2049 Effect of BCL-2 on apoptosis associated with the inhibition of capacitative calcium entry

302

I. J. Radiation

Oncology

l

Biology

l

Physics

Volume

45, Number

3 Supplement

1999

sub-cultured into h-well-plate overnight and irradiated with 10 Gy of X-rays or 2, 3, or 6 neutron Gy (NGy). At 72 hours post irradiation, the surviving cells were washed, trypsinized, and counted using a Coulter Counter. Student t-test was used to compare the mean number of surviving cells with non-irradiated controls. Poly (ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation assays were performed to determine whether cells underwent apoptosis. Results: The surviving fractions for wild type PC3 cells and p” clones 1-5 receiving photon irradiation were 15% and 33%, 36%, 39%, and 54%, respectively. The differences in the mean number of surviving cells between the wild type group and each of the mutant groups were highly significant (p
2048 : Bray

JA’.

Mitochondrial respiratory chain function plays an important role in mediation The data also suggest that MRC may not be essential for neutron cell kill.

PREVENTION OF IRRADIATION-INDUCED VITRO BY OVEREXPRESSION OF THE Epperly

MW’,

Universi~ of Pittsburgh Inc., Burlingame, CA3

Defilippi Cancer

S’, Koe Ga, Liggitt Institute,

Pittsburgh,

APOPTOSIS MANGANESE D3, Luketich

PA, USA’;

JD’, University

IN HUMAN SUPEROXIDE Greenberger

of apoptosis

ESOPHAGUS DISMUTASE

induced

by photon

SECTIONS TRANSGENE

IN

JS’

of Pittsburgh,

Pittsburgh,

PA, USA’;

Valentis,

Objective/Purpose: Esophagitis is a major limiting factor in the treatment of lung cancer by radiation alone or in combination with carboplatin and Taxol. We have previously demonstrated that intraesophageal injections of MnSOD plasmid/liposome complex could prevent esophagitis in C3WHeNsd mice following 3500 cGy (Stickle et al, Fortieth Annual Meeting of ASTRO, Oct. 24-28, 1998, Phoenix, AZ). To determine whether MnSOD transgene protection also was demonstrable in human tissue, normal human esophagus sections were obtained from surgical excision specimens, then transfected with MnSOD plasmid/ liposome complex in vitro, irradiated, and examined for transgene expression and magnitude of irradiation-induced apoptosis. Materials & Methods: Normal esophagus sections were obtained from adjacent tissue in esophagectomy specimens from esophageal cancer patients. The sections were washed in PBS and transfected with either MnSOD (200 ug DNA) or alkaline phosphatase (200 or 20 ug DNA) plasmid/liposome complex by applying 100 ul to each section, followed 10 mins later with a second application. The sections were covered with tissue culture media consisting of a 50/50 mixture of DMEM and F12 media. At 24, 48 or 72 hrs later, sections of the esophagus transfected with alkaline phosphatase plasmid/liposome complex were removed, frozen in OCT, sectioned, and stained for detection of alkaline phosphatase. At 24 and 96 hrs, sections of esophagus treated with MnSOD plasmidlliposome complex were removed and assayed for biochemical MnSOD activity. Also at 24 hrs, control esophagus sections and MnSOD plasmid/liposome complex-treated esophagus sections were irradiated to either 1000 or 2000 cGy, then removed after 0, 24 or 48 hrs, frozen in OCT, sectioned, and stained for apoptosis using Promega’s Apoptosis Detection Kit. Results: Alkaline phosphatase expression in esophageal tissue ranged from (85.8%.55%) of cells in the squamous cell layer at 24 hrs to (28.4%-42.3%) at 72 hrs for sections of esophagus treated with either 200 or 20 ug alkaline phosphatase plasmid/liposome, respectively. There were 4.3 U of MnSOD biochemical activity/mg protein for untreated esophagus sections, compared to 6.7 and 5.0 U/mg at 24 and 96 hrs after MnSOD plasmid/liposome transfection (p=O.OOS and 0.007, respectively). There were fewer apoptotic cells in the esophagus sections treated with MnSOD plasmid/liposome complex at either 24 or 96 hrs. Twenty-four hrs after 1000 or 2000 cGy, control esophagus sections exhibited 53.7% and 65.6% apoptotic cells, compared to 7.5% and 25.0%, respectively, for the MnSOD plasmidiliposome complex-treated sections of esophagus (p
2049

