70 Radiosensitization and chemosensitization of ovarian carcinoma by induction of apoptosis via recombinant adenovirus encoding bax

I. J. Radiation

182

Oncology

l

Biology

l

Physics

Volume

45, Number

3 Supplement

1999

Pain before and after treatment was scored as level I to V (I: no pain, no medications. II: occasional pain, not requiring medication. III: some pain, adequately controlled by medication. IV: pain, not adequately controlled by medication. V: severe pain. no pain relief). At a mean follow-up time of 9.1 months (range 2-23), pain improvement was found in 97.7% (42/43). 34.9% (15/43) had complete pain relief (level I), 7.0% (3/43) level II, 34.9% (15/43) level III, and 20.9% (9/43) level IV. 1 patient reported no pain relief (level V) after GK. The mean time to onset of pain relief was 30.6 days (range O-l 15), and to maximal relief 77.8 days (range O-356). 5 reported complete pain relief within 1 day of GK. The mean improvement in pain score was 2.4 levels. Among the 25 patients remaining on medications (levels III-V)? 36% (9/25) have decreased their medication intake by ~50%. We found no clear correlation between degree of response and prior treatment. 50% (1 l/22) with prior invasive treatment had a dramatic response (level I or II) vs 33.3% (7/21) without prior treatment. Multiple sclerosis (MS) does not appear to adversely influence response. 37.5% (318) of patients with MS had a dramatic response vs 42.9% (15/35) without MS. Complications have been limited to facial sensory loss. 7.0% (3/43) had added facial numbness, rated as bothersome in none. No patient has developed anesthesia dolorosa, cornea1 anesthesia or keratitis. 3 patients, each at pain level IV, have had 2 GK treatments, at a mean interval of 10.7 months. All 3 had additional pain relief following the second procedure. Follow-up in these 3 cases is limited to 4.0 months, but no complications have been encountered. Conclusion: GK radiosurgery is effective treatment for trigeminal neuralgia. either as primary therapy, or for recurrence after prior invasive treatment. It carries a low risk of facial hypesthesia, and, in our experience, is not associated with other morbidity. We have thus recently initiated a protocol using 40 Gy, replacing than the above 35 Gy, to the trigeminal root entry zone. Although longer follow-up will be required for a thorough appraisal, GK appears very promising. We currently include GK among the appropriate options for TN patients who have failed optimal medical management, with or without prior invasive neurosurgical procedures.

69

TAXOL RESTORES RADIATION RESISTANT LYMPHOMA CELL

Nguyen

LN,

The University

Munshi

A, Hobbs

of Texas M.D.

ML,

Story

Anderson

MD, Cancer

INDUCED LINE Meyn

APOPTOSIS

IN A BCL-2

EXPRESSING

RADIATION

RE

Center,

Houston,

TX, USA

Purpose/Objective: To restore radiation induced apoptosis in a bcl-2 expressing, radiation resistant murine lymphoma cell line (LY-ar) by pretreatment with Taxol. Since this cell line also has high intracellular levels of glutathione (GSH), reportedly due to the bcl-2 expression and involved in the cell’s antioxidant functions, Taxol treatment was correlated with GSH levels. Materials and Methods: LY-ar cells were pretreated with Taxol and then were irradiated with 5 Gy. Apoptosis was measured by DNA fragmentation index 6 hrs later. Dose response and time course experiments were performed. Intracellular glutathione levels were measured after Taxol treatment. Cell survival analysis was performed for various Taxol levels ? 5 Gy. Results: LY-ar cells pretreated with 0 nM, 10 nM, 25 nM and 50 nM Taxol for 20 hrs underwent apoptosis at 2%, 15%, 25%, and 33%, respectively. With the addition of 5 Gy irradiation, LY-ar cells apoptosis increased to 4%; 30%, 49%, and 57%. Maximal apoptosis was detected with a Taxol pretreatment time of 20 hrs. Intracellular GSH levels decreased progressively with Taxol pretreatment times: 1.95, 1.88, 0.73 pg/lO’ cells with control, 10 hrs, 20 hrs, respectively. Surviving fraction (SF) with 0 nM, 10 nM, 25 nM, and 50 nM Taxol and 0 Gy were 1.0: 0.33, 0.05, and 0.03, respectively. SF with 0 nM, 10 nM, 25 nM, and 50 nM Taxol and 5 Gy were 0.005, 0.002, 4 x 10m6, and 8 x 10m7, respectively. Conclusion: Radiation induced apoptosis levels of intracellular GSH. Cell survival

70 Arafat DT’

RADIOSENSITIZATION AND INDUCTION OF APOPTOSIS WO’-‘,

Gomez-Navarro

J’, Xiang

in LY-ar cells was restored by pretreatment with Taxol. This correlated with lowered analysis suggests a supra-additive effect from Taxol and irradiation on cell survival.

