Radiation Biology & Oncology

Staff
Funding
Collaborators
Key Publications
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Lab Head: Professor Martin Lavin
martinL@qimr.edu.au

The Radiation Biology and Oncology Laboratory is studying the response of human cells to X-rays (radiation) and has selected the human genetic disorder ataxia-telangiectasia (A-T) to facilitate this investigation. A-T is a useful model system because patients with this disorder are very sensitive to radiation, exhibit a progressive loss of brain function and have elevated risk of developing cancers including leukaemia and lymphoma of blood cells. What we are attempting to do is to determine how the protein product in this disease functions, with a view to solving its role in brain function, radiation sensitivity and susceptibility to develop cancer.

We have designed experiments to isolate the protein defective in A-T and to understand the regulation of the gene specifying this protein. We have generated a mouse model to mimic this disease and this has already provided interesting results confirming the cancer susceptibility in the human disease. This model is also being employed to investigate neurodegeneration. These investigations are important because they will allow us to understand why this disease causes a gradual but irreversible effect on the motor function in patients and will also assist in the design of new therapies to slow down or prevent this loss of function.

A second major area of research activity is the development of new approaches for the early detection of prostate cancer. The emphasis is on identifying genes that may be useful as markers of this form of cancer. This information will assist in detecting prostate cancer and in formulating better treatment protocols for the disease.

In summary, our major area of research interest is centred on the genetics and biology of the human genetic disorder ataxia-telangiectasia (A-T). Research areas include:

• Mouse models for ataxia-telangiectasia
• Role of ATM in DNA damage recognition and radiation signal transduction
• Relationship between A-T and other genetic instability syndromes
• Identification of functional domains on the ATM protein and interacting proteins
• Mutation analysis to study the function of ATM and its role in breast cancer
• Role of ATM in the stress response and effect on neuronal function
• Clinical Radiosensitivity

Additional areas of research include:

• Identification of genes involved in development in the murine asidian Herdmania momus
• Isolation of novel compounds from snake venoms important in haemostasis
• Early detection of prostate cancer

Mouse models for ataxia-telangiectasia
The most debilitating aspect of the A-T syndrome is the progressive loss of neurons in the brain and the resulting loss of neurological function in patients. We have produced a knock-in mouse model in which the Atm gene is non-functional. This model is being used to study neurodegeneration and cancer development with a view to developing therapies for these conditions.

Cancer in Atm "knock-in" mouse heterozygotes
Epidemiological data provide evidence for an increased relative risk of breast and other cancers in carriers of the A-T gene. To date none of the "knock-out" mouse models have reported increased cancer risk in Atm heterozygotes for this gene disruption. We have recently demonstrated that carriers of the "knock-in" mutation develop a variety of tumours. Dominant interference by the mutated Atm protein appears to be responsible. We are investigating mechanism of tumorigenesis and we are generating other Atm missense mutant mice to study cancer predisposition. The effect of low dose radiation on cancer induction is also being investigated as well as the effect on mammary tissue development.

Missense mutations but not allelic variants alter the function of ATM by dominant interference in patients with breast cancer
The human genetic disorder ataxia-telangiectasia (A-T) is characterised by hypersensitivity to ionizing radiation and an elevated risk of malignancy. Epidemiological data support an increased risk for breast and other cancers in A-T heterozygotes. However, screening breast cancer cases for truncating mutations in the ATM gene has largely failed to reveal an increased incidence in these patients. It has been hypothesized that ATM missense mutations are implicated in breast cancer and there is some evidence to support this. The presence of a large variety of rare missense variants in addition to common polymorphisms in ATM makes it difficult to establish such a relationship by association studies. To investigate the functional significance of these changes we have introduced missense substitutions, identified in either A-T or breast cancer patients, into ATM cDNA prior to establishing stable cell lines to determine their effect on ATM function. Pathogenic missense mutations and neutral missense variants were initially distinguished by their capacity to correct the radiosensitive phenotype in A-T cells. Furthermore missense mutations abolished the radiation-induced kinase activity of ATM in normal control cells, caused chromosome instability and reduced cell viability in irradiated control cells, whereas neutral variants failed to do so. Mutant ATM was expressed at the same level as endogenous protein and interference with normal ATM function appeared to be by multimerization. This approach represents a means of identifying genuine ATM mutations and addressing the significance of missense changes in the ATM gene in a variety of cancers including breast cancer.

