Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas.

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.

Effects of cyclic adenosine-monophosphate on growth and PSA secretion of human prostate cancer cell line.

Prolonged increase of cyclic adenosine-monophosphate (cAMP) level in the culture medium of a well differentiated human prostatic cancer cell (LNCaP) inhibits cellular growth and stimulates PSA secretion. The differentiation of the cells tested was documented by their responsiveness to androgens and the ability to synthesize cellular markers of differentiation (PSA). The raise in cAMP level was produced by dibutyryl cyclic AMP (DBcAMP) or by agents acting at distinct levels in the pathway of cAMP generation (forskolin) or degradation (IBMX). Each of these three agents in a range of concentrations between 10-4-10-6 M had an inhibitory effect on the growth which is dose and time-dependent. The inhibition was reversible as demonstrated by complete restoration of cell growth soon after the withdrawal of the substances from the culture medium. When cAMP levels in culture medium was raised, an increase in PSA content was observed. However, the effects of cAMP on PSA content was not due to increase in PSA synthesis, since simultaneous measurement of secreted and cellular PSA indicated that the principal effect of the cyclic nucleotide was to enhance the secretion of stored PSA. Furthermore the inhibition of cellular growth by cAMP suggests new approaches in prostatic carcinoma therapy.

Evaluation of epidermal growth factor receptor, carcinoembryonic antigen and Lewis carbohydrate antigens in human colorectal and liver neoplasias.

The expression of LewisY related carbohydrate antigens and the content of epidermal growth factor receptor (EGF-R), the carcinoembryonic antigen (CEA) and the alpha-fetoprotein (AFP) in colorectal and liver tumors were determined. These included 30 large bowel adenocarcinomas (7 colon, 6 sigma, 5 caecum, 12 rectum), 12 hepatocellular carcinomas and 6 liver metastases. Histologically normal tissue excised along with the tumors were used as controls. All plasma membranes studied showed specific EGF binding, and tumor plasma membranes had an EGF receptor level higher than that of the normal counterpart. However, EGF-R was positive in only a few tumors, and no correlation between clinical stages and grades of differentiation was observed. Cytosol CEA was higher in tumors than in normal counterparts. Tissue AFP and CEA content was different in liver hepatocellular carcinomas and in liver metastases. They are good markers to differentiate between primary and secondary liver neoplasias. The LewisY and related carbohydrate antigens, evaluated by the reactivity of the tissues to monoclonal antibody MAb B3, are expressed in liver metastases from colorectal adenocarcinoma. MAb B3 failed to react with hepatocellular carcinomas and with peritumoral liver tissues obtained from both metastatic and primary tumor lesions. These data suggest that immunoblotting with MAb B3 may be useful to obtain more information on liver carcinomas. Furthermore, MAb B3 or CEA armed with toxin in the form of recombinant immunotoxin or linked to a radionuclide can be useful in new treatments of metastatic lesions, such as immunotherapy, radioimmunotherapy and radioimmunoguided surgery.

Expression of Lewis carbohydrate antigens and chromogranin A in human prostatic cancer.

The prostate specific antigen (PSA) content, the neuroendocrine differentiation and the Lewis(y) and the expression of related carbohydrate antigens in pathological prostatic tissues were determined. These included 13 cancers and 11 benign hyperplasias. PSA is expressed strongly in hyperplastic and poorly in neoplastic tissues. The neuroendocrine differentiation detected by a monoclonal antibody directed against chromogranin A (CgA) is a frequent event in carcinomas and rare in hyperplastic prostate. The Lewis(y) and related carbohydrate antigens, evaluated by the reactivity of the tissues to two monoclonal antibodies MAbB3 and MAbB1, are expressed in a considerable percentage in malignant tissues of prostate and only occasionally in benign lesions. Our results suggest that immunoblotting with antibodies against CgA, B3 and B1 on the tissues, obtained after surgery, may be useful to obtain more information on the neoplastic transformation of human prostate. Furthermore, the expression of Lewis(y) and related carbohydrate antigens on the surface of prostate cancer suggest that, following a clinical trial, an immunotoxin combination of MAbB3 or MAbB1 and Pseudomonas exotoxin, may be used in the treatment of prostate cancer.

