Raymond E. Meyn

Radiation-induced apoptosis: Relevance to radiotherapy

Radiation-induced apoptosis is reviewed in terms of: (a) the identification of apoptotic and necrotic cells, (b) observations in vitro and in vivo of radiation-induced apoptosis, (c) genes controlling apoptosis, (d) evidence that the target may be the plasma membrane or nuclear DNA, (e) quantitative comparisons of apoptotic death and reproductive (clonogenic) death, (f) the importance of radiation-induced apoptosis in radiotherapy, and (g) studies of radiation-induced apoptosis that are needed. High priority should be placed on determining the molecular pathways that are important in the expression and modulation of radiation-induced apoptosis. Specifically, the events that modulate the apoptosis that occurs in interphase before the cell can divide should be distinguished from the events before division that modulate the misrepair of DNA damage, that results in chromosomal aberrations observed in mitotic cells, which in turn cause the progeny of the dividing cell with aberrations to die by either apoptosis or necrosis. Then, molecular events that determine whether a cell that divides with or without a chromosomal aberration will produce progeny that apoptose or necrose need to be identified. These considerations are important for determining how modulation of radiation-induced apoptosis will affect the ultimate clonogenic survival, and possibly genomic instability in the surviving progeny.

Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity

Purpose: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. Experimental Design: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. Results: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined γ-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, γ-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. Conclusions: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that γ-H2AX phosphorylation can be used as a predictive marker of radioresponse.

Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols

The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M−) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. Intracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M− medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M− medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.

Apoptosis in irradiated murine tumors

Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies.

Fifteen different murine tumours were evaluated with respect to the degree of apoptosis development that occurs in the tumour tissue in the first few hours following irradiation in vivo. Animals were killed at 3 or 6 h following irradiation with 0, 2.5, 10 or 25 Gy. Apoptosis was scored as percent aberrant nuclei by microscopic examination of histological sections made from the tumour specimens. Results showed that three of four mammary adenocarcinomas, one ovarian adenocarcinoma, and one lymphoma displayed at least 10% apoptotic cells after 25 Gy, whereas five sarcomas, three squamous cell carcinomas, and a hepatocarcinoma did not. The time courses and dose responses were similar in those tumours that responded. These data were compared with the known response of these same tumours when analysed using conventional assays. The tumours that did respond by significant apoptosis had longer specific growth delays and lower TCD50 (dose to cure 50% of animals) doses, thus suggesting that an acute apoptotic response following irradiation may be a feature of certain tumours that respond well to irradiation. Additionally, this analysis revealed heterogeneity in the apoptotic response both within an individual tumour specimen and among different tumour types. These observations of intra and intertumour heterogeneity are consistent with the idea that the propensity for apoptosis in tumours is genetically regulated.

Tumor Irradiation Increases the Recruitment of Circulating Mesenchymal Stem Cells into the Tumor Microenvironment

Mesenchymal stem cells (MSC) migrate to and proliferate within sites of inflammation and tumors as part of the tissue remodeling process. Radiation increases the expression of inflammatory mediators that could enhance the recruitment of MSC into the tumor microenvironment. To investigate this, bilateral murine 4T1 breast carcinomas (expressing renilla luciferase) were irradiated unilaterally (1 or 2 Gy). Twenty-four hours later, 2 x 10(5) MSC-expressing firefly luciferase were injected i.v. Mice were then monitored with bioluminescent imaging for expression of both renilla (tumor) and firefly (MSC) luciferase. Forty-eight hours postirradiation, levels of MSC engraftment were 34% higher in tumors receiving 2 Gy (P = 0.004) than in the contralateral unirradiated limb. Immunohistochemical staining of tumor sections from mice treated unilaterally with 2 Gy revealed higher levels of MSC in the parenchyma of radiated tumors, whereas a higher proportion of MSC remained vasculature-associated in unirradiated tumors. To discern the potential mediators involved in MSC attraction, in vitro migration assays showed a 50% to 80% increase in MSC migration towards conditioned media from 1 to 5 Gy-irradiated 4T1 cells compared with unirradiated 4T1 cells. Irradiated 4T1 cells had increased expression of the cytokines, transforming growth factor-beta1, vascular endothelial growth factor, and platelet-derived growth factor-BB, and this up-regulation was confirmed by immunohistochemistry in tumors irradiated in vivo. Interestingly, the chemokine receptor CCR2 was found to be up-regulated in MSC exposed to irradiated tumor cells and inhibition of CCR2 led to a marked decrease of MSC migration in vitro. In conclusion, clinically relevant low doses of irradiation increase the tropism for and engraftment of MSC in the tumor microenvironment.

Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol

The kinetics of taxol-induced mitotic arrest and apoptosis in murine mammary carcinoma MCA-4 and ovarian carcinoma OCA-I tumors were determined to establish a possible causative relationship between mitotic arrest and apoptosis and to see whether these cellular effects of taxol would correlate with the extent of its antitumor efficacy. Mice bearing 8-mm tumors in a hind leg were given taxol i.v. at a dose of 10–80 mg/kg. Both tumors responded to taxol by significant growth delay or transient regression; in general, the response was greater as the dose of taxol was increased. For kinetics studies the mice were treated with 60 mg/kg taxol given once when tumors were 8 mm in size or twice, with the second dose being given 3 days after the first. At various times ranging from 1 to 96 h after treatment with taxol, tumors were histologically analyzed to quantify mitotic and apoptotic activity. After a single dose of taxol, mitotic arrest was visible at 1 h, and the mitotic index increased with time to reach peak values of 36% in MCA-4 tumors and 22% in OCA-I tumors at 9 h. The index then declined to a baseline of 1%–3% at 3 days for MCA-4 tumors and 1 day for OCA-I tumors. Apoptosis followed mitotic arrest, beginning at the time of peak mitotic arrest, increasing to the highest level of about 20% at 18–24 h after treatment and gradually declining to the normal level of 3%–6% after 3–4 days. Nuclear material progressively condensed in mitotically arrested cells, culminating in the frank appearance of multiple apoptotic bodies. The change in cell morphology plus the dynamics of apoptosis development imply that a large percentage of tumor cells arrested in mitosis by taxol die by apoptosis. Kinetic analysis undertaken after the second dose of taxol showed a considerably lower percentage of cells arrested in mitosis as compared with that seen after a single dose, and the induction of apoptosis by the second dose was minimal. However, the antitumor efficacy of the second dose of taxol was similar to or better than that of the first dose, implying that in addition to mitotic arrest and apoptosis, there exist other mechanisms by which taxol exerts its antitumor action.

Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of γ-H2AX foci

Vorinostat (suberoylanilide hydroxamic acid) is the prototype of a family of hybrid polar compounds that can induce growth arrest in transformed cells and shows promise for the treatment of cancer. Vorinostat specifically binds to and inhibits the activity of histone deacetylases resulting in acetylation of nucleosomal histones and an activation of gene transcription. Because histone deacetylases modulate chromatin structure and gene expression, both of which can influence radioresponse, this study was designed to examine the capacity of Vorinostat to influence radiation response in human tumor cells and investigate the mechanism underlying these interactions. Vorinostat induced hyperacetylation of histone H4 in a dose-dependent manner. We tested its ability to radiosensitize three human tumor cell lines (A375, MeWo, and A549) using clonogenic cell survival assays. Clonogenic cell survival assay showed that Vorinostat significantly radiosensitized all three tumor cell lines, substantially reducing the surviving fraction at 2 Gy. We examined potential mechanisms that may contribute to the enhanced radiation response induced by Vorinostat. Vorinostat and radiation alone did not induce apoptosis in the melanoma cell line. However, enhanced apoptosis was observed when cells were exposed to both Vorinostat and radiation, suggesting that Vorinostat renders tumor cells more susceptible to radiation-induced apoptosis. Results from DNA damage repair analysis in cultured A375 cells showed that Vorinostat had a strong inhibitory effect on the nonhomologous end joining pathway after radiation. A detailed examination of the involvement of the DNA repair pathway following Vorinostat treatment showed that Vorinostat reduced the expression of the repair-related genes Ku70, Ku80, and Rad50 in A375 cells as detected by Western blot analysis. We also examined γ-H2AX phosphorylation as a predictive marker of radiotherapy response to Vorinostat and observed that the combination of Vorinostat and radiation caused a prolongation of expression of DNA repair proteins such as γ-H2AX. Overall, we conclude that Vorinostat enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation. [Mol Cancer Ther 2006;5(8):1967–74]

TP53 Disruptive Mutations Lead to Head and Neck Cancer Treatment Failure through Inhibition of Radiation-Induced Senescence

Purpose: Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a classification of TP53 mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and radiation sensitivity. Experimental Design: Seventy-four patients with HNSCC treated with surgery and postoperative radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and the in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using short hairpin RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and reactive oxygen species (ROS) assays were carried out. The effect of the drug metformin on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an orthotopic xenograft model. Results: Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly predicted LRR (P = 0.03). Cell lines with disruptive mutations were significantly more radioresistant (P < 0.05). Expression of disruptive TP53 mutations significantly decreased radiation-induced senescence, as measured by SA-β-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence. Examination of our patient cohort showed that LRR was decreased in patients taking metformin. Conclusions: Disruptive TP53 mutations in HNSCC tumors predicts for LRR, because of increased radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC with disruptive TP53, presaging the possibility of personalizing HNSCC treatment. Clin Cancer Res; 18(1); 290–300. ©2011 AACR.

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo.

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.