A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems

Regression of Human Prostate Tumors and Metastases in Nude Mice following Treatment with the Recombinant Oncolytic Vaccinia Virus GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma

Significant Growth Inhibition of Canine Mammary Carcinoma Xenografts following Treatment with Oncolytic Vaccinia Virus GLV-1h68

Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

<p>Abstract</p> <p>Background</p> <p>Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic.</p> <p>Methods</p> <p>Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects.</p> <p>Results</p> <p>We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia.</p> <p>Conclusion</p> <p>Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.</p

Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS

Detection and therapy of human prostate carcinoma metastases with the oncolytic vaccinia virus GLV-1h68

Zurzeit sterben jährlich ca. 11.000 Männer in Deutschland am Prostatakarzinom. Damit stellt dies die zweithäufigste Krebstodesursache von Männern dar. Da das Prostatakarzinom häufig asymptomatisch verläuft, wird die Erkrankung oftmals erst so spät erkannt, dass zum Zeitpunkt der Diagnose bereits eine Metastasierung stattgefunden hat. Durch metastasierende Prostatakarzinomzellen werden Lymphknoten, Knochen und Lungen befallen. Es sind zwei unterschiedliche Verbreitungsarten von metastasierenden Tumorzellen beschrieben. Zum einen kann eine Migration über Lymphgefäße erfolgen, ein Prozess der als lymphatische Metastasierung bezeichnet wird. Zum anderen können Tumorzellen über das Blutsystem im Körper zirkulieren: die hämatogene Metastasierung. In dieser Arbeit wurde die lymphatische Metastasierung der humanen Prostatakarzinomzellline PC-3 im Detail analysiert und Teilaspekte der hämatogenen Verteilung untersucht. Ausgangspunkt der Untersuchungen bildete die Vergrößerung lumbaler und renaler Lymphknoten in PC-3-Tumor-tragenden Mäusen 60 Tage nach der Implantation von PC-3-Zellen. Es wurde daraufhin der zeitliche Verlauf der Vergrößerung untersucht und festgestellt, dass sowohl das Volumen als auch die Anzahl vergrößerter Lymphknoten von Woche zu Woche nach Implantation der PC-3-Tumore zunehmen. Anschließend wurden alle vergrößerten Lymphknoten bezüglich des Vorhandenseins von metastasierenden humanen PC-3-Zellen in den Mäusen untersucht. Dies geschah mit Hilfe einer RT-PCR unter Verwendung von Primern für humanes β-Aktin. Sechs Wochen nach Implantation konnten in 90 % der vergrößerten Lymphknoten PC-3-Zellen nachgewiesen werden. Weiterhin wurde durch lentivirale Transduktion das Gen für das rot fluoreszierende Protein (RFP) in die PC-3-Zellen inseriert, wodurch eine Visualisierung dieser Zellen in der Maus ermöglicht wurde. Es konnten metastasierende PC-3-RFP-Zellen in lumbalen und renalen Lymphknoten PC-3-RFP-Tumor-tragender Mäuse nachgewiesen werden. Ebenso konnte mittels RFP gezeigt werden, dass die Lymphknotenmetastasierung in Abhängigkeit von der Lokalisation des PC-3-RFP-Tumors erfolgt. Es kam zur Metastasierung jener Lymphknoten, in deren Einzugsgebiet sich der PC-3-Tumor befand. Es wurde eine PC-3-RFP-Zellmigration zwischen lumbalen und renalen Lymphknotenmetastasen nachgewiesen und bei immunhistologischen Untersuchungen stellte sich heraus, dass PC-3-RFP-Zellen tatsächlich in lymphatischen Bahnen zwischen lumbalen und renalen Lymphknotenmetastasen migrieren. Außerdem wurde gezeigt, dass es von Woche zu Woche nach Implantation von PC-3-Zellen zu einer Zunahme der Anzahl von Lymphgefäßen in PC-3-Tumoren kommt. Die Zunahme der Lymphgefäßdichte korrelierte hierbei positiv mit der Bildung von Lymphknotenmetastasen. Es konnten weiterhin neben Lymphknotenmetastasen hämatogene Mikrometastasen in den Lungen PC-3-RFP-Tumor-tragender Mäuse beobachtet werden. Da die Haupttodesursache von Prostatakarzinompatienten in der Bildung von Metastasen liegt, ist es von herausragender Bedeutung eine effektive Therapie gegen lymphatische und hämatogene Metastasen zu entwickeln. Aus diesem Grund erlangt die onkolytische Virustherapie große Bedeutung. Deshalb wurde als zweiter Aspekt in dieser Arbeit der Einfluss des onkolytischen Vaccinia-Virus GLV-1h68 auf den Prozess der PC-3-Zellmetastasierung untersucht. Dabei konnte zunächst gezeigt werden, dass GLV-1h68 in der Lage ist, erfolgreich sowohl migrierende PC-3-Zellen als auch metastasierende PC-3-Zellen in Lymphknoten zu kolonisieren. In der Folge wurde deshalb ein möglicher Metastasen-inhibierender Effekt von GLV-1h68 untersucht. Hierbei stellte sich heraus, dass GLV-1h68 drei Wochen nach intravenöser Injektion eine signifikante Reduktion der Anzahl der für PC-3-Zellen positiven Lymphknoten bewirkt. Des Weiteren konnte ein inhibierender Effekt von GLV-1h68 auf die im Blut zirkulierenden PC-3-Zellen und auf hämatogene Metastasen in den Lungen beobachtet werden. Durch intravenöse Injektion von GLV-1h68 in PC-3-RFP-Tumor-tragenden Mäusen konnte gezeigt werden, dass es zu einer präferentiellen Virus-Kolonisierung der Lymphknotenmetastasen im Vergleich zu den Tumoren kommt. Auch nach intraperitonealer und intratumoraler Injektion von GLV-1h68 konnte eine präferentielle Virus-Kolonisierung der Lymphknotenmetastasen gezeigt werden. Darüber hinaus wurden die