Prof Malcolm Taylor
Increased clinical and cellular radiosensitivity
The cellular features of A-T include an increased sensitivity to both ionising radiation and a range of radiomimetic drugs all of which cause cultured cells to die by apoptosis (Taylor et al. 1975, Taylor et al. 1994, Meyn, 1995). This increased radiosensitivity can be measured using either a colony forming assay or chromosomally. There is good evidence for a defective G1/S checkpoint involving abnormal kinetics of p53 accumulation following exposure of A-T cells to ionising radiation (Kastan et al. 1992, Lu and Lane 1993), a defective S phase checkpoint as shown by the failure to inhibit DNA synthesis following exposure to either gamma rays or radiomimetic drugs (Painter and Young 1980) and a G2/M checkpoint abnormality (Beamish and Lavin 1994 ).
Correcting the increased radiosensitivity in cells in culture
There has been some success in expressing ATM protein to complement the increased radiosensitivity phenotype of cultured A-T cells. Ziv et al. (1997) constructed and cloned a recombinant, full length open reading frame of ATM using different vectors and hosts to overcome an instability conferred by the 5' half of the coding sequence. Recombinant ATM protein expressed episomally in an immortalised A-T cell line abolished the increased sensitivity to either gamma rays or neocarzinostatin normally shown by A-T cells. Similarly expression of ATM also abolished the radioresistant DNA synthesis normally associated with A-T cells. Morgan et al. (1997) showed that expression of only the C-terminal half of ATM, containing the PI-3 kinase domain complemented radiosensitivity, radioresistant DNA synthesis and reduced IR induced chromosomal breakage. This may be an indication of the importance of the PI3K domain in this function. Morgan et al. (1997) also reported the dominant negative effect of expression of an ATM fragment containing the leucine zipper motif. Expression of this region in normal cells resulted in RDS, enhanced radiosensitivity and chromosome breakage but not loss of p53 induction (accumulation) or block in G1 and G2 checkpoints in response to damage induction.
C-Abl and ATM association and radiosensitivity
One of the responses of cells following exposure to ionising radiation is the activation of c-Abl. Both A-T cells and cells from atm-/- knockout mice, however, are defective for radiation induced activation of c-Abl. It was shown that when normal cells, with functional ATM, are exposed to ionising radiation ATM phosphorylation of c-Abl is required for c-Abl tyrosine kinase activation. The SH3 domain of c-Abl binds to ATM through a central proline rich domain (aa 1373-1382) in a constitutive way and this association itself is not induced by radiation exposure. In contrast activation of C-Abl tyrosine kinase does require exposure to IR (Baskaran et al. 1997, Shafman et al. 1997). Expression of the ATM kinase domain alone is sufficient to restore activity of c-Abl (Baskaran et al. 1997) and phosphorylation of serine 465 is the critical target within c-Abl tyrosine kinase, for c-Abl activation.
The downstream events of this pathway are not known. Shafman et al. (1997) suggested that the ATM/c-Abl interaction may be part of radiation induced G1 arrest through a pathway involving p53. Activation of c-Abl and stabilisation of p53, however, are both dependent on ATM, but not on each other. These are independent targets downstream of ATM. It had previously been reported that activation of c-Abl by DNA damaging agents including ionising radiation is necessary for radiation induced G1 arrest (Yuan et al. 1996). Baskaran however reported no checkpoint defects in Abl-/- mice. The ATM/c-Abl pathway may not, therefore, be responsible for the cell cycle arrest features of A-T cells. An alternative function suggested by Baskaran et al. (1997) is that ATM/c-Abl modulates the expression of target genes by phosphorylating RNA pol II. The function of the ATM/c-Abl interaction, however, remains to be resolved.
Use of increased radiosensitivity in confirming the diagnosis of A-T and in prenatal diagnosis.
An important practical use of the increased radiosensitivity observed in A-T cells is for confirmation of the diagnosis of the disorder. Peripheral blood lymphocytes from A-T patients show a greatly increased radiosensitivity which can be easily measured chromosomally. A small proportion of A-T patients in the British Isles, however, have a near normal chromosomal radiosensitivity and this must always be borne in mind. In addition prenatal diagnosis for A-T is carried out by examining radiosensitivity in chorionic villus sample cells (CVS).
