Institut für Biologie

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Bedeutung der c-Abl-Aktivität für die Reaktion auf DNA-Schädigung und für die genetische Stabilität Bcr-Abl-negativer Zellen
(2011) Fanta, Silke; Aulitzky, Walter E.
The launch of Imatinib (Glivec®, Gleevec®, STI571) in August 2001 was an important advancement in the therapy of chronic myeloid leukemia (CML). The small-molecule inhibitor directly targets the oncogenic tyrosine kinase Bcr-Abl, which has been identified as the central cause for the development of CML. Treatment with Imatinib is the gold standard in the therapy of CML. However, taking the current state of research, an elimination of the malignant Bcr Abl-positive clone cannot be achieved by treatment with Imatinib. Thus, long-term or even lifelong treatment of patients is necessary. As a consequence, it is of great interest to clarify the biological effects of Imatinib on physiologically normal cells. Previous studies of the group showed that Imatinib treatment of Bcr Abl-positive cells leads to a decreased mutation frequency following DNA damage. Within the scope of the present work, evidence for significantly enhanced mutation rates after DNA damage in non-cancerogenic primary human lymphocytes (PBMC) and murine hematopoietic cell lines (32D and BaF3) after Imatinib treatment was obtained for the first time. Thus, Imatinib treatment of Bcr Abl-negative cells shows opposite effect compared to Bcr Abl-positive cells. It was therefore proven that the Imatinib-related inhibition of Bcr Abl as well as the off-target effects in Bcr Abl-negative cells play an important role in the genetic stability. To determine whether an Imatinib-mediated inhibition of c Abl activity is responsible for effects independent of Bcr Abl, genetic c Abl models were used to assess stress-induced mutation frequency. To this, we employed c Abl-knockout-MEFs (embryonic mouse fibroblasts), which were retransfected with wild type c Abl and a kinase-deficient form, respectively. After DNA damage, there was a significant increase in mutation frequency in the kinase-deficient cells (MEF Abl-KD) when compared to the c-Abl wild type (MEF Abl-wt) cells. Consequently, c-Abl activity is of great importance for the maintenance of genetic stability. Several factors can result in an increased mutation frequency in cells. Examples include altered cell proliferation, impaired DNA repair mechanisms or a delayed induction of cell death. In the latter case, DNA damage is not adequately repaired and passed to the daughter cells. In this study, different hematopoietic cell lines were used to show that neither the pharmacological nor the genetic inhibition of c-Abl activity has an influence on induction of cell death, division rate, cloning efficiency and cell cycle distribution. To investigate how far Imatinib influences the kinetics of DNA strand break repair after irradiation, alkaline comet assays were performed. Imatinib treatment of cells had no influence on induction of strand breaks or constitutive strand breaks prior to irradiation. However, cells treated with Imatinib exhibited a significantly delayed repair of DNA strand breaks. This delay was shown in the same manner in hematopoietic cell line models and in primary human lymphocytes, which were treated with Imatinib as well as with Dasatinib, a second generation Abl-inhibitor. Cell line models with different forms of c-Abl were used to provide evidence that this effect is caused by inhibition of the c-Abl kinase activity. The delayed repair of DNA strand breaks was also seen in cells with a kinase-deficient form of c-Abl (MEF Abl KD). But treatment with Imatinib had no effect on the kinetics of DNA repair in cells that expressed an Imatinib-resistant form of c Abl (c Abl T315I). Double- (DSB) as well as single-strand breaks (SSB) are determined in an alkaline comet assay. By applying neutral conditions, this assay can be modified to exclusively analyze DSB repair. As expected, there was a significantly lower induction of DSB after irradiation when compared to the occurrence of SSB. However, Imatinib did neither influence the induction nor the kinetics of DSB repair. Both pulsed-field gel electrophoresis and the quantification of gamma-H2AX were used to confirm that Imatinib does not affect DSB repair. Rather, the delayed repair kinetics are exclusively caused by an Imatinib-dependent interference with SSB repair. Extensive investigations of the molecular signaling pathways of DNA damage repair show that inhibition of c Abl activity does not affect ATM-Chk2-p53 or ATR-Chk1 signaling. Poly(ADP-ribosyl)ation of proteins is an early event in the processing of the SSB repair. This modification of proteins by addition of long and branched poly(ADP-ribose) chains (PAR) is an essential part of the SSB repair and base excition repair (BER). Both the synthesis and the cleavage of PAR is mediated by the kinases PARP-1 (poly(ADP-ribose) polymerase-1) and PARG (poly(ADP-ribose) glycohydrolase). This activity was determined by quantification of PAR and the percentage of cells, which were PAR-positive at a certain time. Possible effects of an Imatinib-induced inhibtion of c-Abl on poly(ADP-ribosyl)ation were investigated. To this, a method for the measurement of PAR events on a single-cell level was established. Poly(ADP-ribose) residues were marked with a PAR-specific antibody and detection followed by means of a fluorochrome-conjugated secondary antibody. The specificity of the method was proven unequivocally by a complete loss of signal when a specific PARP inhibitor (PJ34) was applied prior to irradiation-induced ribosylation. The advantage of this method is that the simultaneous determination of the DNA content in every cell allows the analysis of ribosylation events in correlation with cell cycle distribution. Based on these experiments it was found that in Imatinib-treated cells both the constitutive and the irradiation-induced poly-ribosylation are significantly enhanced. Furthermore, irradiation does not result in poly-ribosylation of all cells at a certain time: A subpopulation of cells, presumably those in the G0 resting phase, remain PAR-negative before and after irradiation. Thus, a novelty of the work at hand lies in the correlation of ribosylation