Radiotherapy in localized prostate cancer: a multicenter analysis evaluating tumor control and late toxicity after brachytherapy and external beam radiotherapy in 1293 patients.

BACKGROUND AND PURPOSE: Comparing oncological outcomes and toxicity after primary treatment of localized prostate cancer using HDR- or LDR-mono-brachytherapy (BT), or conventionally (CF) or moderately hypofractionated (HF) external beam radiotherapy. MATERIALS AND METHODS: Retrospectively, patients with low- (LR) or favorable intermediate-risk (IR) prostate cancer treated between 03/2000 and 09/2022 in two centers were included. Treatment was performed using either CF with total doses between 74 and 78 Gy, HF with 2.4-2.6 Gy per fraction in 30 fractions, or LDR- or HDR-BT. Biochemical control (BC) according to the Phoenix criteria, and late gastrointestinal (GI), and genitourinary (GU) toxicity according to RTOG/EORTC criteria were assessed. RESULTS: We identified 1293 patients, 697 with LR and 596 with IR prostate cancer. Of these, 470, 182, 480, and 161 were treated with CF, HF, LDR-BT, and HDR-BT, respectively. For BC, we did not find a significant difference between treatments in LR and IR (p = 0.31 and 0.72). The 5­year BC for LR was between 93 and 95% for all treatment types. For IR, BC was between 88% in the CF and 94% in the HF group. For CF and HF, maximum GI and GU toxicity grade ≥ 2 was between 22 and 27%. For LDR-BT, we observed 67% grade ≥ 2 GU toxicity. Maximum GI grade ≥ 2 toxicity was 9%. For HDR-BT, we observed 1% GI grade ≥ 2 toxicity and 19% GU grade ≥ 2 toxicity. CONCLUSION: All types of therapy were effective and well received. HDR-BT caused the least late toxicities, especially GI.

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

Two cellular proteins encoded by the breast and ovarian cancer type 1 susceptibility (BRCA1 and BRCA2) tumor suppressor genes are essential for DNA integrity and the maintenance of genomic stability. Approximately 5-10% of breast and ovarian cancers result from inherited alterations or mutations in these genes. Remarkably, BRCA1/BRCA2-deficient cells are hypersensitive to selective inhibition of poly(ADP-ribose)polymerase 1 (PARP-1), whose primary functions are related to DNA base excision repair; PARP-1 inhibition significantly potentiates the cytotoxicity of various anti-cancer drugs, including inhibitors of topoisomerase I and II. In the present study, we examined the anti-proliferative and pro-apoptotic effects of C-1305, a selective inhibitor of topoisomerase II, on human breast cancer cell lines with different BRCA1 and p53 statuses. BRCA1-competent breast cancer cell lines exhibited different responses to topoisomerase II inhibition. BT-20 cells that express high levels of BRCA1 levels were most resistant to C-1305 than other tested cells. Surprisingly, pharmacological interference with PARP-1 activity strongly inhibited their proliferation and potentiated the efficacy of C-1305 treatment. In contrast, PARP-1 inhibition only weakly affected the proliferation of BRCA1-deficient SKBr-3 cells and was not synergistic with the effects of C-1305. Further experiments revealed that the inhibition of PARP-1 in BT-20 cells caused the accumulation of DNA strand breaks and induced caspase-3 dependent apoptosis. These results seem to indicate that PARP-1 inhibition can potentiate the cytotoxicity of anti-cancer drugs in cancer cells with functional BRCA1 and suggest that mutations in other DNA repair proteins may render cancer cells more sensitive to interference with PARP-1 activity.

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.

Can ex vivo evaluation (testing) predict the sensitivity of CLL cells to therapy with purine analogs in conjunction with an alkylating agent? A comparison of in vivo and ex vivo responses to treatment.

