Long-term efficacy of microbiology-driven periodontal laser-assisted therapy.

Periodontitis represents a highly prevalent health problem, causing severe functional impairment, reduced quality of life and increased risk of systemic disorders, including respiratory, cardiovascular and osteoarticular diseases, diabetes and fertility problems. It is a typical example of a multifactorial disease, where a polymicrobial infection inducing chronic inflammation of periodontal tissues is favoured by environmental factors, life style and genetic background. Since periodontal pathogens can colonise poorly vascularised niches, antiseptics and antibiotics are typically associated with local treatments to manage the defects, with unstable outcomes especially in early-onset cases. Here, the results of a retrospective study are reported, evaluating the efficacy of a protocol (Periodontal Biological Laser-Assisted Therapy, Perioblast™) by which microbial profiling of periodontal pockets is used to determine the extent and duration of local neodymium-doped yttrium aluminium garnet (Nd:YAG) laser irradiation plus conventional treatment. The protocol was applied multicentrically on 2683 patients, and found to produce a significant and enduring improvement of all clinical and bacteriological parameters, even in aggressive cases. Microbiome sequencing of selected pockets revealed major population shifts after treatment, as well as strains potentially associated with periodontitis in the absence of known pathogens. This study, conducted for the first time on such a large series, clearly demonstrates long-term efficacy of microbiology-driven non-invasive treatment of periodontal disease.

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer.

Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorage-independent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorage-independent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells.

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20-45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC(50)) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC(50) : 30-40 microM) compared with MCF-7 (IC(50) : 60-70 microM) and MDA-MB231 (IC(50) : 90-100 microM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC(50) : 45-50 microM) compared with MCF-7 (IC(50) : 1-5 microM) and MDA-MB231 (IC(50) : 5-10 microM) (P<0.02), as well as to paclitaxel (Tax) (IC(50) : >2 microM for HCC1937, 0.1-0.2 microM for MCF-7 and 0.01-0.02 microM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/(WT)BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.

Oxaliplatin (L-OHP) treatment of human myeloma cells induces in vitro growth inhibition and apoptotic cell death.

Oxaliplatin (L-OHP), a diaminocyclohexane platinum derivative, is an active and well tolerated anticancer drug which is presently used in the treatment of gastrointestinal tumours. Since the efficacy of L-OHP in the treatment of multiple myeloma (MM) has not yet been evaluated, we studied the antiproliferative activity of this compound in vitro in a panel of MM cell lines (XG1, XG1a, U266 and IM-9). We found that L-OHP inhibited the growth of MM cells at therapeutically achievable concentrations (IC(50): 5-10 microM after 24 h of exposure) and was more active than Cisplatin (CDDP) or Carboplatin (CBDCA). The activity of L-OHP was apparently not affected by interleukin-6 (IL-6), the major growth and survival factor of MM cells. We also found that L-OHP induced apoptotic cell death. We demonstrated that the combination of L-OHP with Dexamethasone (Dex) resulted in the enhancement of the anti-myeloma effects. L-OHP and Dex both induced poly adenosine diphosphate (ADP)-ribose polymerase (PARP) cleavage and this induction was enhanced by the combined treatment. L-OHP-induced apoptosis correlated with caspase-3 cleavage, but this correlation could not be demonstrated in Dex-treated cells. Taken together, these in vitro results provide a rationale for the experimental use of L-OHP in the treatment of MM patients and suggest therapeutic combinations of potential value.

Rat protein tyrosine phosphatase eta physically interacts with the PDZ domains of syntenin.

The tyrosine phosphatase r-PTPeta is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines. To identify r-PTPeta interacting proteins, a yeast two-hybrid screening was performed and an insert corresponding to the full-length syntenin cDNA was isolated. It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF-alpha, beta-ephrins and neurofascin. We show that r-PTPeta is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine-phosphorylated protein it is not a substrate of r-PTPeta. The integrity of both PDZ domains of syntenin and the carboxy-terminal region of r-PTPeta are required for the interaction between syntenin and r-PTPeta.

Large errors in the perception of vertically are generated by luminance borders (integrated across space) not by subjective borders.