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.

Analysis of glycoproteins in cancers and normal tissues reactive with monoclonal antibodies B3 and B1.

In this study we show by immunoblotting that B1 and B3, two newly isolated monoclonal antibodies, react with a variety of glycoproteins with different molecular weights expressed in stomach, pancreas, colorectal and breast cancers. The pattern of reactivity differed among cancers arising in different tissues, although no correlation has been observed with the histopathological characteristics of the lesion analysed. MAb B3 and MAb B1, have a limited reactivity with peritumoral tissues, whereas react very strongly with metastatic lesion. Because of the limited reactivity of these antibodies with normal tissue, MAbs B3 and B1, armed with toxin in the form of recombinant immunotoxins, can be useful in treating certain kinds of cancer such as metastatic lesions. However, until current clinical trials are completed, we will not know if they will be helpful in cancer treatment.

Increased CA 125 serum levels in patients with advanced acute leukemia with serosal involvement.

BACKGROUND: CA 125 is a tumor marker used for the diagnosis and monitoring of ovarian carcinoma. This marker also has been found to be increased in patients with serosal effusion derived from nonneoplastic inflammatory disease and in a few instances of advanced non-Hodgkin lymphoma with serosal involvement. METHODS: CA 125 levels were tested in the serum of 15 patients with acute myeloblastic leukemia (AML) at the time of diagnosis and in 3 patients with advanced leukemia with serosal involvement. In two patients with elevated serum CA 125 levels, a CA 125 assay was performed on leukemic cells and on the supernatant fluid of short term liquid culture. RESULTS: Increased serum CA 125 was found in the three patients with acute leukemia with extramedullary localization and serosal effusion, whereas it was normal in 15 AML patients tested at the time of diagnosis. CA 125 was not detectable in leukemic cell extracts nor in the supernatant fluid of primary cultures. CONCLUSIONS: These results indicate that leukemic cells were unable to produce CA 125 and suggest that its elevation in the serum is likely due to a serosal inflammatory reaction caused by the leukemic infiltration.

AlphaANP, AVP, and pituitary-thyroid axis in patients with congestive heart failure and acute respiratory failure.

The pituitary-thyroid axis and neurohumoral activation indexes were simultaneously investigated in 16 in-patients hospitalized for cardiac heart failure (CHF), New York Heart Association (NYHA), class II-IV, and Killip clinical scale, class II-III, to evaluate their relationship with CHF morbidity and the relative prognostic value. At entry the patients were divided into two subgroups (A and B), according to the severity of CHF. Patients were further classified into two subgroups, according to the subsequent clinical course (C, poor outcome and D, improved clinical course). Blood samples were obtained every day for the radioimmunoassay measurement of plasma alpha-atrial natriuretic peptide (alphaANP), arginine vasopressin (AVP), and thyroid hormones, and the results were compared with those of 12 control subjects. At admission, alphaANP and 3,3',5'-triiodothyronine (rT3) values were higher, while 3,3',5-triiodothyronine (T3) to rT3 (T3/rT3) ratio was lower in subgroups A and B than in controls (p<0.001), respectively, and in C than in D (p<0.001), respectively, according to the prognosis. Conversely, no differences in other thyroid indexes, nor a significant correlation between alphaANP and either rT3 or T3/rT3 ratio were present in any of the subgroups. AVP plasma levels in subgroup A were not statistically different from those of controls, whereas they were significantly decreased in subgroups B and C (p<0.05) and D (p<0.001). In conclusion, these results indicate that in CHF the pituitary-thyroid axis is not altered, that alphaANP and T3/rT3 ratio are non-invasive and reliable predictors of severity and prognosis, while AVP might be affected by the different pathological processes leading to CHF or by the concomitant use of drugs.

