA damage protein GADD45 and 153, early response protein c-fos and 53 kDA protein tumor suppressor response element P53RE.

The most prominent pathway of cellular response to DNA damage in eukaryotes is an activation of the tumor suppressor and transcription factor p53 through phosphorylation by DNA damage- responsive kinases (Zhou and Elledge 2000, Nature 408, 433, 2000). Activated p53 induces the expression of genes involved in DNA repair, cell cycle arrest, or apoptosis, which then either allows the repair of the damage, or commit cells too extensively damaged to apoptosis (Sionov and Haupt, Oncogene, 18, 6145, 1999). US 6,344,324 and WO 2005/113802 disclose methods for detection of alterations in expression of GADD 153 and GADD 45, genes that are activated by p53, via EGFP reporter protein expression as an indication of the induction of DNA damage. US 6,344,324 describes that cellular injury can be detected by inducting EGFP expression in a cell line. In this document a reporter system is used which combines a reporter gene, enhanced green fluorescent protein (EFGP), with a GADD153 promoter. This construct was stably integrated into the genome of carcinoma cells. It was found that after contact with cell toxins, EGFP expression is detectable, but not in all cells and only after some days, with a peak at day 6. In the tests described in the document the determination was done four days after treatment by flow cytometry. Moreover, substantial differences were detected in the responsiveness of the transfected cells. Although it was found that this system can detect cell injuries, it is time consuming and does not provide reliable results in general.

Recent toxicogenomic studies showed that the DNA damage responsive genes GADD45 and 153, are rather unspecific and are upregulated also as response to non-genotoxic stress (Ellinger-Ziegelbauer et al., Mutat. Res., 575, 61, 2005) and, thus, are not reliable means for measuring genotoxicity. The study showed that only several genes are characteristic for damage by genotoxic carcinogens: cyclin dependent kinase 1A (CDKN1A) inhibitor, cyclin Gl and 06-methylguanine-DNA methyltransferase (MGMT). These are specifically upregulated upon exposure to genotoxic carcinogens.

The cyclin-dependent kinase 1A (CDKN1A) inhibitor p21 (Wafl/Cipl) 1 has been identified as p53 regulated tumor suppressor gene. Several groups have independently cloned p21 cDNA using different screening strategies. Using a yeast two-hybrid screen p21 was identified as CDK binding protein and named CIPl as CDK-interacting protein 1 (Harper et al., Cell, 75, 805, 1993). Microsequencing of the protein interaction with CDK lead to its cloning by PCR (Xiong et al., Nature 366, 701, 1993). It has been found that the expression of p21 is directly induced by p53 as the entire promoter region of p21 is under control of p53. Therefore, it was named wafl (wilde-type p53- activated factor) (El-Deiry et al., Cell, 75, 817, 1993). p53-dependent regulation of p21 has been shown to be critical for the response to DNA damage (Macleod et al., Genes Dev., 9, 935, 1995).

A p21 reporter construct comprising a p21 transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product and a method for screening potential therapeutic agents for the ability to suppress the growth of tumor cells by activating the expression of p21 has been disclosed in US 2002/142442 of Vogelstein and Kinzler (2002). Vogelstein and Kinzler were starting from the assumption that an agent that activates expression of p21 is useful for the treatment of cancer because p21 is involved in cell cycle rest. Therefore, the method disclosed by Vogelstein and Kinzler is concerned with an approach where a potential therapeutic agent is incubated with a cell containing a p21 reporter construct comprising a p21 transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product and the production of the assayable product is measured. An agent increasing the production by the cell of the assayable product is deemed to be a potential therapeutic agent which will activate the expression of p21 thereby suppressing the growth of tumor cells.

However, until now no reliable method for detecting genotoxicity of an agent in a reasonable time period which can be used commercially in high throughput screening has been provided. There is still a need for a simple, rapid, reliable and cost efficient assay for identifying genotoxic agents, i.e. agents that have the ability to cause DNA damage, mutations or other genetic damage in eukaryotic cells, in particularly in human cells. Moreover, there is a need to provide a method for improved toxicity testing and with improved safety of the materials in use.

