Differential Potential of Pharmacological PARP Inhibitors for Inhibiting Cell Proliferation and Inducing Apoptosis in Human Breast Cancer Cells.

BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s).

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.

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.

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.

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.

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.

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.

Triazoloacridone C-1305 abrogates the restriction checkpoint in cells lacking functional p53 and promotes their accumulation in the G2/M phase of the cell cycle.

Triazoloacridone C-1305, a new topoisomerase II inhibitor, exhibits potent cytostatic activity toward various tumours under in vitro and in vivo conditions. Interestingly, mouse cells lacking poly(ADP-ribose) polymerase-1 are much more sensitive to C-1305 than their normal counterparts. In the present study we tested the hypothesis that the functional status of p53 in tumour cells might have an impact on the efficiency of C-1305 in experiments with both p53-deficient human HL-60 promyelocytic leukemia cells and human MCF-7 breast cancer cells harboring a functional p53 pathway. Exposure of both cancer cell lines to C-1305 reduced the number of viable cells in a time- and concentration-dependent manner. Remarkably, however, HL-60 cells were much more strongly affected than MCF-7 cells. Measurements of DNA concentrations in single cells revealed that C-1305 arrested the tested cancer cells at the G/M transition. Analysis of the cell cycle and apoptosis regulators revealed that C-1305 strongly elevated phosphorylation of CDK1 at the inhibitory sites (Thr14/Tyr15) in HL-60 cells. Furthermore, C-1305 increased phosphorylation of pRb protein and CDK2 at Thr160 in HL60 cells, but not in MCF-7 cells. These observations suggest that C-1305 abrogates the restriction checkpoint and promotes G1/S transition in cells lacking functional p53.

Roscovitine, a small molecule CDK inhibitor induces apoptosis in multidrug-resistant human multiple myeloma cells.

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential against rapidly dividing cancers and malignancies, characterized by the accumulation of transformed cells due to deregulation of apoptosis, such as multiple myeloma. In the present study we addressed the possibility that pharmacological CDK inhibitors like Roscovitine (ROSC) may be effective against human multiple myeloma cells that have acquired resistance to doxorubicin (DOX). For this purpose we selected an experimental model of human multiple myeloma-sensitive (RPMI-8226s) and doxorubicin-resistant (RPMI-8226(DOX40)) cell lines. Exposure of RPMI-8826 cells to ROSC markedly increased the proportion of hypoploid cells, representing cells undergoing apoptosis, in both sensitive and resistant cells. Unlike ROSC, DOX at high dosage did not elevate the apoptosis rate in the RPMI-8226(DOX40) cell line. Our results show that ROSC has the capacity to induce apoptosis in the RPMI-8226(DOX40) cells overexpressing the P-gp glycoprotein. Since ROSC not only inhibits cell cycle-related CDKs but also negatively regulates kinases involved in the regulation of transcription, its administration to quiescent multidrug-resistant cells might be advantageous. Inhibition of transcription of pro-survival genes such as BCL2 and MCL-1 as well as destabilization of survivin seems to improve its therapeutic efficacy.

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.

R-roscovitine (Seliciclib) affects CLL cells more strongly than combinations of fludarabine or cladribine with cyclophosphamide: Inhibition of CDK7 sensitizes leukemic cells to caspase-dependent apoptosis.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant, apoptosis-resistant B CD19(+)/CD5(+) cells. Populations of CLL cells are heterogeneous and consist primarily of quiescent cells with a minor subset of dividing cells. In this study the efficacy of a first-line in vivo therapy was compared with treatment by R-roscovitine (ROSC) alone or by purine analogues (cladribine and fludarabine) combined with maphosphamide for 14 CLL patients under ex vivo conditions. ROSC induced the highest reduction in numbers of living B-cells, coinciding with an increased rate of apoptosis. After 24 h the percentage of apoptotic cells in ROSC-treated cultures was markedly higher than in untreated controls. ROSC also induced strong activation of the apoptosome and effector caspases in CLL cells. During progression of apoptosis the plasma membrane became permeable, resulting in the release of activated caspases into the culture medium. Leukemic cells were more sensitive to ROSC than normal mononuclear cells. Treatment with ROSC did not affect the activating phosphorylation of CDK2 or CDK1. However, ROSC decreased phosphorylation of survivin, CDK7, and RNA-Pol II, resulting in inhibition of transcription elongation and subsequent down-regulation of levels of anti-apoptotic factors, thereby facilitating apoptosis. Unlike ROSC, two other purine analogues barely affected the cellular levels of anti-apoptotic proteins and more weakly activated effector caspases. In addition, the efficacies of in vivo and ex vivo therapies were found to be correlated. Marked between-patient differences in expression patterns of apoptosis-regulating factors in CLL cells were observed, explaining the variations in patients' sensitivity to therapy.

