Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Figures & data

Figure 1 Treatment effect on cell cycle and key cell cycle checkpoint proteins and kinases. (A) Cell cycle effects by treatments examined by flow cytometry. Cells were exposed to 1 µM veliparib, 10 nM topotecan alone or in combination for 24 h. Numbers above G₁, S and G₂ peaks indicate the percentage of cells in each phase of the cell cycle. Shown are the representative data of three independent experiments. (B) Protein gel blotting analyses of p21^CDKN1A expression, activation of Chk1 and Chk2, and histone H3 phosphorylation (pH 3) after treatments for 24 h. Shown are the representative results of at least two independent experiments. ABT, veliparib; TPT, topotecan.

Figure 2 Enhancing effects of veliparib on apoptosis and cell death induced by topotecan. (A) Cleaved PARP detected by protein gel blotting 96 h after treatments. (B) Relative % changes in cell death in response to veliparib (gray bar), topotecan alone (white bar) or in combination (black bar) assessed by clonogenic assays. Shown are the representative data of two independent experiments. (C) Relationship between the cell death and G₂ arrest (ratio of drug-treated/vehicle-treated) in response to veliparib, topotecan or in combination in HCT-116 p53^+/+ (dot), HT-29 (diamond), MCF-7 (square), MDA-MB-231 (triangle), and p53^−/− (star). ABT, veliparib; cl, cleaved; CC, correlation coefficient; TPT, topotecan.

Figure 3 Effect of UCN-01 on the cell cycle distribution and cell death in cells treated with veliparib plus topotecan. After 24 h exposure to veliparib plus topotecan, the cultures were washed, and UCN-01 (100 nM) or vehicle was added for an additional 18 h. (A) Cell cycle effects of veliparib in combination with topotecan in the presence and absence of UCN-01. The numbers above G₁ and G₂ peaks indicate the percentage of cells in each phase of the cell cycle. (B) Percent change, relative to vehicle treatment without UCN-01, of cell death by veliparib plus topotecan without UCN-01 (gray bar), and with UCN-01(black bar) assessed by clonogenic assays. Shown are the representative data of two independent experiments. ABT, ABT-888; TPT, topotecan.

Table 1 The differentially expressed genes in the cell cycle

Probe set	Description	Gene symbol	Parametric p value	Geometric mean of intensities (ABT* + TPT†/Vehicle)
A
1565702_at	ElaC homolog 1	ELAC1	0.006	6.427
202284_s_at	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	CDKN1A	0.041	2.380
233288_at	ataxia telangiectasia and Rad3 related	ATR	0.046	2.145
211803_at	cyclin-dependent kinase 2///β-carotene dioxygenase 2	CDK2///BCDO2	0.073	0.311
203084_at	transforming growth factor, β1	TGFB1	0.108	0.320
205899_at	cyclin A1	CCNA1	0.111	2.031
205397_x_at	SMAD family member 3	SMAD3	0.215	2.732
205659_at	histone deacetylase 9	HDAC9	0.354	1.310
207039_at	cyclin-dependent kinase inhibitor 2A (p16, inhibits CDK4)	CDKN2A	0.354	0.579
205675_at	microsomal triglyceride transfer protein	MTTP	0.446	0.424
209644_x_at	cyclin-dependent kinase inhibitor 2A (p16, inhibits CDK4)	CDKN2A	0.526	0.807
242939_at	transcription factor Dp-1	TFDP1	0.859	1.048
B
202284_s_at	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	CDKN1A	0.028	3.027
205397_x_at	SMAD family member 3	SMAD3	0.061	2.806
211803_at	cyclin-dependent kinase 2///β-carotene dioxygenase 2	CDK2///BCDO2	0.084	0.111
205899_at	cyclin A1	CCNA1	0.099	1.946
205675_at	microsomal triglyceride transfer protein	MTTP	0.108	0.360
207039_at	cyclin-dependent kinase inhibitor 2A (p16)	CDKN2A	0.207	1.804
209644_x_at	cyclin-dependent kinase inhibitor 2A (p16)	CDKN2A	0.224	0.434
205659_at	histone deacetylase 9	HDAC9	0.227	0.618
1565702_at	ElaC homolog 1 (E. coli)	ELAC1	0.253	0.528
233288_at	ataxia telangiectasia and Rad3 related	ATR	0.447	1.811
203084_at	transforming growth factor, β1	TGFB1	0.475	1.359
242939_at	transcription factor Dp-1	TFDP1	0.705	0.878
C
237891_at	Mdm2, transformed 3T3 cell double minute 2, p53 binding protein	MDM2	0.002	12.546
202284_s_at	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	CDKN1A	0.028	3.027
1556491_at	Retinoblastoma binding protein 8	RBBP8	0.030	0.160
211832_s_at	Mdm2, transformed 3T3 cell double minute 2, p53 binding protein	MDM2	0.057	2.154
205675_at	microsomal triglyceride transfer protein	MTTP	0.108	0.36
230405_at	RAD50 homolog	RAD50	0.402	1.351
D
237891_at	Mdm2, transformed 3T3 cell double minute 2, p53 binding protein	MDM2	0.002	12.54
202284_s_at	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	CDKN1A	0.028	3.027
211832_s_at	Mdm2, transformed 3T3 cell double minute 2, p53 binding protein	MDM2	0.057	2.154
211803_at	cyclin-dependent kinase 2///β-carotene dioxygenase 2	CDK2///BCDO2	0.084	0.111
203684_s_at	B-cell CLL/lymphoma 2	BCL2	0.106	0.568
1555186_at	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	CDKN1A	0.16	0.614
203685_at	B-cell CLL/lymphoma 2	BCL2	0.201	0.667
201148_s_at	TIMP metallopeptidase inhibitor 3	TIMP3	0.366	0.542
201147_s_at	TIMP metallopeptidase inhibitor 3	TIMP3	0.438	1.343
244035_at	B-cell CLL/lymphoma 2	BCL2	0.59	0.856

G₁/S check point pathway by topotecan alone (A) and veliparib plus topotecan (B); and those in ATM signaling pathway (C) and p53 signaling pathway (D) by veliparib plus topotecan in p53-wild-type lines.

* ABT, veliparib;

† TPT, topotecan.

Table 2 The differentially expressed genes in role of BRCA1, BRCA2 and ATR in cancer susceptibility pathway* by veliparib plus topotecan in p53-mutant lines

Probe set	Description	Gene symbol	Parametric p value	Geometric mean of intensities (ABT+TPT†/Vehicle)
234535_at	RAD50 homolog (S. cerevisiae)	RAD50	0.079	3.922
205848_at	Growth arrest-specific 2	GAS2	0.116	0.515
233288_at	Ataxia telangiectasia and Rad3 related	ATR	0.245	0.352
218689_at	Fanconi anemia, complementation group F	FANCF	0.245	1.916

* The title of this pathway can be interpreted as “misnomer” in this study context.

† ABT, veliparib; TPT, topotecan.