Thus, it was the object of the present invention to provide a method that improves the detection and quantification of genotoxicity of agents. Particularly it is the object of the present invention to provide a system that is safe, rapid and at the same time cost efficient and yields reliable results. These objects are solved by a method as defined in claim 1.

It is one of the aspects of the present invention to exploit alteration of the activation of expression of a specific gene that is activated by p53 as an indication for DNA damage and to use it as a tool for early detection and quantification of DNA damage inflicted by genotoxic agents.

The present invention provides a method for detecting and/or quantifying genotoxicity of an agent comprising the steps of

a) contacting an agent to be tested with a biosensor system comprising

a. a metabolically competent cell transfected with a construct wherein

b. a nucleotide sequence coding for DsRed or a functional derivative thereof is operatively linked in a cis configuration to

c. a transcription regulatory region of p21 or a functionally equivalent variant thereof, wherein the regulatory region of p21 is activated as response to DNA damage;

b) measuring the expression of DsRed and c) determining after a predetermined time period whether there is, in the test cell, a change in DsRed protein expression as a result of contact with said candidate compound as compared to a control culture not in contact with candidate compound or any other genotoxic compound where increase in DsRed expression indicates that said candidate compound causes DNA damage.

In other words the genotoxicity of an agent can be determined by incubating the agent with a cell comprising a reporter-construct where the reporter protein is expressed by activation via p53 as reaction to DNA damage and the expression of the reporter is determined, usually by the fluorescence of the reporter, i.e. the fluorescence index (MFI) is measured, and compared to a standard value that has been obtained in a cell that was not in contact with a tested agent. If the value for the expression, for example the MFI is higher than the standard value this is an indication for genotoxicity of the agent and the extent of genotoxicity can be determined by the difference of both values.

It was found that genotoxic agents can be detected in a reliable but also simple manner in reasonable time by using a biosensor system that comprises a host cell line transfected with a construct comprising a specific regulatory region and a specific reporter gene, in a preferred embodiment the construct is stably integrated in a host cell line. Reliable results can be obtained within 24 to 48 hours.

The term "DNA damage" in the present description refers to any damage, injury or alteration of genomic DNA that result in a disruption of the function of DNA, that gives rise to a lack or partial lack of expression or in incorrect expression of a gene. The damage can be caused by different modifications, for example by breaking of bonds as outlined above.

The term "genotoxic" when used in the present description means any detrimental influence on DNA that causes damage.

The term "transcription regulatory region of p21" refers to a regulatory region of p21 that is responsible for starting expression of p21 and is activated by p53. The sequence of the p21 transcription regulatory region is known in the art (seeEI-Deiry et al., Cell, 75, 817, 1993). A functionally equivalent variant of the transcription regulatory region of p21 is any sequence that has a homology of at least 70 %, preferably at least 80 %, more preferred at least 85 % with the p21 promoter and is able to start the expression of a gene in response to activation by p53. The variant can have silent base changes corresponding to codon-usage preferences and/or can have conservative base exchanges that do not alter the function.

In a preferred embodiment of the present invention a p21 promoter is used as p21 transcription regulatory region which is activated by p53. The promoter region of p21 gene is well known and any variant thereof having the transcription regulatory activities for p21 can be used. Variants or derivatives of the p21 promoter are also well known to the skilled person or can be produced by well known methods.

The reporter protein used in the method of the present invention is DsRed and in the construct the DsRed gene or a functional derivative thereof is present. DsRed is a protein known in the art, it has fluorescence and, therefore, is used as reporter protein. Variants thereof are also known. Functional variants of the DsRed sequence in this description are sequences that code for DsRed and can have silent base changes corresponding to codon-usage preferences and/or can have conservative base exchanges that do not alter the function, i.e. the fluorescence of the DsRed protein.

It was found that with the system of the present invention a reliable and fast detection of genotoxic agents is possible. This is due to the very combination of the host cell, the specific promoter and the specific reporter gene used which only in this combination yield reliable test results.

