Transfected Tumor Cells Research Articles

The PI3 kinase/mTOR blocker NVP-BEZ235 overrides resistance against irreversible ErbB inhibitors in breast cancer cells

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, and gefitinib), complete growth inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drug-exposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled sensitivity in drug-resistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drug-refractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.

New Strategies of Mammary Cancer Vaccination

A new strategy of vaccination against mammary tumors, extendible to tumors of distinct histological origin, based on the administration of tumor cells genetically modified to express major histocompatibility complex (MHC) class II gene products, will be described. Expression of MHC class II molecules in solid tumors, generally lacking these molecules, is achieved by transfecting tumor cells with the MHC class II transactivator (CIITA), the major regulator of the entire family of MHC class II genes. CIITA is encoded by the AIR-1 locus, discovered in our laboratory. The rationale underlying this approach consists in making the tumor cells a sort of surrogate antigen presenting cells for MHC-II-restricted CD4 + T helper (TH) cells. Indeed, it is known that an efficient adaptive immune response against cancer cells can only be achieved if tumor-specific TH cells, the key lymphocyte subpopulation required to trigger both humoral and cellular effector mechanisms, are optimally stimulated. Results from our group show that: (a) CIITA-modified tumor cells can be rejected in vivo by syngeneic immunocompetent mice; (b) this rejection is mediated primarily by CD4 + TH lymphocytes that activate cytolytic CD8 + T cell effectors ; (c) tumor-rejecting mice are resistant to challenge with parental unmodified tumor cells and display long term immune memory; (d) anti-tumor vaccination can be reproduced by using inactivated, nonreplicating CIITA-transfected tumor cells; (e) immune effectors and particularly primed CD4 + TH cells can be used successfully in approaches of immunotherapy of established tumors. These results open the way to envisage a possible use of CIITA-modified mammary tumor cells as a vaccine for increasing both the inducing and the effector phase of the anti-tumor immune response in human settings.

Synthesis of Lamivudine stearate and antiviral activity of stearic acid-g-chitosan oligosaccharide polymeric micelles delivery system

To increase lipophilicity of water-soluble antiviral drug, the prodrug of Lamivudine (LA), Lamivudine stearate (LAS) was synthesized via ester linkage between LA and stearic acid. After the esterification, the octanol–water partition coefficient (log P) of LA increased from −0.95 to 1.82. Stearic acid-g-chitosan oligosaccharide (CSO-SA) micelles have demonstrated fast internalization and accumulation ability to tumor cells. Herein, the CSO-SA with 3.79% amino substitution degree (SD) was prepared for loading LAS. The critical micelle concentration (CMC) of CSO-SA was about 0.032 mg/ml. The micelles with 1 mg/ml CSO-SA concentration had 460.8 nm average diameters with a narrow size distribution and 29.7 mV surface zeta potential. After LAS was incorporated, the micellar size decreased and the zeta potential increased. The LAS loaded CSO-SA micelles (CSO-SA/LAS) possessed high entrapment efficiency and drug loading. LA release from CSO-SA/LAS showed a pH-dependent behavior. The release rate of LA from CSO-SA/LAS increased significantly as the pH of release medium reduced from 7.4 to 6.2. CSO-SA/LAS presented a low cytotoxicity and high cellular uptake percentage of LAS against HBV transfected tumor cells (HepG2.2.15). In vitro anti-HBV activities of CSO-SA/LAS presented more conspicuous inhibitory effects on antigen expression and DNA replication compared with LA and LAS.

Low generation PAMAM dendrimer and CpG free plasmids allow targeted and extended transgene expression in tumors after systemic delivery

Polyplexes consisting of a standard CMV promoter driven luciferase plasmid condensed with PAMAM starburst dendrimers (generation 4 and 5) efficiently transfected tumor cells in vitro. Tail vein injection of PAMAM polyplexes into immune competent mice bearing subcutaneous, well vascularized murine neuroblastoma tumors (Neuro2A) led to predominant luciferase reporter gene expression in the tumor, and negligible transgene expression levels in other organs. Repeated PAMAM polyplex applications were well tolerated and prolonged transgene expression in the tumor. In vivo imaging studies using polyplexes fluorescently labeled with near infrared emitting semiconductor quantum dots (quantoplexes) revealed lung accumulation for both PAMAM and linear PEI (LPEI) based polyplexes, but only LPEI polyplexes induced high luciferase expression in lung, demonstrating that biodistribution and transgene expression of polyplexes does not necessarily correlate. With a luciferase plasmid devoid of immune modulatory CpG sequences and a combination of human CMV enhancer and human elongation factor 1 alpha promoter elements, Neuro2A tumor transgene expression after a single intravenous injection of generation 5 PAMAM polyplexes was observed for up to 1 week as measured by luciferase bioluminescence imaging. Utilizing a human xenograft model (HUH7) in immune compromised nude mice, a low level of luciferase activity in the tumor area was observed after systemic PAMAM polyplex application.

