APOPTOSIS OF CANCER CELLS

Dr. Subhash BasuDepartment of Chemistry and Biochemistry

University of Notre DameNotre Dame, IN USA

Indian Statistical Institute Kolkata, India

February 18th 2008

What are the Characteristic Events of Apoptosis?Membrane BlebbingPhosphatidylserine FloppingApoptotic BodiesCytoplasmic Membrane DamageDNA FragmentationRegulation of GLTs

Apoptosis vs. Necrosis

Normal and Cancer Cell Growth Regulation

24hrs

24hrs

Normal Cells

Undifferentiated Dividing Cells

Differentiated Cells

APOPTOTIC (Programmed) CELL DEATH

24hrs

24hrs 24hrs

Cancer Cells

n+1 2n

1) Apoptotic Program is Ready and Remains Inhibited2)Apoptosis is Inducible

Drug Treatment

APOPTOTIC (Programmed) CELL DEATH

5

QuickTime™ and a decompressor

are needed to see this picture.

Human Breast Carcinoma CellsSK-BR-3, MCF-7, and MDA-468

Derived from pleural effusion of breast carcinoma

Cells HER2 Status

ER PgR p53

SK-BR-3 High - - Mutant

MCF-7 Normal + + Normal

MDA-468 Normal - - Mutant

•HER2 (c-erbB-2/neu) protein

HER2 comes from a proto-oncogene encoding a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. HER2 gives the cells different responsiveness to anti-cancer drugs versus HER2 negative breast cancers cells

•Estrogen receptor (ER) and progesterone receptor (PgR)

L-/D-PPMP

Tamoxifen

cis-Platin

Melphalan

Structures of Anti-Cancer Apoptotic Agents

Betulinic Acid

Morphological Changes of Apoptotic Breast Cancer Cells Treated with L-PPMP

Control MCF-7 MCF-7 (8μM L-PPMP) 48hrs

SKBR-3 (8μM L-PPMP) 48hrs

MDA-468 (8μM L-PPMP) 48hrs

MCF-7 with (16μM) L-PPMP 48h

SKBR-3 (Control)

MDA-468 (Control)

SKBR-3 (16μM L-PPMP) 48hrs

MDA-468 (16μM L-PPMP) 48hrs

Galβ1-4Glc-Cerα2-3

SA

Galβ1-4Glc-Cer GlcNAcβ1-3Lc2SAT-1 GlcNAcT- 1

Glc-CerGalT-2

CeramideGlcTD-/L-PDMP

D-/L-PPMP

GalNAcβ1-4Gal-Glc-Cer

SA

GM3

GalNAcT-1

Galβ1- 3GalNAc-Gal-Glc-Cer

SA

GM2

GM1

GalT-3

Gal-GalNAc-Gal-Glc-Cer

SAα2-3

SA

SAT-4

GD1a

Lc3

Galβ1- 4GlcNAcβ1-3Lc2

GalT-4

nLc4SAT-3

Galβ1- 4GlcNAcβ1-3Lc2α2-3

SALM1

FucT-3

Galβ1-4Glc-CerGD3SA

α2-8SA

SAT- 2

GalNAcβ1-4Gal-Glc-CerSA GD2

SA

GalNAcT-1

Galβ1-3GalNAc-Gal-Glc-CerGD1b

SA

SA

GalT-3

Biosynthesis of Mono- and Di-Sialosyl Glycosphingolipids

GlcNAcT-2

GlcNAcβ1-3nLc4inLc5

GlcNAcT-3

nLc4

Ii

GlcNAcβ1-3

GlcNAcβ1-6

Galβ1- 4GlcNAcβ1-3Lc2

SA

SA-LeX

α1-3Fuc

Galα1-4Lc2

Lc2GalT-5’

GalNAcT-2Gb3

GalNAcβ1-3Gb3GbOsc4Cer

GalT-5

Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2

nLc5

i

I

Hypothesis

Mitochondrion

Fas/TNF

FADD

Pro-Caspase-8

Active -Casp-8

Pro-Caspase-3

Casp-3 (p19/12)

Active Casp-3(p17/12)

Caspase-3Activation

DFF/ICAD/PARP etc.

?

DNA Cleavage

?

