Józefa Węsierska-Gądek

Increased Susceptibility of Poly(ADP-Ribose) Polymerase-1 Knockout Cells to Antitumor Triazoloacridone C-1305 Is Associated with Permanent G2 Cell Cycle Arrest

Triazoloacridone C-1305 is a novel inhibitor of DNA topoisomerase II, which exhibits potent antitumor activity toward solid tumors. In this study, antiproliferative action of C-1305 and its close analog C-1533 was investigated in nontransformed mouse fibroblasts and two mutant cell lines in which the PARP-1 gene was specifically disrupted. Unexpectedly, C-1305 very strongly affected proliferation of cells lacking poly(ADP-ribose) polymerase-1 (PARP-1), whereas the action of less active compound C-1533 toward normal and PARP-1-negative cells was comparable. The IC50 concentration of C-1305 determined for PARP-1 knockout cells was ∼150-fold lower than that determined for cells with functional PARP-1. Both studied triazoloacridones exhibited very low direct cytotoxicity as evidenced by accumulation of 7-amino-actinomycin D, and only low levels of apoptosis were observed after a 24-h exposure to studied drugs. Analysis of DNA damage induced by C-1305 by the Comet assay showed that this drug induced very low levels of DNA strand breaks. C-1305 strongly affected cell cycle progression in normal and PARP-1 mutant cells and arrested both cell types in G2-M phase. However, the G2-M arrest induced by C-1305 was greatly prolonged in PARP-1-deficient cells as compared with normal fibroblasts. Together, these results show that mouse cells lacking PARP-1 are extremely sensitive to C-1305, a new topoisomerase II inhibitor. This is in striking contrast with previous reports in which PARP-1-deficient cells were shown to be resistant to classical topoisomerase II inhibitors. Our data also suggest that the PARP-1 status might be essential for the maintenance of the G2 arrest induced by C-1305.

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells.

Human MCF‐7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase‐3 activity. We reported recently that roscovitine (ROSC), a potent cyclin‐dependent kinase 2 inhibitor, arrests human MCF‐7 breast cancer cells in the G2 phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF‐7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time‐ and dose‐dependent manner. The p53 level increased despite upregulation of Hdm‐2 protein and was attributable to the site‐specific phosphorylation at Ser‐46. The p53 protein phosphorylated at serine 46 causes the up‐regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC‐induced p53 activation. Exposure of MCF‐7 cells to ROSC activated homeodomain‐intereacting protein kinase‐2 (HIPK2). The overexpression of wild‐type but not kinase inactive HIPK2 increased the basal and ROSC‐induced level of p53 phosphorylation at Ser‐46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC‐induced P‐Ser‐46‐p53, thereby stabilizing wt p53 and increasing the efficacy of drug‐induced apoptosis in MCF‐7 cells. These results identify HIPK2 as a component of the ROSC‐induced signaling pathway leading to the stabilization and activation of wt p53 protein. J. Cell. Biochem. 100: 865–874, 2007.

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.

A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death

The efficacy of distinct anti‐cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti‐proliferative and pro‐apoptotic action of a number of anti‐cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti‐proliferative and pro‐apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose‐ and time‐dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases‐3/7 in two separate microtiter plates using the CellTiter‐Glo™ Luminescent Cell Viability Assay and the Caspase‐Glo™ 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter‐Blue™ Cell Viability Assay and Caspase‐Glo™ 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases‐3/7. The new multiplexing assay offers a range of benefits over standard assays.

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.

Selective cyclin-dependent kinase inhibitors discriminating between cell cycle and transcriptional kinases: future reality or utopia?

