A 3-D study of capsular invasion in follicular thyroid tumors. A novel approach to an old dilemma.

OBJECTIVE: The diagnosis of follicular tumors of the thyroid mainly rests on the examination of peri-lesional capsule. Lesions with an intact shell are labeled as adenoma, those with capsular invasion are considered carcinoma and those with doubtful aspects are regarded as tumors of uncertain malignant potential. AIM: To better understand the biology of capsular invasion and its practical implication by applying a peculiar three dimension (3-D) reconstruction. METHOD: Two follicular carcinoma (FC) and one follicular tumour of uncertain malignant potential (FT-UMP) were considered. Areas of true/doubtful capsular invasion were labeled using Tissue Micro Array technology and the corresponding paraffin blocks underwent serial sectioning. H&E slides (range 30-100, mean 70) were captured as pictures, aligned using automated method based on the maximization of mutual information and imported into a 3-D image processing software (AMIRA). RESULTS: The 3-D reconstruction revealed that capsular openings were oval shaped and sized approximately equal to 100-200 microm. In one FC the hole was entirely engaged by a tumor mass. In the remaining cases (1 FC and 1 FT-UMP) the 3-D reconstruction showed a small feeding vessel (approximately equal to 50 micro) passing through the capsule together with the bulge of the lesion [see 3-D reconstruction at http://www.ibfm. cnr.it/ricerca/inv_cap.php]. CONCLUSIONS: Our approach allows a better spatial reconstruction of the exact point of capsular interruption; the results obtained suggest that capsular invasion can be due either by abruptly interruption of the shell or by a protrusion along the path of a small feeding vessel.

Hexamethylene bisacetamide inhibits malignant phenotype in T-ALL cell lines.

T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.

No evidence for SEL1L as a candidate gene for IDDM11-conferred susceptibility.

BACKGROUND: The SEL1L gene is located on human chromosome 14q24.3-31 close to D14S67 which has been previously proposed to be a type 1 diabetes mellitus locus (IDDM11). Sel-1 is a negative regulator of the Notch signalling pathway and SEL1L is selectively expressed in adult pancreas and islets of Langerhans. This suggests that SEL1L may be a candidate gene for IDDM11. METHODS: We have analysed two newly identified CA-repeat polymorphisms within the genomic sequence of the SEL1L locus for association with type 1 diabetes mellitus (T1DM) in 152 Danish T1DM-affected sib-pair families and in 240 Sardinian families (229 simplex and 11 sib-pair families). RESULTS: No evidence for association of the two SEL1L markers with T1DM was observed in either the Danish or the Sardinian families. We have also used allelic sharing methods to analyse linkage with T1DM in the IDDM11 region using the same markers and the Danish collection of affected sib-pair families. No evidence of linkage was observed (Z(max)=0.86). CONCLUSION: Although several lines of evidence suggest that SEL1L might be a candidate for IDDM11-conferred susceptibility to T1DM the present study does not support this hypothesis.

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes.

The pathways of transduction of oxidative stress signals have been studied using the Jurkat T cell model. The oxidative stress was induced by exposure of the cells to 100 microM H(2)O(2). DNA damage was detected within 15 min after commencement of treatment. DNA damage repair occurred within about 1 h in cells exposed to oxidative stress for 15 min. In continuous exposure to stress, DNA repair was slower and control levels of DNA integrity were not reached. DNA repair did not involve gene transcription. H(2)O(2) at 100 microM caused cell death by necrosis as well as by apoptosis. Both these processes were induced by 15 min exposure to the stress stimulus. However, some important differences were found between necrosis and apoptosis. Necrosis was more rapid, began within an hour of treatment and continued to increase during the full duration of the experiment. But apoptosis was seen after 4 h from treatment and was conspicuous between 6 and 20 h after the start of treatment. The necrotic phase preceded apoptosis, although these did show an overlap. In the necrotic phase, Bcl-2, Caspase 8 genes were down regulated. The 6-20 h phase characterised by a marked increase in apoptosis is accompanied by the up regulation of both Bcl-2 and Caspase genes. Expression of the Fas and p53 genes was not altered in either phase. We also analysed the levels of expression of the scavenging genes whose gene products are involved in detoxification. No modulation of the antioxidant enzymes, catalase, Cu/Zn superoxide dismutase and glutathione peroxidase was detectable.