Malfunctions in the regulation of apoptosis cause the accumulation of malignant, long-lived B CD19+/CD5+ cells in chronic lymphocytic leukemia (CLL). The primary goal in CLL therapy is to overcome resistance to apoptosis and efficiently trigger programmed cell death in leukemic cells. This study demonstrated that the in vivo responses of malignant cells from CLL patients after administration of purine analogs (cladribine/fludarabine) with cyclophosphamide vary significantly. For comparative purposes, the sensitivity of leukemic cells obtained from the same CLL patients to conventional purine analogs and the selective CDK inhibitor R-roscovitine (ROSC) was determined, with and without the addition of an alkylating agent, prior to the onset of in vivo therapy. The kinetics and rate of spontaneous and drug-induced apoptosis of CLL cells under ex vivo conditions differed significantly between patients, mirroring the variability observed during in vivo treatment. Interestingly, individual patients' leukemic cells were comparably sensitive to the drugs under both conditions. Of the drugs examined, ROSC exerted the highest therapeutic efficacy under ex vivo conditions. Our results indicate that ex vivo testing might be useful for identifying the most potent first-line therapeutic regimen for specific CLL patients and possibly for the design of therapies tailored for individual CLL patients.

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 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.

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.

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.

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.

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.

Impact of roscovitine, a selective CDK inhibitor, on cancer cells: bi-functionality increases its therapeutic potential.

Increased expression and activity of proteins driving cell cycle progression as well as inactivation of endogenous inhibitors of cyclin-dependent kinases (CDKs) enhance the proliferative potential of cells. Escape of cells during malignant transformation from the proper cell cycle control rendering them independent from growth factors provides rationale for therapeutic targeting of CDKs. Exposure of rapidly growing human MCF-7 breast cancer and HeLa cervix cancer cells to roscovitine (ROSC), a selective inhibitor of CDKs, inhibits their proliferation by induction of cell cycle arrest and/or apoptosis. The outcome strongly depends on the intrinsic traits of the tumor cells, on their cell cycle status prior to the onset of treatment and also on ROSC concentration. At lower dose ROSC primarily inhibits the cell cycle-related CDKs resulting in a strong cell cycle arrest. Interestingly, ROSC arrests asynchronously growing cells at the G(2)/M transition irrespective of the status of their restriction checkpoint. However, the exposure of cancer cells synchronized after serum starvation in the late G(1) phase results in a transient G(1) arrest only in cells displaying the intact G(1)/S checkpoint. At higher dosage ROSC triggers apoptosis. In HeLa cells inhibition of the activity of CDK7 and, in consequence, that of RNA polymerase II is a major event that facilitates the initiation of caspase-dependent apoptosis. In contrast, in the caspase-3-deficient MCF-7 breast cancer cells ROSC induces apoptosis by a p53-dependent pathway. HIPK2-mediated activation of the p53 transcription factor by phosphorylation at Ser46 results in upregulation of p53AIP1 protein. This protein after de novo synthesis and translocation into the mitochondria promotes depolarization of the mitochondrial membrane.

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 G(2) 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 G(1) 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 G(1)-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 G(1) arrest for 4 h. ROSC diminished the frequency of S-phase cells. Exposure of MCF-7 cells released from G(1) arrest to ROSC for 24 h resulted in an increase of the G(1)-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.

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.

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.

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.

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 autoantibodies against components of the nuclear pore complexes: high frequency of anti-p62 nucleoporin antibodies.

Antibodies against nuclear components (ANAs) occur in sera of approximately 50% of patients with primary biliary cirrhosis (PBC). By indirect immunofluorescence (IIF) ANA-positive PBC sera generate most frequently, homogeneous, speckled, centromere, and rim-like staining patterns. A perinuclear staining pattern is indicative for the reactivity of the sera with the components of the nuclear envelope. A substantial subset of PBC patients develops antibodies against constituents of the nuclear pore complexes (NPCs). These autoantibodies target two major autoantigens: gp210 glycoprotein and p62 kDa nucleoporin. Originally, a strong reaction of PBC with a 60 kDa protein of NPCs that was affinity purified on wheat-germ agglutinin (WGA)-Sepharose was described. Recently, using human recombinant p62 nucleoporin the identity of the reactivity was confirmed. In this work we compared by immunoprecipitation the reactivity of 20 PBC sera with the two recombinant autoantigens of the NPCs. Two out of 20 (10%) PBC sera precipitated recombinant gp210 glycoprotein and 11 out of 20 (55%) PBC sera reacted with p62 nucleoporin. These results evidence that anti-p62 antibodies occur more frequently than the autoantibodies against gp210 glycoprotein. Considering the recently reported clinical significance of ANAs in PBC, the prognostic value of the anti-NPC antibodies and their correlation with severity and progression of the disease is under evaluation.