The test system used comprises a p21 transcription regulatory region. It is this specific regulatory region which provides for the high sensitivity and specificity of the system. p21 is a DNA damage responsive gene. Cell viability and apoptosis are regulated in a very sensitive manner in cells. Inactivation of p53 inactivates apoptosis and cell cycle arrest, which is a common event in the development of human neoplasia. As apoptosis is an important part of maintaining genetic stability, the cell reacts to activation of p53 by activating the expression of genes that contain p53 binding sites. p53 induced genes therefore may play a role as tumor suppressors. In this connection p53 induced genes have been in the focus of research. One gene whose expression is activated by p53 is p21 or WAF1 whose entire promoter region is unambiguously under p53 control in mammalian cells. The p21 promoter has been found to be very useful in the construct of the present invention because it reacts in a very sensitive manner to cell injuries. The very reliable and sensitive activation is due to the fact that p53 induces p21 directly. It is assumed that there is at least one strong binding site within the transcription regulatory region. Instead of indirect activation as is the case in the prior art methods, with the present invention a direct activation via p21 promoter is obtained. It has been shown that the p53 dependent regulation of p21 is critical for the response to DNA damage and this property is used for identifying genotoxic agents by the method of the present invention by detecting a change in expression of the gene that is under the control of the p21 promoter.

In the construct used according to the present invention the p21 transcription regulatory region is covalently linked in a cis configuration to a gene encoding an assayable product, namely DsRed.

Although constructs combining a p21 promoter and a gene encoding a reporter protein have been described, for example in US 2002/142442, they were used for a different purpose and used a different reporter proteins, i.e. luciferase activity. The constructs known from USS 2002/142442 were used to screen for potential therapeutic agents that should suppress the growth of tumor cells by activating the expression of p21. However, it was found that these constructs, were not used in a test for determination of genotoxic effects.

Many reporter genes were known in the art, such as β-galactosidase, luciferase, chloramphenicol acetyl transferase, however all of these need a substrate to monitor their expression in terms of their activity. Therefore an additional step is needed for the performance of assays based on these reporters. Reporters based on fluorescent proteins such as green fluorescent protein (GFP) and its derivatives have the advantage, that their expression can be detected and quantified directly. However, it has been found that GFP has an emission in the wavelength range that corresponds to the autofluorescence of many organic compounds. Therefore, when using these known constructs the results are possibly too high or false positive. In contrast thereto, according to the present invention a fluorescent protein produced by a variant of Discosoma sp. is used, DsRed which is encoded by PCLEF35. A variant thereof, DsRed2, that contains a series of silent base pair changes corresponding to human codon usage preferences for high expression in mammalian cells, can also be used. DsRed2 contains six amino acid substitutions: A105V, I161T and S197A which result in the more rapid appearance of red fluorescence in transfected cell lines, and R2A, K5E and K9T, which prevent protein from aggregating.

The excitation maximum of DsRed is 563 nm and the emission maximum is 582 nm and thus is in a wavelength range which does not collide with the autofluorescence of proteins. In contrast thereto the excitation maximum of EGFP is 395 nm, a second excitation peak is at 457 nm and the emission maximum is at 510 nm.

The p21 promoter and the gene for the DsRed reporter protein are operatively linked such that the reporter protein is expressed under the control of the p21 promoter. Both are covalently linked in a cis configuration.

Moreover, it has been found that the best results are obtained if the construct of the present invention is stably inserted in the genome of a hepatocyte cell line, particularly HepG2. Stably transfected HepG2 cells that comprise the construct of the present invention have been found to yield reliable results in short time. Therefore, in a preferred embodiment a HepG2 cell line which is stably transfected with the p21-DsRed gene is used.

This system provides a cost effective test tool for the detection of genotoxicity of agents. This system can be used for high throughput screening of agents to find out those agents that cause DNA damage.

The present invention provides a method for the detection of at least one genotoxic agent wherein the agent is incubated with the detection system of the present invention. The time period for the incubation is long enough to allow the expression of the reporter protein if the agent is genotoxic and the upper limit is not critical. However, it is one of the advantages of the present invention that reliable results can be obtained with the system of the present invention within 24 hours at least 48 hours.

