Bernhard Hemmerlein

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the hosts immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a ‘mixed response’, and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.

Overexpression of Eag1 potassium channels in clinical tumours

BackgroundCertain types of potassium channels (known as Eag1, KCNH1, Kv10.1) are associated with the production of tumours in patients and in animals. We have now studied the expression pattern of the Eag1 channel in a large range of normal and tumour tissues from different collections utilising molecular biological and immunohistochemical techniques.ResultsThe use of reverse transcription real-time PCR and specifically generated monoclonal anti-Eag1 antibodies showed that expression of the channel is normally limited to specific areas of the brain and to restricted cell populations throughout the body. Tumour samples, however, showed a significant overexpression of the channel with high frequency (up to 80% depending on the tissue source) regardless of the detection method (staining with either one of the antibodies, or detection of Eag1 RNA).ConclusionInhibition of Eag1 expression in tumour cell lines reduced cell proliferation. Eag1 may therefore represent a promising target for the tailored treatment of human tumours. Furthermore, as normal cells expressing Eag1 are either protected by the blood-brain barrier or represent the terminal stage of normal differentiation, Eag1 based therapies could produce only minor side effects.

Generation of Functional Cardiomyocytes From Adult Mouse Spermatogonial Stem Cells

Stem cell–based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca2+ channels and responded to Ca2+ channel–modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca2+ in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca2+ release from intracellular Ca2+ stores. By using line-scan mode, we found rhythmic Ca2+ transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.

EXPRESSION OF METALLOPROTEINASE 2 AND 9 AND THEIR INHIBITORS IN RENAL CELL CARCINOMA

Degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Two gelatinolytic matrix metalloproteinase enzymes, MMP-2 and MMP-9, are supposed to be key enzymes in this process. The purpose of this study was to correlate the presence of MMP-2, MMP-9 and their inhibitors with the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 RNA using reverse transcriptase PCR technique with tumor stage in 17 samples of renal cell carcinoma. The ratio of tissues expressing MMP-2 and MMP-9 to those expressing TIMP-1 and TIMP-2 was defined to be 1 in normal kidney tissue. This MMP:TIMP ratio was significantly increased to 2.43 (standard deviation, SD = 0.8) in locally confined renal cell carcinoma and to 4.86 (SD = 1.1) in advanced carcinoma (p <0.01). In primary tumor cell lines the ratio of MMP:TIMP expression was 3.44 (SD = 0.6). These data suggest that the balance of MMP-2 and MMP-9 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of renal cell carcinoma.

Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo

To analyze the biological role of factor XII (FXII, Hageman Factor) in vivo, we generated mice deficient for FXII using a gene targeting approach on two distinct genetic backgrounds, i.e. mixed C57Bl/6J X 129X1/SvJ and inbred 129X1/SvJ. Homozygous FXII knockout (FXII(-)/(-)) mice showed no FXII plasma activity and had a markedly prolonged activated partial thromboplastin time (aPTT). In contrast, coagulation factors XI, VIII, IX, X,VII, V, II and fibrinogen did not differ between FXII(-/-) mice and their wild-type littermates. Heterozygous matings segregated according to the Mendelian inheritance indicating that FXII deficiency does not increase fetal loss. Furthermore, matings of FXII(-/-) males and FXII(-/-) females resulted in normal litter sizes demonstrating that total FXII deficiency in FXII(-/-) females does not affect pregnancy outcome. Also, gross and histological anatomy of FXII(-/-) mice was indistinguishable from that of their wild-type littermates on both genetic backgrounds. Thus it appears that deficiency of murine FXII does not cause thrombophilia or impaired fibrinolysis in vivo. These results indicate that FXII deficiency does not affect hemostasis in vivo and we anticipate that the FXII(-/-) mice will be helpful to elucidate the biological role(s) of FXII in health and disease.

Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up-regulates IGF binding protein-3, and suppresses MMP-2 expression

The type I insulin‐like growth factor receptor (IGF‐IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF‐IR in benign and malignant prostate epithelium. In this study, endogenous IGF‐IR gene expression was reduced in stably transfected PC‐3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC‐3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF‐IR gene expression and either up‐regulation of IGF binding protein (BP)‐3 or down‐regulation of matrix metalloproteinase (MMP)‐2 expression in androgen‐independent PC‐3 cells. Moreover, inhibition of IGF‐IR gene expression in transfected PC‐3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT‐PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF‐IR gene expression was up‐regulated in nine of 12 prostate cancers, whereas IGFBP‐3 gene expression was down‐regulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF‐IR and IGFBP‐3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF‐IR or IGFBP‐3 gene expression in order to improve understanding of the IGF‐IR‐activated signalling pathways and as a potential treatment for prostate cancer. Copyright

Bax Inhibitor-1 Is Overexpressed in Prostate Cancer and Its Specific Down-Regulation by RNA Interference Leads to Cell Death in Human Prostate Carcinoma Cells