Leukaemia, and lymphoma in ataxia-telangiectasia
There is a clear increased risk of lymphoid leukaemia in A-T patients and approximately 10% of A-T patients will develop lymphoid leukaemia as children (Taylor et al. 1996). These include both B and T cell tumours. B cell tumours may be associated more with older children and young adults although T cell tumours may occur at any age and may be T-ALL (T-cell acute lymphocytic leukaemia), T-cell lymphoma or T-PLL (T cell prolymphocytic leukaemia). There may be a 4-5 fold increased frequency of T cell tumours compared with that of B cell tumours in these patients.
Chromosome abnormalities and proliferation of lymphocytes
Nothing is known about the development of the majority of leukaemias or lymphomas in A-T patients although more is understood about the development of T cell prolymphocytic leukaemia (T-PLL). A-T patients show a very high level of chromosome translocations involving the immune system genes, compared with normal. Cells with particular translocations can proliferate to occupy 100% of the T cell compartment. Clonal growth of these same chromosome translocation carrying cells and subsequent clonal selection is associated with development of T-PLL in young adult A-T patients (Taylor et al 1996). The clonal growth takes some years and so there is some warning of the impending tumour. These translocations involve the TCL gene (on chromosome14) and or the MTCP1 gene on the X chromosome and appear to activate expression of either TCL1 or an MTCP1 transcript, although what the precise role of these respective proteins is in the tumourigenic process is still not understood.
ATM mutations and leukaemia in A-T patients
The cloning of the ATM gene has allowed the opportunity to try and correlate the presence of particular ATM mutations with the development of particular leukaemias. The causal relationship between the ATM mutations present in A-T patients and the risk of leukaemia, however, is complex. Some patients without any ATM protein develop leukaemia as also do some A-T patients who express some mutant ATM protein. We can say, however, that the ATM gene may not be the only gene involved in the predisposition to leukaemia in A-T patients. The reason for this is that some families where A-T patients produce no ATM protein develop T-ALL in childhood whereas in other families, also without A-T protein, T-PLL can develop at a much later age ( about 40 years). If none of these A-T patients express any ATM protein then something else must also be influencing the predisposition of these different tumour types at different ages.
ATM mutations and leukaemia in non-A-T patients
Recently a clustering of ATM mis-sense mutations has been described in non A-T patients with the rare tumour, T cell prolymphocyic leukaemia (T-PLL). 2/17 of the mutations have been previously reported in A-T patients. The implication is that somatic inactivation of ATM may be important in the development of T-PLL and that ATM can act as tumour suppressor gene. The presence of ATM mutations seen previously in A-T patients also suggests the possibility that some of these T-PLL patients might be A-T heterozygotes (Vorechovsky et al. 1997).
ATM and breast cancer
Studies of A-T families have suggested that carriers of the ATM gene may have about a 5 fold increased risk of developing breast cancer (Swift et al. 1991, Easton et al. 1994). This would mean that 3-4% of all breast cancers would be attributable to the A-T gene (Easton et al. 1994). The existence of this increased risk of breast cancer in A-T carriers, however, remains controversial. Recently, a study of women with early onset breast cancer concluded that carrying the ATM gene does not confer an increased risk of early onset breast cancer (Fitzgerald et al.1997).
In contrast Athma et al. (1996) tested the A-T carrier status of 775 blood relatives in 99 A-T families by tracing the ATM gene using DNA markers tightly linked to ATM. They observed the significant finding that 25/33 women with breast cancer were also A-T carriers, compared with 14.9 expected. Interestingly, they also showed that the breast cancer risk is not limited to young women but appeared higher at older ages.
Athma P., Rappaport R., Swift M. 1996, Molecular genotyping shows that ataxia telangiectasia heterozygotes are predisposed to breast cancer. Cancer Genet Cytogenet 42, 130-134
Baskaran R, Wood L.D., Whittaker L.L., Canman C.E., Morgan S.E., Xu Y., Barlow C., Baltimore D., Wynshaw-Boris A., Kastan M.B., Wang J.Y.J. (1997). Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature 387, 516-519.