events and cell cycle distribution before and after DNA damage. In this context, the central role of the Imatinib-mediated inhibition of c-Abl could also be established. The inhibited kinase activity of c-Abl seems to cause a delayed degradation of PAR. This is either caused by decreased activity of the PARP-1 antagonist PARG or by increased activity of PARP-1 itself. A disturbance of the spatially and temporally tightly modulated synthesis and degradation of PAR may lead to a prolonged interaction of PARP-1 with proteins related to SSB repair or BER, e.g. XRCC1 and DNA polymerase beta, thus resulting in the observed delay in DNA damage repair. The present study provides new insights into the impact of Imatinib on Bcr Abl-negative cells. The obtained in vitro data suggest that long-term treatment with c-Abl inhibitors may be associated with an increased likelihood of secondary neoplasias. Despite the outstanding success in Imatinib treatment of CML patients in the chronic phase, the complete elimination of the malignant clone should be the primary goal of the treatment of Bcr-Abl-positive leukemias.
Charakterisierung primärer Tumor-assoziierter Fibroblasten und Tumorzellen aus bronchialen Karzinomen und Untersuchung ihrer Reaktion auf zielgerichtete und zytotoxische Therapie
(2013) Schmid, Jens Oliver; Aulitzky, Walter E.
Worldwide lung cancer is the leading cause of death among all malignancies. This is largely due to its high frequency of diagnosis and its poor 5-year survival rate of 15%. As a solid tumor, lung cancer consists of tumor cells and a variable stromal part, that is made up of a cellular and a non-cellular fraction. The stroma influences several processes like growth, invasion of surrounding tissues, metastatic spread as well as tumor supply of oxygen and nutrients. Thereby, the stroma is dominated by the cancer-associated fibroblasts (CAFs), actively shaping the microenvironment through the secretion of soluble factors and the synthesis of extracellular matrix (ECM) components. While there are several studies with the aim of identifying the differences between CAFs and normal fibroblasts (NAFs) of the breast, there is only little information about those differences in the corresponding cells of the lung. One of the aims of the study was the identification of the molecular differences between CAFs and NAFs derived from lung tissue. A further objective was the investigation of therapy effects under conditions that mimic the situation in vivo. Therefore, an ex vivo-model allowing the culture of primary lung tumor tissue had to be evaluated. Afterwards, the effect of the epidermal growth factor receptor (EGFR) inhibitor Erlotinib on tumor cell proliferation was investigated in this model system. To investigate a response of both, tumor cells and their adjacent CAFs to chemotherapy, lung cancer tissue samples were treated with cisplatin. Finally, owing to their important role in tumors, CAFs were chosen as a target for therapy using small molecule inhibitors, with the aim of inhibiting their stimulatory effect. Molecular comparison of isolated CAFs and the corresponding NAFs of 9 lung cancer patients revealed a significantly different expression of 60 genes. The identification of a set of differentially regulated genes is quite surprising because of the assumable activation of NAFs due to culture conditions. This indicates that CAFs are more than just activated fibroblasts, which are found at sites of tissue injury. Rather, they are a distinct cell type showing parallels to activated fibroblasts. Expression data for 46 of the 60 identified genes were available in a Non-Small Cell Lung Cancer collective comprising of 342 patients. As it turned out, a NAF-like expression of the genes was associated with a significantly better survival prognosis. Another central objective of the work was the investigation of the tumor cell response to therapy in an intact tissue. An already established tissue culture system required initial validation. This was done by comparing the tissue which has been cultivated for 4 days with the corresponding tissue, that has been fixed immediately after surgery. No significant changes in morphology and biological function were detected. Thus the model system adequately mimics the situation in the patient and a negative effect of the culture could be excluded. This makes the system an excellent opportunity to investigate the effect of a drug under in vivo-like conditions. Treatment of tissue samples, characterized for EGFR expression, and EGFR, and KRAS gene status, with the small-molecule inhibitor Erlotinib displayed no effect on the proliferation of the analysed tumor cells.This reflects the situation in the clinics quite adequately where only a small proportion of patients benefits from the treatment with the EGFR-inhibitor Erlotinib. To follow-up, the reaction of CAFs and tumor cells on a chemotherapeutical treatment was investigated under in vivo-like conditions. Interestingly, cisplatin led to a parallel accumulation of p53 and induction of cell death in tumor cells and their adjacent CAFs. Thereby, the p53 accumulation of CAFs seems to be dictated by their tumor cells because the same CAFs which do not accumulate p53 in the tissue, respond to cisplatin with the accumulation of p53 in the isolated state. Therefore, it is tempting to speculate that tumor cells modulate the DNA damage response of their microenvironment, with the objective to raise their own chemoresistance. Inhibiting CAF proliferation was examined as a feasible approach to inhibit their tumor-stimulating properties. Screening of a kinase inhibitor library consisting of 160 small molecule inhibitors resulted in the identification of PDGFR signaling as a promising target. Among the FDA approved PDGFR inhibitors Dasatinib turned out to be the most potent inhibitor of CAF proliferation, resulting in a molecular phenotype comparable to that of normal fibroblasts. Furthermore, the Dasatinib-mediated changes in CAFs led to the secretion of factors, inhibiting the proliferation of lung tumor cells. In contrast, the secreted factors of untreated CAFs stimulated their proliferation. Together, these results indicate that Dasatinib treatment is a promising approach to reduce the tumor promoting capacity of CAFs.
