Bacterial artificial chromosomes improve recombinant protein production in mammalian cells

<p>Abstract</p> <p>Background</p> <p>The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production.</p> <p>Results</p> <p>In this work, we explore the efficacy of a Bacterial Artificial Chromosome based vector applied to production of the constant region of the human IgG1. Direct comparison of bulk HEK 293 cell cultures generated with a "conventional" vector or with a BAC-based vector showed that the BAC-based vector improved the protein yield by a factor of 10. Further analysis of stable cell clones harboring the BAC-based vector showed that the protein production was directly proportional to the number of integrated BAC copies and that the protein production was stable for at least 30 passages.</p> <p>Conclusion</p> <p>Generation of stable cell clones for protein production using Bacterial Artificial Chromosomes offers a clear advantage over the use of conventional vectors. First, protein production is increased by a factor of 10; second, protein production is stable overtime and third, generation of BAC-based expression vectors does not imply a significant amount of work compare to a conventional vector. Therefore, BAC-based vectors may become an attractive tool for protein production.</p

Metabolomic evaluation of Mitomycin C and rapamycin in a personalized treatment of pancreatic cancer

In a personalized treatment designed for a patient with pancreatic cancer resistant to other treatments, the success of Mitomycin C (MMC) has been highlighted. This was revealed in a murine xenograft tumor model encompassing pancreatic adenocarcinoma cells extracted from the patient. The patient was found to exhibit a biallelic inactivation of the PALB2 gene, involved in DNA repair in addition to another mutation in the TSC2 gene that induces susceptibility of the tumor to therapeutic targets of the PI3K-mTOR pathway. The aim of the study was to apply metabolomics to elucidate the modes of action of each therapy, suggesting why MMC was so successful in this patient and why it could be a more popular choice in future pancreatic cancer treatment. The effectiveness of MMC compared to rapamycin (RM), another relevant therapeutic agent has been evaluated through liquid- and gas-chromatography mass spectrometry-based metabolomic analyses of the xenograft tumors. The relative concentrations of many metabolites in the xenograft tumors were found to be increased by MMC relative to other treatments (RM and a combination of both), including a number that are involved in central carbon metabolism (CCM). Metabolic fingerprinting revealed statistically significantly altered pathways including, but not restricted to, the pentose phosphate pathway, glycolysis, TCA cycle, purine metabolism, fatty acid biosynthesis, in addition to many significant lipid and amino acid alterations. Given the genetic background of the patient, it was expected that the combined therapy would be most effective; however, the most effective was MMC alone. It is proposed that the effectiveness of MMC is owed to its direct effect on CCM, a vital region of tumor metabolism

Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3(Δm)) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3(Δm) mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3(Δm) CRCs. Moreover, STAT3(Δm) host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3(Δm) mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3(Δm) mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse

ERK inhibitor LY3214996-based treatment strategies for RAS-driven lung cancer

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K and RB pathways. Within the MAPK pathway ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing anti-tumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and - equally important - to identify biomarkers for patient stratification

