Monoclonal antibody to HER-2/neu receptor enhances radiosensitivity of esophageal cancer cell lines expressing HER-2/neu oncoprotein

PURPOSE: The role of HER-2/neu in the response of esophageal cancer to radiation is not well known. The purpose of this study was to evaluate the effect of an anti-HER-2/neu antibody trastuzumab on the proliferation, cell cycle distribution, and radiosensitivity of esophageal cancer cell lines. EXPERIMENTAL DESIGN: Expression of HER-2/neu protein by four esophageal squamous cancer cell lines (KE4, TE8, TE9, and TE10) and an esophageal adenocarcinoma cell line (SKGT4) was assessed using immunohistochemical (IHC) analysis and flow cytometry. We also evaluated HER-2/neu oncogene expression by fluorescence in situ hybridization. As a control for HER-2/neu protein expression and gene amplification, breast cancer cell lines (MCF7, MDA MB175VII, and SKBR3) were also examined. The cytotoxity of trastuzumab (0.1-200 microg/mL) was estimated by the MTT assay, and the cell cycle distribution was determined by flow cytometry. The effect of 10 microg/mL trastuzumab combined with radiation was assessed by a clonogenic assay. RESULTS: Flow cytometry and IHC revealed that two esophageal cancer cell lines (TE9 and SKGT4) showed HER-2/neu expression (IHC 1+ and mean fluorescence intensity of 11-20), while the other esophageal cancer cell lines were negative for HER-2/neu expression. Although trastuzumab alone had no effect on the esophageal cancer cell lines, the combination of 10 microg/mL trastuzumab with radiation showed a synergistic effect on the HER-2/neu expressing cell lines. CONCLUSIONS: This study suggested that trastuzumab plus irradiation may be effective for the treatment of esophageal cancers, including adenocarcinoma and squamous cell cancer with HER-2/neu expression.     

Trichostatin A enhances radiosensitivity and radiation-induced DNA damage of esophageal cancer cells

BackgroundTrichostatin A (TSA) is emerging as a potential component of anticancer therapy. In this study, we aimed to identify the radiosensitizing effects of TSA in esophageal squamous carcinoma cell lines and identify the genomic alteration of histone acetylation associated with TSA treatment.MethodsEC109 and KYSE450 cells were pretreated with TSA (0.1 µM) for 12 hours prior to irradiation, and the cell viability, flow cytometry, and comet assays were performed to analyze cell growth, cell apoptosis, and DNA damage, respectively. Chromatin immunoprecipitation sequencing (ChIP-Seq) was performed to identify the acetylation sites of histone H3 lysine 9 (H3K9), which was altered by TSA.ResultsOur data showed that TSA could sensitize esophageal cancer cells to radiation by inducing cell cycle arrest and increasing cell apoptosis. DNA damage induced by radiation was enhanced by TSA treatment. In addition, a total of 105 differential peak-related genes were found to be associated with TSA treatment, which was identified using ChIP-Seq with specific antibodies against acetylated histone H3K9.ConclusionsOur data suggest that pretreatment with TSA can enhance ionizing radiation-induced DNA damage of esophageal cancer cells, which was associated with the altered histone modification of whole genome. TSA has potential implications for clinical use in increasing the anticancer efficacy of radiation.     

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.     

HER-2/neu oncogene expression in advanced breast cancer

Tumor tissue from patients with advanced breast cancer was analyzed for HER-2/neu and p53 expression. The tissue samples from primary tumor and from axillary lymph nodes or distant metastases from 118 breast cancer patients were obtained. Sections from formalin-fixed, paraffin-embedded materials were immunostained for HER-2/neu and p53 oncoprotein expression. Staining results were correlated with survival times and disease-free survival times, flow cytometric synthesis phase fractions, and DNA ploidy. No correlation could be found between HER-2/neu and p53 or any other tested factor, but grade I primary cancers that were positive for HER-2/neu showed a tendency for better outcome. The HER-2/neu staining of the metastases was independent of the staining of the primary tumor. HER-2/neu can be used as a prognostic marker for advanced breast cancer, when the primary tumor is well differentiated.     