CHEMOSENSITIZATION VIA RECOMBINANT J’: Barnes

M3, Alvarez

OF OVARIAN CARCINOMA ADENOVIRUS ENCODING BAX RD3, Siegal

GP4, Buchsbaum

BY

D’, Stackhouse

M5, Curie1

Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA’; Clinical Oncology Department, Alexandria University, Alexandria , Egyp?; Departments of Obstetrics and Gynecology, University qf Alabama at Birmingham, Birmingham, AL, USA’; Departments of Pathology, Cell Biology, and Surgery, University of Alabama at Birmingham, Birmingham, AL, USA4; Radiation Biology, University of Alabama at Birmingham, Birmingham, AL, USA5 Purpose: Stable integration of pro-apoptotic Bax gene favors death in cells otherwise resistant to ionizing radiation and/or chemotherapy. We hypothesized that transient expression of Bax via adenoviral-mediated gene delivery could sensitize radiationand chemotherapy-refractory cells to radiotherapy and chemotherapy, respectively. Materials and Methods: We have generated an inducible recombinant adenovirus encoding Bax (Ad/Bax) using the cre-loxP system. To determine its radiosensitization effect, human ovarian cancer cells were infected with Ad/Bax and its inducing adenovirus vector encoding Cre recombinase (Ad/Cre), and were irradiated 24 hr later. Cell death was evaluated by crystal violet staining, fluorescence-activated cell sorter (FACS) analysis of Annexin-V and propidium iodide, and colony-formation assay. To further characterize the mechanism of death, cell morphology was examined by nuclear staining with Hoechst 33258. To determine its chemosensitization effect, monolayers of immortalized ovarian cancer cell lines (OVCAR3, OV4, PA-l, SKOV3.ip1, and SW626) and primary ovarian cancer cells obtained from ascites of several patients, were transfected with Ad/Bax and grown in the presence or absence of the drugs cisplatin or paclitaxel. Cell death was evaluated by the MTS quantitative calorimetric assay. Results: Ionizing radiation induced the killing of 7.7?3.7% of treated cells at 24 hr and of 1620.2% at 72 hr. When treatment with Ad/Bax and Ad/Cre was added, killing increased from 22.8i 1.7% at 24 hr to 3 1.1 +0.8% at 72 hr. In a colony-formation assay, the number of colonies after radiation or viral treatment alone was reduced around 50%. In contrast, the combination of

Proceedings

of the 41st Annual

ASTRO

Meeting

183

both achieved a decrease of 98%. The Do was 4.8 Gy for radiation alone, consistent with the radioresistance observed in other epithelial cell lines, In contrast, the Do was 1.2 Gy for the combined treatment of radiation and Bax. Phenotypical changes and FACS analysis suggested that Bax-mediated radiosensitization occurs through induction of both apoptosis and necrosis. In the chemotherapy arm, we obtained similar results. In effect, the combined treatment with both viruses and drugs significantly enhanced cytotoxicity. Specifically, the LD50 for cisplatin and paclitaxel were decreased 5-10 fold. Interestingly, the mechanism associated with Bax-mediated cell death appeared to be p53-independent. In addition, Bax-mediated killing is independent of endogenous Bcl-2 and Bax levels. Conclusion: Radiosensitization and chemosensitization of human ovarian cancer cells via recombinant adenovirus encoding Bax is feasible. The efficiency of adenoviral vectors in the in vivo context may allow exploring the therapeutic potential of this novel gene-based approach for combined multi-modality treatment. Thus, adenoviral-mediated delivery of Bax might provide a way to overcome the resistance to both radiation and chemotherapeutic drugs, and ultimately improve the efficacy of radiotherapy and chemotherapy in ovarian cancer patients.

