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.

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.

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

Two cellular proteins encoded by the breast and ovarian cancer type 1 susceptibility (BRCA1 and BRCA2) tumor suppressor genes are essential for DNA integrity and the maintenance of genomic stability. Approximately 5-10% of breast and ovarian cancers result from inherited alterations or mutations in these genes. Remarkably, BRCA1/BRCA2-deficient cells are hypersensitive to selective inhibition of poly(ADP-ribose)polymerase 1 (PARP-1), whose primary functions are related to DNA base excision repair; PARP-1 inhibition significantly potentiates the cytotoxicity of various anti-cancer drugs, including inhibitors of topoisomerase I and II. In the present study, we examined the anti-proliferative and pro-apoptotic effects of C-1305, a selective inhibitor of topoisomerase II, on human breast cancer cell lines with different BRCA1 and p53 statuses. BRCA1-competent breast cancer cell lines exhibited different responses to topoisomerase II inhibition. BT-20 cells that express high levels of BRCA1 levels were most resistant to C-1305 than other tested cells. Surprisingly, pharmacological interference with PARP-1 activity strongly inhibited their proliferation and potentiated the efficacy of C-1305 treatment. In contrast, PARP-1 inhibition only weakly affected the proliferation of BRCA1-deficient SKBr-3 cells and was not synergistic with the effects of C-1305. Further experiments revealed that the inhibition of PARP-1 in BT-20 cells caused the accumulation of DNA strand breaks and induced caspase-3 dependent apoptosis. These results seem to indicate that PARP-1 inhibition can potentiate the cytotoxicity of anti-cancer drugs in cancer cells with functional BRCA1 and suggest that mutations in other DNA repair proteins may render cancer cells more sensitive to interference with PARP-1 activity.

Effect of anti-estrogen combined with roscovitine, a selective CDK inhibitor, on human breast cancer cells differing in expression of ER.

Roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs) reduces numbers of cancer cells in a concentration-dependent manner. At low doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC efficiently inhibits proliferation of human ER-alpha positive MCF-7 breast cancer cells by inducing G/M arrest and concomitantly initiates apoptosis by a p53-dependent pathway. However, the effect of ROSC is much weaker on MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we have examined the action of ROSC on other breast cancer cell lines differing in ER status and confirmed that tamoxifen (TAM) affects the efficacy of this CDK inhibitor. ROSC was effective against all tested breast cancer cell lines, arresting them at G1/S or G2/M transition and inducing apoptosis in SKBR-3 cells. Interestingly, TAM affected all tested cell lines, irrespective of their ER-a status, and in combination with ROSC it enhanced G1 or G2 arrest. Our results provide evidence that ROSC can be combined with antiestrogen therapy and that the mode of ROSC action strongly depends on the cellular context. The effect of TAM on ER-negative cancer cells indicates that TAM also crosstalks with other steroid hormone receptors.

Tamoxifen enhances the anti-proliferative effect of roscovitine, a selective cyclin-dependent kinase inhibitor, on human ER-positive human breast cancer cells.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, can arrest human ER-positive MCF-7 breast cancer cells in the G2 phase of the cell cycle and concomitantly induce apoptosis. The observed effects of ROSC were diminished in MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we decided to test whether combining ROSC with anti-estrogen therapy would modulate the efficacy of ROSC action. Exposure of MCF-7 cells to tamoxifen (TAM) for 24 h decreased the number of living cells by approximately 10%. This was associated with a ca. 25% increase in the G1 cell population and reduction in the proportion of S-phase cells. Unlike TAM, estrogen had very weak effects on the cell cycle progression of MCF-7 cells within 24 h. The proliferation-promoting effect of estrogen did not become evident until cultivation of cells for 48 h. Addition of estrogen to MCF-7 cells 1 h prior to TAM administration abolished the anti-estrogen-induced G1 arrest. Simultaneous treatment of MCF-7 cells with ROSC and TAM strongly enhanced the anti-proliferative effect of ROSC. This was potentiated after co-treatment with estrogen. These results clearly indicate that the efficacy of treating ER-positive breast cancers by ROSC can be enhanced by combined application of antiestrogens.

Combining an FPTase inhibitor with cisplatin facilitates induction of apoptosis in human A549 lung cancer cells.

Despite great efforts to develop efficacious curative treatments, the prognosis for lung cancer patients is poor. In the present study we compared the effects of cisplatin (CP), a strong DNA damaging compound, with those of roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs), on wt p53-positive human A549 lung adenocarcinoma cells harboring a mutated K-RAS gene. Asynchronously growing A549 cells were relatively resistant to CP treatment for 24 h, but after exposure to CP at sufficiently high doses (> or = 20 microM) an accumulation of S-arrested cells was observed. However, after post-incubation of CP-treated cells in a drug-free medium for a further 48 h the number of living cells was markedly reduced. Combining CP with L-744,832, a small molecule FPTase inhibitor (FTI), slightly enhanced its anti-proliferative effect. Interestingly, FTI sensitized A549 cells to CP-induced apoptosis. ROSC inhibited A549 cells at the G/M transition, resulting in a marked decrease in the number of viable cells within 24 h, and prolonged treatment with ROSC for 48 h reduced the frequency of living cells by inducing apoptosis. The effects of ROSC (unlike those of CP) were more strongly enhanced by inhibition of the Ras protein processing pathway. Our preliminary results indicate that functional p53 contributes to the outcome of the therapy in human A549 cells by certain anti-cancer drugs.

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 G(2) 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 G(1) or G(2) 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.

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 Ser(164/170). This in turn prevented the phosphorylation of the carboxyl-terminal repeat domain of RNA Polymerase II and ER-α at Ser(118), resulting in the down-regulation of the latter. Concomitantly, wt p53 was strongly activated by phosphorylation at Ser(46). 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.

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 p16(INK4A) or p27(Kip1), 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.

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 G(2)-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.