HER-2/neu oncogene protein and prognosis in breast cancer

Amplification of the HER-2/neu oncogene was recently reported to predict poor clinical outcome in node-positive breast cancer patients. Since expression of the oncogene as its protein product might be even more closely related than gene amplification to disease progression, we have now examined levels of the HER-2/neu oncogene protein for its prognostic potential in both node-positive and node-negative breast cancer. Using Western blot analysis, levels of this protein were determined in 728 primary human breast tumor specimens. We examined relationships between this protein and other established markers of prognosis, as well as clinical outcome. In node-negative patients (n = 378), the HER-2/neu protein failed to predict disease outcome. However, in node-positive patients (n = 350), those patients with higher HER-2/neu protein had statistically shorter disease-free (P = .0014) and overall survival (P less than .0001) than patients with lower levels of the protein. Higher HER-2/neu protein was found in tumors without estrogen receptor (ER) (P = .02) or progesterone receptor (PgR) (P = .0003), and in patients with more than three positive lymph nodes (P = .04). A significant correlation between levels of the HER-2/neu gene protein and amplification of the gene itself was also found (n = 48, P less than .001). Multivariate analyses in these patients showed that the HER-2/neu protein is a significant independent predictor of both the disease-free and the overall survival in node-positive breast cancer, even when other prognostic factors are considered.     

Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing human breast epithelial cells

In the multistep progressive pathogenesis of human breast cancer, comedo ductal carcinoma in situ (DCIS) represents a preinvasive precursor lesion for therapy resistant invasive cancer. Human tissue derived cell culture models exhibiting molecular similarities to clinical DCIS facilitate an important preclinical mechanistic approach for evaluation of preventive efficacy of natural and synthetic chemopreventive compounds. Natural phytochemicals present in fresh fruits, vegetables and grain products are likely to offer protection against cancer. The clinical efficacy of these natural phytochemicals, however, depends on extrapolation, and is therefore equivocal. The present study determined whether the natural soy isoflavone genistein (GEN) inhibited aberrant proliferation in 184-B5/HER cells (a model for human comedo DCIS) and identified possible mechanisms responsible for its efficacy. Human reduction mammoplasty derived HER-2/neu oncogene expressing preneoplastic 184-B5/HER cells represented the experimental system. Flow cytometry and cellular epifluorescence based assays were utilized to quantitate the alterations in cell cycle progression, cellular apoptosis, and in the status of cell cycle regulatory and apoptosis-associated gene product expression. The 184-B5/HER cells exhibited specific immunofluorescence to p185HER, p53, EGFR, but not to ERalpha, thus resembling comedo DCIS. Treatment of 184-B5/HER cells with GEN resulted in a dose-dependent decrease in the viable cell population, increase in the G0/G1:S + G2/M ratio and enhancement of sub G0/G1 (apoptotic population). Exposure to the maximum cytostatic 10 microM dose of GEN down-regulated HER-2/neu mediated signal transduction as evidenced by a 73.9% decrease (p=0.001) in p185HER specific, and a 89.8% decrease (p=0.001) in phosphotyrosine specific immunofluorescence. The increase in G0/G1:S + G2/M ratio in response to the treatment with 10 microM GEN was associated with a 85.5% decrease (p=0.001) in immunoreactivity to PCNA and a 128.6% increase (p=0.004) in immunoreactivity to the cyclin dependent kinase inhibitor p16INK4. The induction of apoptosis by GEN was associated with a 52.8% decrease (p=0.001) in the immunoreactivity to antiapoptotic Bcl-2 and with a 195.9% (p=0.001) increase in the immunoreactivity to proapoptotic Bax. Thus, preventive efficacy of GEN in HER-2/neu+/ER- 184-B5/HER cells may be due to its ability to down-regulate HER-2/neu mediated signal transduction, increase the expression of the cyclin dependent kinase inhibitor p16INK4, and induce Bcl-2 dependent apoptosis. These data provide evidence that GEN may be a potential chemopreventive lead compound for human comedo DCIS. The 184-B5/HER cells, may therefore, provide a high throughput mechanistic bioassay to identify new chemopreventive agents for human breast cancer.     

Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells.

Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation.     

Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer

Immunomodulatory strategies, such as antibody therapy and cancer vaccines, are increasingly being considered as potential adjuvant therapies in patients with advanced stage breast cancer to either treat minimal residual disease or prevent relapse. However, little is known concerning the incidence and magnitude of the pre-existent breast cancer specific immune response in this patient population. Using the HER-2/neu oncogenic protein as a model, a well-defined tumor antigen in breast cancer, we questioned whether patients with advanced stage HER-2/neu overexpressing breast and ovarian cancers (III/IV) had evidence of pre-existent immunity to HER-2/neu. Forty-five patients with stage III or IV HER-2/neu overexpressing breast or ovarian cancer were evaluated for HER-2/neu specific T cell and antibody immunity. Patients enrolled had not received immunosuppressive chemotherapy for at least 30 days (median 5 months, range 1–75 months). All patients were documented to be immune competent prior to entry by DTH testing using a skin test anergy battery. Five of 45 patients (11%) were found to have a significant HER-2/neu specific T cell response as defined by a stimulation index 2.0 (range 2.0–7.9). None of eight patients who were HLA-A2 had a detectable IFN secreting T-cell precursor frequency to a well-defined HER-2/neu HLA-A2 T cell epitope, p369-377. Three of 45 patients (7%) had detectable HER-2/neu specific IgG antibodies, range 1.2–8.9g/ml. These findings suggest that patients with advanced stage HER-2/neu overexpressing breast and ovarian cancer can mount a T cell and/or antibody immune response to their tumor. However, in the case of the HER-2/neu antigen, the pre-existent tumor specific immune response is found only in a minority of patients.     

HER-3基因表达对HER-2阳性型乳腺癌细胞放射敏感性的影响及作用机制

目的:探讨人表皮生长因子受体-3（human epidermal growth factor receptor-3,HER-3）表达对HER-2阳性型乳腺癌细胞放射敏感性的影响。方法:使用慢病毒颗粒感染HER-2阳性型乳腺癌细胞系（AU-565、SKBR-3）构建HER-3基因敲低细胞系模型,蛋白质印迹法（Western blot）及实时定量PCR(RT-qPCR)技术检测感染的效率。克隆形成实验测定不同组别乳腺癌细胞放射增敏效果。将HER-3敲低组与对照组细胞分别给予X线照射（6 Gy,6 MV,源皮距100 cm）,流式细胞术测定不同组别细胞凋亡率以及细胞周期分布的变化。免疫荧光法检测细胞辐射后不同时间段的γH2AX焦点数,评估辐射后DNA损伤情况,蛋白质印迹法检测细胞周期调控相关蛋白CyclinB1的表达。结果:克隆形成实验结果显示,HER-3敲低组乳腺癌细胞放射敏感性增强。流式细胞术结果显示,HER-3敲低后,HER-2阳性型乳腺癌细胞辐射后凋亡率明显增加,细胞周期G2期分布比例提高。免疫荧光结果显示,HER-3敲低后,HER-2阳性型乳腺癌细胞γH2AX焦点数目在辐射后先增加后降低,较对照组峰值更高,降低时趋势更慢,提示HER-3敲低可增强辐射诱导的DNA损伤,减少DNA修复。蛋白质印迹法结果显示,细胞周期相关驱动蛋白CyclinB1表达降低。结论:HER-3基因敲低后一方面增加了HER-2阳性型乳腺癌细胞辐射后DNA的损伤并减缓其修复能力,促进细胞凋亡;另一方面通过下调细胞周期蛋白CyclinB1的表达,增加G2期细胞分布,提升了放射敏感性。     

Risk of breast cancer according to the status of HER-2/neu oncogene amplification.