During the incubation period the cell line is exposed to the agent. If the agent is causing a DNA damage, the p53 cascade is started. p53 activates the p21 promoter and under the control of this promoter the reporter protein is produced which results in a fluorescing signal.

Another advantage of using the system of the present invention is that the expression of the reporter protein occurs directly by the DNA damage. Therefore, this system is more sensitive and accurate than known systems. In a preferred embodiment an expression cassette is used that comprises a DNA sequence encoding dsRED as reporter protein, which DNA sequence is operatively linked to a human p21 gene promoter arranged to activate expression of the DNA sequence in response to DNA damage. (Figure 1/4) As recombinant vector of the present invention any type of vector as known in the art can be used such as a plasmid, cosmid or phage. Such recombinant vectors are of great utility when replicating the expression cassette. Furthermore, recombinant vectors are highly useful for transfecting cells with the expression cassette, and may also promote expression of the reporter protein.

The recombinant vector used for the present invention may be designed such that the vector will autonomously replicate in the cytosol of the cell or can be used to integrate into the genome. Vectors and methods for designing them are well-known in the art. Elements that induce DNA replication may be required in the recombinant vector. Suitable elements are well known in the art, and for example, may be derived from different commercially available vectors such as pCEP4 (Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley, PA4 9RF, UK) pEGFPNl (BD Biosciences Clontech UK, 21 In Between Towns Road, Cowley, Oxford, OX4LY, United Kingdom) or pCI and pSI (Promega UK ltd, Delta house, chilworth Science Park, SouthamptonS016 7NS, UK). In a preferred embodiment of the present method the recombinant vector is pp21-dsRed2, as illustrated in Figure 1.

One important part of the biosensor system of the present invention is a metabolically competent host cell. It is preferred that the cell is eukaryotic. Such host cells may be mammalian including human derived cells and cell lines as well as other vertebrate and invertebrate cells and cell lines.

Preferred host cells are human cell lines. Preferably, the host cells are human cell lines having a fully functional p53. Preferably, the host cells are metabolically active expressing phase I and phase II metabolizing enzymes in inducible form.

The inventors have found that human hepatoma HepG2 cells are particularly preferred cell line for use in the method of the invention. While the inventors do not wish to be bound by any hypothesis, they believe that HepG2 cells are most useful because they have a fully functional p53 and express several of phase I and pahse II metabolic enzymes in inducible form.

Host cells used for expression of the reporter protein encoded by the DNA molecule are ideally stably transfected, although the use of unstably transfected (transient) cells is not precluded.

Stably transfected cells may be formed by following procedures described in the Example 1. The cell is ideally a human cell line, for example HepG2. Such transfected cells are preferably used in the method of the present invention to assess whether or not agents induce DNA damage.

In a most preferred embodiment the biosensor system used in the method of the present invention comprises HepG2 cells transformed with the vector p21-DsRed2. These cells are referred to herein as p21- HepG2-DsRed2.

The method of the present invention is preferably performed by growing cells transfected with the recombinant vector p21-DsRed2, incubating the cells with the agent which putatively causes DNA damage for a predetermined time and monitoring the expression of the light emitting reporter protein directly from a sample of the cells.

In the second step of the method of the present invention the expression of the reporter protein is determined which is usually done by measuring the mean fluorescence index (MFI). Methods and devices for measuring MFI are known in the art and can be used in known manner. As the emission wavelength is outside the range of auto-fluorescence, the measured value can be contributed to the reporter protein expression and false positive results are mainly avoided.

The methods of fluorescence detection and quantification are known, and a method is described in the Example 2.

Cells are preferably grown in low fluorescence growth medium. This can obviate the need to wash the cells before measurements are made and therefore reduce the number of steps necessary compared to known methods. For instance, in a preferred embodiment of the invention using human cells the cells are grown in a low fluorescent media, such as minimal essential medium without phenol red. The use of such media reduces the "signal to noise ratio" when measuring fluorescence.

