Manuela Vici

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein.

BackgroundLactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.ResultsWe show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin.ConclusionAdhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein.

Binding of Human Plasminogen to Bifidobacterium

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction.

Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

Primary endocervical extraosseous Ewing's sarcoma/PNET

A 36-year-old woman presented with intermenstrual spotting and was found to have a cystic mass involving the uterine cervix on a pelvic ultrasound examination. A necrotic and hemorrhagic tumor was excised by hysterectomy and processed for light and electron microscopic investigation and molecular analysis. Microscopic examination revealed a small round cell tumor that immunohistochemical studies (including staining for the highly restricted surface antigen p30/32MIC2) and ultrastructural studies indicated was an extraosseous Ewings sarcoma (EES)/primitive neuroectodermal tumor (PNET). This diagnosis was established by detection of EWS/ERG fusion transcript through reverse transcription polymerase chain reaction (RT-PCR) with nested primers. Full body computed tomography failed to detect any extrauterine tumor, and the patient is clinically free of disease 18 months after hysterectomy. This case represents the first report of a primary EES/PNET arising in the uterine cervix.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient human osteosarcoma cell lines.

Abstract.  Objectives: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. Material and methods: RB‐ and p53‐proficient U2OS cells and the RB‐ and p53‐deficient SAOS‐2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. Results: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G1‐S and G2‐M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS‐2 cells. No effect on cell cycle progression after actinomycin D‐induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS‐2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS‐2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS‐2 cells after the long‐term actinomycin D treatment, whereas no modification was observed in U2OS cells. Conclusions: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB‐ and p53‐deficient cells. A daily‐repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Loss of retinoblastoma tumor suppressor protein makes human breast cancer cells more sensitive to antimetabolite exposure.

Purpose: The RB tumor-suppressor activity may influence the therapeutic response in human breast cancers. The effect of adjuvant therapy on clinical outcome of breast cancer patients was analyzed, and the sensitivity to 5-fluorouracil (5-FU) and methotrexate was investigated in MCF-7 and HCT-116 human cancer cells, according to their RB status. Experimental Design: RB protein (pRB) expression was prospectively evaluated by immunocytochemistry in 518 consecutive patients and its predictive value was determined according to the adjuvant therapeutic treatments. MCF-7 and HCT-116 human cancer cells silenced for RB1 expression were treated with 5-FU and methotrexate, at the same concentrations and time exposures as determined in the interstitium of breast cancers of patients treated with adjuvant chemotherapy. Results: Multivariate analysis of disease-free survival, including all the established clinical and histopathologic prognostic variables, indicated that the absence of pRB expression was the only predictive factor of good clinical outcome in patients treated with standard systemic chemotherapy (cyclophosphamide, methotrexate, and 5-FU) but not in patients treated with endocrine therapy alone. 5-FU and methotrexate significantly reduced the growth rate of RB1-silenced but not of control MCF-7 and HCT-116 cells. This was likely due to the absence of a DNA damage checkpoint with accumulation of DNA double-strand breaks in RB1-silenced but not in control cells. Conclusions: The absence of pRB expression renders human breast cancer cells more sensitive to 5-FU and methotrexate and predicts a good clinical outcome for patients treated with adjuvant chemotherapy. We suggest that patients with RB-negative breast cancers should be treated with systemic chemotherapy.

Key role of the achievement of an appropriate ribosomal RNA complement for G1-S phase transition in H4-II-E-C3 rat hepatoma cells

Cell growth is closely related to cell proliferation and an adequate ribosome biogenesis appears to be necessary for cell duplication. In the present study, we have investigated the relationship between rRNA synthesis and cell cycle progression. For this purpose, in a first set of experiments, we evaluated the effect of rRNA synthesis variation on cycle duration in asynchronously growing H4‐II‐E‐C3 rat hepatoma cells. Cells were either treated with insulin or insulin plus actinomycin D (AMD). The hormone stimulated ribosome biogenesis, which was later followed by an increased synthesis of DNA and a shortening of cell doubling time (DT). Bivariate flow cytometry indicated that the reduced length of the cell cycle was mainly due to the shorter G1‐phase. AMD, at the concentration of 0.04 μg/ml, hindered ribosome biogenesis without affecting heterogeneous RNA production. A 12‐h reduction in ribosome biogenesis level by AMD caused a lowering of DNA synthesis and a lengthening of cell DT with a longer G1‐phase. In a second set of experiments, we analyzed the cell content variations of 28S and 18S rRNA transcripts during G1 phase in H4‐II‐E‐C3 cells, synchronized by serum deprivation, and then stimulated by serum, serum plus insulin, and serum plus insulin and AMD. In control cells, a progressive increase in rRNA content occurred until the highest value of rRNA content was reached 21 h after serum stimulation. In insulin‐treated cells, the highest rRNA value was reached at 12 h whereas in AMD‐treated cells, the rRNA quantity was constantly low until 18 h and then sharply increased at 21 h. In the three experimental conditions, the highest values of rRNA amount were reached at the end of G1 phase and were quite similar to one another. We also evaluated, by real‐time RT‐PCR, cyclin E mRNA expression, which appeared to sharply increase at those times in which the maximum increase in the rRNA content was observed. Our results indicated that the achievement of an appropriate amount of rRNA allows G1/S phase transition, probably by modulating the expression of cyclin E mRNA.