PI-3 Kinase

Phospho Akt

pBad

Cyto-c

PTP

Pro-Caspase-9 Active-Casp-9

Apaf-1

Glc-GalSASA

(GD3)

CERAMIDE

CGase

Sphingosine

Sph-1-P

PKC

?

GSL (GD3/GD1b)

?

P-Choline (Sphingomyelin)

Sphase

GSL

L-/D-PDMPL-/D-PPMP

P4

Molecular Apoptotic Changes1. Transport of Phosphatidyl-Serine from inside to

outside of Plasma membranes.

2. DNA fragmentation with DNA Laddering assay.

3. Stimulation of Ceramide generation in the cells.

4. Identification of caspase activations by Western blot.

5. Inactivation of DNA Pol-α and Helicase−ΙΙΙ of the Replication Complexes.

6. Regulation of GSL:GLTs of the SA-LeX Biosynthetic Pathway (studied by DNA-microarray).

PC GSLPSPC

APOPTOTIC REAGENTS

i) L/D-PPMPii) GD3/GM1b/GT1b/GD1biii) cis-Platiniv) Betulinic Acidv) Tamoxifen

1) Blue Fluorescence (Live Cells) – PSS-3802) Red Fluorescence (Permeable Dead Cells)

– Propidium Iodide

GSL GSLPC PC

PS

PSPSPE PE

Mitochondrion

Nucleus

NON-APOPTOTICCARCINOMA CELL

PS

PSPE

APOPTOTICCARCINOMA CELL

Nucleus

GD3

Mitochondrion

GD3GD3

Cytochrome C

Nucleus

APOPTOTIC/DEAD CELL

APOPTOTICREAGENTS

GD3GSL

Mitochondrion

GD3

GD3PS

PSPC

PE

PE

PCPSS

2-48hrs1) PSS-380 Dye2) Propidium Iodide

Me

GD3

PC

PS

PE

= DG-O-P-O-CH2-CH

IO-

=O NH3+

COO-

= DG-O-P-O-CH2-CH2- NH3+

= DG-O-P-O-CH2-CH2- N+ - MeMe

=CER Glc Gal-SA-SA

PSSSmith, B. et.al. (Patent Pending)

IO-

=OIO

-=O

PCPC

PCPSS-

PSS

2 Detection of Phosphatidylserine with PSS-380

PSS-380

Propidium Iodide

Remove Culture Medium

Wash with TES buffer(5mM, pH 7.2, 150mM NaCl)

Stain with TES buffer containing 25 μMPSS-380, 250μg/ml PI for 15 min at 37oC

Remove staining buffer, wash with TES buffer

Observe under fluorescence microscopy following excitation with 350 nm.

Staining Protocol

80 μM GD3 6 h

Control

80 μM GD3 24 h

Staining of Apoptotic Cancer Cells with Fluorescent Dyes

Basu S, Ma R, et al., Glycoconj J. 2004;20(9):563-77. PMID: 15454695Ma R, Basu S, et al., Glycoconj J. 2004;20(5):319-30. PMID: 15229396

NNH

O

HN

NH HN

O

NO O

O-

O-

N2+N

Detection of Innercellular Membranewith Squaraine Rotaxane (AKS-0)

SKBR-3 with 10µM L-PPMP 6 hours/PSS-380 and AKS-0

Control - Phase Contract Control – PSS-380 Control – Rotaxane (AKS-0)

L-PPMP - Phase Contract L-PPMP – PSS-380 L-PPMP – Rotaxane (AKS-0)

SKBR-3 with 10µM L-PPMP 24 hours/PSS-380 and AKS-0

Control - Phase Contract Control – PSS-380 Control – Rotaxane (AKS-0)

L-PPMP - Phase Contract L-PPMP – PSS-380 L-PPMP – Rotaxane (AKS-0)

SKBR-3 with 10µM L-PPMP 6 hours/PSS-380 and AKS-0

Control - Phase Contract Control – PSS-380 Control – Rotaxane (AKS-0)

L-PPMP - Phase Contract L-PPMP – PSS-380 L-PPMP – Rotaxane (AKS-0)

Inhibitors of GlcT

P4D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol

Causing apoptosis by elevating endogenous ceramideconcentration in different cancer cells lines, especially in multidrug-resistant cell lines.

