Nora Zulehner

Whether to target single or multiple CDKs for therapy? That is the question

Complexes consisting of cyclin‐dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK–cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16INK4A or p27Kip1, and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad‐spectrum unspecific, pan‐specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan‐specific and highly selective CDK inhibitors in therapy. J. Cell. Physiol. 226: 341–349, 2011.

Roscovitine, a selective CDK inhibitor, reduces the basal and estrogen‐induced phosphorylation of ER‐α in human ER‐positive breast cancer cells

Roscovitine (ROSC), a selective cyclin‐dependent kinase (CDK) inhibitor, arrests human estrogen receptor‐α (ER‐α) positive MCF‐7 breast cancer cells in the G2 phase of the cell cycle and concomitantly induces apoptosis via a p53‐dependent pathway. The effect of ROSC is markedly diminished in MCF‐7 cells maintained in the presence of estrogen‐mimicking compounds. Therefore, we decided to examine whether ROSC has any effect on the functional status of the ER‐α transcription factor. Exposure of MCF‐7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 in a concentration and time‐dependent manner. This inhibition of site‐specific modification of CDK7 at Ser164/170 prevented phosphorylation of RNA polymerase II and reduced basal phosphorylation of ER‐α at Ser118 in non‐stimulated MCF‐7 cells (resulting in its down‐regulation). In MCF‐7 cells, estrogen induced strong phosphorylation of ER‐α at Ser118 but not at Ser104/Ser106. ROSC prevented this estrogen‐promoted activating modification of ER‐α. Furthermore, we sought to determine whether the activity of ROSC could be enhanced by combining it with an anti‐estrogen. Tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affected breast cancer cell lines irrespective of their ER status. In combination with ROSC, however, it had a different impact, enhancing G1 or G2 arrest. Our results indicate that ROSC prevents the activating phosphorylation of ER‐α and that its mode of action is strongly dependent on the cellular context. Furthermore, our data show that ROSC can be combined with anti‐estrogen therapy. The inhibitory effect of TAM on ER‐negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors. J. Cell. Biochem. 112: 761–772, 2011.

The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101.

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing.

PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells.

Graphical abstract

Interference with ER‐α enhances the therapeutic efficacy of the selective CDK inhibitor roscovitine towards ER‐positive breast cancer cells

In recent years many risk factors for the development of breast cancer that are linked to estrogens have been identified, and roscovitine (ROSC), a selective cyclin‐dependent kinase (CDK) inhibitor, has been shown to be an efficient inhibitor of the proliferation of human breast cancer cells. Therefore, we have examined the possibility that interference with estrogen signaling pathways, using tamoxifen (TAM), a selective estrogen receptor modulator (SERM), could modulate the efficacy of treatment with ROSC. In conjunction with TAM, ROSC exhibited enhanced anti‐proliferative activity and CDK inhibition, particularly in estrogen‐dependent MCF‐7 cells. The interaction between both drugs was synergistic. However, in ER‐α‐negative cells the interaction was antagonistic. Exposure of MCF‐7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 at Ser164/170. This in turn prevented the phosphorylation of the carboxyl‐terminal repeat domain of RNA Polymerase II and ER‐α at Ser118, resulting in the down‐regulation of the latter. Concomitantly, wt p53 was strongly activated by phosphorylation at Ser46. Our results demonstrate that ROSC negatively affects the functional status of ER‐α, making it potentially useful in the treatment of estrogen‐dependent breast cancer cells. J. Cell. Biochem. 112: 1103–1117, 2011.

Reconstitution of human MCF‐7 breast cancer cells with caspase‐3 does not sensitize them to action of CDK inhibitors

Human MCF‐7 breast cancer cells are resistant to pro‐apoptotic stimuli due to caspase‐3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin‐dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase‐3 in MCF‐7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF‐7 cells and a secondary mutant line, MCF‐7.3.28 that stably expresses human caspase‐3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF‐7 cells more strongly than caspase‐3‐proficient counterparts. Both inhibitors arrest human breast cancer cells at the G2‐phase of the cell cycle. Analysis of cell‐cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF‐7 cells lacking caspase‐3, but not in caspase‐3‐proficient cells. Furthermore, reconstitution of caspase‐3 in MCF‐7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF‐7 cells. Cytotoxic agents induce caspase‐3‐dependent apoptosis in caspase‐3‐proficient cells. These results indicate that reconstitution of MCF‐7 cancer cells with caspase‐3 sensitize them to the action of DNA damaging agents but not to ATP‐like pharmacological inhibitors of CDKs. J. Cell. Biochem. 112: 277–288, 2011.

Characterization of the T cell response to Dau c 1, the Bet v 1-homolog in carrot

In contrast to other Bet v 1‐related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process, we sought to characterize the T‐cell response to Dau c 1 and its cross‐reactivity with Bet v 1.

Bet v 1 and homologous food allergens are similarly processed by antigen-presenting cells but differ in T cell reactivity.

Background Various plant foods, e.g. apple and celery, express proteins that are homologues of the major birch-pollen allergen Bet v 1, e.g Mal d 1 and Api g 1. The proteins have 63% and 72% sequence similarity with Bet v 1 and share with it a common 3-dimensional structure. Despite this great molecular similarity, Bet v 1 is the only one among its homologues with the ability to sensitise atopic individuals. The aim of this study was to assess whether differences in the uptake and processing by antigen-presenting cells and in the presentation to T cells could be responsible for Bet v 1’s ability to sensitise.

Abstract 3490: Selective estrogen receptor modulators enhance efficacy of small molecule CDK inhibitors on human breast cancer cells differing in the expression of ER

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Inactivation of cyclin-dependent kinases (CDKs) by pharmacological inhibitors is a very effective weapon against malignant cells. Roscovitine (ROSC), a selective CDK inhibitor primarily affecting CDK2, CDK1, and CDK7, reduces the number of human cancer cells in a concentration-dependent manner. At lower doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC inhibits efficiently proliferation of human ER+-ve MCF-7 breast cancer cells by induction of cell cycle arrest at the G2/M transition [1] and concomitantly initiates apoptosis by a p53-dependent pathway [2]. However, the effect of ROSC was much weaker in MCF-7 cells maintained in the presence of estrogen-mimicking compounds [3]. Therefore, we decided to examine the action of selective CDK inhibitors on other breast cancer cell lines differing in the status of p53 and ER and to prove the impact of selective estrogen receptor modulators (SERMs) on the efficacy of ROSC and olomoucine II (OLO II), a new pharmacological CDK inhibitor. ROSC and OLO II were effective on all tested breast cancer cell lines. They arrested MCF-7 and BT-20 cells at G2/M transition and SKBR3 cells in G1 phase and additionally induced apoptosis. The effect of the inhibition of CDKs on distinct cell cycle regulators and pro-survival factors was studied. Interestingly, SERM strongly affected all tested cell lines irrespective of their ER status. Its combination with CDK inhibitors had a different impact. It enhanced G1 or G2 arrest. Our results provide evidence that both CDK inhibitors exert a strong anti-proliferative and pro-apoptotic effect and that their mode of action depends on the cellular context. Moreover, our findings demonstrate that pharmacological CDK inhibitors can be combined with anti-estrogen therapy. The strong effect of anti-estrogens on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors. [1] Wojciechowski J. et al. 2003. Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells. Int J Cancer 106: 486-95. [2] Wesierska-Gadek J. et al. 2005. Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human breast cancer cells. Mol Cancer Ther 4: 113-124. [3] Wesierska-Gadek J. et al. 2006. Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells. J Cell Biochem. 98: 1369-1379. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3490.