Detection of HPV16 African variants and quantitative analysis of viral DNA methylation in oropharyngeal squamous cell carcinomas.

BACKGROUND: HPV16 is the most prevalent genotype in oropharyngeal squamous cell carcinomas (OPSCCs), but there are few information about intratype variants prevalence and their clinical relevance in these tumors. Moreover, since HPV DNA detection alone is not sufficient to determine a favorable prognosis for OPSCCs, additional diagnostic/prognostic biomarkers are necessary. OBJECTIVES: To assess the prevalence of HPV intratype variants and evaluate the role of HPV16 DNA methylation as prognostic marker in OPSCCs. STUDY DESIGN: We analyzed 81 biopsies of OPSCCs for HPV detection and genotyping with a pyrosequencing-based HPV DNA-test. We also quantified viral DNA methylation frequency in the LCR by pyrosequencing in patients with transcriptionally-active HPV16-infection, analyzing it in function of available clinical/virological data. RESULTS: The overall HPV prevalence was 74.1% and HPV16 was confirmed the most prevalent genotype (51/60, 85.0%). Interestingly, we detected, for the first time, HPV16 African variants in 10/51 cases (19.6%), pointing out their clinical relevance also out of the anogenital district. Regarding viral DNA methylation, the E2BS1 showed a significantly higher mean methylation frequency compared to E2BS3/4, as previously observed in high-grade precancerous lesions of the uterine cervix. On the other hand, patient with a mean methylation frequency in the early promoter >10% showed only a tendency to have a worse prognosis. CONCLUSIONS: Our results highlight both the involvement of HPV16 African variants in OPSCCs development and the necessity of further studies to deepen the role of viral methylation in these tumors, in order to better define similarities/differences compared to cervical cancer.

Human prion diseases in the Netherlands (1998-2009): clinical, genetic and molecular aspects.

Prion diseases are rare and fatal neurodegenerative disorders that can be sporadic, inherited or acquired by infection. Based on a national surveillance program in the Netherlands we describe here the clinical, neuropathological, genetic and molecular characteristics of 162 patients with neuropathologically confirmed prion disease over a 12-year period (1998-2009). Since 1998, there has been a relatively stable mortality of Creutzfeldt-Jakob disease (CJD) in the Netherlands, ranging from 0.63 to 1.53 per million inhabitants per annum. Genetic analysis of the codon 129 methionine/valine (M/V) polymorphism in all patients with sporadic CJD (sCJD) showed a trend for under-representation of VV cases (7.0%), compared with sCJD cohorts in other Western countries, whereas the MV genotype was relatively over-represented (22,4%). Combined PrP(Sc) and histopathological typing identified all sCJD subtypes known to date, except for the VV1 subtype. In particular, a "pure" phenotype was demonstrated in 60.1% of patients, whereas a mixed phenotype was detected in 39.9% of all sCJD cases. The relative excess of MV cases was largely accounted for by a relatively high incidence of the MV 2K subtype. Genetic analysis of the prion protein gene (PRNP) was performed in 161 patients and showed a mutation in 9 of them (5.6%), including one FFI and four GSS cases. Iatrogenic CJD was a rare phenomenon (3.1%), mainly associated with dura mater grafts. Three patients were diagnosed with new variant CJD (1.9%) and one with variably protease-sensitive prionopathy (VPSPr). Post-mortem examination revealed an alternative diagnosis in 156 patients, most commonly Alzheimer's disease (21.2%) or vascular causes of dementia (19.9%). The mortality rates of sCJD in the Netherlands are similar to those in other European countries, whereas iatrogenic and genetic cases are relatively rare. The unusual incidence of the VV2 sCJD subtype compared to that reported to date in other Western countries deserves further investigation.

A second case of Gerstmann-Sträussler-Scheinker disease linked to the G131V mutation in the prion protein gene in a Dutch patient.

A rare case of Gerstmann-Sträussler-Scheinker disease in a 36-year-old Dutch man is reported. The clinical phenotype was characterized by slowly progressive cognitive decline, later followed by ataxia and parkinsonism. Neuropathologic findings consisted of numerous amyloid plaques in the cerebellum, which showed positive staining for the abnormal prion protein (PrP(Sc)). In addition, there were tau accumulations around numerous amyloid deposits in the cerebral cortex, striatum, hippocampal formation, and midbrain. There was no spongiform degeneration. Western blot analysis showed the co-occurrence of 2 distinct abnormal prion protein species comprising an unglycosylated, protease-resistant fragment of approximately 8 kd, which was found to be truncated at both N- and C-terminal ends by epitope mapping, and a detergent-insoluble but protease-sensitive form of full-length PrP(Sc). Sequence analysis disclosed a mutation at codon 131 of the prion protein gene (PRNP), resulting in a valine-for-glycine substitution (G131V). The patient was heterozygous at the polymorphic codon 129 and carried the mutation on the methionine allele. To our knowledge, this is the second family worldwide in which this mutation has been identified. Gerstmann-Sträussler-Scheinker disease should be considered in patients with a clinical diagnosis of familial frontotemporal dementia.

