Sanjeewani T. Palayoor

Modulation of Radiation-Induced Apoptosis and G2/M Block in Murine T-Lymphoma Cells

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137Cs gamma irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of gamma radiation. We studied the effect of several pharmacological agents on the radiation-induced G2/M block and DNA fragmentation. The agents which reduced the radiation-induced G2/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G2/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G2/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G2/M block, and to use this effect in the treatment of patients with malignant disease.

Cell cycle alterations, apoptosis, and response to low-dose-rate radioimmunotherapy in lymphoma cells.

PURPOSE In an attempt to elucidate some aspects of the radiobiological basis of radioimmunotherapy, we have evaluated the in vitro cellular response patterns for malignant lymphoma cell lines exposed to high- and low-dose-rate radiation administered within the physiological context of antibody cell-surface binding. METHODS AND MATERIALS We used two different malignant lymphoma cell lines, a Thy1.2+ murine T-lymphoma line called EL-4 and a CD20+ human B-lymphoma line called Raji. Cells were grown in suspension cultures and exposed to high-dose-rate gamma radiation from an external 137Cs source or low-dose-rate beta radiation from DTPA-solubilized 90Y in solution. In some experiments, cells were pre-incubated with an excess of nonradioactive antibody in order to assess the effects of immunoglobulin surface binding during radiation exposure. Irradiated cells were evaluated for viability, cell-cycle changes, patterns of post-radiation morphologic changes, and biochemical hallmarks of radiation-associated necrosis and programmed cell death. RESULTS The EL-4 line was sensitive to both high-dose-rate and low-dose-rate irradiation, while the Raji showed efficient cell kill only after high-dose-rate irradiation. Studies of radiation-induced cell cycle changes demonstrated that both cell lines were efficiently blocked at the G2/M interface by high-dose-rate irradiation, with the Raji cells appearing somewhat more susceptible than the EL-4 cells to low-dose-rate radiation-induced G2/M block. Electron microscopy and DNA gel electrophoresis studies showed that a significant proportion of the EL-4 cells appeared to be dying by radiation-induced programmed cell death (apoptosis) while the Raji cells appeared to be dying primarily by classical radiation-induced cellular necrosis. CONCLUSION We propose that the unusual clinical responsiveness of some high and low grade lymphomas to modest doses of low-dose-rate radioimmunotherapy may be explained in part by the induction of apoptosis. The unusual dose-response characteristics observed in some experimental models of radiation-induced apoptosis may require a reappraisal of standard linear quadratic and alpha/beta algorithms used to predict target tissue cytoreduction after radioimmunotherapy.

PX-478, an inhibitor of hypoxia-inducible factor-1α, enhances radiosensitivity of prostate carcinoma cells

Overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF‐1α is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF‐1α inhibitor PX‐478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX‐478 for 20 hr, and HIF‐1α protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX‐478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX‐478 decreased HIF‐1α protein in PC3 and DU 145 cells. PX‐478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU‐145. PX‐478 (20 μmol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF‐1α and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 μmol/L concentration. PX‐478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of γH2AX expression in the irradiated cells. PX‐478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX‐478 as a clinical radiation enhancer. Published 2008 Wiley‐Liss, Inc.

Modifiers of Radiation-Induced Apoptosis'

EL4 murine lymphoma cells and F9 murine teratocarcinoma cells undergo apoptosis-like cell death after exposure to ionizing radiation. Apoptosis differs in several ways from classical clonogenic cell killing by radiation. We have tested several modifiers and radiomimetic agents in an effort to determine if the mechanism of induction of apoptosis by radiation differs from the mechanism of classical clonogenic cell killing by radiation, and consequently that these two end points of radiation action might be differentially modifiable. We found that internucleosomal DNA fragmentation, characteristics of apoptosis, can result from treatment of EL4 and F9 cells with agents that have diverse modes of action: tert-butyl hydroperoxide, diazenedicarboxylic acid bis(N,N-piperidide), and etoposide. Hydrogen peroxide did not induce internucleosomal DNA fragmentation at concentrations expected to be produced by the doses of ionizing radiation that we used. Radiation-induced DNA fragmentation could be inhibited by 3-aminobenzamide, dibutryl cyclic AMP, or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, although in this respect there appear to be marked differences between the cell lines.

Fractionated radiation alters oncomir and tumor suppressor miRNAs in human prostate cancer cells.

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity.

Fractionated Radiation Therapy Can Induce a Molecular Profile for Therapeutic Targeting

Abstract To examine the possibility of using fractionated radiation in a unique way with molecular targeted therapy, gene expression profiles of prostate carcinoma cells treated with 10 Gy radiation administered either as a single dose or as fractions of 2 Gy × 5 and 1 Gy × 10 were examined by microarray analysis. Compared to the single dose, the fractionated irradiation resulted in significant increases in differentially expressed genes in both cell lines, with more robust changes in PC3 cells than in DU145 cells. The differentially expressed genes (>twofold change; P < 0.05) were clustered and their ontological annotations evaluated. In PC3 cells genes regulating immune and stress response, cell cycle and apoptosis were significantly up-regulated by multifractionated radiation compared to single-dose radiation. Ingenuity Pathway Analysis (IPA) of the differentially expressed genes revealed that immune response and cardiovascular genes were in the top functional category in PC3 cells and cell-to-cell signaling in DU145 cells. RT-PCR analysis showed that a flexure point for gene expression occurred at the 6th–8th fraction and AKT inhibitor perifosine produced enhanced cell killing after 1 Gy × 8 fractionated radiation in PC3 and DU145 cells compared to single dose. This study suggests that fractionated radiation may be a uniquely exploitable, non-oncogene-addiction stress pathway for molecular therapeutic targeting.

NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells

Cyclooxygenase-2 (COX-2) plays a significant role in tumor development and progression. Nonsteroidal anti-inflammatory drugs (NSAID) exhibit potent anticancer effects in vitro and in vivo by COX-2-dependent and COX-2-independent mechanisms. In this study, we used microarray analysis to identify the change of expression profile regulated by a COX-2-specific NSAID NS-398 (0.01 and 0.1 mmol/L), a nonspecific NSAID ibuprofen (0.1 and 1.5 mmol/L) and RNA interference (RNAi)-mediated COX-2 inhibition in PC3 prostate cancer cells. A total of 3,362 differentially expressed genes with 2-fold change and P < 0.05 were identified. Low concentrations of NSAIDs and COX-2 RNAi altered very few genes (1-3%) compared with the higher concentration of NS-398 (17%) and ibuprofen (80%). Ingenuity Pathway Analysis was used for distributing the differentially expressed genes into biological networks and for evaluation of functional significance. The top 3 networks for both NSAIDs included functional categories of DNA replication, recombination and repair, and gastrointestinal disease. Immunoresponse function was specific to NS-398, and cell cycle and cellular movement were among the top functions for ibuprofen. Ingenuity Pathway Analysis also identified renal and urologic disease as a function specific for ibuprofen. This comprehensive study identified several COX-2-independent targets of NSAIDs, which may help explain the antitumor and radiosensitizing effects of NSAIDs. However, none of these categories were reflected in the identified networks in PC3 cells treated with clinically relevant low concentrations of NS-398 and ibuprofen or with COX-2 RNAi, suggesting the benefit to fingerprinting preclinical drug concentrations to improve their relevance to the clinical setting. [Mol Cancer Ther 2009;8(1):261–73]

Radiation-induced apoptosis in F9 teratocarcinoma cells.

We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis.

Radiation Survivors: Understanding and Exploiting the Phenotype following Fractionated Radiation Therapy

Radiation oncology modalities such as intensity-modulated and image-guided radiation therapy can reduce the high dose to normal tissue and deliver a heterogeneous dose to tumors, focusing on areas deemed at highest risk for tumor persistence. Clinical radiation oncology produces daily doses ranging from 1 to 20 Gy, with tissues being exposed to 30 or more daily fractions. Hypothesizing the cells that survive fractionated radiation therapy have a substantially different phenotype than the untreated cells, which might be exploitable for targeting with molecular therapeutics or immunotherapy, three prostate cancer cell lines (PC3, DU145, and LNCaP) and normal endothelial cells were studied to understand the biology of differential effects of multifraction (MF) radiation of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and phosphoproteome were analyzed. Significant differences were observed in the MF radiation exposures including those from the 0.5 Gy MF that produces little cell killing. As expected, p53 function played a major role in response. Pathways modified by MF include immune response, DNA damage, cell-cycle arrest, TGF-β, survival, and apoptotic signal transduction. The radiation-induced stress response will set forth a unique platform for exploiting the effects of radiation therapy as “focused biology” for cancer treatment in conjunction with molecular targeted or immunologically directed therapy. Given that more normal tissue is treated, albeit to lower doses with these newer techniques, the response of the normal tissue may also influence long-term treatment outcome. Mol Cancer Res; 11(1); 5–12. ©2012 AACR.

Radiation sensitivity of human carcinoma cells transfected with small interfering RNA targeted against cyclooxygenase-2.

Purpose: Cyclooxygenase-2 (COX-2) is considered a potential target for cancer therapy, because COX-2 levels are elevated in the majority of human tumors compared with the normal tissues. COX-2 inhibitors inhibit tumor growth and enhance radiation response in vitro as well as in vivo. However, the precise role of COX-2 in radiation response is not clear. The purpose of the present study was to investigate the in vitro radiosensitivity of tumor cells as a function of COX-2 expression. Experimental Design and Results: PC3 and HeLa cells express COX-2 protein constitutively. We silenced the COX-2 gene in these cells using small interfering RNA (siRNA). Transfection of PC3 cells with 100 nmol/L siRNA targeted against COX-2 resulted in reduction of COX-2 protein by 75% and inhibition of arachidonic acid–induced prostaglandin E2 synthesis by ∼50% compared with the vehicle control. In HeLa cells, 100 nmol/L COX-2 siRNA inhibited COX-2 protein expression by 80%. Cell cycle analysis showed that transfection with COX-2 siRNA did not alter the cell cycle distribution. Radiosensitivity was determined by clonogenic cell survival assay. There was no significant difference in the radiosensitivity of cells in which COX-2 was silenced compared with the cells transfected vehicle or with negative control siRNAs (enhancement ratio = 1.1). Conclusions: These data indicate that the in vitro radiosensitivity of tumor cells is minimally dependent on the cellular COX-2 status. Given that a number of potential mechanisms are attributed to COX-2 inhibitors for radiosensitization, specific intervention of COX-2 by RNA interference could help elucidate the precise role of COX-2 in cancer therapy and to optimize strategies for COX-2 inhibition.