Stat3 is a negative regulator of intestinal tumor progression

Dickdarmkrebs ist die 4. häufigste Krebsart weltweit. Er geht aus gutartigen Polypen hervor, die sich zu einer bösartigen Form entwickeln. Intestinale Erkrankungen wie Colitis ulcerosa oder Morbus Crohn stellen erhebliche Risikofaktoren dar und unterstreichen die Rolle von chronischen Entzündungen bei der Entstehung von Dickdarmkrebs. Der Transkriptionsfaktor Stat3 wird von entzündungshemmenden und -fördernden Cytokinen wie IL-6 und IL-10 aktiviert und eine anormale Aktivierung von Stat3 konnte bei Dickdarmkrebs beobachtet werden. Es liegt der Schluss nahe, dass durch Entzündungsvorgänge aktiviertes Stat3 das Fortschreiten und die Metastasierung von intestinalen Krebsarten fördert. Wir haben Mäuse mit konditionaler Deletion von Stat3 im Darmepithel erzeugt (Stat3deltaIEC) und mit APCmin Mäusen gekreuzt. APCmin Mäuse tragen eine Punktmutation im APC Gen und stellen ein Modelsystem für Darmkrebs dar. Durch die Deletion von Stat3 konnte eine Reduktion an frühen Mikroadenomen beobachtet werden. Trotzdem war die Lebenserwartung von Stat3deltaIEC APCmin/+ Mäusen signifikant reduziert. Histologische Analysen zeigten aggressive und invasive Karzinome in Stat3deltaIEC APCmin/+ Mäusen. Interessanterweise beeinflußte das Fehlen von Stat3 in Tumoren von APCmin/+ Mäusen die Apoptose und die Angiogenese nicht signifikant, förderte aber die Zellproliferation. Eine genomweite Expressionsanalyse in Stat3-defizienten Tumoren deutet darauf hin, dass Stat3 den Verlauf von Darmkrebs über das Zelladhäsionsmolekül Ceacam1 negativ reguliert. Unsere Daten zeigen, daß Stat3 ein negativer Regulator bei der Progression von Darmkrebs ist. Das muß bei Darmkrebstherapien, die auf Stat3 abzielen, berücksichtigt werden, um schädliche Effekte zu vermeiden.Colorectal cancer (CRC) represents the forth most common form of cancer worldwide and is thought to arise from benign adenomatous polyps that progress to malignancy. Diseases such as ulcerative colitis or Crohn's disease are significant risk factors which underlines the impact of chronic inflammation on CRC. The transcription factor Stat3 (signal transducer and activator of transcription 3) is activated by pro- and anti- inflammatory cytokines such as IL-6 and IL-10 and its aberrant activation was observed in CRC. Therefore inflammation mediated activation of Stat3 has been considered to promote progression and metastasis of intestinal cancers. We investigated the role of Stat3 in intestinal tumors using mice with conditional ablation of Stat3 in intestinal epithelial cells (Stat3deltaIEC). In the APCmin mouse model of intestinal cancer, genetic ablation of Stat3 reduced the multiplicity of early microadenomas. However, loss of Stat3 promoted tumor progression at later stages leading to formation of invasive carcinomas which significantly shortened the lifespan of Stat3deltaIEC APCmin/+ mice. Interestingly, loss of Stat3 in tumors of APCmin/+ mice had no significant impact on cell survival and angiogenesis but promoted cell proliferation. A genome-wide expression analysis of Stat3-deficient tumors suggested that Stat3 negatively regulates intestinal cancer progression via the cell adhesion molecule Ceacam1. Our data suggest that Stat3 negatively regulates progression of intestinal tumors. Therefore, detrimental effects on tumor progression have to be considered upon therapeutic targeting of the Stat3 signalling pathway in intestinal cancer.Applicant: Monica Andrea MusteanuAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersAbstract, ZsfassungWien, Med. Univ., Diss., 2009(VLID)171615

Whole Exome Sequencing of Rapid Autopsy Tumors and Xenograft Models Reveals Possible Driver Mutations Underlying Tumor Progression.

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy due to its propensity to invade and rapidly metastasize and remains very difficult to manage clinically. One major hindrance towards a better understanding of PDAC is the lack of molecular data sets and models representative of end stage disease. Moreover, it remains unclear how molecularly similar patient-derived xenograft (PDX) models are to the primary tumor from which they were derived. To identify potential molecular drivers in metastatic pancreatic cancer progression, we obtained matched primary tumor, metastases and normal (peripheral blood) samples under a rapid autopsy program and performed whole exome sequencing (WES) on tumor as well as normal samples. PDX models were also generated, sequenced and compared to tumors. Across the matched data sets generated for three patients, there were on average approximately 160 single-nucleotide mutations in each sample. The majority of mutations in each patient were shared among the primary and metastatic samples and, importantly, were largely retained in the xenograft models. Based on the mutation prevalence in the primary and metastatic sites, we proposed possible clonal evolution patterns marked by functional mutations affecting cancer genes such as KRAS, TP53 and SMAD4 that may play an important role in tumor initiation, progression and metastasis. These results add to our understanding of pancreatic tumor biology, and demonstrate that PDX models derived from advanced or end-stage likely closely approximate the genetics of the disease in the clinic and thus represent a biologically and clinically relevant pre-clinical platform that may enable the development of effective targeted therapies for PDAC

Somatic mutation patterns of primary, metastatic and PDX tumors.

<p>Proportion of somatic SNVs by class (C->A, C->G, C->T, T->A, T->C and T-G) in the primary, metastatic and PDX tumor samples are shown for the ten cancer samples included in this study.</p