Future studies will involve the introduction of additional mutations into ATM cDNA for functional studies and the generation of mouse models with these missense mutations.

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Role of ATM in receptor signalling
While the bulk of ATM is present in the nucleus in growing cells a significant portion is localized in cytoplasmic vesicles. These observations together with a defective mitogenic response suggest that ATM has a wider role than DNA damage recognition. Work is in progress to delineate the role of ATM in signalling from membrane receptors. This work is being carried out in collaboration with Drs Peter Rodemann and Nuri Gueven, Tuebingen, Germany and Dr Dianne Watters, Griffith University.

Interaction of ATM with other proteins
We have already shown that ATM interacts with c-Abl, p53 and other unidentified proteins. One approach here is to employ domains from the protein to search for interacting proteins that may influence or alter the function of ATM. We have demonstrated that several of the ATM substrates including p53, BLM and BRCA1 interact with the N-terminus of ATM as well as the kinase domain. Work is in progress to map the exact sites of binding of these proteins and to establish the importance of this binding. We are also generating mutations in other regions of the ATM protein to investigate interactions with other proteins including c-Abl.

Increasing radiotherapeutic benefit with antisense ATM
A universal characteristic of A-T is sensitivity to ionizing radiation (radiotherapy). This is due to the absence of the ATM protein. We are developing methods to abrogate the function of ATM in tumour cells to increase their sensitivity to ionizing radiation with a view to improving the therapeutic benefit for patients with cancer. The strategy being employed is the cloning of fragments of ATM in the opposite orientation (anti-sense) into lentiviral vectors with a view to reducing ATM protein in radioresistant tumours such as glioblastoma to increase their sensitivity to radiation and thus the therapeutic benefit of this treatment in patients. This work is being carried out in collaboration with Dr Ming Wei, Dr David Walker and Dr Jonathan Ramsay, Brisbane.

Regulation of ATM
It is well established that the kinase activity of ATM is increased in response to radiation without changing the amount of protein. Recent results in collaboration with Drs Naomi Kando and Toshiyuki Fukao, Gifu, Japan, reveal that the ATM protein can be markedly increased in amount in response to mitogenic agents. Radiation has also been shown to alter ATM protein in fresh skin samples (in collaboration with Dr Raymond Clarke, Sydney). We are studying the mechanism of regulation of ATM in response to different agents. This has involved the cloning of the bidirectional promoter shared by ATM and a second gene NPAT. Sites on the promoter important for gene regulation are being investigated using mutagenesis and reporter assays in transfected cells. Work is also in progress to study the mechanism of activation of pre-existing ATM protein. The emphasis is on autophosphorylation, identification of specific sites of phosphorylation and relationship to radiation-induced activation.

ATM localization and expression
The localization and amount of ATM protein are not affected by radiation, however levesl are increased when cells are induced to proliferate. In the later stages of tumourigenesis ATM expression is lost and this correlates with tumour invasiveness. In terminally differentiated cells such as Purkinje cells of the cerebellum, ATM is almost entirely outside the nucleus. Our aim is to investigate how this complex regulation is brought about. Lack of suitable antibodies for immunoflourescence studies with ATM protein has made difficult studies on tissue-specific expression of ATM. To circumvent this we are producing transgenic mice in which reporter genes have been inserted at both ends of the ATM/NPAT bidirectional promoter. This approach will allow us to examine expression of ATM in different tissues and in individual cell types when animals are exposed to different environmental conditions. The methodology to be employed involves the use of biophotonic imaging and immunoflourescence with tissue sections.

