A Nodal- and ALK4-independent signaling pathway activated by Cripto-1 through Glypican-1 and c-Src

Human Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor beta-related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membrane-associated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells.     

Luteinizing hormone increases human endometrial cancer cells invasiveness through activation of protein kinase A

Endometrial cancer (EC) is a hormone-dependent cancer that currently represents the most frequent malignancy of the female reproductive tract. The involvement of steroid hormones in its etiology and progression has been reported. The possibility that even gonadotropins (GT) could play a role in the genesis and establishment of EC is supported by the fact that specific receptors for the GT luteinizing hormone/human chorionic GT (LH/hCG) have been detected in a high percentage of ECs, and their expression is apparently related to the cancer grading. However, the precise mechanisms by which GTs might exert their effect on EC is still obscure. The aim of this study was to determine the effects of LH/hCG on the invasion potential of EC cell lines and primary human EC cells. Human recombinant (hr) LH (and hCG) induced a significant increase in cell invasiveness through Matrigel-coated porous membranes in an EC human cell line Hec1A, which expresses the LH/hCG receptor. This effect turned out to depend on hrLH binding to its specific receptors and to the subsequent activation of protein kinase A (PKA). Moreover the hrLH-induced increase in Hec1A invasiveness relied upon a PKA-dependent functional activation of beta(1) integrin receptors, as well as the subsequent induction of matrix metalloproteinase-2 secretion in its active form. The same mechanisms were also found to be operative in primary EC cells. In fact, a significant percentage of primary ECs expressed the LH/hCG receptor, and hrLH addition to primary EC cells, which expressed the specific receptors produced an increase in cell invasiveness only in those tumor cells possessing the specific receptors. This effect was also dependent on PKA activity. We conclude that LH/hCG can regulate EC cells invasiveness, and this result provides a rationale for the use of inhibitors of LH secretion such as GnRH analogues in the treatment of EC.     

Predictive variables for malignant transformation in 452 patients with asymptomatic IgM monoclonal gammopathy

The natural history of asymptomatic IgM monoclonal gammopathies (MG) and variables predicting evolution to symptomatic lymphoproliferative disorders were investigated in 452 patients diagnosed from 1975 to 2001. Univariate and multivariate Cox models were used to identify possible predictors of disease progression. At a median follow-up of 49 months (range, 12 to 233), 41 cases (9.1%) evolved to symptomatic Waldenstrom's macroglobulinemia (n = 36), non-Hodgkin's lymphoma (n = 2), B-cell chronic lymphocytic leukemia (n = 1), IgM multiple myeloma (n = 1), and primary amyloidosis (n = 1); the median interval from diagnosis was 53 months (range, 12 to 154). The cumulative probabilities of transformation into a symptomatic lymphoproliferative disease at 5 and 10 years were 8% (95% confidence interval [CI], 6% to 12%) and 21% (95% CI, 16% to 29%), respectively. At univariate analysis, monoclonal component size and hemoglobin level as continuous parameters, lymphocytosis (>4 x 10(9)/L), bone marrow lymphoplasmacytoid infiltration (>10%), erythrocyte sedimentation rate (>40 mm/h), and detectable Bence Jones proteinuria were significantly related with evolution probability. At multivariate analysis, paraprotein level (P <.0001), hemoglobin level (P <.05), and lymphocytosis (P <.0001) independently predicted malignant evolution (P <.0001). In conclusion, patients with asymptomatic IgM-MG showing hematological features predictive of progression should be carefully monitored in view of an early treatment of the disease.     

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.     

In situ detection of telomeres by fluorescence in situ hybridization and telomerase activity in glioblastoma multiforme: correlation with p53 status, EGFR, c-myc, MIB1, and Topoisomerase IIalpha protein expression

Aberrations of genes/proteins regulating cell cycle and growth, increased proliferation and telomerase activity (TA) are documentable in glioblastoma multiforme. TA is more frequently detectable in secondary glioblastoma, which is also characterized by p53 mutation/overexpression. Discordant telomere (Te) length values have been reported in glioblastomas with and without TA. In 31 glioblastomas, in which pre-existing astrocytoma was not documented, we compared cases with and without TA for the expression of p53, EGFR, c-Myc, MIB-1 and Topoisomerase IIalpha; p53 mutations were also investigated by SSCP-PCR. Correlations were made with Te parameters [TePs: number (TeNo), length and area] as evaluated by image analysis in interphase nuclei of fluorescence in situ hybridization (FISH)-processed sections. We found no differences in the expression of the proteins evaluated and in TePs, except Te/nuclear area %, which was significantly lower in TA+ cases (p=0.02). TePs were, instead, inversely correlated with TA (p=0.0001). TA was positively correlated with MIB1 staining index in the TA+ cases (p=0.033), which also showed a positive correlation between TeNo and EGFR expression (p=0.042), and a trend towards a negative correlation between TeNo and p53 expression (p=0.05). Tumors overexpressing EGFR had a significantly shorter lifetime (p=0.0001). TeNo seems to be inversely correlated to tumor proliferation and lifetime in glioblastoma multiforme.     

The incidence of varicella: correction of a common error

BACKGROUND: Interest in the incidence of varicella (chickenpox) has increased since the discovery of an effective vaccine, but calculations to date have incorrectedly ignored the question of susceptibility. METHODS: We studied the occurrence of varicella in Italy on the basis of 33,343 children (age 0-14 years) cared for by 35 pediatricians between 1 October 1997 and 30 September 1998. The life-table technique was used to calculate the number of susceptible children. On this basis, we estimated the corrected age-specific and cumulative incidence. RESULTS: We identified 1749 cases among the estimated 21,783 susceptible children, for a crude incidence of 8.0% (95% confidence interval [CI] = 7.7-8.4). The rate age-standardized to the Italian population 0-14 years old was 6.8 (CI = 6.5-7.2). The incidence was more than 16% among children age 3-4 years and more than 4% for those age 1-10 years. Comparison of the usual method and our corrected method showed that the uncorrected method underestimates the crude annual incidence (5.2% 8.0%), shifts the peak incidence to earlier ages, and underestimates cumulative incidence (at age 14, 49% 67%). CONCLUSIONS: The use of our corrected method provides more valid estimates of the incidence of varicella than the ones that are currently available. Corrected estimates should be preferred to uncorrected ones in models to study the cost-effectiveness of universal vaccination against varicella.     

A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis

Binding affinity at the [3H]-BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild-type or point-mutated human B2 receptors (hB2R) expressed in CHO cells. The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F. As the wild-type receptor-binding affinity of FR190997 was 40-fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK. Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400- and 150-fold, and that of FR190997 was reduced by 400- and 25-fold, at the W86A and F259A mutant B2 receptors, respectively. Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103-fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant. L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high-affinity antagonist in blocking the IP production induced by BK. The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand. Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.