Inhibition of CRIPTO expression and tumorigenicity in human colon cancer cells by antisense RNA and oligodeoxynucleotides.

CRIPTO is an epidermal growth factor-related gene expressed in a majority of human colorectal tumors. To assess the role of CRIPTO in the growth control of human colon cancer, we have treated human colon carcinoma GEO and CBS cells, that possess high levels of CRIPTO, and WIDR colon cancer cells, that are negative for CRIPTO expression, with two antisense phosphorothioate oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. Both antisense oligodeoxynucleotides significantly reduced endogenous CRIPTO protein levels and inhibited GEO and CBS cell growth in monolayer and in semisolid medium, whereas they did not affect WIDR cell growth. In addition, GEO, CBS and WIDR cells were infected with a recombinant retroviral vector containing the hygromycin-resistance gene and a 900 bp EcoRI-EcoRI coding fragment of the human CRIPTO cDNA oriented in the 3' to 5' direction. GEO and CBS CRIPTO antisense infectants exhibited a 60 to 70% reduction in CRIPTO protein expression, in monolayer growth and in soft agar cloning efficiency as compared to parental noninfected cells. In contrast, infection of WIDR cells with the CRIPTO antisense retrovirus did not alter their growth. Finally, GEO CRIPTO antisense infectants formed tumors in nude mice that were significantly smaller and had a larger latency period as compared to noninfected GEO cells.

Activated platelets and leucocytes cooperatively stimulate smooth muscle cell proliferation and proto-oncogene expression via release of soluble growth factors.

BACKGROUND: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression, such as c-fos. METHODS: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of 3H-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070, a TxA2 receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes + lymphocytes) was also determined in additional experiments. RESULTS: SMC proliferation was significantly increased by activated platelets to 360 +/- 9% of control values (P < 0.05). This effect was reduced by ketanserin, R68070, BN 52021 or trapidil. Whole leucocytes, neutrophils or lymphocytes + monocytes also increased SMC proliferation with respect to control experiments. Simultaneous stimulation of SMCs by platelets and whole leucocytes was associated with a significant greater increase in SMC proliferation as compared to SMC stimulated with platelets or leucocytes alone. c-fos expression, almost undetectable in unstimulated SMCs, was markedly increased by activated platelets or leucocytes. CONCLUSIONS: Activated platelets promote SMC proliferation in vitro via release of soluble mediators, including serotonin, thromboxane A2 PAF and PDGF; activated leucocytes also induce a significant SMC proliferation and exert an additive effect when activated together with platelets; SMCs stimulated with activated platelets and leucocytes show an early expression of the proto-oncogene c-fos.

Thyroglobulin binding and TSH regulation of the RHL-1 subunit of the asialoglycoprotein receptor in rat thyroid.

The ability of asialo-thyroglobulin to bind the thyroid RHL-1 subunit of the asialoglycoprotein receptor has been investigated. Ligand blot assays show that the recombinant carbohydrate recognition domain of the thyroid RHL-1 subunit specifically interacts with rat desialated thyroglobulin. Moreover, RT-PCR and Western blot assays show that TSH deprivation decreases RHL-1 expression in PC C13 thyroid differentiated cells whereas insulin deprivation does not have any effect. The simultaneous absence of both TSH and insulin dramatically decreases the level of RHL-1 expression.

The expression of the sarco/endoplasmic reticulum Ca2+-ATPases in thyroid and its down-regulation following neoplastic transformation.

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.

Inguinal hernia repair in patients with coagulation problems: prevention of postoperative bleeding with human fibrin glue.

BACKGROUND: Our purpose was to establish the efficacy of human fibrin glue (HFG) in preventing coagulative complications after inguinal hernia repair in patients with coagulation disorders. METHODS: A randomized controlled trial of 50 patients with coagulation disorders undergoing hernia repair was performed. Patients had concurrent coagulopathies as a consequence of liver disease or long-term treatment with anticoagulants. Coagulopathies were defined according to the following criteria: prothrombin time < 10.5 seconds, activated partial thromboplastin time < 21 seconds, and fibrinogen < 230 mg/dL. Patients were randomized in a 1:1 ratio with (group A) or without (control group B) use of HFG. RESULTS: Postoperative hemorrhagic complications were significantly reduced in group A (4%) compared with group B (24%). CONCLUSION: This study shows that HFG is effective in preventing local hemorrhagic complications after herniorrhaphy in patients with concurrent coagulation disorders. This implies that the use of HFG reduces the costs of prolonged hospitalization related to such complications.