Phenotypic variability of sporadic human prion disease and its molecular basis: past, present, and future.

Human prion diseases are rare neurodegenerative disorders related to prion protein misfolding that can occur as sporadic, familial or acquired forms. In comparison to other more common neurodegenerative disorders, prion diseases show a wider range of phenotypic variation and largely transmit to experimental animals, a feature that led to the isolation and characterization of different strains of the transmissible agent or prion with distinct biological properties. Biochemically distinct PrP(Sc) types have been demonstrated which differ in their size after proteinase cleavage, glycosylation pattern, and possibly other features related to their conformation. These PrP(Sc) types, possibly enciphering the prion strains, together with the naturally occurring polymorphism at codon 129 in the prion protein gene have a major influence on the disease phenotype. In the sporadic form, the most common but perhaps least understood form of human prion disease, there are at least six major combinations of codon 129 genotype and prion protein isotype, which are significantly related to distinctive clinical-pathological subgroups of the disease. In this review, we provide an update on the current knowledge and classification of the disease subtypes of the sporadic human prion diseases as defined by molecular features and pathological changes. Furthermore, we discuss the molecular basis of phenotypic variability taking into account the results of recent transmission studies that shed light on the extent of prion strain variation in humans.

Genetic Creutzfeldt-Jakob disease and fatal familial insomnia: insights into phenotypic variability and disease pathogenesis.

Human prion diseases are a group of rare neurodegenerative disorders characterized by the conversion of the constitutively expressed prion protein, PrP(C), into an abnormally aggregated isoform, called PrP(Sc). While most people who develop a prion disease have no identifiable cause and a few acquire the disease through an identified source of infection, about 10-15% of patients are affected by a genetic form and carry either a point mutation or an insertion of octapeptide repeats in the prion protein gene. Prion diseases show the highest extent of phenotypic heterogeneity among neurodegenerative disorders and comprise three major disease entities with variable though overlapping phenotypic features: Creutzfeldt-Jakob disease (CJD), fatal insomnia and the Gerstmann-Sträussler-Scheinker syndrome. Both CJD and fatal insomnia are fully transmissible diseases, a feature that led to the isolation and characterization of different strains of the agent or prion showing distinctive clinical and neuropathological features after transmission to syngenic animals. Here, we review the current knowledge of the effects of the pathogenic mutations linked to genetic CJD and fatal familial insomnia on the prion protein metabolism and physicochemical properties, the disease phenotype and the strain characteristics. The data derived from studies in vitro and from those using cell and animal models are compared with those obtained from the analyses of the naturally occurring disease. The extent of phenotypic variation in genetic prion disease is analyzed in comparison to that of the sporadic disease, which has recently been the topic of a systematic and detailed characterization.

Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP.

Stop codon mutations in the gene encoding the prion protein (PRNP) are very rare and have thus far only been described in two patients with prion protein cerebral amyloid angiopathy (PrP-CAA). In this report, we describe the clinical, histopathological and pathological prion protein (PrP(Sc)) characteristics of two Dutch patients carrying novel adjacent stop codon mutations in the C-terminal part of PRNP, resulting in either case in hereditary prion protein amyloidoses, but with strikingly different clinicopathological phenotypes. The patient with the shortest disease duration (27 months) carried a Y226X mutation and showed PrP-CAA without any neurofibrillary lesions, whereas the patient with the longest disease duration (72 months) had a Q227X mutation and showed an unusual Gerstmann-Sträussler-Scheinker disease phenotype with numerous cerebral multicentric amyloid plaques and severe neurofibrillary lesions without PrP-CAA. Western blot analysis in the patient with the Q227X mutation demonstrated the presence of a 7 kDa unglycosylated PrP(Sc) fragment truncated at both the N- and C-terminal ends. Our observations expand the spectrum of clinicopathological phenotypes associated with PRNP mutations and show that a single tyrosine residue difference in the PrP C-terminus may significantly affect the site of amyloid deposition and the overall phenotypic expression of the prion disease. Furthermore, it confirms that the absence of the glycosylphosphatidylinositol anchor in PrP predisposes to amyloid plaque formation.