Evaluation of goiter endemia by ultrasound in schoolchildren in Val Sarmento (Italy).

Ultrasonography is an excellent and objective method for assessing thyroid volume, especially in children where clinical evaluation is inaccurate. The aim of this study was to evaluate the presence of goiter by thyroid ultrasound and palpation in 244 schoolchildren, 6 to 14 years old, living in some rural villages of Val Sarmento, a mountain area of Basilicata, Italy. In 1996 we revealed the presence of endemic goiter in 25% of the schoolchildren evaluated by palpation, according to World Health Organization (WHO) criteria, and in 15.9% of the schoolchildren evaluated by ultrasonography (7.5 MHz linear probe). The median urinary iodine excretion, taken from an extemporaneous sample of the first urines in the morning, was 62.2 microg/l. This study includes Val Sarmento, an area with mild-moderate grade (Grade I) of iodine deficiency, suggesting the need for iodine prophylaxis. Furthermore, it proves that the measurement of thyroid volume by ultrasonography is an essential instrumental method for a correct epidemiological study of endemic goiter, particularly in areas where there is mild iodine deficiency.

Central precocious puberty: a single blood sample after gonadotropin-releasing hormone agonist administration in monitoring treatment.

This study was designed to evaluate whether a single blood sample drawn after the home injection of a long-acting gonadotropin-releasing hormone (GnRH) agonist (GnRHa) in patients treated for central precocious puberty (CPP) could be a more simple and inexpensive test with respect to the conventional GnRH stimulating test in assessing adequate suppression of the pituitary-gonadal axis. The response to the first therapeutic injection of the GnRHa triptorelin was studied in 14 newly diagnosed untreated females with CPP. The results were compared with the response that the same patients had to the conventional GnRH stimulation test performed at the time of diagnosis. A significant increase in LH, FSH and E2 levels was observed 12 h after the triptorelin intramuscular injection; serum peak values of LH (70.3 +/- 58.5 IU/l), FSH (44.2 +/- 21.7 IU/l) and E2 (489.7 +/- 263.9 pmol/l) were significantly greater than those obtained with the conventional GnRH test (LH 31.4 +/- 21.7, p = 0.002; FSH 19.8 +/- 10. 7, p = 0.001; E2 83.3 +/- 25, p < 0.001). In particular, the E2 response, 12 h after triptorelin injection, was clearly consistent with gonadal activation compared to the modest E2 increase in response to the GnRH test. Thereafter 22 girls who were already being treated with triptorelin for CPP were evaluated to see whether a single blood sample drawn 12 h after the therapeutic home injection of GnRHa could be informative in assessing adequate suppression of the pituitary-gonadal axis. This response was also compared to the conventional GnRH stimulation test performed 2 days before the therapeutic triptorelin injection. In 7 girls with evidence of pubertal progression, the E2 response following the GnRHa injection (136.3 +/- 44.4 pmol/l) was significantly higher with respect to the response after the GnRH stimulation test (73.0 +/- 0.0; p < 0.02) indicating an inadequate suppression of the pituitary-gonadal axis. The present data suggest that a single blood sample drawn 12 h after the therapeutic home administration of triptorelin provides a simple, comfortable and inexpensive means of monitoring pituitary as well as gonadal function in girls treated for CPP.

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

Transient dwarfism and hypogonadism in mice lacking Otx1 reveal prepubescent stage-specific control of pituitary levels of GH, FSH and LH.