The rat hepatic lectin-1 subunit of the asialoglycoprotein receptor is upregulated by thyrotropin and downregulated by neoplastic transformation of thyroid cells.

We have recently shown that the rat hepatic lectin (RHL)-1 subunit of the asialoglycoprotein receptor (ASGPr) is expressed in the PC C13 differentiated thyroid cell line. To investigate in vivo the expression of RHL-1 and the ability of thyrotropin (TSH) to modulate its expression, reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot assays have been performed on thyroid extracts from rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels. It is shown that RHL-1 expression is down-regulated by T4 (which decreases serum TSH) and upregulated by PTU (which increases serum TSH), at both mRNA and protein levels. The sensitivity of RHL-1 to neoplastic transformation of thyroid cells has been investigated. The RHL-1 expression pattern has been studied in PC C13 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Western blot assays show that RHL-1 expression progressively decreases as PC C13 cells acquire a more transformed phenotype. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a housekeeping gene used as internal control to normalize RHL-1 mRNA content, exhibits no variations in the different PC C13 cell lines used. In addition, we show that both native and asialo-thyroglobulin (Tg) bind RHL-1 in vitro, and native Tg binds RHL-1 on the surface of PC C13 cells. After thyroid cells transformation, the surface expression of RHL-1 is inhibited in a measure that correlates with the mRNA and protein levels. Therefore, the RHL-1 inhibition at the mRNA, protein and plasma membrane expression follows a gradient that parallels the progressive acquisition of the fully transformed phenotype in the PC C13 system. The results reported in the present article, together with our previous data, suggest that RHL-1 expression could be regulated, at least in part, by the same transcription factors involved in the expression of the other molecules characteristic of the thyroid differentiated state.

Diagnostic and surgical approaches to recurrent varicose veins of lower limbs.

A consecutive series of 82 patients (98 legs) suffering from recurrent varicose veins underwent surgical treatment. In all patients clinical and hand-held US Doppler preoperative examinations were performed, but a phlebography was necessary in 33 legs to certainly visualize the anatomy of venous system and the potential sites of recurrent deep to superficial reflux. The causes of recurrence were: incompetent saphenofemoral junction in 59 legs, saphenopopliteal reflux in 6 legs, incompetence of perforator veins in 18 legs, both insufficiencies of great saphena and perforators in 15 legs. Seventy-four legs with saphenofemoral reflux underwent groin redissections through transversal (44 legs) or vertical (30 legs) incisions; the approach to the saphenopopliteal junction was vertical in two legs and transversal in four legs; the interruption of incompetent perforator veins was performed through incisions in 29 legs and according to Linton's technique in 4 legs. Clinical and US Doppler follow-up was performed every 6 months and no recurrent reflux was demonstrated; seven patients were affected from new small varices that were treated by injection sclerotherapy. This study indicates that more than 1/3 of recurrent varices need phlebography to be clearly studied: only diagnostic accuracy may assure a correct surgical approach, but the strategy of treatment must be adapted to the single patient.

Prevalence of varicose veins in an Italian elderly population.

The prevalence of varicose veins (VV) in the elderly population of the Campania Region, in Southern Italy, was estimated. A random sample of the people aged more than 65 years was drawn by means of a stratified multistage sampling design warranting that observed percentages were direct estimates of population percentages. The investigation covered 1319 subjects, 560 (42.5%) men and 759 (57.5%) women, their ages ranging from 66 to 96 years with an average value of 74.2 years, who were interviewed and visited by trained physicians. VV were defined as any reticular or truncal visible varicosities of the lower limbs, and investigated symptoms were heaviness, pain, nightly cramps, edema, eczema, hyperpigmentation, and ulceration. Some variables were studied as risk factors: age, sex, lifetime occupation, smoking, alcohol, hypertension, diabetes, and obesity; previous treatment and use of elastic stockings were also studied. Statistical associations were evaluated by Chi-square test, a two-tailed P value of 0.05 being assumed as significance level. In total, 391 (29.6%) subjects were reported to be affected by VV, but the clinical examination was positive in only 362 (27.4%) with a good correspondence between answers and clinical findings. Prevalence was greatly affected by sex, the percentage being two times higher in women (35.2%) than in men (17%). VV developed after a pregnancy in 40.5% of women, but a high percentage of women (38.2%) also reported menopause as a time starting point. No significant association between reported risk factors and VV was found among men, whereas obesity was strongly related to VV in women. One or more symptoms were reported in 92.1% of persons affected by VV, but no previous therapy was reported by 58.9% of subjects. Only 16.9% of patients used elastic stockings with a significant difference between men (7.4%) and women (20.2%).

