Margarita Maurer

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.

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G2/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G1/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G1 phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G1 block after serum starvation, but respond with a slight increase of the ratio of G1 population. Exposure of G1‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G1‐phase, but increases the ratio of S‐ and G2‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G1‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G1/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009.

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

Graphical abstract

Roscovitine Differentially Affects Asynchronously Growing and Synchronized Human MCF‐7 Breast Cancer Cells

Roscovitine (ROSC), a selective blocker of cyclin‐dependent kinases (CDKs) efficiently inhibits proliferation of exponentially growing human MCF‐7 breast cancer cells by induction of cell cycle arrest and p53‐mediated apoptosis. ROSC blocks MCF‐7 cells in G2 phase in a time‐ and concentration‐dependent manner. However, ROSC exerts a much weaker antiproliferative effect on human diploid fibroblasts. Therefore, in this study we questioned whether and to what extent the antiproliferative effect of ROSC depends on the cell cycle status of cancer cells exposed to the drug. We investigated the action of ROSC on asynchronous, exponentially growing, and on synchronized human MCF‐7 breast cancer cells. MCF‐7 cells were arrested in G1 phase after serum withdrawal and in S phase by hydroxyurea. After serum refeeding, synchronized cells started to reenter the active cell cycle after 12 h. Exposure of G1‐synchronized cells to ROSC prolonged the cell cycle arrest and was accompanied by a decrease in S‐phase cells after 24 h. A similar but weaker trend occurred after ROSC administration, to cells released from G1 arrest for 4 h. ROSC diminished the frequency of S‐phase cells. Exposure of MCF‐7 cells released from G1 arrest to ROSC for 24 h resulted in an increase of the G1‐cell population by 20%. Exposure to ROSC of MCF‐7 cells released from S‐phase block increased the ratio of S‐phase cells. These results indicate that the cell cycle status of cancer cells prior to the onset of therapy determines the outcome of treatment.

Cellular and organismal ageing: Role of the p53 tumor suppressor protein in the induction of transient and terminal senescence.

In recent years, an impact of the p53 tumor suppressor protein in the processes of cellular and organismal ageing became evident. First hints were found in model organisms like Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster where a clear connection between ageing phenotypes and pathways that are regulated by p53, were found. Interestingly, pathways that are central to the ageing process are usually also involved in energy metabolism and are highly conserved throughout evolution. This also supports the long known empiric finding that caloric restriction has a positive impact on the life span of a wide variety of organisms. Within the last years, on the molecular level, an involvement of the insulin‐like growth factor and of the histone deacetylase SRIT1 could be shown. Insight on the impact of p53 on ageing at the organismal level came from mice expressing aberrant forms of the p53 protein. Obviously, the balance of the full length p53 protein and of the shorter p44/ΔNp53 isomer bear a strong impact on ageing. The shorter isoform regulates full length p53 and in cases where there is too much of the longer isoform, this leads to elevated apoptosis resulting in decreased tumor incidence but also in premature ageing due to exhaustion of the renewal potential. Therefore, modulating the expression of the truncated p53 isoform accordingly, might lead to increased health‐span and elevated life‐span. J. Cell. Biochem. 101: 1355–1369, 2007.

Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.

Comprehensive comparative and semiquantitative proteome of a very low number of native and matched epstein-barr-virus-transformed B lymphocytes infiltrating human melanoma.

Melanoma, the deadliest form of skin cancer, is highly immunogenic and frequently infiltrated with immune cells including B cells. The role of tumor-infiltrating B cells (TIBCs) in melanoma is as yet unresolved, possibly due to technical challenges in obtaining TIBCs in sufficient quantity for extensive studies and due to the limited life span of B cells in vitro. A comprehensive workflow has thus been developed for successful isolation and proteomic analysis of a low number of TIBCs from fresh, human melanoma tissue. In addition, we generated in vitro-proliferating TIBC cultures using simultaneous stimulation with Epstein-Barr virus (EBV) and the TLR9 ligand CpG-oligodesoxynucleotide (CpG ODN). The FASP method and iTRAQ labeling were utilized to obtain a comparative, semiquantitative proteome to assess EBV-induced changes in TIBCs. By using as few as 100 000 B cells (∼5 μg protein)/sample for our proteomic study, a total number of 6507 proteins were identified. EBV-induced changes in TIBCs are similar to those already reported for peripheral B cells and largely involve changes in cell cycle proliferation, apoptosis, and interferon response, while most of the proteins were not significantly altered. This study provides an essential, further step toward detailed characterization of TIBCs including functional in vitro analysis.

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.