Genetic and molecular approaches have enabled the identification of regulatory genes critically involved in determining cell types in the pituitary gland and/or in the hypothalamus. Here we report that Otx1, a homeobox-containing gene of the Otx gene family, is postnatally transcribed and translated in the pituitary gland. Cell culture experiments indicate that Otx1 may activate transcription of the growth hormone (GH), follicle-stimulating hormone (betaFSH), luteinizing hormone (betaLH) and alpha-glycoprotein subunit (alphaGSU) genes. Analysis of Otx1 null mice indicates that, at the prepubescent stage, they exhibit transient dwarfism and hypogonadism due to low levels of pituitary GH, FSH and LH hormones which, in turn, dramatically affect downstream molecular and organ targets. Nevertheless, Otx1-/- mice gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with restored growth and gonadal function at 4 months of age. Expression patterns of related hypothalamic and pituitary cell type restricted genes, growth hormone releasing hormone (GRH), gonadotropin releasing hormone (GnRH) and their pituitary receptors (GRHR and GnRHR) suggest that, in Otx1-/- mice, hypothalamic and pituitary cells of the somatotropic and gonadotropic lineages appear unaltered and that the ability to synthesize GH, FSH and LH, rather than the number of cells producing these hormones, is affected. Our data indicate that Otx1 is a new pituitary transcription factor involved at the prepubescent stage in the control of GH, FSH and LH hormone levels and suggest that a complex regulatory mechanism might exist to control the physiological need for pituitary hormones at specific postnatal stages.

[Urinary iodine excretion in the Campania region]. / Escrezione urinaria di iodio nella regione Campania.

Urinary iodine excretion (UIE) was measured on urine samples collected in the morning from 3480 subjects (both adults and children) living in an extended territory of Campania region including urban and extraurban areas, in the period 1993-96. UIE was measured by an autoanalyzer (Bran + Luebbe) and expressed as microgram/l. Means UIE were significantly lower in the patients living in the villages of Avellino (65 +/- 48), Benevento (44 +/- 44) and Caserta (78 +/- 53) respect to the control subjects from the area of Naples (102 +/- 66). These data demonstrate that in many areas of our region a mild-moderate iodine deficiency is still present as indicated by a low urinary iodine excretion and therefore an effective program of iodoprophylaxis is fundamental in this region.

[Widespread endemic goiter and iodine deficiency in the province of Avellino]. / Diffusione del gozzo endemico e della carenza iodica in provincia di Avellino.

1352 schoolchildren between 6-14 years old (699 males and 653 females) and 943 adults (176 males and 767 females) from eight villages of the province of Avellino were studied. All subjects were examined for thyroid size by at least two expert examiners. In most of them urine samples were collected for iodine determinations. 387 schoolchildren and 161 adults from Flumeri and Villanova were evaluated by thyroid echography. The prevalence of goiter was from 23.5 to 52.2% and the median urinary iodine excretion was from 42.3 to 66.2 micrograms/l in schoolchildren. In adults the prevalence of goiter was from 41.2 to 86.7% and the median urinary iodine excretion was from 37.1 to 53.7 micrograms/l. Our data showed a degree of iodine deficiency from low to moderate. The echography permitted to point out a greater prevalence of nodules than the thyroid palpation.

Long-term interferon-alpha therapy does not affect sex hormones in males with chronic hepatitis C.

The effects of interferon-alpha (IFN-alpha), given at a dosage of 6 MU thrice weekly for 12 months, on gonadal function were investigated in 18 males affected by chronic hepatitis C. Periodically, all patients were clinically monitored and questioned about sexual function. Gonadotropin and serum androgen concentrations (follicle-stimulating hormone, luteinizing hormone, total testosterone, free testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and sex hormone binding globulin) were tested every 3 months. Ten of 18 patients (55%) responded to IFN-alpha therapy. Serum total testosterone and sex hormone binding globulin values decreased slightly at the third month of treatment, then returned to baseline values. Serum free testosterone and other sex hormones remained essentially unchanged during IFN-alpha therapy. Four patients (22.2%) complained of sexual dysfunction (impaired libido, erectile failure, and impaired ejaculation), which was unrelated to any significant hormonal change and resolved after IFN therapy was stopped. Serum sex hormones values did not differ between responders and nonresponders to IFN-alpha. This study indicates that 12 months treatment with 6 MU of IFN-alpha thrice weekly does not significantly affect gonadal function in men with chronic hepatitis C. The sexual dysfunction observed could be ascribed to such other side effects of IFN as asthenia, fatigue, or anxiety, or it could have a psychologic basis.