Lymph- und Blutgefäße von PC-3-Tumoren und Lymphknotenmetastasen analysiert. Hierbei wurde gezeigt, dass es sieben Tage nach intravenöser Injektion von GLV-1h68 zu einer signifikanten Abnahme von beiden Gefäßarten kam. Es wurde in dieser Arbeit somit gezeigt, dass GLV-1h68 in der Lage ist, sowohl lymphatische als auch hämatogene Metastasen der Prostatakarzinomzelllinie PC-3 erfolgreich zu eliminieren. Folglich dürften onkolytische Vaccinia-Viren ein vielversprechendes Therapeutikum für die Behandlung des fortgeschrittenen Prostatakarzinoms darstellen.Every year about 11,000 men in Germany are dying because of prostate carcinoma. Thus, prostate carcinoma represents the second leading cause of cancer related death in men. Since the prostate carcinoma usually proceeds asymptomatically the diagnosis is often made when metastases have already formed. Human prostate cancer usually spreads to lymph nodes, bones and lungs. There are two ways for tumor cells to migrate to other parts of the body: through lymphatic vessels, a process called lymphatic metastasis, or through the blood system, the hematogenous metastasis. In this thesis the lymphatic metastasis of the human prostate carcinoma cell line PC-3 was analyzed in detail while the hematogenous spread was only partially investigated. The initial point of these investigations was the enlargement of lumbar und renal lymph nodes in PC-3 tumor-bearing mice 60 days post implantation of PC-3 cells. Thereafter the time course of the enlargement was assessed. It turned out that the volume as well as the number of enlarged lymph nodes increased from week to week post implantation of PC-3 tumors. Subsequently, all enlarged lymph nodes were tested for the presence of human PC-3 cells in mice. This was done with the help of an RT-PCR using primers for human β-actin. Six weeks post implantation 90% of all enlarged lymph nodes were positive for PC-3 cells. Furthermore, the gene of the red fluorescent protein (RFP) was inserted into PC-3 cells via lentiviral transduction. By using fluorescence microscopy PC-3-RFP cells could be detected in lumbar and renal lymph nodes of PC-3-RFP tumor-bearing mice. With the help of RFP it could also be shown that lymph node metastases depend on the PC-3 tumor location. Metastases occurred in draining lymph nodes next to the tumor. Moreover, a PC-3-RFP cell migration between lumbar and renal lymph node metastases was shown. In the following immunohistochemical analysis it was proven that PC-3-RFP cells are indeed migrating in lymphatic vessels between these lumbar and renal lymph node metastases. Additionally, an increasing number of lymphatic vessels in PC-3 tumors was shown from week to week post implantation of PC-3 cells. This enhancement positively correlates with the formation of lymph node metastases. Besides lymph node metastases hematogenous micro metastases in the lungs of PC-3-RFP-tumor-bearing mice could be detected, too. The major cause of death in prostate cancer patients is the formation of metastases. Therefore, the development of effective therapies for lymphatic and hematogenous metastases is of major importance. One of the most promising novel cancer therapies for humans is oncolytic virotherapy. According to that, the second aspect of this thesis was to investigate the influence of the oncolytic vaccinia virus GLV-1h68 on the process of PC-3 cell metastasis. Thereby, it was initially shown that GLV-1h68 can efficiently colonize both migrating PC-3 cells and metastasized PC-3 cells in the lymph nodes. Ensuing, a possible metastasis inhibiting effect of GLV-1h68 was analyzed. It was shown that GLV-1h68 reduces the volume and the number of enlarged lymph nodes in PC-3 tumor-bearing mice three weeks after intravenous injection. It could also be shown that GLV-1h68 significantly reduces the number of lymph nodes that are positive for PC-3 cells. Additionally, GLV-1h68 has an inhibiting effect on PC-3 cells that are circulating in the blood of PC-3 tumor-bearing mice and on hematogenous metastases of the lungs. In analysing the intravenous injection of GLV-1h68 in PC-3-RFP tumor-bearing mice it turned out that there is a preferential viral colonisation of lymph node metastases compared to the tumors. At early points after injection renal lymph node metastases were colonized more thoroughly by GLV-1h68 than lumbar ones. The same preferential viral colonisation of lymph node metastases was shown upon intraperitoneal und intratumoral viral injection. Further, lymphatic and blood vessels of PC-3 tumors and lymph node metastases were analyzed. There was a significant reduction of lymphatic and blood vessels seven days post intravenous injection of GLV-1h68. This could explain the effect of GLV-1h68 on the reduction of the number of lymph node metastases, because the supply of nutrients as well as of oxygen is reduced due to the decrease of blood vessel density. Also, migration of PC-3 cells is minimized upon the reduction of lymphatic vessels. Thus, it was shown that GLV-1h68 has a great potential in eliminating lymphatic and hematogenous metastases of the human prostate carcinoma PC-3. Therefore, oncolytic vaccinia viruses apparently represent promising therapeutic agents for the treatment of advanced human prostate carcinoma