Overlap with other genetic instability syndromes
There is good evidence that A-T and Nijmegen Breakage Syndrome overlap in the recognition of radiation damage to DNA. Recent results from this laboratory provide evidence for overlap with another cancer predisposition syndrome, Bloom's Syndrome. We have shown that ATM and BLM (the product of the Bloom's gene) interact. Investigations are underway to understand the functional significance of this interaction and the predisposition to cancer in the two syndromes.

Staff

Funding

• National Health and Medical Research Council
• Queensland Cancer Fund
• Australian Research Council
• A-T Children's Project
• National Institutes of Health; Washington
• University of Queensland

Collaborators

• Associate Prof. R.A. Gardiner/Dr Michelle Burger, Dept of Surgery, University of Qld
• Dr Dianne Watters, Griffith University
• Dr R. Clarke / Professor J. Kearsley, The St George Hospital, Sydney
• Dr Zvi Fuks, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Centre
• Dr Yosef Shiloh, Sackler School of Medicine, Tel Aviv University
• Dr J. Ramsay, QRI Mater Hospital
• Professor N. Kondo / Dr T. Fukao/ Dr H. Kaneko, Gifu University School of Medicine
• Dr Susan Lees-Miller, The University of Calgary, Alberta, Canada
• Dr Richard Gatti, UCLA School of Medicine, Los Angeles
• Dr Nathan Ellis, Memorial Sloan Kettering Cancer Centre, New York

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Key Publications

Shafman, T., Khanna, K.K.,, Kedar, P., Spring, K., Kozlov, S., Yen, T., Hobson, K., Gatei, M., Zhang, N., Watters, D., Egerton, M., Shiloh, Y., Kharbanda, S., Kufe, D. and Lavin, M.F. (1997). Interaction between ATM protein and c-Abl in response to DNA damage Nature 387, 520-523. [pubmed abstract]

Zhang, N., Song, Q., Lu, H. and Lavin, M.F. (1996). Induction of p53 and increased sensitivity to cisplatin in ataxia-telangiectasia. Oncogene 13, 655-659. [pubmed abstract]

Zhang, N., Chen, P., Khanna, KK., Scott, S., Gatei, M., Kozlov, S., Watters, D., Spring, K., Yen, T. and Lavin. M.F. (1997) Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype. Proc. Natl. Acad. Sci. USA. 94, 8021-8026. [pubmed abstract]

Hislop, A., Good, M.F., Mateo, L., Gardner, J., Gatei, M.H., Daniel, R.C.W., Meyers, B.V., Lavin, M.F. and Suhrbier, A.S. (1998) Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge. Nature Medicine 4, 1193-1196. [pubmed abstract]

Liao, W-C., Haimovitz-Friedman, A., Persaud, R.S., McLaughlin, M., Ehleiter, D., Zhang, N., Gatei, M.,Lavin, M.F., Kolesnick, R., and Fuks, Z. (1999) ATM inhibits DNA damage-induced apoptosis via ceramide synthase. J. Biol. Chem. 274, 17908-17917. [pubmed abstract]

Chen, P., Gatei, M., O'Connell, M.J., Khanna, K.K., Bugg, S.J., Hog, A., Scott, S.P., Hobson, K., and Lavin, M.F. (1999) Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells. Oncogene 18, 249-256. [pubmed abstract]

Lavin, M.F., and Shiloh Y. (1999). Ataxia-Telangiectasia (A-T). Primary Immunodeficency Diseases, A Molecular and Genetic Approach.(Eds: Hans D. Ochs, Edvard Smith and Jennifer M. Puck) Oxford University Press. pp 306-323.

