Note for: Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation

00609_Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation

(doi: 10.1038/sj.onc.1203172)

Points

- belong to phosphatidylinositol-3 kinase family

- Using DT40 to study the function of ATM

DT40 does not express p53

- ATM(-/-) - retard in growth, defect in G2/M checkpoint, radio-resistant DNA synthesis

- sensitive to ionizing radiation

- higher in spontaneous chromosomal aberrations

- higher in radiation-induced chromosomal aberrations

- slight reduction in targeted integration frequency

- all above properties indicated p53-independent ATM functions

p53 is the key player for

1. cell cycle checkpoint control

2. apoptosis

3. DNA repair

(work through the downstream of ATM phosphorylation -- p53 is the substrate of ATM)

DT40 lacks p53 expression, but

ATM(-/-)DT40 - defect in

1. cell cycle checkpoint control

2. DNA repair

(this means there is another p53 independent mechanisms control all these processes)

Kinase domain between human and chicken is 88% homology

WT-DT40 cell cycle analysis;

1. accumulate at G2-M phase (p53 responsible for G1-S phase) after IR

2. ATM(-/-) - abrogate the S and G2/M checkpoint after IR --> leading to chromosomal aberration --> cell death

Growth rate is slow - two possibilities;

1. spontaneous cell death (using annexin)

2. cell division slows

--

explore on how cell death

1. increased in frequencies of spontaneous chromosomal breaks

2. lesion in break leading to cell death

Plating efficiency of ATM(-/-) in methyl -- 100%

As characteristic in DT40 at S-G2 phase;

1. HR more dominant than NHEJ

2. defect in ATM - could affect the HR

p53 available;

ATM(-/-) --> grow slow bc it stuck at G1-S phase

ATM(-/-)p53(-/-) --> grow faster --> using another route control proliferation

But in case of DT40 which does not express p53 -->growth rate slow should be bc of inability to repair-->leading to higher apoptotic cells

In general, p53 plays role in both G1-S and G2-M. But it appears that p53 played a minor role in G2-M cell cycle arrest.

ATM plays role in p53-independent at G2-M phase due to WT-DT40 stuck at G2-M phase.

ATM does not have a role in S-M checkpoint (test by treating the cell with hydroxyurea)

ATR does have a role in S-M checkpoint --> show sensitivity toward hydroxyurea