Nicholson KM, et al., Br J Cancer. 1999 Oct;81(3):423-30. PMID: 10507766 Basu S, Ma R, et al., Glycoconj J. 2004;20(9):563-77. PMID: 15454695Basu S, Ma R, et al., Glycoconj J. 2004;20(3):157-68. PMID: 15090729

Galβ1-4Glc-Cerα2-3

SA

Galβ1-4Glc-Cer GlcNAcβ1-3Lc2SAT-1 GlcNAcT- 1

Glc-CerGalT-2

CeramideGlcTD-/L-PDMP

D-/L-PPMP

GalNAcβ1-4Gal-Glc-Cer

SA

GM3

GalNAcT-1

Galβ1- 3GalNAc-Gal-Glc-Cer

SA

GM2

GM1

GalT-3

Gal-GalNAc-Gal-Glc-Cer

SAα2-3

SA

SAT-4

GD1a

Lc3

Galβ1- 4GlcNAcβ1-3Lc2

GalT-4

nLc4SAT-3

Galβ1- 4GlcNAcβ1-3Lc2α2-3

SALM1

FucT-3

Galβ1-4Glc-CerGD3SA

α2-8SA

SAT- 2

GalNAcβ1-4Gal-Glc-CerSA GD2

SA

GalNAcT-1

Galβ1-3GalNAc-Gal-Glc-CerGD1b

SA

SA

GalT-3

BIOSYNTHESIS OF MONO- AND DI-SIALOSYL GLYCOSPHINGOLIPIDS

GlcNAcT-2

GlcNAcβ1-3nLc4nLc5

GlcNAcT-3

nLc4

Ii

GlcNAcβ1-3

GlcNAcβ1-6

Galβ1- 4GlcNAcβ1-3Lc2

SA

SA-LeX

α1-3Fuc

Galα1-4Lc2

Lc2GalT-5

GalNAcT-2Gb3

GalNAcβ1-3Gb3GbOsc4Cer

GalT-5

Galβ1- 4Galβ1- 4GlcNAcβ1-3Lc2

inLc5

Ceramide and Apoptosis

•Second messenger involved mitochondrial-related apoptosis

• Instantly produced by the action of sphingomyelinase (SMase) and ceramide glycanase (CGase)

•Open mitochondrial permeability transition pore (MPTP) to relasecytochrome C

•Breakdown to sphingosine as another messenger regulator

Siskind LJ, et al., Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins, J BiolChem. 2000 Dec 8;275(49):38640-4.

Hypothesis

Mitochondrion

Fas/TNF

FADD

Pro-Caspase-8

Active -Casp-8

Pro-Caspase-3

Casp-3 (p19/12)

Active Casp-3(p17/12)

Caspase-3Activation

DFF/ICAD/PARP etc.

?

DNA Cleavage

?

PI-3 Kinase

Phospho Akt

pBad

Cyto-c

PTP

Pro-Caspase-9 Active-Casp-9

Apaf-1

Glc-GalSASA

(GD3)

CERAMIDE

CGase

Sphingosine

Sph-1-P

PKC

?

GSL (GD3/GD1b)

?

P-Choline (Sphingomyelin)

Sphase

GSL

L-/D-PDMPL-/D-PPMP

P4

Identification of Caspase-3 activations by Western blot (SKBR-3)

Basu S, Ma R, et al., Glycoconj J. 2004;20(9):563-77. PMID: 15454695

Ma R, Basu S, et al., Glycoconj J. 2004;20(5):319-30. PMID: 15229396

Basu S, Ma R, et al., Glycoconj J. 2004;20(3):157-68. PMID: 15090729

Identification of Caspase-8 activations by Western blot SKBR-3/cis-Platin

Extrinsic pathway can activate the intrinsic pathway; Extrinsic (Death Receptor)Intrinsic (Mitochrondrial)Extrinsic pathway: Caspase-8and Caspase-3Intrinsic pathway: activation of Caspase-9 and Caspase-3

Apoptosis Signal Transduction Pathway

Rossi et al. Messengers of cell death: apoptotic signaling in health and disease. Haematologica 2003; 88:212-218.

