Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation.

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

IgG and IgA response to Simkania negevensis in sera of patients with respiratory and gastrointestinal symptoms.

The presence of IgG and IgA antibodies to Simkania negevensis in adult Italian patients with respiratory or gastrointestinal symptoms was investigated by the microimmunofluorescence test. In patients with respiratory infections, IgG (50%) and IgA (13%) seropositivity was consistent with previous data. In patients with gastrointestinal disorders, IgG (68%) and IgA (18%) seroprevalence was significantly higher than in healthy controls. These results, in association with the previously described detection of S. negevensis in water sources, could suggest an oral route of infection other than droplets or close contact, and a possible association of S. negevensis with gastrointestinal infections.

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.

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

BACKGROUND: Lactic 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. RESULTS: We 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. CONCLUSION: Adhesion 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.

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.

Esophageal cell proliferation in gastroesophageal reflux disease: clinical-morphological data before and after pantoprazole.

AIM: To evaluate esophageal mucosal defense mechanisms at an epithelial level to establish if pantoprazole treatment can induce ultrastructural healing and improvement in the proliferation activity of the esophageal epithelium in gastroesophageal reflux disease (GERD). METHODS: This was a single-blinded study for pH-monitoring, and histological, ultrastructural and MIB1 immunostaining evaluation. Fifty eight patients with GERD were enrolled and underwent 24 h pH-monitoring and endoscopy. Patients were treated for 12 and 24 mo with pantoprazole. Esophageal specimens were taken for histological and ultrastructural evaluation, before and after the treatment. RESULTS: With transmission electron microscopy, all patients with GERD showed ultrastructural signs of damage with dilation of intercellular spaces (DIS). After 3 mo of therapy the mean DIS values showed a significant reduction and the mean MIB1-LI values of GERD showed an increase in cell proliferation. A further 3 mo of therapy significantly increased cell proliferation only in the erosive esophagitis (ERD) group. CONCLUSION: Three months of pantoprazole therapy induced ultrastructural healing of mucosal damage in 89% and 93% of ERD and non-erosion patients, respectively. Moreover, long-term pantoprazole treatment may be helpful in increasing the capability for esophageal cell proliferation in GERD, particularly in ERD patients.

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.

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.

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 microg/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.