In a preferred embodiment the incubation of the cells and the agent to be tested is made in a microplate and the fluorescence and absorbance is measured from the well of a microplate. Microplates that are suitable for this type of tests are well known and commercially available. One example of a suitable microplate is a 96 well, black, clear bottomed microplate. The fluorescence and absorbance measurements may be recorded using a suitable microplate reader. Microplate readers are also well known to the skilled person and are commercially available. One example is Tecan Infinite 2000 (Tecan UK) microplate reader. The MFI is measured and then is compared to a standard value to determine if the agent has genotoxic capacity or not and, if necessary, a quantification of the genotoxicity can also be made. The potential for genotoxicity can be evaluated by the value of (x/y) where x is the MFI of the agent to be analyzed and y is a standard value obtained by measuring cells that are not in contact with the tested substance. If the MFI of the agent exposed cells is statistically significantly different from MFI of the control non-exposed cells and this value is greater than 1.5 the analyzed agent is deemed to be genotoxic, whereas if the value is less than 1.5 and the difference in MFI is not statistically significant, the agent is deemed to be not genotoxic.

The value of MFI depends not only on the fluorescence excited by the fluorescent reporter protein as a response to genotoxic injury but is also dependent from the cell density. The higher the cell density, the more fluorescent the culture and the lower the cell density, the less fluorescent the culture. Therefore, in order to correct this dependence, the fluorescence data are divided by cell density data to give "fluorescence units" i.e. the average fluorescence per cell. This value is independent from culture density, and, thus, is more reliable.

The cell density can be determined in a manner well known to the skilled person, for example by an MTS assay. The MTS assay is a colorimetric method to identify the number of living cells and to determine the cytotoxic potential of a test item. The assay measures the formation of a soluble formazan product which is directly proportional to the number of live cells in culture. The absorbance values (determined at 492 nm), which reflect cell density are then used for normalization of fluorescence signals. As at the setup of the experiment the number of non-treated control cells and exposed cells are equal, therefore formazan absorbance values of exposed cells against non- treated cells gives also information on the effect of the exposure on cell viability.

The invention is further explained in the following examples. Protocols for measuring fluorescence and cell density are outlined in the examples. Example 1

Vector construction and cell transfection:

The following Example outlines the components that have been used in the construction of p21- DsRed2 vector according to the first and second aspects of the invention, describes the construction of the biosensor (transfected cells according to the third aspect of the invention), and selection of responsive clones.

1. System components:

i) The promoter - from human p21 (CDKN1A; CAP20; CDKN1; CIPl; MDA-6; P21; SDI1; WAF1; p21CIPl) gene:

The source of the p21 promoter was the WWP-LUC plasmid, which was a gift from Prof. Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, Maryland, ZDA). The plasmid has pBluescript (KS+) vector as a backbone was first described by El-Deiry et al. (Cell, 75, 817, 1993). ii) The fluorescence protein -DsRed2:

The source of DsRed2 is the plasmid pCLEF35 DsRed2 (Invivogen). PCLEF35 DsRed2 encodes rapidly maturing variant of Discosoma sp. red fluorescent protein (DsRed). DsRed2, contains a series of silent base-pair changes corresponding to human codon-usage preferences for high expression in mammalian cells (Excitation maximum = 563 nm; emission maximum = 582 nm). In addition to these changes, DsRed2 contains six amino acid substitutions: A105V, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating.

2. Construction of biosensor reporter cassettes (Figure 1)

The construction of recombinant vectors containing p21 promoter reporter cassette was done in several stages using the Clontech pEGFP-Nl plasmid as a back bone. pEGFP is mammalian expression vector that uses the cytomegalovirus (CMV) immideate early enhancer/promoter for high level transcription of downstream genes. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. Between the CMV promoter and the EGFP coding sequences is the multiple cloning site (MCS). SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418.

Step 1: (Removal of CMV promoter from pEGFP-Nl plasmid) - CMV promoter was removed from pEGFP-Nl using Nhel and Asel restriction enzymes and blunt end ligation. This promoterless pEGFP- Nl was named pEGFP-Nl no CMV.