Progression of the cell cycle is controlled by activating and inhibiting cellular factors. The delicate balance between these positive‐ and negative‐acting regulators warrants proper cell cycle progression in normal cells and facilitates cellular response to a variety of stress stimuli. The increased activity of the positive regulators of the cell cycle in cancer cells is frequently accompanied by the loss or inactivation of the inhibitors of cyclin‐dependent kinases (CDKs). The supplementation of the cellular CDK inhibitors by the pharmacological counterparts is a very promising therapeutic option. The generated pharmacological inhibitors of CDKs belong to different classes of compounds and display various CDK inhibitory features. In this article the action and specificity of CDK inhibitor roscovitine, belonging to the group of purine analogues, is reviewed and the rationale for dissecting the inhibitory action on cell cycle and transcriptional CDKs is discussed.

Functional p53 in cells contributes to the anticancer effect of the cyclin‐dependent kinase inhibitor roscovitine

Inhibitors of cyclin‐dependent kinases (CDKs) undergoing clinical trials as anticancer agents usually target several CDKs in cells. Some of them are also able to increase cellular levels of p53 protein and to activate p53‐regulated transcription. To define the role of p53 in the anticancer effect of selective CDK inhibitors, two related compounds roscovitine and olomoucine II were studied. Roscovitine differs functionally from its congener olomoucine II only in the selectivity towards transcriptional CDK9. Action of both compounds on proliferation, cell‐cycle progression, and apoptosis was examined in RPMI‐8226 cells expressing the temperature‐sensitive mutant of p53 and in MCF‐7 cells with wild‐type p53. Both compounds blocked proliferation, decreased phosphorylation of RNA polymerase II, downregulated antiapoptotic protein Mcl‐1 in both cell lines in a dose‐dependent manner, and also activated p53 in MCF‐7 cells. Moreover, we showed that the anticancer efficiency of CDK inhibitors was enhanced by active p53 in RPMI‐8226 cells kept at permissive temperature, where downregulation of Mcl‐1, fragmentation of PARP‐1, and increased caspase‐3 activity was detected with lower doses of the compounds. The results confirm that functional p53 protein may enhance the anticancer activity of roscovitine that could be beneficial for anticancer therapy. J. Cell. Biochem. 107: 428–437, 2009.

Pleiotropic effects of selective CDK inhibitors on human normal and cancer cells.

Escape from the proper control of the cell cycle by up-regulation of cyclins or aberrant activation of cyclin-dependent kinases (CDKs) as well as by inactivation of cellular inhibitors of CDKs (CKI) leads to malignant transformation. Loss of cellular CKIs in cancers provided a rationale for development of pharmacological inhibitors of CDKs. Recently synthesized CKIs, e.g., purine derivatives such as olomoucine (OLO) and roscovitine (ROSC) are non-genotoxic and exhibit increased selectivity towards CDK2 and CDK7/9. Interestingly, both drugs induce additional effects. Recently, a new, unexpected action of OLO on normal human cells was observed. OLO strongly up-regulates CLIMP-63, a 65 kD protein that mediates the anchoring of the ER to microtubules. Moreover, ROSC induces in human MCF-7 cells phosphorylation of p53 protein at Ser-46 which in turn initiates caspase-independent apoptosis. In the present contribution we raised the question whether both CKIs would be able to block cell cycle progression and to reactivate p53 protein in human HPV-positive HeLa cervix carcinoma cells. We also addressed the question whether exponentially growing cancer cells are more susceptible to the inhibitory action of CKIs than normal cells. Our results show that HeLa cells are much more sensitive to ROSC than normal fibroblasts. ROSC induces G(2) arrest and apoptosis in HeLa cells. It also reactivates and stabilizes wt p53 protein. The increase of p53 protein coincides with down-regulation of E6 oncoprotein. Thus, the biological action of substituted purines is not restricted to the inhibition of CDKs and open new perspectives for their therapeutic applications.

Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells.

We reported recently that roscovitine (ROSC), a selective cyclin‐dependent kinase (CDK) inhibitor, arrested human MCF‐7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC‐induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF‐7 cells cultivated in medium with and without phenol red. The kinetics of MCF‐7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF‐7 cells cultivated in phenol red‐deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti‐proliferative and pro‐apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC‐induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER‐positive breast cancers by CDK inhibitors is diminished in the presence of estrogen‐mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors. J. Cell. Biochem.