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Low levels of ATM in breast cancer patients with clinical radiosensitivity.

Fang Z, Kozlov S, McKay MJ, Woods R, Birrell G, Sprung CN, Murrell DF, Wangoo K, Teng L, Kearsley JH, Lavin MF, Graham PH, Clarke RA - Genome Integr (2010)

Bottom Line: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues.In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity.The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, St George Clinical School of Medicine University of NSW, St George Hospital, Kogarah, NSW 2217, Australia. r.clarke@unsw.edu.au.

ABSTRACT

Background and purpose: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein.

Results: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls.

Conclusions: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

No MeSH data available.


Related in: MedlinePlus

ATM kinase activity for LCLs 1 hour after an in vitro IR exposure of 5 Gy for breast cancer patients and controls. Obligate heterozygote ATH5ABR ~ the father of an A-T patient and his two siblings had high ICA counts of 2.18, 3.24, 2.50 & 2.48, respectively. The AT1ABR cell line, used here as a positive control, was originally derived from an A-T patient homozygous for a small (3 amino acid) in-frame deletion that destabilises the protein and which was reported previously with very low levels of mutant ATM protein [18].
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Figure 7: ATM kinase activity for LCLs 1 hour after an in vitro IR exposure of 5 Gy for breast cancer patients and controls. Obligate heterozygote ATH5ABR ~ the father of an A-T patient and his two siblings had high ICA counts of 2.18, 3.24, 2.50 & 2.48, respectively. The AT1ABR cell line, used here as a positive control, was originally derived from an A-T patient homozygous for a small (3 amino acid) in-frame deletion that destabilises the protein and which was reported previously with very low levels of mutant ATM protein [18].

Mentions: To determine the functional significance of low levels of the ATM protein we tested all low expressing patient cell lines for ATM kinase activity [20] and IR-induced chromosomal aberrations (ICA). In vitro ATM kinase activity was measured using ATM immunoprecipitation as described previously [18] followed by immunoblotting with the anti-pS1981 antibody to determine autophosphorylation, and also by using p53 as a substrate for ATM (Figs. 4, 5 &6). We found that 3/4 of the low expressers (patients 11, 13 & 16) had reduced ATM kinase activity (Figs. 4, 5, 6 &7 and reviewed in Table 1). Patient 16 showed no detectable level of ATM kinase activity (Fig. 6). The very low level of ATM protein for patient 13 (Fig. 4, 5, 6) was associated with an equally low level of ATM autophosphorylation. This was confirmed using the GST-p53 substrate which showed a small level of radiation-induced phosphorylation that was only evident at the higher dose of 10 Gy (Fig. 4, lower panels), see longer exposure. After immunoprecipitation, low expresser patient 11 also showed a small degree of ATM autophosphorylation, however, this induction of ATM kinase activity was not evident when p53 was used as the substrate (Fig. 5). Low expresser patient 10 (35% of the ATM protein) showed no reduction in kinase activity which suggested a possible false positive status, however, ATM kinase assays are not strictly quantitative. The only A-T obligate heterozygote in this study to carry a missense mutation, ATH5ABR, had the lowest level of the ATM protein (17%) when compared to the other ATH (Fig. 2 and see also Fig. 1A lane 10 and Fig. 7) and low ATM kinase activity (Fig. 7). A larger study could help here to determine the stoichiometric relationship between ATM protein level and detectable reductions in kinase activity. The A-T affected child of ATH5ABR (ATH18ABR) had no ATM protein and no ATM kinase activity (Fig. 7).


Low levels of ATM in breast cancer patients with clinical radiosensitivity.

Fang Z, Kozlov S, McKay MJ, Woods R, Birrell G, Sprung CN, Murrell DF, Wangoo K, Teng L, Kearsley JH, Lavin MF, Graham PH, Clarke RA - Genome Integr (2010)

ATM kinase activity for LCLs 1 hour after an in vitro IR exposure of 5 Gy for breast cancer patients and controls. Obligate heterozygote ATH5ABR ~ the father of an A-T patient and his two siblings had high ICA counts of 2.18, 3.24, 2.50 & 2.48, respectively. The AT1ABR cell line, used here as a positive control, was originally derived from an A-T patient homozygous for a small (3 amino acid) in-frame deletion that destabilises the protein and which was reported previously with very low levels of mutant ATM protein [18].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914013&req=5

Figure 7: ATM kinase activity for LCLs 1 hour after an in vitro IR exposure of 5 Gy for breast cancer patients and controls. Obligate heterozygote ATH5ABR ~ the father of an A-T patient and his two siblings had high ICA counts of 2.18, 3.24, 2.50 & 2.48, respectively. The AT1ABR cell line, used here as a positive control, was originally derived from an A-T patient homozygous for a small (3 amino acid) in-frame deletion that destabilises the protein and which was reported previously with very low levels of mutant ATM protein [18].
Mentions: To determine the functional significance of low levels of the ATM protein we tested all low expressing patient cell lines for ATM kinase activity [20] and IR-induced chromosomal aberrations (ICA). In vitro ATM kinase activity was measured using ATM immunoprecipitation as described previously [18] followed by immunoblotting with the anti-pS1981 antibody to determine autophosphorylation, and also by using p53 as a substrate for ATM (Figs. 4, 5 &6). We found that 3/4 of the low expressers (patients 11, 13 & 16) had reduced ATM kinase activity (Figs. 4, 5, 6 &7 and reviewed in Table 1). Patient 16 showed no detectable level of ATM kinase activity (Fig. 6). The very low level of ATM protein for patient 13 (Fig. 4, 5, 6) was associated with an equally low level of ATM autophosphorylation. This was confirmed using the GST-p53 substrate which showed a small level of radiation-induced phosphorylation that was only evident at the higher dose of 10 Gy (Fig. 4, lower panels), see longer exposure. After immunoprecipitation, low expresser patient 11 also showed a small degree of ATM autophosphorylation, however, this induction of ATM kinase activity was not evident when p53 was used as the substrate (Fig. 5). Low expresser patient 10 (35% of the ATM protein) showed no reduction in kinase activity which suggested a possible false positive status, however, ATM kinase assays are not strictly quantitative. The only A-T obligate heterozygote in this study to carry a missense mutation, ATH5ABR, had the lowest level of the ATM protein (17%) when compared to the other ATH (Fig. 2 and see also Fig. 1A lane 10 and Fig. 7) and low ATM kinase activity (Fig. 7). A larger study could help here to determine the stoichiometric relationship between ATM protein level and detectable reductions in kinase activity. The A-T affected child of ATH5ABR (ATH18ABR) had no ATM protein and no ATM kinase activity (Fig. 7).

Bottom Line: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues.In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity.The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, St George Clinical School of Medicine University of NSW, St George Hospital, Kogarah, NSW 2217, Australia. r.clarke@unsw.edu.au.

ABSTRACT

Background and purpose: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein.

Results: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls.

Conclusions: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

No MeSH data available.


Related in: MedlinePlus