EFFECT CALCIUM

OF BCL-2 ENTRY

Williams

SS, French

JN, Gilbert

Stanford

University,

Stanford,

ON APOPTOSIS M: Wallecaek

ASSOCIATED J, Knox

WITH

THE

MnSOD plasmid/liposomes and resulted in into the clinical use of MnSOD plasmid/

INHIBITION

OF CAPACITATIVE

SJ

CA, USA

Purpose: To define and characterize the role of capacitative calcium entry in the induction and to determine the effect of the anti-apoptotic protein, Bcl-2, on this process.

of apoptosis

in cultured

cell lines,

Introduction: Many apoptotic inducers such as thapsigargin and gamma-irradiation lead to elevations of cytoplasmic calcium. During calcium signaling, the initial rise in cytoplasmic calcium is due to release of intracellular stores. The potentiation of the calcium signal and replenishment of stores is dependent on the influx of extracellular calcium via capacitative calcium entry triggered by store depletion. Prolonged store depletion, induced by the ER membrane calcium ATPase inhibitor thapsigargin (Tg), induces apoptosis in HL60 and PW cell lines. Since capacitative calcium entry is essential for store refilling, studies were performed to determine whether or not apoptosis could be induced by SKF-96365 (SKF), a known inhibitor of the plasma membrane channels responsible for capacitative calcium entry. Furthermore, we have previously shown that Bcl-2 overexpression causes plasma membrane hyperpolarization, potentially altering trans.membrane ion fluxes. We therefore investigated the effect of Bcl-2 on capacitative calcium entry.

Proceedings

of the 41st Annual

ASTRO

Meeting

303

Materials and Methods: HL60 (promyelocytic leukemia) and PW (B-lymphoma) cell lines were transfected via retroviral gene transfer with the cos MSV-tk-Neor-hBcl-2 vector to produce Bcl-2 overexpressing transfectants as previously described (Naumovski and Cleary, Blood. 83:2261-2267,1994). Apoptosis was measured by flow cytometry of fixed, propidium iodide labeled cells to determine the fraction of cells with sub-diploid DNA content. Intracellular calcium measurements were performed using Fura2-AM loaded cells analyzed on a Hitachi F2000 Fluorescence spectrophotometer running specific calcium analysis software (Hitachi Instruments. Tokyo, Japan) by the method described by Hitachi. Results: In these experiments, SKF at 10.100 uM was demonstrated to be a potent inducer of apoptosis in the above cell lines. At 100 uM SKF, (15% viability was seen at 24 hours. The percentage of viable cells was increased by 3 to 20 fold in cells overexpressing the anti-apoptotic oncogene Bcl-2. In addition, spectrofluorometric calcium release assays showed that SKF-mediated inhibition of capacitative calcium entry was also partially inhibited by the overexpression of Bcl-2. Furthermore, ouabain, a specific inhibitor of the Na+/K+-ATPase pump, which normalizes the membrane potential of hyperpolarized Bcl-2 overexpressing cells, inhibited Tg-induced calcium influx to a greater extent in Bcl-2 transfected cell lines compared to control cell lines. The observation that inhibition of capacitative calcium entry can induce apoptosis may provide important insights into the roles of calcium homeostasis and Bcl-2 in apoptotic pathways, and may explain, in part, the association between hyperpolarization and radioresistance in Bcl-2 overexpressing cells. Conclusions: (1) Inhibition of capacitative calcium entry by SKF markedly decreased cell viability, with significantly (p < 0.05) more killing of control than Bcl-2 overexpressing cells. (2) In calcium release assays, SKF markedly reduced capacitative calcium influx to a greater extent in control as compared to Bcl-2 transfected cells. (3) Ouabain-induced membrane depolarization reduced capacitative calcium entry to a lower level in control cells compared to Bcl-2 overexpressing cells. Bcl-2 transfected cells, in the presence of ouabain, had levels of capacitative calcium entry which were similar to parental cells. (4) Pharmacologic modulation of capacitative calcium entry may provide a novel strategy for developing new anti-cancer therapies and/or increasing the efficacy of current chemo- and radiotherapeutic regimens.