Hyperthermie steigert die Zytotoxizität von Cisplatin und Doxorubicin durch Hemmung der Poly(ADP-Ribosyl)ierung und der damit einhergehenden Verhinderung der Replikationsblockade
(2015) Schaaf, Lea; Aulitzky, Walter E.
Peritoneal carcinomatosis describes widespread metastases of cancerous tumors in the peritoneal cavity particularly arising from ovarian and colon cancers. For long time this far advanced tumor disease was considered extremely difficult to treat. Therefore, only palliative measures were carried out in most cases. This has changed significantly in recent years with the introduction of multimodal treatment options consisting of a complete macroscopic tumor reduction (CRS) followed by an intraoperative intraperitoneal hyperthermic chemotherapy (HIPEC). It is undisputed that this local chemotherapy application allows treatment with much higher drug concentrations as compared to the systemic therapy. However, the role of hyperthermia in this multimodal approach has not been fully clarified so far. There is still no clinical study available showing a clear benefit of elevated temperature in the intraperitoneal chemotherapy. Largely unknown is also which level of temperatures are really needed for an elevated cytotoxicity of chemotherapeutics. Furthermore, the molecular mechanisms behind this synergistic effect are poorly investigated. To answer these questions, an in vitro model was established mimicking the situation of the HIPEC procedure as closely as possible. This model allowed to define a very precise temperature threshold of 40°C. An effective increase in cytotoxicity of cisplatin and doxorubicin was only observed at temperatures of 40°C or above. Importantly, this temperature threshold was also of clinical relevance. Patients who reached this temperature over at least 40 minutes at two sites in the abdominal cavity, namely omental bursa and pelvis, showed a significantly increased overall and progression free survival. In-vitro hyperthermia leads to an increased intracellular concentration of doxorubicin. Interestingly, however, the synergy of hyperthermia with doxorubicin was observed even after reducing the drug concentrations to values which are also reached at 37° C. Together with the finding that hyperthermia had no effect on the amount of DNA-cisplatin adducts, these results clearly indicate that the increased intracellular drug accumulation is not the predominant mechanism behind the synergistic effects of chemotherapeutics and hyperthermia. Rather a compromised repair of cisplatin and doxorubicin induced DNA damages upon hyperthermia treatment could be identified to be important for the observed effects. This significantly delayed DNA repair depends on inhibition of the poly(ADR-ribosyl)ation (PARylation) by hyperthermia. Interestingly, hyperthermia selectively increased the efficiency of cytotoxic agents that induce PARylation. The hypothesis that inhibition of PARylation plays a key role for the synergy between hyperthermia and chemotherapy is further supported by the finding that the treatment with specific pharmacological PARP inhibitors resulted in a comparable elevation of cisplatin and doxorubicin induced cytotoxicity. In this respect PARylation could be identified as a molecular marker for a preclinical substance-screen to identify drugs acting together with hyperthermia. In addition, these in-vitro results for the first time show that there are alternatives to the hyperthermic treatment, which is associated with considerable side effects. Thus, intraperitoneal chemotherapy could be combined with systemic PARP inhibitor pre-treatment. Clinically approved specific PARP inhibitors are far better tolerated by patients than hyperthermia. Hyperthermia mediated inhibition of PARylation led to an increase in the percentage of cells with DNA double-strand breaks (DSB). Interestingly, the results of this work indicate a hyperthermia-induced switch from HR to the error-prone NHEJ. In combination with hyperthermia there was a significant increase in P-53BP1 foci formation, while both the percentage of cells with Rad51 foci and Rad51 protein level remained unchanged. The finding that these DSBs occur only in S-phase cells points to a replication-associated mechanism. In fact, in the framework of this work it could be demonstrated for the first time that drug-induced stalled replication forks are circumvented by the hyperthermia-mediated inhibition of PARylation. The unhindered progression of replication forks upon inhibition of PARylation by hyperthermia or pharmacological PARP inhibition presumably results in the increase of DNA DSBs as well as in the significantly reduced long-term-survival

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