Incidence and spectrum of sporadic Creutzfeldt-Jakob disease variants with mixed phenotype and co-occurrence of PrPSc types: an updated classification.

Six subtypes of sporadic Creutzfeldt-Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrP(Sc), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrP(Sc) types has been reported but with inconsistencies among studies in both results and their interpretation. The issue currently complicates diagnosis and classification of cases and also has implications for disease pathogenesis. To explore the issue in depth, we carried out a systematic regional study in a large series of 225 cases. PrP(Sc) types 1 and 2 concurrence was detected in 35% of cases and was higher in MM than in MV or VV subjects. The deposition of either type 1 or 2, when concurrent, was not random and always characterized by the coexistence of phenotypic features previously described in the pure subtypes. PrP(Sc) type 1 accumulation and related pathology predominated in MM and MV cases, while the type 2 phenotype prevailed in VVs. Neuropathological examination best identified the mixed types 1 and 2 features in MMs and most MVs, and also uniquely revealed the co-occurrence of pathological variants sharing PrP(Sc) type 2. In contrast, molecular typing best detected the concurrent PrP(Sc) types in VV subjects and MV cases with kuru plaques. The present data provide an updated disease classification and are of importance for future epidemiologic and transmission studies aimed to identify etiology and extent of strain variation in sporadic Creutzfeldt-Jakob disease.

Magnetic resonance diagnostic markers in clinically sporadic prion disease: a combined brain magnetic resonance imaging and spectroscopy study.

The intra vitam diagnosis of prion disease is challenging and a definite diagnosis still requires neuropathological examination in non-familial cases. Magnetic resonance imaging has gained increasing importance in the diagnosis of prion disease. The aim of this study was to compare the usefulness of different magnetic resonance imaging sequences and proton magnetic resonance spectroscopy in the differential diagnosis of patients with rapidly progressive neurological signs compatible with the clinical diagnosis of sporadic prion disease. Twenty-nine consecutive patients with an initial diagnosis of possible or probable sporadic prion disease, on the basis of clinical and electroencephalography features, were recruited. The magnetic resonance protocol included axial fluid-attenuated inversion recovery-T2- and diffusion-weighted images, and proton magnetic resonance spectroscopy of the thalamus, striatum, cerebellum and occipital cortex. Based on the clinical follow-up, genetic studies and neuropathology, the final diagnosis was of prion disease in 14 patients out of 29. The percentage of correctly diagnosed cases was 86% for diffusion-weighted imaging (hyperintensity in the striatum/cerebral cortex), 86% for thalamic N-acetyl-aspartate to creatine ratio (cutoff 1.05 (100% specificity and 100% positive predictive value). Univariate logistic regression analysis showed that the combination of thalamic N-acetyl-aspartate to creatine ratio and diffusion-weighted imaging correctly classified 93% of the patients. The combination of thalamic proton magnetic resonance spectroscopy (10 min acquisition duration) and brain diffusion-weighted imaging (2 min acquisition duration) may increase the diagnostic accuracy of the magnetic resonance scan. Both sequences should be routinely included in the clinical work-up of patients with suspected prion disease.

Creutzfeldt-Jakob disease with E200K PRNP mutation: a case report and revision of the literature.

Creutzfeldt-Jakob disease (CJD) is typically characterized by rapidly progressive dementia and myoclonus, and it is caused by a conformational change of the prion protein. The heritable forms are associated with mutation in the gene encoding the prion protein (PRNP). We report a 63-year-old Italian woman harboring the E200K PRNP mutation. Electroencephalogram, cerebrospinal fluid analysis, PRNP gene sequencing, histopathologic examination, immunohistochemical studies, and Western blotting analysis confirmed the diagnosis of CJD. Pyramidal involvement was the first sign and the prominent clinical feature. Later on, she developed also myoclonus, ataxia, spastic tetraplegia, and at last dementia with akinetic mutism. Usually, signs of degeneration of the pyramidal tracts occur in a small number of patients as the disease advances. Our report supports the variability of the clinical expression of the E200K genetic CJD. Further studies are needed to understand the molecular basis underlying the phenotypic variability among patients carrying this mutation.

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease.

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.

A refined method for molecular typing reveals that co-occurrence of PrP(Sc) types in Creutzfeldt-Jakob disease is not the rule.

Molecular typing in Creutzfeldt-Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrP(Sc) types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrP(Sc) isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrP(Sc) types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the 'type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrP(Sc) type 2 is not a PK-resistant PrP(Sc) core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrP(Sc). Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3-5% of the total PrP(Sc) signal (ie, weak band of one type/total PrP(Sc)). Our results show that there are many pitfalls associated with the use of 'type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrP(Sc) types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.

Brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a BrainNet Europe Study.

The present study was designed to reveal protein modifications in control cases related with postmortem delay and temperature of storage in 3 paradigms in which the same postmortem tissue sample (frontal cortex) was frozen a short time after death or stored at 1 degrees C, 4 degrees C, or room temperature and then frozen at -80 degrees C at different intervals. No evidence of protein degradation as revealed with monodimensional gel electrophoresis and Western blotting was observed in samples artificially stored at 1 degrees C and then frozen at different intervals up to 50 hours after death. However, the levels of several proteins were modified in samples stored at 4 degrees C and this effect was more marked in samples stored at room temperature. Two-dimensional gel electrophoresis and mass spectrometry further corroborated these observations and permitted the identification of other proteins vulnerable or resistant to postmortem delay. Finally, gel electrophoresis and Western blotting of sarkosyl-insoluble fractions in Alzheimer disease showed reduced intensity of phospho-tau-specific bands with postmortem delay with the effects being more dramatic when the brain samples were stored at room temperature for long periods. These results emphasize the necessity of reducing the body temperature after death to minimize protein degradation.

Contribution of MEK/MAPK and PI3-K signaling pathway to the malignant behavior of Ewing's sarcoma cells: therapeutic prospects.

Insulin-like growth factor receptor I (IGF-I)-mediated circuit is a major autocrine loop for Ewing's sarcoma (ES) cells and appears to be particularly important in the pathogenesis of this tumor. In this study, we analyzed the contribution of the 2 major pathways of the intracellular IGF-IR signaling cascade to the overall effects elicited by IGF-I in ES. Both the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K) signaling pathways appeared to be constitutively activated in ES, likely due to the presence of the IGF-IR-mediated autocrine loop. We demonstrated that both MEK/MAPK (PD98059 or U0126) and PI3-K inhibitors (LY294002) profoundly impaired ES cell growth in monolayer and soft agar basal conditions. Both PD98059 and LY294002 inhibited ES cell cycle progression by inducing G1 blockage, whereas only LY294002 significantly affected the survival of ES cells. Exogenous IGF-I completely reverted LY294002-induced growth inhibition by abrogating antiproliferative and proapoptotic effects of the PI3-K inhibitor. By contrast, IGF-I could not rescue cells from growth inhibition induced by PD98059. MEK/MAPK blockade also significantly reduced the migratory ability of ES cells, both in basal and IGF-I-induced conditions, and increased chemosensitivity to doxorubicin, a leader drug in the treatment of ES patients. Our findings therefore identify MAPK pathway as a promising target for pharmacologic intervention in ES.

C-kit receptor expression in Ewing's sarcoma: lack of prognostic value but therapeutic targeting opportunities in appropriate conditions.

PURPOSE: Autocrine/paracrine stimulation of c-kit has been recently observed in Ewing's sarcoma (ES) cell lines. In this study, we tested the prognostic and therapeutic role of the receptor in this tumor. METHODS: One hundred one ES tumor biopsies were evaluated for the expression of c-kit by the avidin-biotin-peroxidase procedure. Effectiveness of STI-571 (Gleevec; Novartis, Basel, Switzerland), a selective inhibitor of specific tyrosine kinases, was analyzed with respect to in vitro growth and migration inhibition, as single agent or in combination with doxorubicin. RESULTS: Approximately 30% of patients expressed c-kit in their primary tumors. No significant association between the expression of the receptor and the clinical outcome was observed. In vitro growth of ES cell lines showing high levels of c-kit demonstrated limited inhibition by exposure to STI-571 (10 micromol/L is required to obtain 40% to 50% of growth inhibition). A decrease of stem-cell factor-mediated ES cell migration was also found. The drug acted additively with doxorubicin in inhibiting ES cell growth. CONCLUSION: The negative prognostic findings and the limited in vitro therapeutic activity of STI-571 indicate that the putative aberrant signaling provided by c-kit overexpression may be dispensable for ES development and unlikely to constitute a critical therapeutic target. Accordingly, the dose of STI-571 required to give a significant ES growth inhibition is much higher than for those tumors in which mutations of c-kit constitute a relevant pathogenetic event. Nevertheless, in the subset of ES patients showing a high level of c-kit expression, the activity of the drug may be exploited in combination with standard therapy.

Effectiveness of Ecteinascidin-743 against drug-sensitive and -resistant bone tumor cells.