2050

FUNCTIONAL DOMAIN DISEASE, NIJMEGEN

Ito A’, Tauchi Hiroshima Hiroshima

HZ, Matsuura

University University,

OF THE BREAKAGE

S2, Fujita

UNDERLYING SYNDROME

K’, Morishima

School of Medicine, Hiroshima, Japan’

Hiroshima,

GENE

K’, Nakamura Japanl;

FOR

A’, Akagi

Research

Institute

RADIATION Y’, Hirokawa Radiation

SENSITIVE Y’, Ito K’, Biology

HUMAN Komatsu

K2

and Medicine,

Background: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, combined immunodeficiency, and a high incidence of lymphoid cancer. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell cycle regulation after irradiation. The NBS1 protein consists of 754 amino acids and it shows a weak homology to the yeast Xrs2 protein in the N-terminus region, It has been reported that the NBS1 protein interacts with MREll and that a RADSO/MREl l/NBS1 complex or foci can be seen in the nucleus after irradiation. It has also been suggested that this complex may be active in processing the end of DNA double-strand breaks to permit non-homologous end-joining and also for homologous recombination. Purpose:

We have

assayed

for functionally

important

domains

of the NBS1

protein,

Materials and methods: Full-length or deleted NBS1 cDNA was transfected into NBS patient fibroblasts by electroporation. Stable transfectants were selected and the surviving fraction after y-ray-irradiation was determined by colony formation assay, Immunohistochemical analysis was also performed using human NBS1 antibody. Results: We found that several C-terminus region of the protein

deletion mutants was deleted.

were

able to restore

radiation

resistance

in NBS

cells

when

a part

of

Conclusion: Our results suggest that whole NBS 1 cDNA is not necessary to complement the radiation sensitivity and that there must be a functionally important domain in C-terminus region of the protein. The relationship deletion mutants, restoration of radiation resistance and NBS 1 foci formation will be discussed.

of NBS cells. between NBS

205

1

IN

Casper

D, Pidel A, Tribius

Montejiore

ATM PROTEIN EXPRESSION PRIMARY GLIOBLASTOMA

Medical

Center,

CORRELATES CELLS

WITH

INTRINSIC

RADIOSENSITIVITY

S Bronx,

NY, USA

Objective: Glioblastoma multiforme (GBM) is one of the most resistant malignancies to radiation. In contrast, cells derived from individuals with ataxia telangiectasia (AT), possessing mutations in the ATM gene, demonstrate increased sensitivity to ionizing radiation. The aim of our study was to determine whether ATM protein expression plays a role in glioblastoma cells in response to radiation. Materials & Methods: Fresh tumor specimens obtained from the operating room at the Montefiore Medical Center were adapted to tissue culture conditions, and characterized by immunohistochemistry for glial markers. All experiments were performed in triplicate with cells of low passage number (< 10). Clonogenic survival experiments were performed in 6 well plates, where cells were seeded at an appropriate density and irradiated with 2 Gy, resulting in about 50 colonies per well after 3 to 4 weeks of incubation. Clonogenic capacity in the absence of radiation ranged from 3 to 5%, consistent with the mitotic index of glioblastomas observed in situ. Thymidine incorporation assays were performed in 12 well plates with a 15.hour 3H-thymidine incubation period. Western blots were performed and probed with antibodies to ATM, ~53, ~21, and actin (Oncogene Sci.). Blots were processed using standard procedures and developed with ECL plus (Amersham). Signals were quantified on a BioImager (Millipore). Results: Western Blot analysis demonstrated that ATM protein was detected in all primary gliomas. When normalized against values for actin we also showed differential levels of expression. We observed that established human glioma cell lines demonstrated much lower levels of ATM protein expression compared to the primary gliomas (mean optical density of ATM/actin: 0.29 vs. 0.74). Clonogenic assays yielded a wide range of SF,‘s (0.47.0.94), consistent with the radioresistant