Other Apoptotic Agents

Betulinic Acid

Melphalan

1. 2.0 μM L-PPMP2. Control3. 0.5 μM L-PPMP4. 2.0 μM L-PDMP5. Control6. 0.5 μM L-PDMP

Basu S, Ma R, et al., Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP. Glycoconj J. 2004;20(3):157-68. PMID: 15090729

Basu S, Ma R, et al., Apoptosis of human carcinoma cells in the presence of potential anti-cancer drugs: III. Treatment of Colo-205 and SKBR3 cells with: cis-platin, Tamoxifen, Melphalan, Betulinic acid, L-PDMP, L-PPMP, and GD3 ganglioside.Glycoconj J. 2004;20(9):563-77. PMID: 15454695

2 DNA fragmentation with DNA Laddering assay

[Kelly, T., Moghaggas, S., Bose, R., and Basu, S.(1993)Cancer Biochem. And Biophys., 13, 135-146.]

MODEL FOR CISPLATIN INHIBITION OF POL-α

C C

M

C C

HUMAN- LTDEEKYRDCERFKCPCPTCGTEN IYDNVFDGSGTDMEPSLYRCSN IDCKASPLTFYEAST- I TDVERFKDTVT LELS CPSCDKRFPFGGIVSS NYYRVSYNGLQCKHCEQLFT PLQL

HUMAN- TVQLSNKLIMDIRRFIKKYYDGWL ICEEPT CRNRTRHL PLQFSRTGPL CPACMKATLYEAST- TSQI EH- - - -S IRAHISLYYAGWLQCDDSTCGI V TRQVSVFGKRCLNDGCT- - - -GVM

HUMAN- QPEYSDKSLYTQYEAST- RYKYSDKQLYNQ

COMPARISON OF HUMAN DNA-POLYMERASE-α & YEASTPol-I ZINC BINDING DOMAIN SEQUENCES

[Bose, R. N., Li, D., Yang, W. W., and Basu, S. (1999) J. Biomol. Struct. & Dynamics, 16, 1075-1085 ]

RP-A

PCNA

Pol-α / Primase

DNA Helicase

Pol-δ

MODEL FOR A FUNCTIONAL REPLICATION COMPLEX

30% 10%

Pol-α

Hel-III

DETECTION OF DNA POL-α &HELICASE-III PROTEINS BY SPECIFIC ANTIBODIES

(SKBR3 Human Breast Carcinoma Cells)[Pol-α / SJK 237-70 Mab / Helicase-III Pab]

0

0.2

0.4

0.6

0 25 150

[cisplatin] ( μM)

EFFECT OF cis-PLATIN TREATMENT ON MDA-468 DNA POLYMERASE-α ACTIVITY

0

0.3

0.6

0.9

1.2

1.5

0 25 150

[cisplatin] ( μM)

EFFECT OF cis-PLATIN TREATMENT ON SKBR3 DNA POLYMERASE-α ACTIVITY

ATP

ADP + Pi

Replication Repair Recombination

Helicase

BIOLOGICAL ROLES OF DNA HELICASES

Effect of cis -Platin Treatment on MDA-468 Pol- α and Helicase Activities

0

50

100

150

0 10 20 80

[cis-platin] ( μM)

pol alpha helicase

ROME Assay of Helicase Activities(ROME = Radioactive Oligonucleotide in Membrane filtration Effluent)

5'

5'3'3'

* * *

* * * * * *

* * *

100 mM Tris-HCl pH 8.5 / 8 mM DTT / 1mM MgCl2 / 1 mM ATP / 30 mM KCl / sucrose / 400 μg/ml BSA / enzyme

Filtered through Centricon-30

Descending chromatography with 100ml 0.1 M K-PO4 pH 6.8, 60 g (NH4)2SO4, 2ml n-propanol

Origin (oligonucleotides) counted in toluene based liquid scintillation counting system

Incubated for 2hrs. at 37°C

Filtrate spotted on Whatman-1MM Paper

= [3H] ACT-DNA and [3H] oligonucleotides

= [3H] oligonucleotides [3H]

oligonuc.

[3H] mononuc.

