Oxana Komina

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.

Action of resveratrol alone or in combination with roscovitine, a CDK inhibitor, on cell cycle progression in human HL-60 leukemia cells

Results of a number of epidemiological and experimental studies indicate that polyphenols (e.g. resveratrol (RES), epicatechins etc.), antioxidant agents and abundant micronutrients in our food could have strong anti-mitotic as well as pro-apoptotic effects. In this study we raised the question whether roscovitine (ROSC), an inhibitor of cyclin-dependent kinases (CDKs) with increased selectivity towards CDK2, could be able to affect human leukemia HL-60 cells in which the p53 gene is inactivated and whether ROSC-induced effects could be additionally modulated by compounds of natural origin, especially by polyphenols e.g. RES. Exposure of HL-60 cells to ROSC for 24 h inhibited their proliferation. Flow cytometric analyses revealed that unlike MCF-7 cells, HL-60 cells were arrested in G(1) upon ROSC treatment. Furthermore, ROSC also induced apoptosis in HL-60 cells. After treatment with 40 microM ROSC for 24 h the frequency of hypoploid cells representing cells undergoing apoptosis reached approximately 50%. In the next step the action of RES alone or in combination with ROSC was examined. Interestingly, synergistic effects were observed after combined treatment for 24 h and sequential post-incubation for 48 h in the presence of RES. Such combined treatment resulted in a marked reduction of the frequency of the S- and G(2)/M-phase cells and simultaneously increased the G(1) cell population up to 80% at a fourfold lower ROSC dose. Further analyses revealed that the combined treatment strongly activated caspase-3. These results clearly evidence that RES strongly potentiates ROSC-induced apoptosis.

Synergistic effects of erlotinib and everolimus on bronchial carcinoids and large-cell neuroendocrine carcinomas with activated EGFR/AKT/mTOR pathway.

Background: Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear. Material and Methods: Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting. Results: Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib. Conclusions: Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted.

Characterization of the Antibodies to p62 Nucleoporin in Primary Biliary Cirrhosis Using Human Recombinant Antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat‐germ agglutinin (WGA)‐Sepharose, was previously detected. Recently, clinical significance of the anti‐NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti‐NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti‐p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N‐acetylglucosamine residues. More than 50% of tested PBC sera precipitated 35S‐radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti‐p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62‐positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti‐p62 in PBC. J. Cell. Biochem. 104: 27–37, 2008.

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context.

It is generally accepted that exposure of cells to a variety of DNA‐damaging agents leads to up‐regulation and activation of wild‐type (wt) p53 protein. We investigated the (re)‐activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS3 cervix carcinoma and MCF‐7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) or different anti‐cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site‐specific phosphorylation of H2AX. Unlike in MCF‐7 cells, in HeLaS3 cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS3 cells. After exposure of these cells to MNNG, the ATM‐dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG‐induced DNA breakage was recognized and properly signaled. In HeLaS3 cells wt p53 protein is not functional due to E6‐mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS3 cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS3 cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS3 cells primarily depends on their inhibitory effect on expression of E6 oncoprotein. J. Cell. Biochem. 103: 1607–1620, 2008.

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.