Fac

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Life

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manchester.ac.uk/lifesciences

Effect of mutating regulatory elements on the induction of GADD45a in response to genotoxic stress

Donna Johnson1, Richard Walmsley2

1. FLS, University of Manchester (donna.johnson@postgrad.manchester.ac.uk), 2.Gentronix, Ltd, Manchester (www.gentronix.co.uk)

Aims

Genome damage constitutes a major cancer risk and as a consequence, novel pharmaceutical compounds are assessed for their genotoxic potential using a battery of in vitro and in vivo tests. A recent review of the regulatory in vitro mammalian genotoxicity tests (chromosome aberration, mutation) has revealed a surprising lack of accuracy. Our lab has recently developed a new and more accurate in vitro test exploiting the regulatory elements (RE) of the GADD45a gene in a human lymphoblastoid cell line - GreenScreen HC. Preliminary work has suggested one of these elements, p53RE, was a pre-requisite for accuracy.

The aim of this research was to assess the effect of p53 on the induction of GADD45a. In order to do this, a range of mechanistically grouped chemicals were tested using GreenScreen HC in the wild-type p53 cell line (TK6), and the mutated p53 TK6 derived WI-L2-NS, each with either an unmutated reporter plasmid or one carrying either a mutated p53RE or a mutated WT1RE.

Introduction

GreenScreen HC is a novel pre-regulatory genotoxicity screening assay. It uses GFP expression under the control of human GADD45a regulatory sequences (see figure 1) to indicate if a compound causes DNA/genome damage. GADD45a is involved in the cellular response to DNA damage - cell cycle control, activation of DNA repair or apoptosis; it can be induced in a p53-dependent or independent way. P53 is able to regulate the induction of GADD45a through it’s response element in intron 3 or indirectly through the promoter via WT1.

Figure 1. RE within the GADD45a gene.

p53 is an important molecule in the cellular response to stress. It has been named the ‘Guardian of the Genome’ due to its part in maintaining genomic stability. It primarily acts as a transcription factor, activating/repressing target genes in response to a number of signals such as DNA damage & hypoxia. Once activated, p53 rapidly accumulates & is transported to the nucleus where it is able to exert its effects. Most regulatory genotoxicity tests use cell lines that have mutant p53 & this is thought to contribute to the high number of false predictions of in vivo hazard or positives that may be of low biological relevance. In mutant p53 cells, DNA damage that would normally trigger the induction of DNA repair mechanisms & delay the cell cycle to allow repair before division, instead persists, leading to replication errors & strand breaks GreenScreen HC, however, uses a p53 competent cell line & it’s thought that this contributes to the relatively low number of false positives generated by this assay.

Promoter*

TREOct 1 & CAAT

WT1FOXO

Potential c-Myc & ZBRK1

CDC2B23

ZBRK1 AP1p53

Nf-kB

CDC2B23

Exon 3*

5’UTR* Intron 1 3’UTR*

Exon 2Exon 1 Exon 4*

Intron 2 Intron 3

References - Levy et al., 1968; Oren et al.; 1999 Ryan et al., 2001; Shu et al., 2007

Acknowledgements

This project is funded by the BBSRC & Gentronix Ltd. Many thanks to Louise & Adam & everyone in the lab who has given their assistance

Results Table

Table 1 shows the tes results for GreenScreen HC. A positive is shown here if a compound was positive at 24 or 48 hours or both.

In all cases, the mutant reporters/p53 mutant cells show reduced induction of GADD45a.

In a number of cases, e.g. cisplatin and 5’fluorouracil, the mutation of the WT1 site in the presence of functioning p53 gives a slightly higher induction compared with that of the p53RE mutation.

In the case of aphidicolin, where the LEC is the same for both the p53m and the WT1m, the induction for the WT1 mutant in presence of p53 is slightly higher than that for the p53 mutant, though only at 48h. The same is true of the response to benomyl, though the induction was not enough to cross the threshold.

In some cases there appears to be little difference between the induction of the different mutants, e.g. MMC

Table 1. Results for each compound tested in GreenScreen HC, LEC and the carcinogenicity data where available.

LEC – lowest effective concentration

For key to strains see top right corner.

ND No Data IEInsufficient evidence

LE Limited evidence + Positive   Negative

Conclusions

• There appeared to be no mechanistic grouping in the shapes of the induction graphs or the responses of the various mutant reporter/cell line combinations

• There may be a number of reasons why there is no mechanistic grouping• Potency of each of the compounds may be different• Compounds may induce different response pathways

•p53 dependent and independent

• When p53 regulation is interfered with, either directly through the mutation of the p53RE or by using mutant p53 cells, or indirectly through mutation of the WT1RE, the induction of GADD45a is lessened

• The induction seen by TKw being lesser than that for TK, but higher than that for TKm, suggests that with TKw p53 is able to bind to the RE but not to elements within the promoter or to a lesser extent than with a normal WT1RE and therefore not produce maximum induction.