We examined risk factors for breast cancer after subdividing cases based on the presence of HER-2/neu oncogene amplification in their tumors. Data were from the Carolina Breast Cancer Study, a population-based, case-control study of 577 invasive breast cancer patients, diagnosed during 1993-1996 and ages 20-74 years, and 790 controls frequency-matched on race and age. Information on breast cancer risk factors was obtained from structured personal interviews. About 20% of paraffin-embedded tissues from the breast cancers of cases were identified as positive for HER-2/neu amplification (HER-2/neu+) by differential PCR. Early age at menarche, higher waist:hip ratio, and family history of breast or ovarian cancer were associated with elevated odds ratios (ORs) for both HER-2/neu+ and HER-2/neu- breast cancers. Breastfeeding for at least 1 year was inversely associated with HER-2/neu+ breast cancer [OR, 0.3; 95% confidence interval (CI), 0.1-0.7] more so than HER-2/neu- breast cancer (OR, 0.8; 95% CI, 0.5-1.2). Most of the remaining risk factors had ORs around 1.0 for both HER-2/neu+ and HER-2/neu- breast cancers, although a few exhibited possible associations with one disease subtype in analyses stratified by menopausal status. These study results suggest that most recognized breast cancer risk factors do not operate through HER-2/neu amplification in breast carcinogenesis. Differential effects of long-term breastfeeding by HER-2/neu amplification status have been observed in earlier studies and are provocative; however, the direction and magnitude of the associations have not been consistent.     

Overexpression of HER-2/neu enhances the sensitivity of human bladder cancer cells to urinary isoflavones

Soybean foods have been suggested to be practical chemopreventives for human urinary tract cancers. Recently, we demonstrated that the co-operative action of isoflavones results in an increased dose-dependent growth inhibition and apoptosis than any single isoflavone compound. This study aimed to examine the potential of HER-2/neu as a biological target for soy isoflavones. The sensitivity of the bladder cancer cell lines (n=7) to the isoflavones was inversely related to the amount of HER-2/neu expressed. By using HER-2/neu transfection experiments, all three stable transfectants showed a significant growth inhibition by the isoflavone mixture at concentrations attainable in normal adult urine. An increased inhibition of tyrosine phosphorylation of proteins immunoprecipitated by HER-2/Neu was observed in the neu-transfectants compared with controls. The results of this study suggest that HER-2/neu may be a practical biochemical target for urinary isoflavones in vivo.     

FISH detection of HER-2/neu oncogene amplification in early onset breast cancer

HER-2/neu (c-erbB-2) gene amplification based on Southern blotting or immunohistochemistry has been shown to be predictive of poor outcome in breast cancer occurring in women over 40, but there is little data on the role of HER-2/neu in young women with breast cancer, many of whom may have inherited BRCA1 or other predisposing genes. The present study used fluorescent in situ hybridization (FISH) on archival specimens of breast cancer from 37 women under the age of 40 to evaluate the role of HER-2/neu amplification in this cohort, and to also evaluate the efficacy of FISH for quantifying amplification. The frequency of primary tumors with a greater than fourfold increase in gene copy number was found to be 38%, which is similar to the frequency of amplification reported in Southern blot studies in older women. However, the greater sensitivity of FISH enabled detection of low level amplification (more than 2 but less than 8 gene copies), which was found in an additional 30% of the tumors. Patients with low level amplification demonstrated a 54% recurrence rate, compared to 86% in those with high amplification and 17% in those with no amplification. HER-2/neu amplification appeared to be more prognostic of recurrence than nodal status, with 45% of node negative tumors recurring compared to 62% of those which were node positive, nor was tumor size predictive of recurrence in this cohort since tumors of 2 cm or less recurred in 44% of cases compared to 57% of those larger than 2 cm. Thus, this study demonstrates that FISH is a reproducible and sensitive technique for detecting HER-2/neu amplification, and that amplification of the oncogene is the strongest independent indicator of recurrence of breast cancer in young women.     

Antisense Bcl-2 and HER-2 oligonucleotide treatment of breast cancer cells enhances their sensitivity to anticancer drugs