Electron Microscopic and Immunocyto- chemical Profiles of Human Subcutaneous Fat Tissue Microvascular Endothelial Cells

The ultrastructural and immunocytochemical characteristics of microvascular cells from human subcutaneous fat tissue were studied after the addition of collagenase and Percoll density gradient, respectively. Monoclonal and polyclonal antibodies directed against antigens specific for endothelial cells (factor VIII,Ulex europeaus, CD31, and CD34), pericytes (muscle-specific actin and desmin), adipocytes (S-100 protein), and monocytes-macrophages (MAC 387 and 150.95 protein) were demonstrated by alkaline phosphatase monoclonal antialkaline phosphatase and protein A-gold techniques. In addition, to determine whether the harvesting method interfered with microvascular cell function, DOT immunoassays of factor VIII and CD34 were conducted on solutions recovered at collagenase incubation as well as after nylon filtration and Percoll administration, respectively. After the collagenase step, the vast majority of microvascular cells had the typical ultrastructural and immunophenotypical features of endothelial cells. In sharp contrast, following the Percoll step, only 1% to 18% of microvascular cells stained with factor VIII,Ulex europeaus, and CD31, whereas 90% of them expressed the CD34 antigen. Surprisingly, DOT immunoassay revealed the presence of factor VIII in the washing buffer recovered after the Percoll step only. Consequently the decreased expression of common endothelial cell markers (factor VIII,Ulex europaeus, and CD31) observed at the end of the cell isolation procedure was related to the adverse effects of Percoll on endothelial cell function. The CD34 surface molecule, being highly resistant, is particularly well suited for unequivocal characterization of microvascular cells as true endothelium.

The p53-mediated sensitivity of cancer cells to chemotherapeutic agents is conditioned by the status of the retinoblastoma protein.

Despite the well‐established function of p53 in determining cell cycle arrest and/or apoptosis in response to cytostatic/cytotoxic stresses, the role of the p53 status in the response to chemotherapeutic agents in human cancers has been not clearly defined. We wondered whether this was due to the fact that the p53‐mediated response to chemotherapy drugs might be conditioned by the status of the retinoblastoma protein (pRb), a downstream factor of the pathway activated by p53 stabilization, which is frequently disrupted in cancer. The dependence of p53‐mediated chemosensitivity on pRb status was first investigated in a prospective study on the prognostic relevance of p53 in breast cancer patients treated with adjuvant chemotherapy (5‐fluorouracil, methotrexate and cyclophosphamide). Univariate analysis of disease‐free survival (DFS) indicated that the p53 status, immunohistochemically evaluated, had no predictive value if considered independently of the pRb status. However, in patients with cancer with pRb neither lost nor hyperphosphorylated, p53 was significantly associated with the prognosis and, in a multivariate analysis of DFS including the established clinical and histopathological prognostic parameters, was found to be the only factor predicting the progression of the disease. We then studied the role of pRb status in the p53‐mediated response to 5‐fluorouracil and methotrexate or doxorubicin treatment in three human cancer cell lines. We found that in these cells the chemosensitivity was strictly dependent on the p53 status. However, either RB1 silencing or pRb hyperphosphorylation, caused by p16(INK4a) silencing, strongly reduced the p53‐mediated response to chemotherapeutic agents. These results demonstrated that: (a) the p53‐mediated response to chemotherapeutic agents induces a cytostatic/cytotoxic effect only in cancers with unaltered pRb pathway; and (b) the p53 status can actually predict the clinical outcome in this group of cancer patients. Copyright