Jozefa Wesierska-Gadek

Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis

Although there have been significant advances in understanding the clinical and biochemical features of primary biliary cirrhosis (PBC), there is still a paucity of data on the usefulness of biomarkers as prognostic indicators. This is particularly important at the time of initial diagnosis. Indeed, the widespread use of antimitochondrial antibody testing has led to an earlier diagnosis of asymptomatic PBC and it is difficult to predict which patients will experience a benign versus a rapidly progressive course. To address this issue, we examined a unique population of 127 newly diagnosed patients with PBC during a 15‐year period of observation that began in January 1990. Sera from these patients were analyzed for antimitochondrial, antinuclear, and anti–smooth muscle antibodies, and immunoblotting was performed for nuclear pore complex (NPC). The patients were then followed up longitudinally using biochemical liver function tests. No patient was under any medical therapy for PBC at the time of the initial sera collection. Data were analyzed based not only on the clinical features, but also the Mayo score and specific outcome measures, including time to death, need for liver transplantation, and complication free survival. Among patients with early disease, bilirubin increased to >2 mg/dL in the anti‐NPC(+) patients (26% vs. 5%, P = .019). Anti‐NPC antibodies remained stable or slightly increased over the period of observation. In conclusion, anti‐NPC identifies patients likely to experience an unfavorable clinical course and more rapid disease progression. (HEPATOLOGY 2006;43:1135–1144.)

Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis

BACKGROUND/AIMS Antibodies against nuclear pore complexes (NPCs) have been detected in primary biliary cirrhosis (PBC), but their clinical relevance is still unsettled. METHODS We tested sera from 171 consecutive PBC patients and 230 control subjects (149 with autoimmune or viral liver diseases, 28 with systemic lupus erythematosus, and 53 healthy) by immunoblotting for antibodies against purified human NPCs. RESULTS Antibodies to NPCs were detected in 27% of the patients with PBC, were highly specific (97%), and were not associated with antimitochondrial antibodies. Their prevalence was higher in symptomatic patients (36 vs. 16%, P < 0.01) and was associated (P < 0.001) with more severe disease, as assessed by the presence of cirrhosis or its complications (13% prevalence in patients without cirrhosis, 31% in uncomplicated, and 54% in complicated cirrhosis), or by the application of the Mayo prognostic model (12% in the lowest, 21% in the median, 47% in the highest score tertile). Positive patients had higher levels of serum bilirubin (2.2 +/- 3.7 vs. 1.0 +/- 1.1 mg/dl, P < 0.01) and more marked inflammatory infiltrates on liver biopsy (P < 0.05). CONCLUSIONS Autoantibodies to NPCs are more prevalent in PBC patients than in controls and are strongly associated with more active and severe disease.

Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells.

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. Inhibition of DNA synthesis and accumulation of G2/M‐arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP‐1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF‐7 cells with caspase‐3 did not change the kinetics of ROSC‐induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC‐induced changes. Cell cycle arrest precedes the main wave of apoptosis.

ADP‐ribosylation of p53 tumor suppressor protein: Mutant but not wild‐type p53 is modified

Poly(ADP‐ribosyl)ation of mutant and wild‐type p53 was studied in transformed and nontransformed rat cell lines constitutively expressing the temperature‐sensitive p53135val. It was found that in both cell types at 37.5°C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP‐ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence studies with affinity purified anti‐poly(ADP‐ribose) transferase (pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 37.5°C, where p53 adopts wild‐type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti‐p53 and anti‐pADPRT antibodies. A poly(ADP‐ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tighly complexed to pADPRT and becomes modified. At 32.5°C binding to DNA of p53 or its temperature‐dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein.

Antibodies to nuclear lamins in autoimmune liver disease

Antibodies to nuclear lamins were detected in sera of patients with autoimmune liver disease. In indirect immunofluorescence tests, these sera revealed staining of the nuclear periphery. Using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes, and chromatographically purified lamins as antigen source, the nuclear lamins A, B, and C were identified as reactive antigens in immunoblotting experiments. The lamins were also identified by 2-D gel electrophoresis. Antibodies to nuclear lamins occurred in 12 of 16 cases of active lupoid hepatitis, but not in 35 patients with the disease in remission. However, only 3 of 37 sera of patients with primary biliary cirrhosis contained anti-lamin antibodies. Autoimmune liver disease sera reacted preferentially with lamins A/C and less frequently with lamin B or lamins A/B/C.