Imyanitov, E.N., Birrell, G.W., Filippovich, I., Sorokina, N., Arnold, J., Mould, M.A., Wight, K., Walsh, M., Mok, S.C., Lavin, M.F., Chenevix-Trench, G., and Khanna, K.K. (1999) Frequent loss of heterozygosity at 1p36 in ovarian adenocarcinomas but the gene encoding p73 is unlikely to be the target. Oncogene 18, 4640-4642. [pubmed abstract]

Lavin, M.F. and Khanna, K.K. (1999) ATM: The protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int. J. Rad. Biol. 75, 1201-1214. [pubmed abstract]

Fukao, T., Kaneko, H., Birrell, G., Gatei, M., Tashata, H., Kasahara, K., Cross, S., Kedar, P., Watters, D.,.Khanna, K.K., Misko, I., Kondo, N., and Lavin, M.F. (1999) ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells. Blood 94, 1998-2006. [pubmed abstract]

Kairouz, R., Clarke, R.A., Marr, P.J., Watters, D., Lavin, M.F., Kearsley, J.H., and Soon-Lee, C. (1999) ATM protein expression in sporadic breast cancer. Mol. Pathol. 52, 252-256. [pubmed abstract]

Lavin, M.F., Concannon, P., and Gatti, R.A. (1999) Eight International Workshop on Ataxia-telangiectasia (ATW8). Cancer Res. 59, 3845-3849. [pubmed abstract]

Watters, D., Kedar, P., Spring, K., Bjorkman, J., Chen, P., Gatei, M., Birrell, G., Garrone, B., Srinivasa, P., Crane, D.I., and Lavin, M.F. (1999) Localization of a portion of extranuclear ATM to peroxisomes. J. Biol. Chem. 274, 34277-34282. [pubmed abstract]

Eri, R., Arnold, J.M., Hinman, V.F., Green, K.M., Jones, M.K., Degnan, B.M., and Lavin, M.F. (1999) Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis. Development 126, 5809-5818. [pubmed abstract]

Girjes, A.A., Carrick, F.N., and Lavin, M.F. (1999) Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms. Res Microbiol. 150, 483-489. [pubmed abstract]

Lavin MF. (1999) ATM: the product of the gene mutated in ataxia-telangiectasia. Int J Biochem Cell Biol. 31, 735-740. [pubmed abstract]

Adeeb, A.G., Carrick, F.N., and Lavin, M.F. (1999) Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms. Res Microbiol. 150, 483-489. [pubmed abstract]

Chan D.W., Son, S.C., Block, W., Ye, R., Khanna, K.K., Wold, M.S., Douglas, P., Goodarzi, A.A., Pelley, J., Taya, Y., Lavin, M.F. and Lees-Miller,
S.P. (2000) Purification and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive serine/threonine
protein kinase. J Biol Chem. 275(11), 7803-10. [pubmed abstract]

Imyanitov, E.N., Togo, A.V., Suspitsin, E.N., Grigoriev, M.Y., Pozharisski, K.M., Turkevich, E.A., Hanson, K.P., Hayward, N.K., Chenevix-Trench, G., Theillet, C., and Lavin, M.F. (2000). Evidence for Microsatellite Instability in Bilateral Breast Carcinomas. Cancer Letters 154, 9-17. [pubmed abstract]

Gatei, M, Young, D., Cerosaletti, K.M., Desai-Mehta, A., Spring, K., Kozlov, S., Lavin, M.F., Gatti, R.A., Concannon, P., and Khanna, K. (2000) ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nature Genet. 25, 115-119. [pubmed abstract]

Regueiro, J.R., Porras, O., Lavin, M.F., and Gatti, RA. (2000) Ataxia-telangiectasia: A primary immunodeficiency revisited. In: Immunology and Allergy Clinics of North America, Primary T-cell immunodeficiencies. W.B. Saunders Company. Vol 1, pp177-205.