PURPOSE: The identification of new drugs is strongly needed for bone tumors.Ecteinascidin-743 (ET-743), a highly promising antitumor agent isolated from the marine tunicate Ecteinascidia turbinata, is currently under Phase II clinical investigation in Europe and the United States for treatment of soft tissue sarcoma. In this study, we analyzed the preclinical effectiveness of this drug in osteosarcoma and Ewing's sarcoma. EXPERIMENTAL DESIGN: The effects of ET-743 were evaluated against a panel of human osteosarcoma and Ewing's sarcoma cell lines characterized by different drug responsiveness and compared with the effects of standard anticancer agents. In addition, combination treatments with ET-743 and the other standard chemotherapy agents for sarcoma were analyzed to highlight the best drug-to-drug interaction RESULTS: A potent activity of ET-743 was clearly observed against both drug-sensitive and drug-resistant (multidrug-resistant, methotrexate- and cisplatin-resistant) bone tumor cells at concentrations that are easily achievable in patients (pM to nM range). Ewing's sarcoma cells appeared to be particularly sensitive to the effects of this drug. The analysis of the effects of ET-743 on cell cycle, apoptosis, and differentiation indicated that both osteosarcoma and Ewing's sarcoma cells had a slower progression through the different phases of the cell cycle after treatment with ET-743. However, the drug was able to induce a massive apoptosis in Ewing's sarcoma but not in osteosarcoma cells. In the latter neoplasm, ET-743 showed a differential effect, as indicated by the significant increase in the expression and activity of alkaline phosphatase, a marker of osteoblastic differentiation. Concurrent exposure of cells to ET-743 and other chemotherapeutic agents resulted in greater than additive interactions when doxorubicin and cisplatin were used, whereas subadditive effects were observed with methotrexate, vincristine, and actinomycin D. CONCLUSIONS: Overall, these results encourage the inclusion of this drug in the treatment of patients with bone tumors, although a careful design of new regimens is required to identify the best therapeutic conditions.

Effectiveness of insulin-like growth factor I receptor antisense strategy against Ewing's sarcoma cells.

The insulin-like growth factor I receptor (IGF-IR) plays an essential role in the establishment and maintenance of transformed phenotype of Ewing's sarcoma (ES) cells, and interference with the IGF-IR pathways by a neutralizing antibody causes reversal of the malignant potential of this neoplasm. In this paper, we stably transfected an IGF-IR antisense mRNA expression plasmid in an ES cell line to determine the effectiveness of antisense strategies against the in vitro and in vivo growth of ES cells. Doxorubicin sensitivity of TC-71 cells expressing antisense targeted to IGF-IR mRNA was also examined. Cells carrying antisense IGF-IR had a reduced expression of the receptor, a modest decrease in cell proliferation, a significant increase in anoikis-induced apoptosis, and a severely reduced ability to form colonies in soft agar. Moreover, TC/AS cells showed a marked reduction in their motility. In vivo, when cells carrying antisense IGF-IR were injected subcutaneously in nude mice, tumor formation was delayed and survival increased. Metastatic ability of ES cells was also significantly reduced. Furthermore, TC/AS clones showed a significantly higher sensitivity to doxorubicin - a major drug in the treatment of ES. These results indicate that inhibiting IGF-IR by antisense strategies may be relevant to the clinical treatment of ES patients by reducing the malignant potential of these cells and enhancing the effectiveness of chemotherapy.

The expression of ccn3(nov) gene in musculoskeletal tumors.

The CCN3(NOV) protein belongs to the CCN [cysteine-rich CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed gene (Nov)] family of growth regulators, sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Although previous studies have revealed an expression of CCN3 protein in several normal tissues, including kidney, nervous system, lung, muscle, and cartilage, less is known about its expression in tumors. In this study, we analyzed the expression of CCN3 in musculoskeletal tumors, using a panel of human cell lines and tissue samples. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas, in Ewing's sarcoma, the expression of this protein seemed to be associated with a higher risk to develop metastases. CCN3 expression was found in 15 of 45 Ewing's sarcoma tissue samples. In particular, we did not observe any expression of CCN3 in the 15 primary tumors that did not develop metastases. In contrast, 15 of the 30 primary tumors that developed lung and/or bone metachronous metastases showed a high expression of the protein (P < 0.001, Fisher's test). Our studies indicate that CCN3 is generally expressed in the cells of the musculoskeletal system. This protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. Thus, as reported for other CCN genes, the biological properties and regulation of expression of CCN3 are dependent on the cellular context and the nature of the cells in which it is produced. Further studies will help to clarify the biological role of this protein in musculoskeletal neoplasms.

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein.

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.