Boyle, P.J., et al . (2003) FASEB J. 17(4), A600

ConclusionDNA Degradation & Deregulation of DNA Biosynthesis in

Apoptotic Cancer cells

1. The Novel ROME assay proved to be an effective measure of DNA helicase activity.

2. Helicase-III complexes with DNA Pol-α in breast and colon carcinoma cells.

3. In apoptotic cells, the activities of both DNA Pol-α and Helicase-III are modified (decreased).

Thank You

Acknowledgement

Prof. Jin-ichi Inokuchi(Hokkaido University)Prof Sipra Banerjee(Cleveland Clinic)Dr.M. BasuRui MaPatrick BoyleHui Liang

Morphological Changes of Apoptotic Breast Cancer Cells Treated with L-PPMP

Control MCF-7 MCF-7 (8μM L-PPMP) 48hrs

SKBR-3 (8μM L-PPMP) 48hrs

MDA-468 (8μM L-PPMP) 48hrs

MCF-7 with (16μM) L-PPMP 48h

SKBR-3 (Control)

MDA-468 (Control)

SKBR-3 (16μM L-PPMP) 48hrs

MDA-468 (16μM L-PPMP) 48hrs

Attachments of Neutrophils and Leukocytes to Endothelial CellsDuring Inflammation

NNH

O

HN

NH HN

O

NO O

O-

O-

N2+N

Detection of Innercellular Membranewith Squaraine Rotaxane (AKS-0)

• Symptoms of endothelial damage immune vasculitis+ Hepatic inflammation

spontaneous phenotype

• Neutrophils in endothelial inflammation (copper cuff) and CLPinduced phenotype

/ p-carboxypept.HKa

gC1qR

ACTIVATION OF PROTEASES DURING INFLAMMATION

Serine protease (trypsin family)released duringinflammation

ElastaseCathepsinTryptaseTrypsinKallikreinThrombin PlasminFactors VII & X

Neutrophils, Monocytes,Eosinophils Natural killer cells Macrophages Mast cells

Cellular responseto inflammation

Innate immune cell responseto inflammation

Epithelial cell, Endothelial cell,Smooth muscleFibroblastPlateletsNeoplastic

BikuninH2

H1

Inter-α-Inhibitor

ActivationBikunin

Uristatin

Uristatin 2Uristatin 1

Inhibition

Additional fragmentation

Bik anti inflammatory system.

H

Increasedproteinexpression

Replication and transcription

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Increasedhormoneproduction

Cell proliferation and differentiation

PAR mediated cell proliferation during inflammation.

Nucleus

Extra cellular

Plasmin

uPARActivation

uPA

MembraneSynthesis

PAR Activation

DNA pol-αHelicase

uPAR

MAPK/ERK

PKC

PLA2PLCAC

RAS

PKA

TrypsinThrombin

Cytoplasm

Cytokine &growth factor

release

Extrinsic death factor & NF-KB

activation

Cell shrinking and

membrane blebbing

Growth factor & cytokine

stress signalingImmune cell

Elastase

Extra cellular

TNF-αIL-1βIL-18TGF α/ β

IL-1βEGFR

TNFRFasL

Transcriptionof cytokineproduction

Caspase 9

NF-κB

CytoC

MAPK p38 Caspase 3,6,7

Mitochondria Caspase 8 Granzyme B

Apoptosis

DNA Fragmentation and no repair

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Nucleus

Cytoplasm

DNA pol-αHelicase

MKK3/6

Immune cell mediated apoptosis

Ca+

Extra cellular

DNA Fragmentation and no repair

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Nucleus

DNA pol-αHelicase

Caspase 9No Activation

observed

NF-κB

Caspase 3

Caspase 8No Activation

observed

No apoptosisobserved

NoPAR

activation

MAPK/ERKNot tested

MAPK p38Activationobserved

ReducedCytokine &

growth factor release

Inhibitedgrowth factor

signaling

Bik-RCD44 Bik

Potential change inintracellularcalcium

Reduced cell proliferationobserved

ReduceduPAR

activation

uPARLP

BikuPA

Impact of Bik on normal cell signaling.