Beamish H., Lavin M.F., 1994, Radiosensitivity in ataxia telangiectasia: anomalies in radiation induced cell cycle delay. Int J Radiat Biol 65, 175-184.
Easton, D.F. (1994) Cancer risk in A-T heterozygotes. Int J Radiat Biol 66, S177-S182
FitzGerald M.G., Bean J.M., Hegde S.R., Unsal H., MacDonald D.J., Harkin D.P., Finkelstein D.M. et al. 1997, Heterozygous ATM mutations do not contribute to early onset of breast cancer. Nature Genet 15, 307-310
Kastan M.B., Zhan Q.M., Eldeiry W.S., Carrier F., Jacks T,. Walsh W.V. Plunkett B.S., Vogelstein B., Fornace A.J. 1992, A mammalian cell cycle checkpoint pathway utilising p53 and GADD 45 is defective in ataxia telangiectasia. Cell 71, 587-597.
Lu, X., Lane, D.P. (1993). Differential induction of transcriptionally active p53 following UV or ionising radiation; defects in chromosome instability syndromes? Cell 75, 765-778.
Meyn M.S. (1995). Ataxia telangiectasia and cellular responses to DNA damage. Cancer Res. 55: 5991-6001.
Morgan S.E., Lovly C., Pandita T.K., Shiloh Y., Kastan M.B. (1997) Fragments of ATM which have dominant-negative or complementing activity. Mol Cell Biol 17:2020-2029
Painter R.B., Young B.R. (1980). Radiosensitivity in ataxia telangiectasia; a new explanation. Proc Natl Acad Sci (USA) 77: 7315-7317.
Shafman T., Khanna K.K., Kedar P., Spring K., Kozlov S., Yen T., Hobson K., Gatel M., Zhang N., Watters D., Egerton M., Shiloh Y., Kharabanda S., Kufe D. Lavin M.F. (1997). Interaction between ATM protein and c-Abl in response to DNA damage. Nature 387, 520-523.
Swift M., Morrell D., Massey R.B. Chase C.L. (1991) Incidence of cancer in 161 families affected by ataxia-telangiectasia. New Engl J Med 325: 1831-1836
Taylor A.M.R., Harnden D.G., Arlett C.F., Harcourt S.A., Lehmann A.R., Stevens S., Bridges B.A. (1975). Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity. Nature 258: 427-428.
Taylor.A.M.R., Byrd P.J., McConville C.M., Thacker S. (1994). Genetic and cellular features of ataxia telangiectasia Int. J. Radiat. Biol. 65, 65-70
Taylor A.M.R., Metcalfe J.A., Thick J., Mak Y.F. (1996) Leukaemia and lymphoma in ataxia telangiectasia. Blood 87: 423-438
Vorechovsky I. Luo L., Dyer M.J.S., Catovsky D., Amlot P., Yaxley J.C., Foroni L., Hammarstrom L., Webster A.D.B., Yuille M.A.R. (1997). Clustering of missense mutations in the ataxia telangiectasia gene in a sporadic T cell leukaemia. Nature Genet. 17: 96-99
Yuan Z. M., Role of c-Abl tyrosine kinase in growth arrest response to DNA damage. Nature 382; 272-274
Ziv, Y., Bar-Shira, A., Pecker, I., Russell, P., Jorgensen, T.J., Tsarf, I., Shiloh, J. (1997) Recombinant ATM protein complements the cellular A-T phenotype. Oncogene 15, 159-167
Prof A M R Taylor
CRC Laboratories
Department of Cancer Studies
University of Birmingham
Edgbaston
BIRMINGHAM
OTHER PRESENTATIONS:
Ataxia Telangiectasia: 1926 - 1997 - An Introduction
The Neurological Aspects of Ataxia-Telangiectasia
Immunodeficiency and Infections Associated with Ataxia-Telangiectasia
Ataxia-Telangiectasia and ATM: The Next Stage
Why it is Important to Identify Cases of Ataxia-Telangiectasia