[Biological activity of poly(ADP-ribose)polymerase-1]. / Biologiczna aktywnosc polimerazy poli(ADP-rybozy)-1.

Poly(ADP-ribose)polymerase-1 (PARP-1) catalyzes the polymerization of ADP-ribose units from NAD+ modules on target proteins, resulting in the attachment of linear or branched polymers. PARP-1 and its product poly(ADP-ribose)--PAR have recently received considerable attention because of their involvement in a wide range of cellular processes including chromatin modification, metabolism of nucleic acids, transcription regulation, and cell death. This review summarizes recent work on modular structure of six functional domains (A-F) of PARP-1 molecule in the context of three classic domains, i.e., DNA binding (DBD), automodification (AD) and catalytic (CD) released by proteolytic enzymes. A special attention is paid to subcellular localization and molecular mechanisms of PARP-1 posttranslational modifications, such as: poly(ADP-ribosylation), phosphorylation, acetylation and sumolyation. In addition, main functions of PARP-1 are discussed, focusing on the activity of this enzyme in DNA damage detection and repair, genome stability, and cell death.

p53-mediated regulation of cell cycle progression: pronounced impact of cellular microenvironment.

Data on the biological effects of some overexpressed oncogenes and their cooperation with cellular factors are, at least partially, contradictory. There are reports on the strong pro-apoptotic action of temperature-sensitive (ts) p53(135val) in transformed cells at permissive temperature. However, in our experience very high levels of p53(135val) induce in transformed rat cells at permissive temperature cell cycle arrest but not apoptosis. Comparison of the experimental protocols reveals that cells used for transfection strongly differ. Therefore, we decided to explore the impact of primary cells used for generation of cell clones on the biological effects evoked by p53 and c-Ha-Ras. In the present study, we used primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o). We immortalized rat cells using ts p53(135val) mutant and additionally generated transformed cells after co-transfection with oncogenic Ras. The RECs were transfected with a constitutively activated Ha-Ras protein, a mutation that is found in a wide variety of human tumors. The ts p53(135Val) mutant, switching between wild-type (wt) and mutant conformation, offers the possibility to study the escape from p53-mediated cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non-permissive temperature and that of cell cycle arrest at 32 degrees C strongly differed between cell clones established from yRECs and oRECs, thereby indicating that overexpression of genes such as ts p53(135Val) mutant and oncogenic-Ha-Ras does not fully override the intrinsic cellular program.

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 G(2)/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 G(1)/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 G(1) 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 G(1) block after serum starvation, but respond with a slight increase of the ratio of G(1) population. Exposure of G(1)-enriched HeLa cells to ROSC after re-feeding does not block their cell-cycle progression at G(1)-phase, but increases the ratio of S- and G(2)-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 G(1)-phase. These results show that inhibition of CDKs by ROSC in cells lacking the G(1)/S restriction checkpoint has different outcomes depending on the cell-cycle status prior to the onset of treatment.

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.

A combined treatment of HeLa cells with the farnesyl protein transferase inhibitor L-744,832 and cisplatin significantly increases the therapeutic effect as compared to cisplatin monotherapy.

Activating mutations of Ras that frequently occur during malignant transformation, enhance growth-promoting signal transduction, allowing cells to bypass stringent control of cell cycle progression, thereby rendering them highly proliferative. Abundantly expressed c-Ha-ras protein in human cervical HeLa cells is farnesylated and attached to the plasma membrane, inducing enhanced signal transduction. Exposure of HeLa cells to cisplatin very efficiently inhibits cell proliferation and induces apoptosis. Unfortunately, high doses of cisplatin are strongly cytotoxic, therefore, an alternative therapeutic strategy allowing dose reduction of cisplatin by inhibition of farnesylation could increase the curative effects of cisplatin, thereby benefiting cancer patients. We used two inhibitors of farnesyl protein transferase (FPTase), FTI, and L-744,832, to sensitize HeLa cells to the action of cisplatin. The combined administration of cisplatin and inhibitors of FPTase increased the cytostatic potency of cisplatin. L-744,832 exhibited a stronger synergistic effect in combination with cisplatin than FTI. Moreover, the efficiency of the combined therapy strongly depended on the treatment regimen: The highest efficiency was achieved after combined treatment for 24 h and post-incubation with an inhibitor of FPTase for 48 h. Following this optimized treatment, apoptosis was induced in approximately 50% of HeLa cells treated with 1 microM cisplatin, representing approximately a threefold increase as compared to cisplatin monotherapy. Combined treatment of HeLa cells with cisplatin and inhibitors of FPTase significantly increases the efficacy of the therapy and allows to reduce the dose of cisplatin. Importantly, best therapeutic effects can be achieved by post-treatment with inhibitors of FPTase.

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: HeLaS(3) 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 HeLaS(3) 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 HeLaS(3) 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 HeLaS(3) 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 HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) 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 HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.

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 (35)S-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.

Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells.

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.