The rod-and-frame illusion shows large errors in the judgment of visual vertical in the dark if the frame is large and there are no other visible cues (Witkin and Asch, 1948 Journal of Experimental Psychology 38 762-782). Three experiments were performed to investigate other characteristics of the frame critical for generating these large errors. In the first experiment, the illusion produced by an 11 degrees tilted frame made by luminance borders (standard condition) was considerably larger than that produced by a subjective-contour frame. In the second experiment, with a 33 degrees frame tilt, the illusion was in the direction of frame tilt with a luminance-border frame but in the opposite direction in the subjective-contour condition. In the third experiment, to contrast the role of local and global orientation, the sides of the frame were made of short separate luminous segments. The segments could be oriented in the same direction as the frame sides, in the opposite direction, or could be vertical. The orientation of the global frame dominated the illusion while local orientation produced much smaller effects. Overall, to generate a large rod-and-frame illusion in the dark, the tilted frame must have luminance, not subjective, contours. Luminance borders do not need to be continuous: a frame made of sparse segments is also effective. The mechanism responsible for the large orientation illusion is driven by integrators of orientation across large areas, not by figural operators extracting shape orientation in the absence of oriented contours.

The highly malignant phenotype of anaplastic thyroid carcinoma cell lines is recessive.

OBJECTIVE: The aim of our studies was to determine whether the phenotype of the anaplastic thyroid carcinomas is dominant or recessive. In fact, it is hypothesized, on the basis of epidemiological and pathological data, that undifferentiated thyroid carcinomas are derived from differentiated tumours through a mechanism of tumour progression. DESIGN: Cell hybrids have been generated by cell fusion of anaplastic thyroid carcinoma cell lines, which show a highly malignant phenotype, to cell lines deriving from differentiated thyroid carcinoma, which show a non-tumorigenic or a poorly tumorigenic phenotype. All of the parental cell lines showed impaired p53 gene function. RESULTS: The cell hybrids contained alleles from the parental cell lines. All of the cell hybrids showed a lower growth rate compared with the parental undifferentiated carcinoma cell lines and were unable to grow in soft agar and to induce tumours after injection into athymic mice. CONCLUSION: Taken together, these findings suggest that the highly malignant phenotype of the anaplastic thyroid carcinoma is achieved by the impairment of gene functions that negatively regulate cell growth, rather than by the activation of dominant oncogenes.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma.

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Hierarchical organisation in perception of orientation.

According to Rock [1990, in The Legacy of Solomon Asch (Hillsdale, NJ: Lawrence Erlbaum Associates)], hierarchical organisation of perception describes cases in which the orientation of an object is affected by the immediately surrounding elements in the visual field. Various experiments were performed to study the hierarchical organisation of orientation perception. In most of them the rod-and-frame-illusion (RFI: change of the apparent vertical measured on a central rod surrounded by a tilted frame) was measured in the presence/absence of a second inner frame. The first three experiments showed that, when the inner frame is vertical, the direction and size of the illusion are consistent with expectancies based on the hierarchical organisation hypothesis. An analysis of published and unpublished data collected on a large number of subjects showed that orientational hierarchical effects are independent from the absolute size of the RFI. In experiments 4 to 7 we examined the perceptual conditions of the inner stimulus (enclosure, orientation, and presence of luminance borders) critical for obtaining a hierarchical organisation effect. Although an inner vertical square was effective in reducing the illusion (experiment 3), an inner circle enclosing the rod was ineffective (experiment 4). This indicates that definite orientation is necessary to modulate the illusion. However, orientational information provided by a vertical or horizontal rectangle presented near the rod, but not enclosing it, did not modulate the RFI (experiment 5). This suggests that the presence of a figure with oriented contours enclosing the rod is critical. In experiments 6 and 7 we studied whether the presence of luminance borders is important or whether the inner upright square might be effective also if made of subjective contours. When the subjective contour figure was salient and the observers perceived it clearly, its effectiveness in modulating the RFI was comparable to that observed with luminance borders.

Protein tyrosine phosphatase-eta expression is upregulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells.

We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.

Frequent dysregulation of the c-maf proto-oncogene at 16q23 by translocation to an Ig locus in multiple myeloma.

