The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Congenital and acquired resistance to chemotherapy and radiotherapy has been a major obstacle for successful cure of human malignancies. Therefore, alternative drugs intended to overcome this clinical hindrance are searched. 1 The inhibitors of cyclindependent kinases (CDKs) represent a well-defined group of biologically active compounds suitable for nonconventional treatments. 2,3 One potential adjunct of the CDK inhibitors to classical cytostatics is direct induction of programmed cell death (PCD). The CDK inhibitors stimulate cell death from any stage of the cell cycle, 4 whereas the DNA-damaging drugs kill more efficaciously S-phase cells. 1 Roscovitine (ROSC) belongs to chemical inhibitors of CDK; the mechanism turns out to be a competitive inhibition of ATP binding. 3,5,6 ROSC occupies the purine-binding pocket, located in the cleft between the small and large lobes of the kinase. The kinase specificity of ROSC was investigated with 25 highly purified kinases. 3 Most kinases were not significantly inhibited by ROSC. However, cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 were substantially inhibited by ROSC. 3 Considering this unique selectivity for some CDK, ROSC clearly provides a useful reagent not only for cell cycle studies 3 but also offers a potent drug for efficient anticancer therapy. 1 The effect of ROSC on the cell cycle progression is independent of the tumour-suppressor protein p53, a primary controller of the endogenous cell-cycle inhibitor p21 waf1/Cip1 , because an inhibition was observed irrespective of p53 status: normal vs. mutated/deleted. Recently, ROSC has been shown to drive the estrogen-negative MDA-MB-231 human breast cancer cells in apoptotic cell death. 7 Therefore, it appeared interesting to address the question whether ROSC's capability to initiate the apoptosis in breast cancer cells is dependent on the estrogen-receptor status. Furthermore, it also seemed attractive to examine whether wt p53 in human breast carcinoma cells can be activated by ROSC. The p53 protein, a product of a tumour suppressor gene, is known to exert in response to various types of stress not only antiproliferative effects including a transient cell cycle arrest or terminal cellular senescence, but also...
We previously reported that therapy of human cervical carcinoma HeLa cells with CP induced segregation of nucleoli and changes of nuclei characteristic of apoptosis. We raised the question of whether p53 can be reactivated by chemotherapy in HeLa cells despite the presence of HPVencoded E6 activity. Cellular levels of p53 protein increased after CP treatment, reaching a maximum after 6 hr. p53 protein accumulated preferentially in the nucleoli, with a peak after 15 hr. CP-induced nucleolar targeting of p53 appears to be selective because p73, another member of the p53 gene family, accumulated primarily in nuclei in response to CP. Monitoring of the intranuclear distribution of Hdm-2, a negative regulator of p53, revealed this protein in the nucleoli of untreated controls translocated into chromatin during CP therapy. Interestingly, p14ARF showed an inverse intranuclear redistribution. Proteasome inhibitors were not able to mimic the effect of CP on p53 levels. Since the reduced stability of wild-type p53 protein in HeLa cells is a consequence of its enhanced ubiquitination by virally encoded E6 protein, resulting in its accelerated degradation, we checked the cellular level of E6 during CP therapy. Six hours after application of CP, E6 protein expression was markedly reduced. This coincided with the increase of cellular p53 and preceded the nucleolar accumulation of p53 protein, indicating that repression of virally coded E6 protein by CP contributes to the restoration of p53 expression.
Activation of the p53 tumour suppressor protein by distinct forms of stress leads to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. The cyclin-dependent kinase inhibitor roscovitine has been shown to induce nuclear accumulation of wild-type p53 in human untransformed and tumour-derived cells. We analyzed the response of different human tumour cell lines to roscovitine treatment with respect to their p53 status. Striking induction of wild-type p53 protein and dramatic enhancement of p53-dependent transcription, coinciding with p21WAF1 induction, was observed in wildtype, but not mutant, p53-bearing tumour cells after treatment with roscovitine. The transcriptional activity of p53 was substantially higher in roscovitine-treated cells than in cells irradiated with ultraviolet C or ionizing radiation, even though all these agents induced a similar amount of p53 accumulation. These results highlight the therapeutic potential of roscovitine as an anticancer drug, especially in tumours retaining a functional wild-type p53 pathway.
The detection of doxorubicin-induced apoptosis in individual cardiomyocytes was performed on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells their lysis in an alkaline environment and the separation of released apoptotic DNA fragments. The fragments with typical 180 base pairs ladder pattern were electrophoretically resolved in a 2% solution of linear polyacrylamide with 0.1 M NaOH on a migration distance of 6 mm. The laser-induced fluorescence of fragments labeled by ethidium bromide was monitored by a photomultiplier tube mounted on a confocal microscope. The causal relation between the enhanced doxorubicin concentration and the extent of DNA fragmentation in a single cell was confirmed. The results show that the extent of DNA fragmentation is proportional to the time of a cell treatment. Onset of necrosis was evident in cardiomyocytes treated by doxorubicin for more than 24 h. The adverse effect of doxorubicin, an important cytostatics used for the treatment of many solid tumors, leads to the destruction of cardiomyocytes and, consequently, may result in the heart failure of treated individuals. Therefore, the monitoring of the extent of apoptotic DNA damage of cardiac myocytes represents critical step toward understanding of the development of chronic doxorubicin-induced cardiomyopathy.
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