Role of PARP on iNOS pathway during endotoxin-induced acute lung injury

Objective Excessive nitric oxide (NO) and especially peroxynitrite may cause pulmonary tissue damage, e.g., through lipid peroxidation and/or exhaustion of cellular energy depletion induced by activation of poly (ADP-ribose) polymerase (PARP). Furthermore, PARP seems to aggravate tissue destruction by regulating the expression of respective genes.Design Prospective animal study.Setting University research laboratory.Intervention We investigated the effect of competitive PARP inhibition by 3-aminobenzamide (3-AB) on the pulmonary iNOS pathway after infusion of lipopolysaccharide (LPS).Measurements and results The pretreatment of rabbits with 3-AB attenuated the LPS-induced iNOS mRNA and protein expression analyzed by RT-PCR and Western blot, and plasma nitrite concentrations quantified by Griess reaction (71±6%, 93±6% vs baseline). Electromobility shift assay showed an enhanced NF-κB and attenuated AP-1 activation after 3-AB vs LPS alone. Lipid peroxidation determined as levels of thiobarbituric acid reactive substances in plasma and lung tissue was reduced by 50% in the LPS+3-AB in comparison to LPS alone. Simultaneously, 3-AB was able to inhibit correspondingly the LPS-induced extravasation of gold-labeled albumin and increase of alveolo-arterial oxygen difference.Conclusion PARP regulates the pulmonary NO pathway during endotoxemia via AP-1 and not NF-κB. Thus, pharmacological inhibition of PARP might be an effective intervention to prevent endotoxin-induced lung injury, interrupting the vicious circle of NO production and PARP activation.

Dual action of the inhibitors of cyclin-dependent kinases: targeting of the cell-cycle progression and activation of wild-type p53 protein

The inhibition of cyclin-dependent kinases (CDKs) represents a novel approach to the therapy of human malignancies. Already in clinical trials, recently developed CDK inhibitors very efficiently target the rapidly proliferating cancer cells and inhibit their cell-cycle progression. Interestingly, some CDK inhibitors additionally affect the stability and activity of the tumour-suppressor protein p53, thereby enhancing their antiproliferative action towards cancer cells. Considering the fact that the p53 protein is mutated or inactivated in ~ 50% of all human cancers, the efficacy of CDK inhibitor therapy could differ between cancer cells depending on their p53 status. Moreover, recent reports demonstrating that some cancer cells can proliferate despite CDK2 inhibition questioned the central role of CDK2 in the cell-cycle control and suitability of CDK2 as a therapeutic target; however, the p53 activation that is mediated by CDK inhibitors could be essential for the efficacy of CDK inhibitors in therapy of CDK2-independent cancers. Furthermore, there is also reason to believe that CDK2 inhibitors could be used for another purpose, to protect normal cells from the effects of chemotherapy.

Roscovitine up‐regulates p53 protein and induces apoptosis in human HeLaS3 cervix carcinoma cells

Exposure of human HeLaS3 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 HeLaS3 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 G2/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 HeLaS3 cells. These results indicate that therapy of HeLaS3 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. J. Cell. Biochem. 105: 1161–1171, 2008.

Nucleolus and apoptosis.

Abstract: The nucleolus represents a highly dynamic nuclear compartment of the interphase nucleus. It plays a key role in ribosome biogenesis. The number of nucleoli, their size, and their activity increase in exponentially growing cells; therefore these parameters reflect the proliferating activity of the cells. A variety of staining techniques have been employed to vizualize nucleolar changes in malignant cells. Staining of so‐called nucleolar organizer regions (NORs), based upon a strong avidity of nucleolar proteins to bind silver ions, represents the technique most frequently used by pathologists. Nucleolar changes and pleomorphism associated with overt proliferation of tumor cells have also been documented by immunohistochemical and ultrastructural studies. Contrary to cell proliferation, cytostatics‐induced changes of nucleolar phenotype in malignant cells point to a potential role of nucleolar components in the execution of active cell death. Recent studies have provided direct clues that so‐called death domains and other apoptosis‐related proteins are accumulated in nucleoli upon induction of active cell death. It can be concluded that the plurifunctionality of nucleoli regarding cell proliferation and apoptosis could open new vistas toward understanding dysregulation in malignant cells.

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1

We recently characterized the interaction between poly(ADP‐ribose) polymerase‐1 (PARP‐1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP‐1 and of human wild‐type (wt) p53 were involved in this protein–protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP‐1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy‐terminal fragments of p53 were sufficient to confer binding to PARP‐1, whereas the amino‐terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino‐terminal and central fragments of PARP‐1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post‐translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two‐dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho‐serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull‐down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP‐1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy‐terminal part of p53 undergoes complex formation with PARP‐1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP‐1 and overexpression of PARP‐1. Our results unequivocally show that the presence of PARP‐1 regulates the basal expression of wt p53 in unstressed cells.