71

INHIBITION

Sarkaria Mayo

OF THE

JN. Busby Foundation,

EC, Kamitz Rochester,

CHECKPOINT

KINASE,

CHKl,

BY THE

RADIOSENSITIZING

AGENT

UCN-01

LM MN,

USA

Purpose/Objective: Cells with an intact p53-signaling pathway are resistant to the radiosensitizing effects of the kinase inhibitor, 7-hydroxystaurosporine (UCN-01). Combined with the limited toxicity observed with UCN-01 in single-agent Phase I clinical trials. these data suggest that UCN-01, or a related compound, may be useful radiosensitizers that preferentially sensitize tumors with non-functional p53 while sparing normal tissues. The abrogation of the G2 checkpoint by UCN-01 presumably underlies the mechanism of radiosensitization by this drug. Recent studies have identified several kinases that are involved in control of the G2 checkpoint in human cells, including ATM, hChk1 and hChk2. The purpose of this study was to determine whether the activities of any of these checkpoint kinases were sensitive to UCN-01 at concentrations associated with abrogation of the G2 checkpoint and radiosensitization. Materials and Methods: The kinase activities of recombinant hChk1 and hChk2 and endogenous ATM were measured in immune-complex kinase assays in the presence of graded concentrations of UCN-01. The integrity of the gamma-radiationinduced G2 checkpoint was assessed by flow cytometry in A549 lung adenocarcinoma cells treated with UCN-01. Cells were pulsed with bromodeoxyuridine (BrdU) immediately prior to irradiation and drug treatment and harvested 14 hours later. Cells were stained with propidium iodide and a FITC-conjugated anti-BrdU antibody prior to analysis by flow cytometry. The effect of UCN-01 on the electrophoretic mobility of Cdc25C was assessed in irradiated K562 erythroblastoid leukemia cells. Following exposure to UCN-01, cells were lysed in a 1% NP-40.containing buffer. Detergent-soluble proteins ‘were separated by SDS-PAGE, transferred to a PVDF membrane and immunoblotted with a polyclonal Cdc25C antisera. Results: UCN-01 was a potent inhibitor of recombinant hChk1 in immune complex kinase assays (IC50 = 10 &I). In contrast, up to 1 PM UCN-01 had no effect on either hChk2 or ATM kinase activities. UCN-01 was equally effective at abrogating the gamma-radiation-induced G2 arrest of A549 cells irradiated in S-phase (BrdU-positive cells). Fourteen hours following exposure to 5 Gy and 30 nM UCN-01, 9% of the BrdU-positive cells remained in G2/M compared to 76% of BrdU-positive cells treated with radiation alone. To assess the integrity of the G2 checkpoint signaling pathway downstream of Chkl, we examined the effect of UCN-01 on the electrophoretic mobility of the cyclinBl/cdc2-activating phosphatase, Cdc25C. Treatment of K562 cells with 1 FM UCN-01, 16 hours following exposure to 8 Gy, resulted in release of cells from a G2 arrest and disappearance of a Cdc25C species with reduced mobility on SDS-PAGE. We suggest that loss of this mobility shift is due to the inhibition of hChk1 phosphorylation of Cdc25C at Ser-216. The results of ongoing studies addressing this hypothesis and the dose-response of this effect will be presented. Conclusion: UCN-01 inhibits hChk1 kinase activity at concentrations associated with abrogation of the radiation-induced G2 checkpoint. These data suggest that hChk1 may be the relevant molecular target responsible for the radiosensitizing effects of the checkpoint inhibitor UCN-01.

72 Curry

HEAT SHOCK INHIBITS RADIATION-INDUCED INHIBITION OF I-kB KINASE (IKK): POSSIBLE RADIOSENSITIZATION HA.

Washington

Botero

A, Clemens

Univer-sity

School

RA,

Shah S, Gius

of Medicine,

ACTIVATION OF NF-LB DNA-BINDING NOVEL MECHANIM OF HYPERTHERMIC

VIA

D

St. Louis,

MO,

USA

Purpose: Mammalian cells activate several specific early response genes that function as transcription factors that are thought to be directly involved in the cellular response to IR. We have previously shown that heat shock (hyperthermia) also modifies the activity of some of the same transcription factors as IR. Furthermore, heat shock significantly alters how cells respond to IR and the addition of heat to a regimen of radiotherapy effectively sensitizes cells to the lethal effects of IR. Thus, we hypothesize that one mechanism whereby heat shock alters the cellular response to IR involves heat alterations in IR-induced signaling pathways regulating nuclear transcription factors. The objective of the proposed work is to determine if there is a coupled interaction between IR, heat shock, and the regulation of signal transduction pathways activating the nuclear transcription factor, NF-kB. Materials and Methods: HeLa cells were maintained in a MEM media with 5% FCS. Electromobility shift were performed with a 30.bp oligomer containing a NF-kB binding site. Subcellular fraction was preformed NP-40 lysis method and Western analysis was performed using anti-NF-kB and I-kB antibodies on the nuclear cell fractions. Immunoprecipitation (IP) Westerns were preformed using protein A and anti-I-kB followed by with an anti-phosphoserine antibody. IKK assays were preformed by IP the IKK complex with an anti-IKKa

assays (EMSA) using a standard and cytoplasmic immunoblotting antibody and