Gatei, M., Scott, S.P., Filippovich, I., Sorokina, N., Lavin, M.F., Weber, B., and Khanna, K.K, (2000). Role for ATM in DNA damage-induced phosphorylation of BRCA1. Cancer Res. 60, 3299-3304. [pubmed abstract]

Yan, J., Khanna, K.K., and Lavin, M.F. (2000) Defective radiation signal transduction in ataxia-telangiectasia cells. Int. J. Radiat Biol. 76,1025-1035. [pubmed abstract]

Masci, P.P., Whitaker, A.N., Sparrow, L.G., de Jersey, J., Winzor, D.J., Watters, D.J., Lavin, M.F., and Gaffney, P.J. (2000). Textilinins from Pseudonaja textilis textilis. Characterization of two plasma inhibitors which reduce bleeding in an animal model. Blood Coagulation and Fibrinolysis 11, 385-393. [pubmed abstract]

Christie, D., Lavin, M.F., Tan, L. (2000). Clinical application of in vitro radiohypersensitivity testing. Australas Radiol. 44, 333-335. [pubmed abstract]

Lavin, M.F. (2000) An unlikely player joins the ATM signaling network. Nat Cell Biol. 2, 215-217. [no abstract available]

Clements, J.A., Merritt, T., Devoss, K., Swanson, C., Hamlyn, I., Scells, B., Rohde, P., Lavin, M.F., Yaxley, J., and Gardiner, R.A. (2000) Inactive free: total prostate specific antigen ratios in ejaculate from men suspect and known prostrate cancer, compared with young control men. BJU Int. 86, 453-458. [pubmed abstract]

Chan, D.W., Son, S.C., Block, W., Ye, R., Khanna, K.K., Wold, M.S., Douglas, P., Goodarzi, A.A.R., Pelley, J., Taya, Y., Lavin, M.F., and Lees-Miller, S.P. (2000) Purification and characterization of ATM from human placenta. A managanese-dependent, wortmannin-sensitive serine/threonine protein kinase. J. Biol. Chem. 275, 7803-7810. [pubmed abstract]

Gatei, M., Shkedy, D., Khanna, K.K., Uziel, T., Shiloh, Y., Pandita, T.K., Lavi, M.F., and Rotman, G., (2001) Ataxia-telangiectiasia: chronic activation of damage-response functions is reduced by alpha-lipoic acid. Oncogene 20, 289-294. [pubmed abstract]

Fang, Z.M., Lee, C.S., Sarris, M., Kearsley, J.H., Murrell, D., Lavin, M.F., Keating, K., and Clarke, R.A. (2001). Rapid radiation-induction of ATM protein levels in situ. Pathology 33, 30-36. [pubmed abstract]

Birrell, G.W., Ramsay, J.R., Tung, J.J., and Lavin, M.F. (2001). Exon skipping in the ATM gene in stored blood samples. Human Mutation 17, 75-76. [pubmed abstract]

Fillippovich, I., Sorokina, N., Gatei, M., Haupt, Y., Karen, H., Moallem, E., Spring, K., Mould, M., McGuckin, M.A., Lavin, M.F., and Khanna, K.K. (2001) Transactivation-deficient p73 alpha (p73 exon2) inhibits apoptosis and competes with p53. Oncogene 20(4), 514-22. [pubmed abstract]

Keating, K., Gueven, N., Watters, D., Rodemann, P., and Lavin, M.F. (2001) Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein. Oncogene 20(32), 4281-90. [pubmed abstract]

Spring, K., Cross, S., Li, C., Watters, D., Scott, S., Ben-Senior, L., Waring, P., Ahangari, F., Liu, S., Chen, P., Misko, I., Paterson. C., Kay, G., Smorodinsky, N.I., Shiloh, Y., and Lavin, M.F. (2001) Atm knock-in-mice harbouring an in-frame detection corresponding to the human ATM 7636del9 mutation exhibit a variant A-T phenotype. Cancer Res. 61(11), 4561-8. [pubmed abstract]

Ye, R., Bodero, A., Zhou, B.B., Khanna, K.K., Lavin, M.F., and Lees-Miller, S.P. (2001) The plant isoflavenoid, genistein, activates p53 and Chk2 in an ATM-dependent manner. J. Biol. Chem. 276(7), 4828-33. [pubmed abstract]

Gueven, N., Keating, K., Chen, P., Fukao, T., Khanna, K., Watters, D., Rodemann, P.H. and Lavin, M.F. (2001) Epidemermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia. Journal of Biol. Chem. 276, 8884-8891. [pubmed abstract]

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