Role of complement activation component C5a on tumor progression (100.9)

Abstract Enhancement of anti-tumor mAb immunological mechanisms for tumor destruction may be obtained by promoting complement activation. However, complement activation components, such as C5a, trigger inflammation and chronic inflammation promotes tumor growth. Recently, it has been shown that C5a in the tumor could recruit and activate myeloid-derived suppressor cells to inhibit the anti-tumor CD8 T cell response. However, this effect was observed in only one tumor model. We chose to address the role of C5a on tumor growth by transfecting tumor cells with C5a. A human ovarian carcinoma, SKOV-3, and a murine lymphoma, RMA, were transfected which allows us to study C5a in an immunocompromised xenograft model and an immunocompetent syngeneic model. In vitro growth kinetics observed revealed no significant difference between C5a, control vector (CV), or wild-type cells. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the xenograft model, CD11b+DX5+ NK cells were significantly more and VEGF and arginase levels were significantly less in C5a expressing tumors. The syngeneic lymphoma model revealed C5a modulates tumor-infiltrating CD11b+ cells to produce more IL-12 and there is a greater influx of NK and CD4 T cells. Thus our studies demonstrate C5a chemoattracts innate NK cells and downregulates negative mediators in the xenograft model while in the syngeneic model C5a promotes shift to a Th1 response.

Photodynamic therapy-driven induction of suicide cytosine deaminase gene

Photodynamic therapy (PDT) of tumors is associated with induction of hypoxia that results in activation of hypoxia-inducible factors (HIFs). Several observations indicate that increased HIFs transcriptional activity in tumor cells is associated with cytoprotective responses that limit cytotoxic effectiveness of PDT. Therefore, we decided to examine whether this cytoprotective mechanism could be intentionally used for designing more efficient tumor cell cytotoxicity. To this end we transfected tumor cells with a plasmid vector carrying a suicide cytosine deaminase gene driven by a promoter containing hypoxia response elements (HRE). The presence of such a genetic molecular beacon rendered tumor cells sensitive to cytotoxic effects of a non-toxic prodrug 5-fluorocytosine (5-FC). The results of this study provides a proof of concept that inducible cytoprotective mechanisms can be exploited to render tumor cells more susceptible to cytotoxic effects of prodrugs activated by products of suicide genes.

Conditioning Vaccination Site With Irradiated MIP-3α–transfected Tumor Cells Enhances Efficacy of Dendritic Cell-based Cancer Vaccine

Macrophage inflammation protein-3alpha (MIP-3alpha) is a chemokine expressed in inflamed tissue and capable of inducing migration of immature dendritic cells (DCs) or Langerhans cells. We postulated that conditioning vaccination sites with MIP-3alpha might enhance the efficacy of subsequently administered DC-based cancer vaccines. Our results demonstrate that subcutaneously injection of irradiated tumor cells expressing MIP-3alpha induces substantial cell infiltration to the injection site. Vaccination of irradiated tumor cells expressing MIP-3alpha followed by DCs pulsed with irradiated tumor cells can effectively suppress tumor growth in animals, which is significantly better than vaccination with irradiated MIP-3alpha-producing tumor cells or DCs pulsed with tumor cells alone. The protective effect was most evident when the MIP-3alpha-producing tumor cells and DC-based vaccines were injected at the same site. These results support the notion that this combination vaccination strategy might generate a more effective immune response to suppress the growth of tumor cells in animals.

Adenovirus-Mediated ING4 Expression Suppresses Pancreatic Carcinoma Cell Growth via Induction of Cell-Cycle Alteration, Apoptosis, and Inhibition of Tumor Angiogenesis

Recent studies have demonstrated that ING4, as a novel member of the ING (inhibitor of growth) family, has a potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in the application of gene therapy for pancreatic carcinoma has not been reported. To explore its therapeutic effect on human pancreatic carcinoma, we constructed a recombinant adenoviral vector, Ad-ING4, expressing the green fluorescent protein (GFP) marker gene and the tumor-suppressor gene, humanized ING4 derived from murine ING4 with two amino-acid modifications at residues 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of PANC-1 human pancreatic carcinoma cells inhibited cell growth, altered the cell cycle with S-phase reduction and G2/M phase arrest, induced apoptosis, and downregulated interleukin (IL)-6 and IL-8 expression of transfected tumor cells. In athymic mice bearing the PANC-1 human pancreatic tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth, downregulated CD34 expression, and reduced the tumor microvessel formation. Therefore, this study will provide a framework for future clinical application of Ad-ING4 in human pancreatic carcinoma gene therapy.

PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

Construction of murine CCL21 eukaryotic expression plasmid and chemotactic function of its products

To construct the murine CCL21 eukaryotic expression plasmid, and to investigate the chemotactic function of its products. Murine CCL21 cDNA was amplified by RT-PCR from murine total RNA, and was inserted into eukaryotic expression plasmid pcDNA3.1 after confirmation of sequencing. The recombinant CCL21 plasmid was transferred into mouse forestomach carcinoma (MFC) cells and the chemotactic function of expressed products was detected by chemotaxis assay. Gene sequencing, gel electrophoresis of PCR products and restrictive digestion proved the successful construction of CCL21, and its expression was confirmed by Western Blot. The transfected tumor cells had a significant chemotactic function to DC. The recombinant murine CCL21 eukaryotic expression plasmid has been successfully constructed, and its expression products in tumor cells have a marked chemotactic function to DC.

Hydrogel‐mediated DNA delivery confers estrogenic response in nonresponsive osteoblast cells

Oligo(polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photoencapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for coencapsulation of DNA and bone cells and examined whether coencapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), coencapsulated with bone tumor cells, was capable of transfecting the cells, and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the coencapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.

S100A2 Induces Metastasis in Non–Small Cell Lung Cancer

S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non-small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

Direct Inhibitory Effects of Lenalidomide on the Proliferation and VEGF Production of Non-Hodgkin Lymphoma Cells Are Associated with Increased SPARC Expression

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug approved for the treatment of Low- or Intermediate-risk myelodysplastic syndrome associated with a del(5q) cytogenetic abnormality, and in combination with dexamethasone for previously treated multiple myeloma. Early clinical results suggest potential clinical efficacy in B-cell non-Hodgkin lymphoma (NHL). In this study, we investigated lenalidomide-mediated inhibition of cell proliferation and angiogenic factors in the following NHL subtypes: mantle cell lymphoma (MCL), diffuse large-B-cell lymphoma, and follicular lymphoma (FL). The effect of lenalidomide on these tumor cells was determined after 1–3 days of treatment by 3H-thymidine incorporation, Luminex-based microbead array, and real-time qRT-PCR. We also assessed the expression of tumor suppressor genes such as p21cip1 and secreted protein acidic and rich in cysteine (SPARC). We found that lenalidomide induced direct anti-proliferative effects on each NHL subtype, with MCL cells being the most sensitive. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) were expressed at a high level in all lymphoma cell lines. Lenalidomide inhibited VEGF production at much lower concentrations than required for anti-proliferative effects, particularly in MCL and FL cell lines. Addition of recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on MCL cell proliferation, suggesting that these effects are independent. Mechanistic studies indicated that lenalidomide strongly increased the gene expression of the tumor suppressor genes p21cip1 and SPARC to varying degrees. Elevation of SPARC mRNA significantly correlated with both the anti-proliferative and the VEGF-suppressive effects of lenalidomide on MCL cells (p < 0.05). Transfection of tumor cells with SPARC siRNA led to significant resistance to lenalidomide suggesting that this effect is mediated at least in part through the up-regulation of SPARC. In conclusion, lenalidomide demonstrates anti-proliferative activity against multiple NHL cell subtypes with greatest potency against MCL. The potent anti-VEGF activity of lenalidomide supports the anti-angiogenic potential of the drug. Thus, lenalidomide-induced up-regulation of SPARC mRNA correlates with MCL sensitivity and may have biomarker potential.

Long-term progression and therapeutic response of visceral metastatic disease non-invasively monitored in mouse urine using β-human choriogonadotropin secreting tumor cell lines

Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human beta-chorionic gonadotropin (beta-hCG) that can be measured in urine. We describe examples of beta-hCG-transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used beta-hCG-tagged mouse B16 melanoma and erbB-2/Her-2-expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2-targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2-targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine beta-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by beta-hCG levels. Our results also suggest that beta-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that beta-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease.

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice.

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4(+) T cells and antibodies are important components.

Adenovirus-mediated ING4 expression suppresses lung carcinoma cell growth via induction of cell cycle alteration and apoptosis and inhibition of tumor invasion and angiogenesis

Previous studies demonstrated that ING4 as a novel member of ING (inhibitor of growth) family has potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in inhibition of human tumors has not been reported. To explore its therapeutic effect on human lung carcinoma, we constructed a recombinant adenoviral vector Ad-ING4 expressing the humanized ING4 gene derived from murine ING4 with two amino acid modifications at residue 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of A549 human lung carcinoma cells induced cell apoptosis, altered cell cycle with S phase reduction and G2/M phase arrest, suppressed cell invasiveness, and down-regulated IL-6, IL-8, MMP-2, and MMP-9 expression of transfected tumor cells. In athymic mice bearing A549 lung tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth and reduced the tumor microvessel formation. Therefore, Ad-ING4 may be useful in gene therapy of human lung carcinoma.