• Without WT1 there may be nothing at the promoter for p53 to interact with

• The absence of WT1 may alter the binding positions of proteins bound at the promoter, meaning p53 is not able to bind where it normally would

• The lower response for TKm suggests p53 is unable to bind and the binding of p53 to promoter elements may not be possible

• For those compounds that produced very little difference with TKw compared to the other mutants, the induction of GADD45a may be reliant on the interaction of another protein bound at the promoter that requires a functioning WT1RE or an interaction of WT1 with another protein that may not be possible with the inability of WT1 to bind to it’s mutated RE

BenomylAv GFP Induction 24h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 0.1172 0.4688 1.875 7.5

Concentration mg/ml

Flu

ore

scen

ce I

nd

ucti

on

TK

TKm

WI

WIM

WIw

TKw

Threshold

AphidicolinAv GFP Induction 24h

0

0.5

1

1.5

2

2.5

3

0 0.1563 0.625 2.5 10

Concentration mg/ml

Flu

ore

scen

ce I

nd

ucti

on

TK

TKm

WI

WIM

WIw

TKw

Threshold

Figure 1 - Cisplatin

Maximal induction is only seen when p53 and the p53RE are present as in the TK6/unmutated combination.

The induction for the reporter with WT1m RE is much higher compared to that produced by the TK6/p53m, WI/unmutated and WI/WT1m combinations.

Figure 2 and 3 - Benomyl and Aphidicolin

At 24h the induction in response to benomyl in the mutant reporters/p53m cells are very similar and much lower than maximum. However, at 48h the induction from the TKw combination is higher than that of the other mutants.

The response to benomyl shows a greater difference between TKw and the other mutants and the shape matches that of TK more than the other mutants. However, most of the other compounds more closely matched the response of aphidicolin – TKw induction was slightly higher compared to the other mutants but of a similar shape.

Figure 4 - Mitomycin C

Induction for mitomycin c at 48 hours.

There appeared to be no difference between the response of the mutants/p53m cells at either 24 or 48 hours.

Key :

TK = TK6 cells & normal reporterTKm = TK6 cells & p53m reporterTKw = TK6 cells & WT1m reporterWI = WI-L2-NS cells & normal reporterWIm = WI-L2-NS cells & p53m reporterWIw = WI-L2-NS cells & WT1m reporter

Cisplatin

0

0.5

1

1.5

2

2.5

3

3.5

0 0.1172 0.4688 1.875 7.5

Concentration mg/ml

Flu

ores

cenc

e In

duct

ion

TK

TKm

WI

WIM

WIw

TKw

Threshold

AphidicolinAv GFP Induction 48h

0

0.5

1

1.5

2

2.5

3

3.5

0 0.1563 0.625 2.5 10

Concentration mg/ml

Flu

ore

scence I

nduction

TK

TKm

WI

WIM

TKw

WIw

Threshold

BenomylAv GFP Induction 48h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.1172 0.4688 1.875 7.5

Concentration mg/ml

Flu

ore

scen

ce I

nd

ucti

on

TK

TKm

WI

WIM

WIw

TKw

Threshold

MMCAv GFP Induction 48h

0

0.5

1

1.5

2

2.5

3

3.5

0 0.0625 0.25 1 4

Concentration mg/ml

Flu

ore

scen

ce I

nd

uct

ion TK

TKm

WI

WIM

WIw

TKw

Threshold

Compound CarcMechanism

of action

Overall GreenScreen HC Result

LEC mg/ml

TK TKm TKw WI WIm WIw

Cisplatin +Direct acting

3.75 15 1.88      

MMS + 12.5 50 50 50 50 50

Mitomycin C + 0.13 2 0.5 4 2 1

Colchicine IE

Aneugen

1.56 100 200 200 200 100

Benomyl IE 1.88          

Thiabendazole -            

Acetaminophen LEFalse

positive

15          

1,2-DCB -            

Sulfisoxazole - 250          

Aphidicolin ND Interferes with

nucleotide synthesis

0.16 0.31 0.31      

5-Fluorouracil + 1.88 7.5 1.88     15

Hydroxyurea IE 4.69          

Figure 1

Figure 3A Figure 3B

Figure 4

Figure 2BFigure 2A