[Ureteral duplication and ectopic ureterocele]. / Duplicazione ureterale ed ureterocele ectopico.

An ectopic ureterocele and unilateral ureteral duplication in a young women is reported. It is a rare congenital malformation of the urinary tract. In this typical case, the aspecific symptomatology and diagnostic imaging are discussed. The pathology was not diagnosed preoperatively. The excretory urography, sonography and CT scanning did not show neither the duplication and the ureterocele, and diagnosis of retroperitoneal mass was made. The data of literature are analyzed and the importance of surgical therapy is underlined.

[Giant benign and malignant pathology of the ovary]. / Patologia gigante benigna e maligna dell'ovaio.

After a discussion of ovarian tumour classification, two cases of giant neoplasm, one benign the other malignant are reported. Ultrasonography and CA125 research represent two irreplaceable investigations, for preoperative balance and for therapeutic strategy.

NF-κB-dependent cytokine secretion controls Fas expression on chemotherapy-induced premature senescent tumor cells.

Induction of a senescent phenotype in tumor cells has been linked to anticancer immune response, however, the molecular mechanisms mediating these phenomenon have not yet been determined. In this study, we present evidence that induction of premature senescence in human cancer cell lines induces Fas expression, and loss of resistance to Fas-induced apoptosis. Triggering of Fas by using the agonistic antibody CH11 or the recombinant ligand APO010, activates an apoptotic pathway responsible for cell death. Secretion of pro-inflammatory cytokines by the senescent cells, particularly TNF-α and IFN-γ, mediates Fas upregulation. Indeed, treatment of proliferating cancer cell lines with TNF-α and IFN-γ, upregulates Fas expression, while blocking TNF-α and IFN-γ by using neutralizing antibodies, decreases Fas expression in senescent cells. We also demonstrate that NF-κB has a central role in controlling the senescence-associated secretory phenotype (SASP) by the premature senescent cells, and that TNF-α and IFN-γ, transcriptionally controlled by NF-κB, are the main mediators of Fas upregulation. Our data suggest the existence of an NF-κB-dependent autocrine loop, mediated by TNF-α and IFN-γ, responsible for expression of Fas on the surface of senescent cells, and for their killing.

The murine cripto gene: expression during mesoderm induction and early heart morphogenesis.

The murine cripto gene encodes a 171-aminoacid epidermal growth factor-related protein, with 93% similarity to its human counterpart in the 'EGF-like' domain. The murine cripto mRNA contains two B1 repeats in its 3' non-coding region and a 163-nucleotide homology to the human mRNA. The mouse cripto gene is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. In situ hybridization analysis during murine embryogenesis (day 6.2 to day 10.5) reveals a very restricted expression pattern. cripto transcripts are first detected in a few epiblastic cells at day 6.5. During gastrulation, the transcripts are expressed in the forming mesoderm and later during development cripto gene expression is restricted to the truncus arteriosus of the developing heart. This expression pattern suggests a role for cripto gene in the determination of the epiblastic cells that subsequently give rise to the mesoderm.

The rat asialoglycoprotein receptor binds the amino-terminal domain of thyroglobulin.

We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRD(RHL-1)) and the anti-rCRD(RHL-1) antibody markedly inhibited (125)I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRD(RHL-1) was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRD(RHL-1) on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRD(RHL-1) specifically recognized a thyroglobulin fragment with an apparent M(r) of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role.

Serum withdrawal induces apoptotic cell death in Ki-ras transformed but not in normal differentiated thyroid cells.

Thyroid cells transformed by the Kirsten-ras oncogene become tumorigenic in syngeneic animals. Their growth is no longer dependent on TSH but becomes dependent on serum. Combining morphological and biochemical evidence, we show that serum withdrawal induces apoptotic cell death in Kirsten and Harvey-ras transformed thyroid cell. On the other hand, neither serum nor TSH withdrawal induce apoptosis in differentiated FRTL-5 cells. The induction of apoptosis by serum withdrawal is rapid and not triggered at a specific phase of the cell cycle. We suggest that induction of apoptosis following growth factor deprivation is an additional important characteristic, besides TSH-independence for growth and dedifferentiation, of the thyroid transformed phenotype.