Regulation of the human glut4 gene expression in tumor RD18 cell line.

We tested if glut4, the gene for muscle-specific glucose transporter, underwent some variations of expression in neoplastic cells. Our model was a rhabdomyosarcoma cell line (RD18) which retains the ability to differentiate along the myogenic pathway. Any definable changes of expression of glut4 in normal and RD18 cells were revealed by Northern blot analysis. In order to identify the transcriptional regulatory regions of the glut4 gene we performed a deletion analysis of the 5' flanking region. The downregulation which we found in the expression of this gene in RD18 cells could be related with the activity of a negative regulatory element.

Is mts1(S100A4) gene involved in the metastatic process modulated by gamma-interferon?

This study investigates the involvement of mts1 (S100A4) in the metastatic process. We have evaluated the levels of mts1 gene in murine cancer cells following modulation of the metastatic ability. We report here that modulation of the expression of mts1 gene in cells derived from a breast murine adenocarcinoma (TS/A) did not correlate with metastatic ability either when pretreated in vitro with gamma-interferon (gamma-IFN) or following transfection with gamma-IFN gene. In another series of experiments we treated the cell lines B16-A and B78H1, both derived from B16 melanoma, with gamma-IFN. An increased expression of mts1 was found only in B16-A, but not B78H 1, correlated with a strong increase in metastatic activity of B16-A clone upon gamma-IFN treatment.

Computer sequence analysis of human highly conserved zinc finger modules.

We defined a sub-family of zinc finger proteins by computer analyses and comparisons of five new finger domains against protein databases. This subclass of the cysteine-cysteine/histidine-histidine motif shows additional well conserved amino acid patterns and belongs to the human kox and gli-Kruppel gene family, sharing also the same stretches of regulatory zinc finger-containing proteins of mouse and Xenopus. We particularly describe ZF6 cDNA which contains the most interesting sequence, encoding a putative multi-domain regulatory protein.

P1GF-saporin fusion protein: a potential anti-angiogenic agent.

Vascularization is an important step in tumor growth and metastasis. Tumor neovascularization can be considered, therefore, as a good target for antineoplastic therapy. In order to target saporin, a powerful plant toxin, in proximity of the tumor we fused the saporin coding sequence to that for placental growth factor-2 (P1GF-2). P1GF is an angiogenic factor involved in tumor neovascularization. The fusion protein P1GF-2-saporin was obtained by transient transfection of mammalian cells and released in the culture medium as a 57.5 kDa polypeptide. Selectivity and cytotoxic activity are reported as a preliminary step towards the evaluation of its in vivo antitumor activity.

Over-expression of hepatocyte growth factor in human Kaposi's sarcoma.

Kaposi's sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposi's-sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion-derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte-growth-factor receptor (c-MET). Moreover, elevated levels of transforming growth factor beta 1 (TGF beta 1) mRNA were found in lesions of human sporadic Kaposi's sarcoma and in lesion-derived spindle cells which also over-express urokinase. Since HGF, TGF beta 1 and urokinase are all involved in capillary-vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposi's sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.

Senescence-dependent regulation of type 1 plasminogen activator inhibitor in human vascular endothelial cells.

Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor plasminogen activator and has been found to be increased in a number of clinical conditions generally defined as prothrombotic. Since in aging and in atherosclerosis the changes observed in the endothelium resemble those of in vitro aged endothelial cells, we have examined the expression of PAI-1 in cells at different population doublings. In senescent endothelial cells, PAI-1 mRNA and protein are constitutively high, but uninducible by exogenous interleukin 1 alpha as well as by the phorbol ester TPA. Interestingly the increase of PAI-1 levels correlates with the upregulation of interleukin 1 alpha, which characterizes endothelial cell senescence. Since PAI-1 expression is not increased in young cells made nondividing by contact inhibition, we anticipate that PAI-1 expression can be used as an appropriate marker of endothelial senescence. Moreover, PAI-1 was not upregulated in senescent or in progeric human fibroblasts, which do not overexpress interleukin 1 alpha, thus suggesting that multiple pathways may exist to regulate aging of human fibroblasts and endothelial cells.

The protective role of high-density lipoprotein on oxidized-low-density-lipoprotein-induced U937/endothelial cell interactions.