Bik Ca+

KCa ChannelK+

K+

K+

K+

Ca+

Ca+

Ca+

Ca Channel

Bik

Bik

ReducedK + influx

Decreasedproteinexpression

Inhibited extrinsic death

factor & NF-KB signaling

TNFRFasL

Inflammation markers Cut-off Specificity Sensitivity P

Uri (mg/L) >=7.8 mg/L 96% 20% P<0.05

uTi strip(mg/L) >=7.8 mg/L 91% 25% P<0.05

Bik(mg/L) >=2.5 mg/L 100% 19% NS

I-α-I (mg/L) <=36 mg/L 100% 8% NS

CRP(mg/L) >=2 mg/L 97% 10% P<0.05

WBC total count (cell/nL)2 >=10cell/ nL 95% 29% P<0.01

Granulocyte (cell/nL) >=7cell/ nL 100% 27% P<0.05

Lymphocyte (cell/nL) >=3cell/ nL 100% 19% P<0.05

Bik or Uri same as above 92% 36% P<0.01

Bik or Uri or I-α-I same as above 89% 42% P<0.01

Predictive value of inflammation markers for vascular stenosis.

i) Phase Contrast ii) PSS-380 iii) Propidium Iodide

10mg/L Bik

50mg/L Bik

0 mg/L Bik

a) 4 h

Microscopic images of Bik treated normal kidney cells(NHMC).

10mg/L Bik

50mg/L Bik

0 mg/L Bik

i) Phase Contrast ii) PSS-380 iii) Propidium Iodide

b) 24 h

ACKNOWLEDGEMENTSAt Notre Dame Outside Collaboration

Dr. Manju Basu

Patrick J. Boyle 1.Dr. Jin-ichi Inokuchi (Hokkaido University)Rui Ma 2.Dr. Sipra Banerjee (Cleveland Clinic)

3. Dr. Rathin N. Bose (Kent State Univ.)4. Dr. Narendra Tuteja (ICGEB,New Delhi,INDIA)

Chris MoultonBrett CampbellBrian MikkulaMatthew BradleyJames VranishKim St. Jean

NIH/NCI and Bayer Corporation

Human DNA Helicases

Helicase SourceI HeLa Tuteja et al 1990II (Ku) HeLa Tuteja et al 1994III HeLa Tuteja et al 1992IV (nucleolin) HeLa Tuteja et al 1991V (FBP) HeLa Tuteja et al 1993

VI HeLa Tuteja et al 1995VIII (G3BP) HeLa Costa et al 1999

DIFFERENT DNA HELICASES ISOLATED FROM HUMAN CELLS

Hypothesis

Mitochondrion

Fas/TNF

FADD

Pro-Caspase-8

Active -Casp-8

Pro-Caspase-3

Casp-3 (p19/12)

Active Casp-3(p17/12)

Caspase-3Activation

DFF/ICAD/PARP etc.

?

DNA Cleavage

?

PI-3 Kinase

Phospho Akt

pBad

Cyto-c

PTP

Pro-Caspase-9 Active-Casp-9

Apaf-1

Glc-GalSASA

(GD3)

CERAMIDE

CGase

Sphingosine

Sph-1-P

PKC

?

GSL (GD3/GD1b)

?

P-Choline (Sphingomyelin)

Sphase

GSL

L-/D-PDMPL-/D-PPMP

P4

- ORC

- Cdt1

- Cdc6

- MCM10

- MCM

- DDK

- Cdc45

- Dpb11

- Pol-α / Primase

- Pol-ε

- RP-ATakisawa, H, Mimura, S., and Kubota, Y. Eukaryotic DNA Replication: from pre-replication complex to initiation complex. Curr. Opin. Cell Biol., 200, 12:690-696.

PROPOSED MODEL FOR FOMATION OF A REPLICATION INITIATION COMPLEX

1. Inhibition of chromosomal DNA replication.( cis-platin in Colo-205)

2. Ceramide-generated from inhibition of the first committed step in the biosynthesis of glycosphingolipids(L/D-PPMP; L/D-PDMP in Colo-205/SKBr3)

3. Ceramide-generated from breakdown of glycosphingolipids.(GD3 & GD1b Gangliosides in SKBr3)

4. Ceramide-generated from breakdown of sphingomyelin.

5. Deregulation of the transcription factors.

WHAT PROVOKES APOPTOSIS ?

