Uwe Zangemeister-Wittke

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

AbstractSmall cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5′-aza-2-deoxycytidine and IFNγ partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNγ.

Circulating DNA: a new diagnostic gold mine? ☆

The recent discovery that cell-free DNA can be shed into the bloodstream as a result of tumour cell death has generated great interest. Numerous studies have demonstrated tumour-specific alterations in DNA recovered from plasma or serum of patients with various malignancies, a finding that has potential for molecular diagnosis and prognosis. The implication is that tumour-derived nucleic acids of human or viral origin can be retrieved from blood by a minimally invasive procedure, and used as a surrogate tumour marker to monitor the course of the disease or aid in early diagnosis. The present review will describe the main areas of ongoing investigation, with particular emphasis on technical issues and available data of clinical relevance.

Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines.

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5 EFFECTS ON TUMOR TARGETING

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

A novel antisense oligonucleotide inhibiting several antiapoptotic Bcl-2 family members induces apoptosis and enhances chemosensitivity in androgen-independent human prostate cancer PC3 cells

Bcl-2 and Bcl-xL are associated with treatment resistance and progression in many cancers, including prostate cancer. The objective of this study was to determine whether a novel bispecific antisense oligonucleotide targeting both Bcl-2 and Bcl-xL induces apoptosis and enhances chemosensitivity in androgen-independent PC3 prostate cancer cells. An antisense oligonucleotide with complete sequence identity to Bcl-2 and three-base mismatches to Bcl-xL selected from five antisense oligonucleotides targeting various regions with high homology between Bcl-2 and Bcl-xL was found to be the most potent inhibitor of both Bcl-2 and Bcl-xL expression in PC3 cells. This selected Bcl-2/Bcl-xL bispecific antisense oligonucleotide reduced mRNA and protein levels in a dose-dependent manner, reducing Bcl-2 and Bcl-xL protein levels to 12% and 19%, respectively. Interestingly, Mcl-1 was down-regulated as well, although levels of Bax, Bad, or Bak were not altered after treatment with this bispecific antisense oligonucleotide. Indirect down-regulation of inhibitor of apoptosis (IAP) family, including XIAP, cIAP-1 and cIAP-2, via second mitochondria-derived activator of caspases was also observed after bispecific antisense oligonucleotide treatment. Executioner caspase-3, caspase-6, and caspase-7 were shown to be involved in apoptosis induced by bispecific antisense oligonucleotide. This Bcl-2/Bcl-xL bispecific antisense oligonucleotide also enhanced paclitaxel chemosensitivity in PC3 cells, reducing the IC50 of paclitaxel by >90%. These findings illustrate that combined suppression of antiapoptotic Bcl-2 family members using this antisense oligonucleotide could be an attractive strategy for inhibiting cancer progression through alteration of the apoptotic rheostat in androgen-independent prostate cancer.

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.

Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.

Over‐expression of the anti‐apoptotic protein bcl‐xL is frequently found in lung cancer where it potentially contributes to tumor development, progression and drug resistance. To override the apoptotic block in lung‐adenocarcinoma and small‐cell‐lung‐cancer (SCLC) cells caused by over‐expression of bcl‐xL, an anti‐sense oligodeoxynucleotide was designed targeting a sequence unique to the bcl‐xL coding region and not shared by the pro‐apoptotic splice variant bcl‐xS. Moreover, to improve the biophysical properties of the anti‐sense compound, 2`‐methoxy‐ethoxy modifications were made to selected deoxy‐ribose residues. The resulting gapmer oligonucleotide 4259 was tested on lung‐adenocarcinoma and SCLC cell lines in vitro. Treatment of the adenocarcinoma cell lines A549 and NCI‐H125 and the SCLC cell lines SW2 and NCI‐H69 with 600 nM 4259 reduced bcl‐xL levels by 70 to 90%. In the lung‐adenocarcinoma cell lines, apoptosis was induced, as indicated by caspase‐3‐like protease activation and nuclear condensation and fragmentation. In contrast, in the SCLC cell lines, no induction of apoptosis could be demonstrated. These findings imply that bcl‐xL is a more critical survival factor for lung adenocarcinomas than for SCLC, and suggest the use of oligonucleotide 4259 for therapy of this major sub‐type of lung cancer. Int. J. Cancer 86:570–576, 2000.

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

The inhibitor of apoptosis protein (IAP) survivin is overexpressed in many tumors but is absent in most normal adult tissues. We report high levels of survivin expression in small cell lung cancer (SCLC), and describe the role of the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway in survivin upregulation. Moreover, the cytoprotective function of survivin in response to the anti‐cancer agent cisplatin (CDDP) was investigated. Negative modulation of PI3K/Akt using pharmacological inhibitors or dominant negative Akt (DN‐Akt) decreased Akt kinase activity and resulted in decreased survivin expression and phosphorylation on Thr34, whereas transfection of constitutively active Akt (CA‐Akt) increased survivin expression and phosphorylation. Interestingly, we found that treatment of SCLC cells with CDDP further increased survivin expression in a cell cycle independent manner by activation of Akt. CA‐Akt or lentiviral survivin also inhibited apoptosis induced by CDDP, whereas DN‐Akt or survivin‐specific RNA interference sensitized cells to CDDP. We identified survivin as an anti‐apoptotic protein in SCLC cells that is regulated by Akt, and demonstrate that treatment with the DNA damaging agent CDDP activates the PI3K/Akt/survivin pathway that in part protects cells from drug‐induced apoptosis.

Bcl-xl antisense treatment induces apoptosis in breast carcinoma cells.

Upregulated expression of bcl‐xL is involved in the initiation and progression of breast cancer by inhibiting tumor cell apoptosis. Here we describe the use of the 2`‐O‐methoxy‐ethoxy antisense oligonucleotide 4259 targeting nucleotides 687–706 of the bcl‐xL mRNA, a sequence that does not occur in the pro‐apoptotic bcl‐xS transcript, to restore apoptosis in estrogen‐dependent and independent breast carcinoma cells. The antisense effect of oligonucleotide 4259 was examined on the mRNA and protein level using real‐time PCR and Western blot analysis, respectively, and the induction of cell death was investigated in viability and apoptosis assays. Treatment of MCF7 cells with oligonucleotide 4259 at a concentration of 600 nM for 20 hr decreased bcl‐xL mRNA and protein levels by more than 80% and 50%, respectively. This resulted in the induction of apoptosis characterized by mitochondrial cytochrome c release, decrease of mitochondrial transmembrane potential, and the appearance of condensed nuclei in approximately 40% of cells. Moreover, oligonucleotide 4259 efficiently downregulated bcl‐xL expression and decreased cell growth in the breast carcinoma cell lines T‐47D, ZR‐75‐1, and MDA‐MB‐231. Our data emphasize the importance of bcl‐xL as a survival factor for breast carcinoma cells and suggest that oligonucleotide 4259 deserves further investigations for use in breast cancer therapy. Int. J. Cancer 87:582–590, 2000.