On the seismic response of instable rock slopes based on ambient vibration recordings

Abstract Rock slope failures can lead to huge human and economic loss depending on their size and exact location. Reasonable hazard mitigation requires thorough understanding of the underlying slope driving mechanisms and its rock mass properties. Measurements of seismic ambient vibrations could improve the characterization and detection of rock instabilities since there is a link between seismic response and internal structure of the unstable rock mass. An unstable slope near the village Gondo has been investigated. The unstable part shows strongly amplified ground motion with respect to the stable part of the rock slope. The amplification values reach maximum factors of 70. The seismic response on the instable part is highly directional and polarized. Re-measurements have been taken 1 year later showing exactly the same results as the original measurements. Neither the amplified frequencies nor the amplification values have changed. Therefore, ambient vibration measurements are repeatable and stay the same, if the rock mass has not undergone any significant change in structure or volume, respectively. Additionally, four new points have been measured during the re-measuring campaign in order to better map the border of the instability. Graphical abstract

Ambient vibration classification of unstable rock slopes: A systematic approach

In this paper, we are comparing the seismic response of 25 different rock slope instabilities with diverse geological properties, activity level, failure mechanism, fracturing and volumes. We classify them according to their dynamic behaviour. In the dataset, we found two main classes of unstable rock slopes: depth-controlled sites and volume-controlled sites. For depth-controlled instabilities, the seismic wavefield is controlled by horizontally propagating surface waves in the highly fractured and weathered material. At such sites, surface-wave dispersion curves can be retrieved and inverted into velocity profiles of the underground. The lateral borders of depth-controlled instabilities are not obvious and the seismic properties mainly change with depth. The seismic response of volume-controlled sites is dominated by the eigenvibrations of the rock mass itself. Such instabilities have clear lateral and vertical borders and show highly amplified ground motion in limited frequency bands. The observed polarisation of the ground motion is perpendicular to deep open fractures that do not allow surface waves to propagate. A special case of the volume-controlled sites is represented by tower-like rock masses showing strong amplification and directionality. Their dynamic behaviour might not only be related to the internal structure, but also to their geometry that is similar to high-rise buildings. Another type of rock instabilities is represented by block structures, which are difficult to identify by the proposed ambient-vibration methods. A clear relation between geological properties and seismic response was not found.ISSN:0013-7952ISSN:1872-691