dNTP Supply Gene Expression Patterns in Microarrays after P53 Loss

Tom Radivoyevitch

Collaborative work with: John Pink, Charles Kunos, Gina Ferris, Ian Lent, Mark Jackson, Damian Junk

Pre-Rx 30’ 1 2 4 6 24

X X X X X XX X X X X

2X X X X X X XX X X X X

2X X X X X X X

48R-shp16 + 2 Gy

48R-shp16 + 2 Gy & 3-AP

CaSki + 2 Gy

CaSki + 2 Gy & 3-AP

TIME (hrs)

48R-shp16-shp53 + 2Gy

32 U219 CHIPS CaSki Cells

human mammary epithelial cells (HMEC)

9.4

10.2

RRM1 average of 4

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

10.0

11.5

RRM2 average of 2

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

7.0

8.5

RRM2B average of 3

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

P53 out so less p53R2

R1 compensates for p53R2 loss

No R2 compensation for p53R2 loss

Averaging probesets with aves > 6

911

CDKN1A average of 2

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

7.5

8.5

DCK average of 5

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

9.8

10.2

DCTD average of 5

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

P21 is p53 dependent, so there is a big loss in expression without it

Compensate for loss of de novo with increase in salvage

Lower average is consistent lower DCTD in next slide, i.e. less de novo dTMP

Lower levels is consistent with lower DCTD

Salvage compensates for less p53R2?Since R2 did not increase, not likely an S-fraction change

HPV does more than take down p53 9.

010

.5TK1 average of 2

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

10.5

11.5

TYMS average of 2

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

10.2

10.8

DGUOK average of 1

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

5.5

6.5

7.5

PPARG average of 2

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

6.0

7.0

TK2 average of 3

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

More Warburg => less mtDNA => less TK2

8.5

10.0

RRM1 average of 2

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

610

RRM2 average of 2

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

6.5

8.0

RRM2B average of 1

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

Herbert et al. Oncotarget. 2010, 1(6): 405–422 (Normal, Li-Fraumeni, & LF-Like) 3 people, 4 replicates, 2 tissues (Breast epithelial and stromal cells)

Herbert et al. Oncotarget. 2010, 1(6): 405–422 (Normal, Li-Fraumeni, & LF-Like) 3 people, 4 replicates, 2 tissues (Breast epithelial and stromal cells)

911

CDKN1A average of 1

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

7.5

9.0

DCK average of 1

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

9.9

10.3

DCTD average of 3

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

68

10TK1 average of 2

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

79

11

TYMS average of 2

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

9.0

10.0 DGUOK average of 2

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

5.0

6.5

8.0 PPARG average of 1

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

7.6

8.2

TK2 average of 4

Epithelial StromalNormal Li-Fraumeni Li-Frau-Like Normal Li-Fraumeni Li-Frau-Like

p53R2 Inhibits the Proliferation of Human Cancer Cells in Association with Cell-Cycle Arrest Keqiang Zhang, Jun Wu, Xiwei Wu, Xiaochen Wang, Yan Wang, Ning Zhou, Mei-ling Kuo, Xiyong Liu, Bingsen Zhou, Lufen Chang, David Ann, and Yun Yen Mol Cancer Ther; 10(2) 2011Affy Gene ST 1.0. GEO gse25238 (p53R2 siRNA of oropharyngeal carcinoma KB cells)

8.2

8.6

CDKN1A

Cntrls siP53R2

6.70

6.80

6.90

DCK

Cntrls siP53R2

7.8

8.0

8.2

8.4

DCTD

Cntrls siP53R2

10.2

510

.35

10.4

5 RRM1

Cntrls siP53R2

8.2

8.6

9.0

RRM2

Cntrls siP53R2

5.0

6.0

7.0

RRM2B

Cntrls siP53R2

9.90

10.0

0

TK1

Cntrls siP53R2

9.0

9.1

9.2

9.3

TYMS

Cntrls siP53R2

7.5

8.0

8.5

9.0

DCK Medians: 8.3 (p16); 9.12 (p53) Wilcoxon P = 9.83e-06

9.6

10.0

10.4

UCK2 Medians: 10.2 (p16); 10.5 (p53) Wilcoxon P = 0.0199

=> Rationale for using Deoxy-Aza-Cytidine (DAC) instead of aza-cytidine in p53 minus myelodysplastic syndrome (MDS) patients

Ongoing Active Research Projects1. Optimize triapine timing relative to irradiation of p53- cervical cancers (Charles Kunos).

2. Treatment of MDS with DAC (Yogen Saunthararajah, Cleveland Clinic). Goal is to understand dNTP supply system dependence on gender, age, and p53 status.

0 5 10 15 20

0.8

1.2

time

3 Gy10 Gy

RRM1

0 5 10 15 20

0.4

0.7

1.0

time

RRM2

0 5 10 15 20

1.0

1.4

1.8

time

TYMS

0 5 10 15 20

0.6

1.0

time

MTHFD1

0 5 10 15 200.

71.

01.

3

time

TK1

0 5 10 15 20

1.0

2.5

4.0

time

CDKN1A

GEO data GDS479Jen, K. Y. and V. G. Cheung (2003). Transcriptional response of lymphoblastoid cells to ionizing radiation. Genome Res 13(9): 2092-100.

Xiao-Peng Zhang, Feng Liu, and Wei Wang, (2011) PNAS 108, 8990–8995

Nor

mal

ized

to t=

0

Hours after irradiation

46

8

NME4 average of 1

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

57

NME5 average of 1

Ca.Pre

Ca.1 Ca.2 Ca.4 Ca.6Ca.2

4

CaAP.1

CaAP.2

CaAP.4

CaAP.6

CaAP.24

shp1

6

p16.P

re

p16.3

0Mp1

6.1p1

6.2p1

6.4p1

6.6

p16.2

4

p16A

P.1

p16A

P.2

p16A

P.4

p16A

P.6

p16A

P.24sh

p53

p53.P

re

p53.3

0Mp5

3.1p5

3.2p5

3.4p5

3.6

p53.2

4

NDPK expressed in NonMEtastatic cells, decreasing as cells become more cancerous

X48R Ca.1 Ca.2 Ca.24 Ca.4 Ca.6 Ca.Pre CaAP.1 CaAP.2 CaAP.24 CaAP.4 CaAP.6 Ep MIR

68

12

MYC OSM.Emt OSM.Ep p16.1 p16.2 p16.24 p16.30M p16.4 p16.6 p16.Pre p16AP.1 p16AP.2 p16AP.24

68

12

p16AP.4 p16AP.6 p53.1 p53.2 p53.24 p53.30M p53.4 p53.6 p53.Pre shp16 shp53 Stem1 Stem2

68

12

UDP

CDP

GDP

ADP

dTTP

dCTP

dGTP

dATP

dT

dC

dG

dADNA

dUMP

dU

TS

DCTD

dCK

DNA

poly

mer

ase

TK1

cytosol

mitochondria

dT

dC

dG

dA

TK2

dGK

dTMPdCMP

dGMP

dAMP

dTTPdCTP

dGTP

dATP

5NT

NT2

cytosol

nucleus

dUDP

dUTPdUTPase

dN

dN

dCK

flux activation inhibition

ATPordATP

RNR

dCK

dNTP Supply