Differential expression of the asialoglycoprotein receptor in discrete brain areas, in kidney and thyroid.

The asyaloglycoprotein receptor is a dimer formed by two polypeptide chains abundantly expressed in the liver (RHL-1 and RHL-2). Using specific primers for the two polypeptide chains we measured, by semiquantitative reverse PCR (RT-PCR), the corresponding mRNAs in different rat tissues. We found that both RHL-1 and RHL-2 mRNAs are expressed in the liver, kidney, brain and thyroid. Under the same conditions we did not detect any specific mRNA in the spleen. In the brain these sequences are expressed along a posterior-anterior gradient. Cerebellum and brainstem display the highest expression of the brain RHL-1 and RHL-2 mRNAs. Tissues and regional distribution of this receptor suggest that other body districts besides liver may participate in the clearance of serum glycoproteins.

Characterization of the mouse Tdgf1 gene and Tdgf pseudogenes.

Cripto protein is a member of the "EGF family" of growth factors present in colon tumors and in human and mouse undifferentiated teratocarcinoma cells. During gastrulation in the mouse, cripto-encoding transcripts are expressed in the forming mesoderm and later in the truncus arteriosus of the developing heart. As a necessary step prior to investigating the in vivo role of cripto through gene disruption, we have isolated all the genomic cripto-related sequences in the mouse. One gene (Tdgf1) and two pseudogenes (Tdgf2 and Tdgf3) have been isolated and characterized. The mouse Tdgf1 (coding for cripto), like the human gene, is divided into six exons. Comparison of the human and mouse genomic sequences reveals that mouse exons 1 and 3 are shorter than the corresponding human exons. The pseudogene Tdgf2 corresponds to about 1 kb of the mRNA and contains five base substitutions in the coding region that represent both silent and replacement substitutions. The pseudogene Tdgf3 corresponds only to the coding portion of Tdgf. Many mutations have been introduced in this pseudogene, suggesting its early origin. Alignments of the Tdgf3, human and mouse mRNA sequences, shows that this pseudogene has retained the 33 nucleotides of the human exon 3 that are missed in the Tdgf1 gene. Taken together, these data suggest that Tdgf3 is derived from an ancestral gene and that the human and mouse genes are probably evolving separately.

A truncated form of teratocarcinoma-derived growth factor-1 (cripto-1) mRNA expressed in human colon carcinoma cell lines and tumors.

The human teratocarcinoma-derived growth factor (TDGF)-1 gene encodes a 188-amino acid protein, cripto-1. The TDGF-1 gene is overexpressed in the majority of human primary colorectal carcinomas and hepatic metastases, in breast carcinomas and in testicular nonseminoma germ cell embryonal carcinomas. In the human embryonal carcinoma cell line NTERA-2 clone D1, a 2-kb TDGF-1 mRNA transcript is expressed. The present study shows that a 1.7-kb mRNA transcript lacking the first two exons of the TDGF-1 gene is expressed in the human colon carcinoma cell line GEO. This shorter mRNA is the only TDGF-1 transcript that is present in the majority of primary human colorectal carcinomas and hepatic metastases and in adult human tissues such as the pancreas, heart, stomach, mammary gland, skeletal muscle, liver and placenta. In contrast, in the kidney, brain, testis, ovary and spleen, the longer 2-kb TDGF-1 mRNA transcript is expressed. The putative shorter protein starts at a CUG codon 129 nucleotides downstream of the starting AUG codon of the longer protein. These data indicate the potential for differential transcriptional regulation of the TDGF-1 gene in different normal and tumor tissues.

Inhibition of c-ras reactivates thyroglobulin synthesis in middle-T transformed thyroid cells.

Differentiated thyroid cells expressing polyoma Middle-T became transformed and tumorigenic when injected into syngenic animals. The expression of thyroglobulin was greatly reduced and no longer responsive to thyrotropin (TSH) and to cAMP. Inhibition of endogenous c-ras by the expression of two transdominant negative mutant H-ras genes, Asn17 and Leu61-Ser186, reactivated thyroglobulin synthesis. Reactivation of thyroglobulin synthesis by c-ras inhibition was not observed in the absence of TSH. These findings indicate that MT elicits dedifferentiation of thyroid cells by activating endogenous c-ras and that c-ras interferes with TSH or cAMP signaling.

Follicular thyroglobulin (TG) suppression of thyroid-restricted genes involves the apical membrane asialoglycoprotein receptor and TG phosphorylation.

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A. , and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.