H

Increasedproteinexpression

Replication and transcription

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Increasedhormoneproduction

Cell proliferation and differentiation

Fig 1a. PAR mediated cell proliferation during inflammation.

Nucleus

Extra cellular

Plasmin

uPARActivation

uPA

MembraneSynthesis

PAR Activation

DNA pol-αHelicase

uPAR

MAPK/ERK

PKC

PLA2PLCAC

RAS

PKA

TrypsinThrombin

Cytoplasm

Cytokine &growth factor

release

Extrinsic death factor & NF-KB

activation

Cell shrinking and

membrane blebbing

Growth factor & cytokine

stress signaling Immune cell

Elastase

Extra cellular

TNF-αIL-1βIL-18TGF α/ β

IL-1βEGFR

TNFRFasL

Transcriptionof cytokineproduction

Caspase 9

NF-κB

CytoC

MAPK p38 Caspase 3,6,7

Mitochondria Caspase 8 Granzyme B

Apoptosis

DNA Fragmentation and no repair

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Nucleus

Cytoplasm

DNA pol-αHelicase

MKK3/6

Fig 1b. Immune cell mediated apoptosis

Normal Cells

24h

24hn+1

G2

S

G1

M

24h

Cancer Cells

24h

24h2n

24h

Cell cycle

a) b) c)

Figure 2. Normal and Cancer Cell Growth Regulation.

50 KDa

25 KDa

150 KDa

75 KDa

100 KDa

NormalKidney

Cell(VERO)

Cancer cellLine

(MCF-7)

Cancer cellLine

(MDA-468)

NormalKidney

Cell(VERO)

Cancer cellLine

(MCF-7)

Cancer cellLine

(MDA-468)

50 KDa

25 KDa

150 KDa

75 KDa

100 KDa

Fig 3. Bik and ADBP-26 in Normal Kidney and Cancer Cells

a) Bik b) ADBP-26

1 2 3 4 1 2 3 4

0

50

100

150

200

250

300

0 5 10 15 20

Hours

Avg

Cel

l N

um

ber

(10^

4)

050

100150200250300350400

0 5 10 15 20Hours

Avg

CPM

/10^

4 c

ells

a)

d)c)

b)Figure 4. Cell Culture Synchronization.

50 KDa

25 KDa

150 KDa

75 KDa

100 KDa

NormalKidney

Cell(VERO)

Cancer cellLine

(MCF-7)

Cancer cellLine

(MDA-468)

NormalKidney

Cell(VERO)

Cancer cellLine

(MCF-7)

Cancer cellLine

(MDA-468)

50 KDa

25 KDa

150 KDa

75 KDa

100 KDa

Fig 3. Bik and ADBP-26 in Normal Kidney and Cancer Cells

a) Bik b) ADBP-26

1 2 3 4 1 2 3 4

Ca+

Extra cellular

DNA Fragmentation and no repair

Nuclear receptors

Transcriptioninitiation complex

Cell cycle apparatus

Nucleus

DNA pol-αHelicase

Caspase 9No Activation

observed

NF-κB

Caspase 3

Caspase 8No Activation

observed

No apoptosisobserved

NoPAR

activation

MAPK/ERKNot tested

MAPK p38Activationobserved

Reducedcytokine &

growth factor release

Inhibitedgrowth factor

signaling

Bik-RCD44 Bik

Potential change inintracellularcalcium

Reduced cell proliferationobserved

ReduceduPAR

activation

uPARLP

BikuPA

Fig 7. Impact of Bik on normal cell signaling.

Bik Ca+

KCa ChannelK+

K+

K+

K+

Ca+

Ca+

Ca+

Ca Channel

Bik

Bik

ReducedK + influx

Decreasedproteinexpression

Inhibited extrinsic death

factor & NF-KB signaling

TNFRFasL

0

2.5

5

7.5

10

12.5

15

17.5

20

22.5

Kid

ney

cell

x106

/mL

0 10 20 30Bik (mg/L)

40 50 60

0

50

100

150

200

250

300

350

400

450

AD

BP-

26 (n

g/m

L)

Figure 6. Kidney cell loss and ADBP-26 release uponexposure to Bik.