Step 2: (Insertion of p21 promoter) The 5' flanking region of the p21 gene was cut out of the plasmid Wwp-Luc by EcoRI and Sail restriction enzymes, and about 2,4 bp length gene fragment containing the promoter region of p21 gene with p53 recognition site was obtained. This was inserted into the multiple cloning site of the plasmid resulting from step 1.

Step 3: (Replacement of the EGFP sequence with DsRed2 sequence) DsRed2 encoding sequence was cut out of the pCLRF35DsRed2 with Ncol and Nhel and cloned into pORF-mlL-12 (Invivogen) plasmid to obtain new restriction sites needed for subsequent cloning. DsRed encoding sequence was then cut out again, this time using Sail and Xbal restriction enzymes, and GFP encoding sequence in p21- EGFP plasmid was replaced with DsRed. The resultant plasmid was named pp21-DsRed.

3. Cell line:

The HepG2 (85011430 ECACC, Wiltshire, UK) cells were isolated from hepatocellular carcinoma of 15 years old boy. The cells excrete plasma proteins and express number liver specific enzymes (Science, 1980 209:497-9). HepG2 cells are one of the very few cell lines that have retained activities of xenobiotic metabolising enzymes, which are normally lost during in vitro cell cultivation. The cells retain wild type p53, which is essential for the cell line to respond to genotoxic damage in a way that would allow the required response of the reporter. Cells were grown in advanced minimum essential medium without phenol red (to avoid eventual interference with DsRed2 fluorescence) supplemented with 10% heat inactivated foetal calf serum.

4. Cell line transfection and selection of the transfected clones

HepG2 cells were trypsinized at 70% confluence and centrifuged. The cell pellet was resuspended in 125 mM saccharose in PBS buffer to a density of 3.3*10⁵ cells/μΐ.. 20 μg of plasmid p21-dsRED2 was added per 1*10⁶ cells. A 50 μί. drop containing cells and plasmid was then placed in the 2 mm gap between the electrodes of the electroporator (developed at Faculty of Electrical Engineering, Ljubljana, Slovenia). For each electroporation, 8 600 V/cm square electric pulses, 5 ms long, were used. After electroporation, the cells were placed in a well of a 6-well plate. 5 min after electroporation, 3 mL of antibiotic-free culture medium was added to each well. 48 hours after transfection, the medium was exchanged for complete medium with 1 mg/mL of G418 (Sigma, St. Louis, USA). After 14 days of selection, positive colonies were picked and transferred to 96 well plates. The isolated clones were expanded in 0.5 mg/ml G418, frozen at -80 °C and transferred to liquid nitrogen for longterm storing. Clones with visible changes in morphology and proliferation rate were not used for further screenings.

5. Screening of the clones for positive response to a known genotoxic agent MMS

- Selected clones of p21-dsRED2 transfected cells were seeded on 96-well plates (50000 cells/well) and left overnight to attach. The next day, they were treated with 10 μg/mL methylmethane sulphonate (MMS), a known genotoxic agent. The fluorescence of the clones was measured after 24 and 48 hours using a spectrofluorimeter (Tecan, Austria). Cell density/cytotoxicity was determined after 48 hours using the MTS assay. The induction of dsRED was normalised to cell density. Clone 1 of p21-DsRed2 transfected cells was chosen for further screening, as it had the best response to MMS genotoxicity. The new cell line was named HepG2-p21-DsRed.

Example 2

Genotoxins Induce Measurable DsRed Expression in the HepG2-p21-DsRed cell - Based Biosensor The cells p21-HepG2-DsRed were exposed to the following genotoxins: methyl methanesulphonate (MMS), cisplatin (CP), and benoz(a)pyrene (BaP). The chosen genotoxins represent monofunctional S_N2 alkylating agent, bifunctional alkylating agent, and indirectly acting genotoxic carcinogen, which requires metabolic activation to nucleophylic intermediate that forms DNA adducts.

Assay:

The following protocol may be followed.