Overexpression of the HER-2 correlates with drug-resistance and a poor prognosis in breast cancer, however the mechanism(s) of HER-2-mediated drug-resistance are unknown. We examined the effects of antisense Bcl-2 and HER-2 oligonucleotides (ODN) to assess the mechanism(s) through which down-regulation of Bcl-2 and HER-2 enhances drug-sensitivity. Using two human breast cancer cell lines, MDA-MB-231 and BT-474, the antitumor effects of a combination of antisense ODN and anticancer drugs, including mitomycin C (MMC), adriamycin (ADM), paclitaxel (TXL), and docetaxel (TXT) was evaluated. The expression of Bcl-2 protein was suppressed by treatment with antisense Bcl-2 ODN in a dose-dependent manner. An enhanced drug-sensitivity to MMC and TXL upon pretreatment with antisense Bcl-2 ODN was observed, with the IC50 values increasing 1.9- and 2.0-fold, respectively. Treatment of BT-474 cells with antisense HER-2 at 1.0 micro M suppressed HER-2 overexpression by 60.5%. Pretreatment with antisense HER-2 ODN increased the sensitivity of these cells to ADM and TXL 20.8- and 10.8-fold, respectively. In vivo experiments using a combination of antisense HER-2 and TXL showed the similar enhancement of antitumor effect of TXL as compared to that of antisense HER-2 or TXL alone (p=0.068). Enhancement of drug-sensitivity was associated with the induction of apoptosis. Of interest, treatment with antisense HER-2 ODN also suppressed the expression of Bcl-2 and pAkt. These results indicate that down-regulation of Bcl-2 and HER-2 increased drug-sensitivity by modulating drug-induced apoptotic pathways in breast cancer cells, and that antisense ODN therapy, targeting Bcl-2 and HER-2 may be a useful strategy to enhance drug-sensitivity.     

Decreased response to paclitaxel versus docetaxel in HER-2/neu transfected human breast cancer cells

Taxanes are effective in the treatment of metastatic breast cancer. Docetaxel has been shown to be more potent than paclitaxel in inducing bcl-2 phosphorylation and apoptosis and is clinically active in some paclitaxel-resistant breast tumors. HER-2/neu overexpression has been shown to correlate with resistance to hormonal therapy as well as chemotherapy. Using a HER-2/neu transfected MCF-7 human breast cancer cell line, we investigated the role of HER-2/neu overexpression on resistance to paclitaxel and docetaxel treatment. A control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were treated with various concentrations of docetaxel or paclitaxel. Cell number was assessed using the MTT tetrazolium dye assay. In the control vector transfected MCF/neo cell line, paclitaxel and docetaxel gave similar dose-dependent growth inhibition ( p = 0.175). In HER-2/neu transfected MCF/18 cells, docetaxel treatment resulted in a dose-dependent inhibition similar to that seen in MCF/neo cells. Paclitaxel, however, gave significantly less growth inhibition than docetaxel in the HER-2/neu overexpressing MCF/18 cells (p = 0.0003). These data suggest that HER-2/neu overexpression may contribute to paclitaxel resistance. In contrast, the cytotoxic effects of docetaxel in these breast carcinoma cells are not affected by HER-2/neu expression. Therefore, docetaxel may be the preferred taxane therapy in HER-2/neu overexpressing breast tumors.     

敲降HER-2和TOP2A增强乳腺癌细胞放射敏感性研究

目的 研究HER-2和TOP2A对乳腺癌细胞系SK-Br-3的放射敏感性影响,并探讨二者联合作用,为临床研究奠定基础。方法 分别构建表达HER-2或TOP2A siRNA的重组质粒,并使用lipofectamine 2000将其转染至乳腺癌细胞SK-Br-3中,敲降HER-2和TOP2A的基因表达。2 d后利用蛋白印迹法检测干扰效果,利用流式细胞仪和MTT法分别检测放射处理后细胞凋亡和增殖变化,并检测凋亡和增殖相关蛋白Caspase-3、Bcl-2、Ki-67表达变化。克隆形成实验检测放射敏感性。结果在乳腺癌细胞SK-Br-3中敲降HER-2和TOP2A的表达,放射处理后细胞凋亡率升高,增殖率降低。凋亡标志蛋白活化的Caspase-3表达升高,凋亡抑制蛋白Bcl-2表达降低。细胞增殖标志蛋白Ki-67表达降低。同时敲降HER-2和TOP2A两个基因时促进凋亡和抑制增殖及克隆形成的作用增强,表明这两个基因存在协同效应。结论 敲降HER-2和TOP2A基因在乳腺癌细胞SK-Br-3中的表达后,乳腺癌细胞的放射敏感性提高,细胞凋亡率升高,细胞增殖率降低,克隆形成率降低,且2基因存在协同效应,作用机制可能与Caspase-3、Bcl-2有关的凋亡通路及细胞增殖蛋白Ki-67有关。     

Inhibition of IGF1R activity enhances response to trastuzumab in HER-2-positive breast cancer cells