Dysregulation of oncogenes by translocation to an IgH (14q32) or IgL (kappa, 2p11 or lambda, 22q11) locus is a frequent event in the pathogenesis of B-cell tumors. Translocations involving an IgH locus and a diverse but nonrandom array of chromosomal loci occur in most multiple myeloma (MM) tumors even though the translocations often are not detected by conventional cytogenetic analysis. In a continuing analysis of translocations in 21 MM lines, we show that the novel, karyotypically silent t(14;16)(q32.3;q23) translocation is present in 5 MM lines, with cloned breakpoints from 4 lines dispersed over an approximately 500-kb region centromeric to the c-maf proto-oncogene at 16q23. Another line has a t(16;22)(q23;q11), with the breakpoint telomeric to c-maf, so that the translocation breakpoints in these 6 lines bracket c-maf. Only these 6 lines overexpress c-maf mRNA. As predicted for dysregulation of c-maf by translocation, there is selective expression of one c-maf allele in 2 informative lines with translocations. This is the first human tumor in which the basic zipper c-maf transcription factor is shown to function as an oncogene.

Thyroid cell transformation inhibits the expression of a novel rat protein tyrosine phosphatase.

We have isolated a rat thyroid cDNA encoding a novel rat receptor-type tyrosine phosphatase protein. This gene, on the basis of its homology to another tyrosine phosphatase, the recently isolated human DEP-1/HPTPeta, has been named r-PTPeta. In rat thyroid cells the r-PTPeta gene acts as a differentiation marker. Indeed, the block of thyroid cell differentiation induced by viral and cellular oncogenes is associated with the inhibition or marked reduction of the expression of this gene, and its expression is positively regulated by thyrotropin, the physiological stimulator of thyroid cell growth.

Frame-of-reference and hierarchical-organisation effects in the rod-and-frame illusion.

Two hypotheses proposed as alternatives by Rock--frame of reference and hierarchical organisation of perception--were tested in a series of experiments with the use of the rod-and-frame illusion. This illusion produces errors in the apparent vertical due to the presence of a tilted frame surrounding the test rod. The apparent vertical is shifted in the direction of the frame tilt. When an upright square was added inside the tilted frame, rod-setting errors varied according to the visual characteristics of the display. In the case of a large display presented in the dark (experiment 1), there continued to be large errors in the direction of the outer-square tilt. This finding supports the frame-of-reference hypothesis, which proposes that the orientation of all objects in the visual field is dominated by the most peripheral reference. In the case of a small display presented in a lit environment (experiments 2 and 3), the direction of errors was the opposite. This latter finding was taken to indicate that the rod was set with reference to the perceived tilt of the inner upright square. Thus, according to a hierarchical-organisation hypothesis, the orientation of an object in the visual field is influenced by objects in the immediate surroundings not by outermost reference. Overall, the results confirm the presence of two qualitatively different classes of orientational phenomena: one is concerned with the definition of egocentric coordinates and one with an object-centred visual representation.

The v-erbA oncogene selectively inhibits iodide uptake in rat thyroid cells.

v-erbA is the oncogenic form of the c-erbA proto-oncogene, which encodes the receptor for thyroid hormones. The expression of the v-erbA oncogene in thyroid differentiated cells, PC Cl 3, inhibits iodide uptake and thyrotropin-dependent growth, whereas it has no effect on the expression of the other thyroid specific markers, i.e. thyroglobulin, thyroperoxidase and thyrotropin receptor. The activity of transcription factor AP-1, evaluated by a specific DNA binding assay and by transcription of AP-induced promoter (TRE) is enhanced in PC v-erbA cells. v-erbA mutants in the DNA binding domain do not affect the iodide uptake of thyroid cells nor AP-1 activity. We suggest that this transcriptional activation mediates the selective effects of v-erbA on the expression of thyroid specific markers.

Low frequency of p53 mutations in human thyroid tumours; p53 and Ras mutation in two out of fifty-six thyroid tumours.

OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.

Block of c-myc expression by antisense oligonucleotides inhibits proliferation of human thyroid carcinoma cell lines.

Although elevated c-myc expression seems to be related to an unfavorable prognosis of human thyroid neoplasias, the role of c-myc overexpression in the process of thyroid carcinogenesis is still unknown. We analyzed c-myc expression in 7 human thyroid carcinoma cell lines, originating from different histotypes, and in 50 fresh thyroid tumors and found a higher level of c-myc mRNA in all the thyroid carcinoma cell lines and in several fresh thyroid tumors compared with normal thyroid. The highest increases occurred in the most malignant cell lines and in undifferentiated human thyroid carcinomas. The block of c-MYC protein synthesis with myc-specific antisense oligonucleotides reduced the growth rate of the thyroid carcinoma cell lines significantly. Our results indicate that c-myc overexpression plays a critical role in the growth of thyroid cancer cells, which supports the hypothesis that the myc proto-oncogene might be involved in the neoplastic progression of thyroid carcinogenesis.

