Biomedical Research Centre

Colin J Henderson Cancer Research UK Staff Scientist & Honorary Senior Lecturer Transgenic Group, Cancer Research UK Molecular Pharmacology Unit

E-mail: c.j.henderson@dundee.ac.uk   Tel. 01382 632623

Transgenic technology has the potential to revolutionise the field of drug metabolism; the ability to manipulate genetically the expression of individual drug metabolising enzymes in order to better understand their function(s) could lead to the development of new drugs for the treatment of diseases such as cancer, as well as the optimisation of treatment regimes with existing drugs [1].

The Transgenic Group has been interested for several years in the expression and regulation of drug metabolising enzymes and the role of such enzymes in cellular processes such as carcinogenesis and drug resistance. Recently, we have developed several transgenic models to enable us to study how drug metabolising enzymes protect us from the chemically challenging environment in which we live. We have deleted a Phase II drug metabolising enzyme, glutathione S-transferase Pi (GstP), from mice. GstP has been found to be expressed at elevated levels in a number of animal and human tumours, and in cell lines made resistant to a variety of drugs. Little, however, is known about the endogenous role of this enzyme. Mice lacking GstP are apparently phenotypically normal, with no change in morbidity or mortality. However, GstP null mice are found to have significantly higher levels of tumour formation, in the skin or lungs, when challenged chemically. Furthermore, such mice also have altered sensitivity to the effects of drugs such as acetaminophen, involving a mechanism(s) which have not yet been fully elucidated, but which may involve the recent discovery that GstP can act as an inhibitor of Jun kinase, thus effecting cellular signalling cascades and be involved in the regulation of stress response genes in this manner.

As part of the the CRUK Molecular Pharmacology Unit the Transgenic Group has been studying genes which determine cellular sensitivity to chemical agents, their relation to the aetiology and prevention of cancer and the development and use of anti-cancer drugs.

We have developed several transgenic models to enable us to study how drug metabolising enzymes protect us from the chemically challenging environment in which we live.

We have deleted a Phase II drug metabolising enzyme, glutathione S-transferase Pi (GstP), from mice. GstP has been found to be expressed at elevated levels in a number of animal and human tumours, and in cell lines made resistant to a variety of drugs. Little, however, is known about the endogenous role of this enzyme. Mice lacking GstP are apparently phenotypically normal, with no change in morbidity or mortality. However, GstP null mice are found to have significantly higher levels of tumour formation, in the skin or lungs [2], when challenged chemically. Furthermore, such mice also have altered sensitivity to the effects of drugs such as acetaminophen [3], involving a mechanism(s) which have not yet been fully elucidated, but which may involve the recent discovery that GstP can act as an inhibitor of Jun kinase, thus effecting cellular signalling cascades and be involved in the regulation of stress response genes in this manner[4,5].

The cytochrome P450 supergene family (CYP) represents a group of Phase I drug metabolising enzymes catalysing the insertion of an atom of molecular oxygen into a huge range of diverse substrates. A number of CYPs have been 'knocked out' in the mouse; deletion of those CYPs which carry out essentially 'house-keeping' reactions tend to result in embryonic lethality, or sever welfare problems, whereas deletion of P450s which carry out metabolism of xenobiotics tend to display little or no obvious phenotypic changes. This is at least in part due to the overlapping substrate specificity demonstrated by CYPs, and the ability of other enzymes to at least partially compensate for the absence of individual enzymes. In order to overcome this, we have deleted the sole electron donor to microsomal cytochrome CYPs, cytochrome P450 reductase (POR); as anticipated, this deletion was embryonic lethal [6]. However, our targeting strategy allowed us to conditionally delete POR when used in conjunction with a mouse line carrying Cre recombinase under the control of a tissue-specific promoter.

We have now generated the mice which lack hepatic POR, and thus the CYP system in the liver is essentially inactivated [7]. Despite this, Hepatic Reductase Null (HRN) mice grow and develop normally, demonstrating that a functional hepatic CYP system is not essential for survival in the postnatal period. We have employed the HRN mouse to study the role of hepatic CYPs in drug metabolism and disposition, for example with the anti-tumour agent cyclophosphamide, demonstrating that changing the mode of administration can reduce the myelotoxic side-effects of this drug without altering anti-tumour efficacy [8,9].

References

1. Henderson, C.J. and Wolf, C.R., Transgenic analysis of drug metabolising enzymes: precilinical drug development and toxicology. Mol Intervent, (2003) 3, 331 -343.
2. Henderson, C.J., et al., Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases. Proceedings of the National Academy of Sciences of the United States of America, (1998) 95, 5275-5280.
3. Henderson, C.J., et al. Increased resistance to acetominophen hepatotoxicity in mice lacking glutathione S-transferase Pi. Proc Natl Acad Sci U S A, (2000) 97, 12741-5.
4. Adler, V., et al., Regulation of JNK signaling by GSTp. Embo J, (1999) 18, 1321-34.
5. Elsby, R. et al., Increased constitutive c-Jun n-terminal kinase signaling in mice lacking glutathione S-transferase Pi. J Biol Chem, (2003) 278, 22243-9.
6. Otto, D.M., et al., Identification of novel roles of the cytochrome P450 system in early embryogenesis: effects on vasculogenesis and retinoic Acid homeostasis. Mol Cell Biol, (2003) 23, 6103-16.
7. Henderson, C.J., et al., Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase. J Biol Chem, (2003) 278, 13480-6.
8. Pass, G.J., et al., Role of hepatic cytochrome p450s in the pharmacokinetics and toxicity of cyclophosphamide: studies with the hepatic cytochrome P450 reductase null mouse. Cancer Res, (2005) 65, 4211-7.
9. Henderson, C.J., G.J. Pass, and C.R. Wolf, The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity. Toxicol Lett, 2005.

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