To analyze differential gene expression of putative prostate tumor markers we compared the expression levels of more than 400 cancer-related genes using the cDNA array technique in a set of capsule-invasive prostate tumor and matched normal prostate tissue. The overexpression of Bax inhibitor-1 (BI-1) in prostate carcinoma and prostate cancer cell lines was confirmed by using Northern blot and Western blot analyses. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) on intact RNAs from 17 paired laser-captured microdissected epithelial tissue samples confirmed up-regulated BI-1 expression in 11 of 17 prostate tumors. In addition, it was demonstrated that BI-1 expression is down-regulated in stromal cells as compared to matched normal epithelial cells of the prostate. In situ hybridization experiments on prostate sections also revealed that BI-1 expression is mainly restricted to epithelial cells. Furthermore, quantitative RT-PCR on RNAs derived from five benign prostate hyperplasia (BPH) samples showed no significant difference in BI-1 expression as compared to normal epithelial prostate tissue. To determine the function of BI-1 in vitro, human PC-3, LNCaP, and DU-145 prostate carcinoma cells were transfected with small interfering double-strand RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression. Furthermore, transfection of PC-3, LNCaP, and DU-145 cells with BI-1 sequence-specific siRNAs caused a significant increase in spontaneous apoptosis in all cell lines. Taken together, our results indicate that the human BI-1 gene contains the potential to serve as a prostate cancer expression marker and as a potential target for developing therapeutic strategies for prostate cancer.

Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas

Rapidly growing tumors often develop necrosis. In the present study the expression of vascular endothelial growth factor (VEGF) was investigated and compared to microvessel density and necrosis of renal cell carcinomas. In the tumor-host interface the microvessel density was significantly increased compared to central tumor areas. Tumor necrosis was associated with a decrease of microvessel density and an increase of the VEGF protein expression within the perinecrotic rim. VEGF protein was focally upregulated in vital tumor tissue. An increase of the apoptotic rate of endothelia and vital tumor tissue in tumors with necrosis could not be detected. VEGF(121,165) mRNA was decreased in proliferatively active carcinomas compared to less proliferative tumors. Multicellular renal cell cancer spheroids as a model of chronic hypoxia developed central apoptosis but no necrosis. VEGF was upregulated in the spheroid. Tumor microvessels expressed matrix metalloproteinase -2 and -9 and an incomplete pericyte covering in comparison to tumor-free tissue indicating immature active angiogenesis. We conclude that highly proliferative renal cell carcinomas outgrow their vascular supply and develop chronic hypoxia inducing a decrease of proliferation and an increase of VEGF expression. However, chronic hypoxia does not cause significant necrosis or apoptosis. Tumor necrosis is more likely induced by acute hypoxia due to immature microvessels. Furthermore, VEGF expression associated with concomitant tumor necrosis may help identify renal cell carcinomas susceptible to antiangiogenic therapy.

Expression and functional analysis of Bax inhibitor-1 in human breast cancer cells.

Recently, deregulated expression of the anti‐apoptotic protein Bax inhibitor‐1 (BI‐1) has been shown in several human cancers. In this report, we show that BI‐1 is expressed at various levels in six different human breast cancer cell lines. In order to investigate the function of BI‐1 in oestrogen‐dependent MCF‐7, T‐47D and oestrogen‐independent MDA‐MB‐231 breast cancer cells, the RNA interference technique was used to knock down BI‐1 expression specifically. Suppression of BI‐1 expression caused a significant increase in spontaneous apoptosis in MDA‐MB‐231 cells, whereas MCF‐7 and T‐47D cells remained almost unaffected. Furthermore, BI‐1 expression analysis using a cancer profiling array showed up‐regulation of BI‐1 expression in cancer samples of breast, uterus and ovary, whereas down‐regulated BI‐1 expression was identified in stomach, colon, kidney, lung and rectal cancer. In addition, immunohistochemical studies using a BI‐1‐specific antibody on human breast cancer specimens also revealed that BI‐1 is expressed in the majority of cases. Moreover, to analyse whether BI‐1 expression is oestrogen receptor‐dependent, tumour cells were treated with oestradiol, ICI and tamoxifen: this showed no significant changes in BI‐1 expression. Taken together, our results demonstrate that BI‐1 expression is differentially deregulated in different cancers and that BI‐1 plays an important role in preventing certain breast cancer cells from undergoing apoptosis. Thus, the development of novel therapeutic strategies based on targeting BI‐1 gene expression in breast cancer could be restricted to selected individual cancer types. Copyright

Molecular characterization of Patched-associated rhabdomyosarcoma

Mutations in the human homologue of Drosophila Patched1 (PTCH1) have been found in several common tumours including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (RMS). Medulloblastoma and RMS are also present in the murine model for Ptch1 deficiency. Tumours in heterozygous Ptch1neo67/+ mice consistently exhibit elevated transcript levels of the proto‐oncogene Gli1, of Ptch1 itself, and of the insulin‐like growth factor 2 (Igf2). The present study has investigated additional molecular changes in RMSs of Ptch1 mutant mice by means of microarray analysis and protein expression analysis. The data show activation of the cell survival‐promoting Akt/protein kinase B (Pkb). Furthermore, RMSs express increased levels of the anti‐apoptotic protein Bcl‐2 and of genes and proteins known to inhibit cell proliferation, including Gadd45a and p27kip1. Taken together, the data suggest that the formation of RMSs in Ptch1 mutants is associated with the ability of tumour cells to resist apoptosis. Copyright