A mutated p53 gene alters thyroid cell differentiation.

p53 is the gene most frequently found mutated in human neoplasias. In the majority of tumors, p53 mutations contribute to the progression towards stages of increasing malignancy with the appearance of an undifferentiated phenotype. Also in thyroid cancerogenesis, p53 mutations correlate with the loss of the differentiated phenotype. The results presented here, suggest a direct involvement of p53 in the molecular mechanisms regulating cellular differentiation in thyroid since a mutated p53 gene markedly affects the growth potential and differentiated functions of the rat thyroid cell line PC Cl 3. Blockage in the expression of the PAX-8 transcription factor seems to be a key event in the loss of thyroid differentiated functions induced by the mutated p53 gene. Thyroid cells carrying a mutated p53 gene did not form colonies in soft agar or tumors in athymic mice, suggesting that a mutation of the p53 gene is not sufficient for the induction of the malignant phenotype and probably a cooperation with other oncogenes is necessary to accomplish full malignancy. No effect on either growth or differentiation of thyroid cells was exerted either by overexpression of the wild-type p53 gene, or by the vector alone.

Expression of galectin-1 in normal human thyroid gland and in differentiated and poorly differentiated thyroid tumors.

We previously reported that galectin-1 gene expression increases up to 100-fold in oncogene-transformed rat thyroid cells compared with their normal counterparts and that the relative mRNA levels correlate with the degree of malignancy. In the present study we investigated whether galectin-1 is differentially expressed in human thyroid neoplasms, which range from well-differentiated tumors to undifferentiated anaplastic carcinomas. We analyzed 74 human thyroid specimens of neoplastic, hyperproliferative and normal tissues and several tumor cell lines. Galectin-1 mRNA and protein levels were higher in 6 thyroid carcinoma-derived cell lines than in normal thyroid primary cultures and adenoma cells. Galectin-1 mRNA levels increased in 28/40 papillary carcinomas and in 6/7 anaplastic carcinomas compared with normal or hyperplastic thyroid. Conversely, galectin-1 expression was unaffected in follicular carcinomas and benign adenomas. Immunohistochemical analysis of normal thyroid and papillary carcinoma sections revealed a higher content of galectin-1 protein in neoplastic follicular cells than in normal cells.

Cloning of the rat tissue inhibitor of metalloproteinases type 2 (TIMP-2) gene: analysis of its expression in normal and transformed thyroid cells.

We have recently reported that the introduction of the E1A gene of the adenovirus 5 gene into the rat thyroid PC Cl 3 epithelial cell line causes loss of the differentiated thyroid phenotype without the appearance of the typical markers of neoplastic transformation. It is well known that the E1A gene is able either to induce or to repress the transcription of various endogenous cellular genes. In order to characterize the genes involved in the transformation of PC Cl 3 cells by the E1A gene, we have isolated several cDNA clones, whose expression was induced by E1A. One of these clones was found to be the rat homologue of the human tissue inhibitor of metalloproteinase type 2 (TIMP-2) gene. Here we show the complete sequence of the rat TIMP-2. Its homology to the human TIMP-2 is 98% at the amino acid level and, like its human counterpart, the rat TIMP-2 is transcribed in two different mRNAs of 1.0 and 3.5 kb. The expression of TIMP-2, in PC Cl 3 cells, was significantly induced by the E1A gene and also by other oncogenes. Finally TIMP-2 was shown to be significantly overexpressed in human thyroid neoplastic cell lines and some tumoral thyroid samples.

Involvement of RET oncogene in human tumours: specificity of RET activation to thyroid tumours.

Non-thyroid neoplasia were analysed by Southern blot of genomic DNA and DNA prepared by reverse transcription and amplification by polymerase chain reaction (RT/PCR) for the activation of the RET oncogene. It is known that the rearrangement of RET occurs in about 10%-20% of human thyroid papillary carcinomas. None of 528 non-thyroid tumours showed rearrangement of the RET proto-oncogene, whereas three out of 30 thyroid papillary carcinomas were positive for RET activation. Therefore the activation of RET seems to be a somatic cell mutation specific to human thyroid carcinomas.