Novel enrichment of tumor cell transfectants expressing high levels of type 4 glutathione peroxidase using 7α-hydroperoxycholesterol as a selection agent

A novel approach for selecting high expressing cells out of a general population that had been transfected with a construct encoding cytosolic type 4 glutathione peroxidase (GPx4) is reported. The approach is described for GPx4-null COH-BR1 breast tumor cells and is based on use of a highly specific GPx4 substrate, 7α-hydroperoxycholesterol (7α-OOH), as a selection agent. Cells recovering from a highly toxic dose of liposomal 7α-OOH were found to be substantially more resistant to a second 7α-OOH challenge than cells recovering from a less toxic dose, but were much less resistant to t-butyl hydroperoxide ( t-BuOOH) or H 2O 2. Several clones isolated from the general transfectant population exhibited variable, relatively low GPx4 activities. However, clones from the 7α-OOH-selected population exhibited uniformly high GPx4 activities (each ∼3-fold higher than that of the starting transfectant population) and elevated steady-state mRNA levels. t-BuOOH could also be used for selecting high GPx4-expressing cells, but consistent recovery from toxic doses was more difficult than with 7α-OOH. Compared with conventional “hit or miss” cloning procedures, the 7α-OOH approach we describe affords a uniform population of high GPx4-activity cells in a relatively rapid manner. This approach should prove valuable for investigators interested in the peroxide regulatory properties of GPx4, in the context of both cytoprotection and redox signaling.

Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells

Hyaluronan (HA) is a major glycosaminoglycan in the extracellular matrix whose expression is tightly linked to multidrug resistance and tumor progression. In this study we investigated HA-induced interaction between CD44 (a HA receptor) and Nanog (an embryonic stem cell transcription factor) in both human breast tumor cells (MCF-7 cells) and human ovarian tumor cells (SK-OV-3.ipl cells). Using a specific primer pair to amplify Nanog by reverse transcriptase-PCR, we detected the expression of Nanog transcript in both tumor cell lines. In addition, our results reveal that HA binding to these tumor cells promotes Nanog protein association with CD44 followed by Nanog activation and the expression of pluripotent stem cell regulators (e.g. Rex1 and Sox2). Nanog also forms a complex with the "signal transducer and activator of transcription protein 3" (Stat-3) in the nucleus leading to Stat-3-specific transcriptional activation and multidrug transporter, MDR1 (P-glycoprotein) gene expression. Furthermore, we observed that HA-CD44 interaction induces ankyrin (a cytoskeletal protein) binding to MDR1 resulting in the efflux of chemotherapeutic drugs (e.g. doxorubicin and paclitaxel (Taxol)) and chemoresistance in these tumor cells. Overexpression of Nanog by transfecting tumor cells with Nanog cDNA stimulates Stat-3 transcriptional activation, MDR1 overexpression, and multidrug resistance. Down regulation of Nanog signaling or ankyrin function (by transfecting tumor cells with Nanog small interfering RNA or ankyrin repeat domain cDNA) not only blocks HA/CD44-mediated tumor cell behaviors but also enhances chemosensitivity. Taken together, these findings suggest that targeting HA/CD44-mediated Nanog-Stat-3 signaling pathways and ankyrin/cytoskeleton function may represent a novel approach to overcome chemotherapy resistance in some breast and ovarian tumor cells displaying stem cell marker properties during tumor progression.

High-dose γ-irradiation enhances the expression of a transgene controlled by the immediate-early CMV promoter in stably transfected tumor cells

γ-irradiation is commonly used to inactivate whole-cell anticancer vaccines containing viable tumor cells. To evaluate the effect of γ-irradiation on transgene expression in tumor cells, human and mouse cell lines were stably transfected with constructs expressing the granulocyte macrophage colony-stimulating factor (GM-CSF) or green fluorescent protein (GFP) gene under the control of the immediate-early CMV promoter. Irradiation of cells at 20–100 Gy caused a loss of proliferation capacity and gradual cell death, with the survival depending on the irradiation dose. G2/M cells accumulated in irradiated cultures, while the portion of S-phase cells was reduced. Surviving cells displayed activation of β-galactosidase and morphological changes associated with cell senescence. Mitochondrial dehydrogenase activity did not change with the irradiation dose. Irradiated cells retained transgene expression. Moreover, the amount of secreted GM-CSF and GFP production significantly increased after γ-irradiation, up to tenfold in cells exposed to 100 Gy. Transgene expression increased gradually and positively correlated with the total irradiation dose. The results demonstrate that γ-irradiation at 100 Gy is optimal for whole-cell anticancer vaccine inactivation.