The adherence of monocytes to the endothelium is an early event in atherogenesis. We have investigated this process by examining whether native and oxidized low-density and high-density lipoproteins could modulate this process. Only oxidized low-density lipoprotein caused a significant dose-dependent and time-dependent increase in U937 monocyte-like cell line binding to human endothelial cells, by a process which required de novo protein synthesis. Interestingly, E-selectin, intercellular adhesion molecule-1, vascular cell-adhesion molecule or P-selectin induction was not apparent in this system suggesting the presence of an alternative system for the interaction of endothelial cells with monocyte-like cells in response to oxidized low-density lipoprotein. High-density lipoprotein completely suppressed oxidized low-density-lipoprotein-induced adhesion of U937 cells to the endothelial monolayer, while oxidized high-density lipoprotein did not. These data suggest that the balance between native and oxidized lipoproteins may play a role in the formation of the atherosclerotic lesion by modulating monocyte endothelial interactions.

Identification of the 5' end of the gene encoding a human insulin-responsive glucose transporter.

The 5' end of a gene (GLT4) encoding a human insulin-responsive glucose transporter was isolated from a human lambda Fix genomic library and sequenced. The human sequence, as compared to the mouse GLT4 promoter [Kaestner et al., Proc. Natl. Acad. Sci. USA 87 (1990) 251-255], revealed an overall homology of 81%. Putative transcription controlling sequences were identified.

Tumorigenic activity of a rearranged c-myc gene from a human T-cell leukemia line.

The T-lymphoma cell line Hut78 contains a rearranged c-myc oncogene derived from a translocation between the long arms of chromosomes 8 and 2; the event deletes the 3' end of the gene, causing the loss of the transcribed AT-rich sequence. It has recently been shown that the mutant c-myc mRNA is several-fold more stable than normal c-myc mRNA. We have assessed the tumorigenicity of the mutant c-myc allele by transfecting this gene and its normal counterpart into NIH3T3 cells, together with a neomycin resistance gene. Following selection for G-418 resistance, the cells were injected into nude mice. Tumors containing integrated c-myc arose in animals injected with cells transfected by the mutated, but not by the normal, allele. The results suggest that this rearranged c-myc bears a tumorigenic activity not observed in other naturally occurring mutated c-myc alleles and may have directly contributed to the tumorigenic event in the Hut78 cell line.

Characterization of a subfamily of zinc finger genes expressed in human hematopoietic cell lines.

We isolated by low stringency screening of a human erythroleukemia cDNA library (K562) 45 independent clones hybridizing to a Krüppel-like (HF.10) zinc finger cDNA. The expression of 15 such cDNAs in human hematopoietic cell lines was investigated. Preliminary sequence analysis of the zinc finger motifs in these cDNAs indicate that they belong to a subclass of the Cys-Cys/His-His motif, showing the highest homology to the Wilm's tumor and EGR1, EGR2 cDNAs.

Characterization of a subfamily of zinc finger genes expressed in human hematopoietic cell lines.

We isolated by low strigency screening of a human erythroleukemia cDNA library (K562) 45 indipendent clones hybridizing to a Kruppel-like (HF.10) zinc finger cDNA. The expression of 15 such cDNAs in human hematopoietic cell lines was investigated. Preliminary sequence analysis of the zinc finger motifs in these cDNAs indicate that they belong to a subclass of the Cys-Cys/His-His motif, showing the highest homology to the Wilm's tumor and EGR1, EGR2 cDNAs.

The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer.

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.

An erythroid specific nuclear factor binding to the proximal CACCC box of the beta-globin gene promoter.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemoglobin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess gamma-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural beta-thalassemic mutation (-87, C----G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of beta-globin transcription.

A frequent A gamma-hereditary persistence of fetal hemoglobin in northern Sardinia: its molecular basis and hematologic phenotype in heterozygotes and compound heterozygotes with beta-thalassemia.

A survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the A gamma type; compound heterozygotes with beta-thalassemia have 20%-26% HbF and run a very mild clinical course. The sequence analysis of the cloned A gamma gene linked to the HPFH determinant revealed the presence of a G----A substitution at position -117 of the A gamma-globin gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (alpha 2 A gamma 2) level and a mild thalassemia phenotype in Sardinia is associated with the -196C----T substitution in the A gamma-globin gene promoter (Sardinian delta beta-thalassemia). Population studies using oligonucleotides complementary both to the -117 G----A and -196C----T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and delta beta-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter.

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3' end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.