AACR: CLL Cells Undergo Apoptosis with Halogenated ATP

[Guest guest]

AACR Abstract Number: 2070

Bioenergetically compromised and transcriptionally challenged chronic

lymphocytic leukemia cells undergo apoptosis with halogenated ATP

Kumudha Balakrishnan, Ayers, J. Keating, Varsha Gandhi.

UT M. D. Cancer Center, Houston, TX.

Chronic lymphocytic leukemia (CLL), an incurable disease, results

from the accumulation of small mature, slowly dividing, monoclonal B-

lymphocytes.

Accumulation of these leukemic lymphocytes is due to the presence of

antiapoptotic factors such as Mcl-1. The drug strategy of CLL is

therefore challenging and considerable effort has been directed

towards novel therapeutic agents, ultimately aimed on the critical

survival factors.

CLL cells are quiescent, but are dependent on cellular bioenergy and

transcription of survival genes. Hence, dissipation of cellular

bioenergy and inhibition of RNA synthesis may impose a lethal effect

on quiescent cells.

Our previous investigations with multiple myeloma cell lines

demonstrated that incubation with halogenated adenosine analog (8-Cl-

Ado) results in accumulation of 8-Cl-ATP with a concomitant decline

in intracellular ATP pool. The analog was preferentially incorporated

into mRNA, which inhibits RNA synthesis by premature transcriptional

chain termination.

In the present study, primary leukemic lymphocytes were isolated by

ficoll gradient method from peripheral blood obtained from 32

patients. Cells were incubated with 8-Cl-Ado at different dose and

time periods and the accumulation of triphosphate as well as the

concomitant decline in ATP were measured.

When cells from CLL patients (n=12) were incubated up to 4 hr with

different concentrations (1, 3, 10, 30 µM) of 8-Cl-Ado there was a

significant dose-dependant increase in 8-Cl-ATP accumulation;

concentrations were 15, 45, 113 and 188 ìM, respectively. At 10 µM 8-

Cl-Ado, there was a time-dependant increase in the accumulation of 8-

Cl-ATP, with parallel depletion of ATP. At 7 hr, the intracellular

level of analog triphosphate was median 379 µM and ATP was a median

1100 µM, a decline of 40-80% of control value (n=7).

To determine if accumulation of 8-Cl-ATP reflects on the analog-

mediated inhibition of global RNA synthesis, leukemic cells from 5

patients were tested for uridine incorporation. There was a 60-70%

inhibition of RNA synthesis at 4 hr. Real time RT-PCR assay was used

to test the effect of 8-Cl-Ado on survival genes at mRNA and protein

levels. There was a decline in Mcl-1 protein starting at 3 hr with 10

ìM of 8-Cl-Ado. In contrast, no effect was observed on Bcl-2 protein;

this differential effect may be due to a relatively short half-life

of Mcl-1 protein (t1/2 = 20 min-3 hr) compared to Bcl-2 protein (t1/2

=10 –14 hr). As expected these aspects were associated with the

induction of apoptosis starting as early as 2 hr (PARP cleavage)

after incubation with 10 µM 8-Cl-Ado.

In conclusion, 8-Cl-Ado affects cellular bioenergy and targets RNA

transcription. These bioenergetically compromised and

transcriptionally challenged quiescent leukemia cells undergo

apoptosis after loss of survival proteins. We plan to test this

strategy for the treatment of patients with CLL.

Presenter: Kumudha Balakrishnan

Affiliation: UT M. D. Cancer Center, Houston, TX; E-mail:

kbalakr@...

Copyright © 2004 American Association for Cancer Research. All rights

reserved. Citation information: Proceedings of the AACR, Volume 45,

March 2004.