Dr. Steven GrantDr. Steven Grant

Dr. Roberto RosatoDr. Roberto Rosato

Jorge AlmenaraJorge Almenara

Stefanie CoeStefanie Coe

Dr. Allison Johnson, HHMI CoordinatorDr. Allison Johnson, HHMI Coordinator

APOPTOSIS : Programmed cell death

HDAC : Histone deacetylases are a class of enzymes

HDACI : HDAC Inhibitors are involved in anticancer activity

LAQ824 : HDAC Inhibitor

FLUDARABINE :A nucleoside analog

U937 CELLS : Leukemia cell lines

• A class of enzymes that remove acetyl groups fromA class of enzymes that remove acetyl groups from

lysine amino acid on a histone.lysine amino acid on a histone.

•Altered expression and mutations of genes encoding Altered expression and mutations of genes encoding

HDACs have been linked to tumor developmentHDACs have been linked to tumor development

• Inhibits cell growth in a dose- and time–dependent mannerInhibits cell growth in a dose- and time–dependent manner

• Induces upregulation of p21, caspase cascade activation and Poly Induces upregulation of p21, caspase cascade activation and Poly (ADP) ribose polymerase (PARP) cleavage(ADP) ribose polymerase (PARP) cleavage

• Inhibits Inhibits DNA synthesis

• Active against both dividing and resting cells. Active against both dividing and resting cells.

• One of the newer chemotherapy drugs which has been around One of the newer chemotherapy drugs which has been around and in use for a few years. and in use for a few years.

Dose response of LAQ824 and Fludarabine to U937

cancer cells

Study of Proteins in U937 cells treated with LAQ824 and Study of Proteins in U937 cells treated with LAQ824 and FludarabineFludarabine

• Treatments :Treatments :

- Control- Control

- LAQ824 (40 nM)- LAQ824 (40 nM)

- FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM)- FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM)

- L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7- L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7

• All LAQ824 treatments for 48 hoursAll LAQ824 treatments for 48 hours

• All Fludarabine (FD) treatments for 24 hoursAll Fludarabine (FD) treatments for 24 hours

• Cells and Cell CultureCells and Cell Culture

• Drugs and ChemicalsDrugs and Chemicals

• Flow CytometryFlow Cytometry

Percentage of Apoptosis in U937 cells induced by LAQ824 and Fludarabine

using DIOC6 dye

0102030405060708090

100

Treatment

Pe

rce

nta

ge

of

Ap

op

tos

is

Percentage of Apoptosis in U937 Cells induced by LAQ824 and Fludarabine

using 7AAD dye

0102030405060708090

Treatment

Per

cen

tag

e o

f Ap

op

tosi

s

(p21, PARP, Caspase-3, Caspase-7 and Caspase-9)(p21, PARP, Caspase-3, Caspase-7 and Caspase-9)

• TreatmentsTreatments

- Control- Control

- LAQ824 40 nM- LAQ824 40 nM

- FD 0.4 µM- FD 0.4 µM

- L/F 0.4- L/F 0.4

• Cells and Cell CultureCells and Cell Culture

• Drugs and ChemicalsDrugs and Chemicals

• Flow CytometryFlow Cytometry

• Western Blot AnalysisWestern Blot Analysis

p21

Control LAQ824 FD 0.4 L/F 0.4

PARP

C L40 4h 8h 16h 24h 28h 32h 40h 48h

C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h

Cleavage

Fragment

C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h

C L40 4h 8h 16h 24h 28h 32h 40h 48h

C L40 4h 8h 16h 24h 28h 32h 40h 48h

C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h

Cleavage

Fragment

LAQ824 and Fludarabine interact synergistically LAQ824 and Fludarabine interact synergistically

in U937 cancer cells inducing cell death by in U937 cancer cells inducing cell death by

apoptotic events.apoptotic events.