Endemic goiter in Calabria: etiopathogenesis and thyroid function.

An endemic goiter study was carried out in a vast territory of the Calabria region, including the provinces of Catanzaro and Cosenza. About 50% of 7231 school-children, aged between 6 and 12 years, examined in 34 villages, presented a thyroid enlargement. The high prevalence of goiter seems to be related to a moderate iodine deficiency intake indicated by a mild urinary iodine excretion (lower than 100 micrograms/g creatinine). In a few villages, in which a two year voluntary iodine prophylaxis was carried out, an increase in the urinary iodine excretion with a decreased goiter prevalence was observed. A slight increase in TT3, FT3 and TSH in one endemic area studied, compared to the control area, was also observed. These data suggest that an effective program of iodoprophylaxis is fundamental in this region as well.

Serum and tissue levels of CEA, TPA, CA 125 and CA 15.3 in patients with lung cancer.

Forty-nine healthy subjects (Group I), 24 patients with benign lung diseases (Group II) and 48 patients surgically treated for lung cancer (Group III): 28 with squamous cell carcinoma (SCC) and 20 with adenocarcinoma (adenoca), were tested for the presence of carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), cancer antigen CA 125 and antigen CA 15.3. The four markers were measured in the serum of the patients of the three groups and in the cytosol extract of tumoral and peritumoral tissues of Group III subjects. The mean levels of serum CEA and TPA were significantly higher in squamous cell carcinoma and in adenocarcinoma patients than in normal subjects. In benign lung disease serum CEA was equal and TPA slightly higher than in normal subjects. CA 125 was higher in the serum of patients with malignant diseases compared to normal or benign lung diseases but this difference was not statistically significant. Serum CA 15.3 levels were similar in all subjects studied. CA 125 in squamous cell carcinoma cytosol was much higher than in peritumoral cytosol whereas the other three markers were not significantly different in tumor cytosol or peritumoral cytosol. A direct correlation between serum and cytosol values was observed for CEA, but not for the other markers. The levels of the four markers in serum and cytosol did not correlate with the stage or grade of the tumors.

[Laboratory diagnosis of growth hormone]. / Diagnostica di laboratorio dell'ormone della crescita.

The role of Clinical Pathology Laboratory in normal and altered growth hormone secretion is discussed. In particular, it is reported that the normal GH secretion can be studied by serum and urine GH determinations whereas the diagnosis of GH deficiency rests upon the demonstration of an inadequate rise serum GH after provocative stimuli and serum measurement of somatomedins (IGF-I) by radioimmunoassay method. As it concerns increase GH secretion the diagnosis is clinically made (acromegaly and gigantism) and the laboratory has only the role to confirm it by the assessment of basal and stimulated GH secretion.

Epidermal growth factor receptor and lipid membrane components in human lung cancers.

The binding of 125I-epidermal growth factor (EGF) to the plasma membranes of 54 samples of human lung tumors was determined. These included 34 squamous cell carcinomas and 20 adenocarcinomas. Twenty samples of histologically normal lung excised surgically along with the tumors were used as controls. Most of the plasma membranes showed an EGF receptor level higher than that of normal tissue. A moderate increase in the amount of 125I-EGF bound (2-5 fold) was observed in the majority of the tumors. Only a few cases (5-10% of the total) showed a large increase (> than 10 fold). The binding of 125I-EGF was compared with clinical stages and grades of differentiation. No correlation between the stage of the tumor and 125I-EGF binding was observed. However, the highest levels of EGF receptor (EGF-R) were found in poorly differentiated squamous cell carcinomas. The total amount and the distribution pattern of gangliosides and phospholipids were analyzed in individual tumors. A decrease in GD1b, GD1a and sphingomyelin and an increase in GM1 and GM3 was observed. No correlation was detected when tumors with the highest or lowest levels of gangliosides or phospholipids were compared with tumors exhibiting the highest binding of 125I-EGF.