Background: Although trastuzumab has improved the prognosis for HER-2-positive breast cancer patients, not all HER-2-positive breast tumours respond to trastuzumab treatment and those that initially respond frequently develop resistance. Insulin-like growth factor-1 receptor (IGF1R) signalling has been previously implicated in trastuzumab resistance. We tested IGF1R inhibition to determine if dual targeting of HER-2 and IGF1R improves response in cell line models of acquired trastuzumab resistance.Materials and methods: HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by enzyme-linked immunosorbent assays in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using both small interfering RNA (siRNA) and the tyrosine kinase inhibitor, NVP-AEW541.Results: IGF1R levels were significantly increased in the trastuzumab-resistant model, SKBR3/Tr, compared with the parental SKBR3 cell line. In both the SKBR3/Tr and BT474/Tr cell lines, inhibition of IGF1R expression with siRNA or inhibition of tyrosine kinase activity by NVP-AEW541 significantly increased response to trastuzumab. The dual targeting approach also improved response in the parental SKBR3 cells but not in the BT474 parental cells.Conclusions: Our results confirm that IGF1R inhibition improves response to trastuzumab in HER-2-positive breast cancer cells and suggest that dual targeting of IGF1R and HER-2 may improve response in HER-2-positive tumours.     

Inhibition of DNA repair by a herpes simplex virus vector enhances the radiosensitivity of human glioblastoma cells

Expression of the herpes simplex virus (HSV) protein, ICP0, from the viral genome, rendered two radioresistant human glioblastoma multiforme cell lines more sensitive to the effects of ionizing radiation. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic survival assays, U87-MG and T98 cell survival was more greatly decreased as a function of ionizing radiation dose when ICP0 was preexpressed in cells compared with when ICP0 was not expressed. Consistent with previous results, we found that the catalytic subunit of DNA-dependent protein kinase was degraded as a function of ICP0 in both cell types. This most likely resulted in the inhibition of DNA repair as inferred by the persistence of gammaH2AX foci or DNA double-strand breaks. Enhanced apoptosis was also found to occur following irradiation of U87-MG cells preinfected with the ICP0-producing HSV-1 mutant, d106. Our results suggest that expression of ICP0 in human glioblastoma multiforme cells inhibits the repair of DNA double-strand breaks after ionizing radiation treatment, decreasing the survival of these cells in part by induction of apoptosis.     

Soluble HER-2/neu neutralizes biologic effects of anti-HER-2/neu antibody on breast cancer cells in vitro

Amplification and over-expression of the HER-2/neu proto-oncogene are associated with poor prognosis in women with both node-positive and node-negative breast cancer. Therefore, the encoded surface glycoprotein represents an attractive target for cancer immunotherapies. Furthermore, the extracellular domain of HER-2/neu is released from the cell surface by proteolytic cleavage. In the present experiments, we investigated the potential biologic effects of soluble HER-2/neu with particular emphasis on its interaction with anti-HER-2/neu antibodies. A monoclonal antibody specific for the extracellular domain of HER-2/neu dose dependently inhibited the proliferation of highly HER-2/neu-expressing SK-BR-3 and BT-474 breast cancer cells but had no effect on the proliferation of weakly to moderately HER-2/neu-expressing MCF-7, HBL-100 and ZR-75-1 breast cells. Addition of SK-BR-3 or BT-474 cell supernatants with high concentrations of soluble HER-2/neu led to a neutralization of anti-HER-2/neu antibody–mediated inhibition of proliferation due to a binding of soluble HER-2/neu by the antibody, which could be demonstrated by immunoprecipitation. Furthermore, the ability of anti-HER-2/neu antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC) by lymphokine-activated killer cells was assessed. Cytolysis of SK-BR-3 tumor cells was increased significantly in the presence of anti-HER-2/neu antibodies. Similar to the proliferation inhibition, ADCC was neutralized by addition of soluble HER-2/neu-containing supernatants. Our data suggest that tumors rich in HER-2/neu might thus escape certain steps of immunologic control by neutralizing biologic activities of anti HER-2/neu antibodies due to the presence of soluble HER-2/neu. Int. J. Cancer 73:875–879, 1997. © 1997 Wiley-Liss, Inc.