A suspension of exponentialy growing cells: p21-HepG2-DsRed at the cell density of 3x10^s cells/mL was prepared in minimum essential medium without phenol red with 10 % fetal calf serum. The suspension was then distributed in 3 ml aliquots to platic test tubes. To each tube 30 μΙ of test chemical of appropriate concentration (100 fold higher concentrations form final treatment concentrations) and 30 μΙ of vehicle for control cells were added. The following final concentrations were used: MMS: 5, 10, 20, 40, 50 jig/ml; CP: 0.4125, 0.825, 1.65, 3.3, 6.6 Mg/ml; BaP: 30, 20, 10, 5, 2, 1, 0,5 and 0,2 μΜ. From each tube 100 μΙ aliqotes were distributed to 6 wells of 96 well black microtiter plates with clear bottom (Greiner) and incubated at 37 °C in an incubator containing humidified atmosphere and 5% C0₂ for 24 and 48 hours.

After 24 h incubation fluorescence was measured with Tecan Infinite 200 (Tecan UK) microplate reader: for DSRed fluorescence: excitation 560 nm/emission 590 nm; Then the plates were incubated for another 24 hours after which the second fluorescence measurement was performed. Viability was measured after 48 h by adding that 20 μΙ of MTS reagent (Promega) was added to each well and further incubated for 2 hours. Thereafter the absorbance was measured through 492 nm filter to detect formazan product, which was used as an indicator of cell density and chemical cytotoxity. The fluorescence and MTS absorbance data were transferred to Excel worksheet and are graphically presented. The fluorescence data were divided by MTS aborbance data to get the measure of average DeRed induction per cell ("fluorescence units"). The data were normalised to untreated control (=1) to give relative "DsRed induction ratio" The MTS absorbance data were normalized to untreated control to give relative viability data.

Figure 2 shows viability (A) and relative induction of DsRed expression (B) of p21-HepG2- DsRed cells after exposure to MMS for 24 and 48 hours.

Figure 3 shows viability (A) and relative induction of DsRed expression (B) of p21-HepG2- DsRed cells after exposure to CP for 24 and 48 hours.

Figure 4 shows viability (A) and relative induction of DsRed expression (B) of p21-HepG2- DsRed cells after exposure to BaP for 24 and 48 hours.

Claims

1. A method for detecting and/or quantifying genotoxicity of an agent comprising the steps of a) contacting an agent to be tested with a biosensor system comprising

i) a metabolically competent cell transfected with a construct wherein

ii) a nucleotide sequence coding for DsRed or a functional derivative thereof is operatively linked in a cis configuration to

iii) a transcription regulatory region of p21 or a functionally equivalent variant thereof, wherein the regulatory region of p21 is activated as response to DNA damage;

b) measuring the DsRed protein expression and

c) determining after a predetermined time period whether there is, in the test cell, a change in the MFI, wherein increased DsRed protein expression as a result of contact with said candidate compound as compared to a control culture not in contact with candidate compound or any other genotoxic compound indicates that said candidate compound causes DNA damage.

2. The cell system for rapid detection and quantification of genotoxicity of claim 1, wherein the metabolically competent cell is a human cell line.

3. The cell system for rapid detection and quantification of genotoxicity of claim 2, wherein the metabolically competent cell is a host cell line is HepG2

4. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the metabolically competent cell is a cell stably transfected with the construct.

5. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the biosensor system comprises a cell line that is stably transfected with an expression vector comprising the coding sequence for DsRed protein operatively linked to the p21 promoter.

6. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the biosensor system comprises p21-HepG2- DsRed cells that are stably transfected with an expression vector comprising the coding sequence of DsRed operatively linked to the p21 promoter and wherein expression of said Ds ed protein is induced in response to DNA damage.

7. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the expression of DsRed is detected by spectrofluorometry.

8. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the expression of DsRed is detected by flow cytometry.

9. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the expression of DsRed is detected by fluorescenece microscopy.

10. The cell system for rapid detection and quantification of genotoxicity of the preceding claims, wherein the system comprises a metabolically competent cell transfected with a construct comprising a nucleotide sequence coding for DsRed or a functional derivative thereof operatively linked to a transcription regulatory region of p21 or a functionally equivalent variant thereof, wherein the regulatory region of p21 is activated as response to DNA damage.