Recent developments in the investigation of thyroid regulation and thyroid carcinogenesis

This review covers new mechanistic information spanning the past 10 years relevant to normal and abnormal thyroid growth and function that may assist in the risk assessment of chemicals inducing thyroid follicular cell neoplasia. Recent studies have shown that thyroid regulation occurs via a complex interactive network mediated through several different messenger systems. Increased thyroid-stimulating hormone (TSH) levels activate the signal transduction pathways to stimulate growth and differentiation of the follicular cell. The important role of TSH in growth as well as in function helps to explain how disruptions in the thyroid-pituitary axis may influence thyroid neoplasia in rodents. New investigations that couple mechanistic studies with information from animal cancer bioassays (e. g., sulfamethazine studies) confirm the linkage between prolonged disruption of the thyroid-pituitary axis and thyroid neoplasia. New initiation/promotion studies in rodents also support the concept that chronic stimulation of the thyroid induced by goitrogens can result in thyroid tumors. Some of these studies confirm previous suggestions regarding the importance of chemically induced thyroid peroxidase inhibition and the inhibition of 3,3',5, 5'-tetraiodothyronine (T4, thyroxine) deiodinases on disruption of the thyroid-pituitary axis leading to thyroid neoplasia. Some comparative physiologic and mechanistic data highlight certain differences between rodents and humans that could be expected to confer an increased vulnerability of rodents to chronic hypersecretion of TSH. New data from epidemiologic and molecular genetic studies in humans contribute further to an understanding of thyroid neoplasia. Acute exposure to ionizing radiation, especially in childhood, remains the only verified cause of thyroid carcinogenesis in humans. Iodine deficiency studies as a whole remain inconclusive, even though several new studies in humans examine the role of dietary iodine deficiency in thyroid cancer. Specific alterations in gene expression have been identified in human thyroid neoplasia, linked to tumor phenotype, and thus oncogene activation and tumor-suppressor gene inactivation may also be factors in the development and progression of thyroid cancer in humans. An analysis by the U.S. EPA Risk Assessment Forum, prepared as a draft report in 1988 and completed in 1997, focused on the use of a threshold for risk assessment of thyroid follicular tumors. New studies, involving several chemicals, provide further support that there will be no antithyroid activity until critical intracellular concentrations are reached. Thus, for chemically induced thyroid neoplasia linked to disruptions in the thyroid-pituitary axis, a practical threshold for thyroid cancer would be expected. More information on thyroid autoregulation, the role of oncogene mutations and growth factors, and studies directly linking persistently high TSH levels with the sequential cellular development of thyroid follicular cell neoplasia would provide further confirmation.     

Oncogenes and signal transduction

Cancer biologists now have a unified concept to guide their search for specific genetic abnormalities and new therapeutic approaches: The genes and proteins that participate in the conversion of normal into malignant cells are also involved in the key process that converts extracellular signals into intracellular events that culminate in division and growth.     

Dietary interventions of human carcinogenesis

The diet is a complex mixture that is associated with approximately 30% of human cancer in the U.S. Extensive laboratory studies indicate that the diet is composed of many mutagens/carcinogens as well as antimutagens/anticarcinogens. Overwhelming evidence from epidemiological studies indicates that a diverse diet that is high in fruits and vegetables and low in certain fats, along with moderate caloric intake and exercise, is most closely associated with reduced cancer risk. Dietary intervention studies using complex food items (fruits, vegetables, and fats) support these epidemiological observations; dietary interventions using single compounds (vitamins, antioxidants, etc.) have generally not. Estimates suggest that appropriate dietary changes could reduce the percentage of deaths due to prostate, colorectal, pancreatic, and breast cancer by >/=50%.     

Oncogenes and tumor suppressor genes in photocarcinogenesis

A case of coup de sabre, a linear form of scleroderma, is presented. Treatment consisted of soft-tissue expansion and autologous bone grafting to the forehead, a composite graft for alar reconstruction, and a scalp graft for eyebrow reconstruction. None of the linear scleroderma cases reported in the literature consisted of bony reconstruction.     

DNA adducts in human carcinogenesis: etiological relevance and structure-activity relationship

Sensitive methods for quantifying DNA adducts from (i) benzo[a]pyrene (BP), (ii) alkylation exposure, and (iii) etheno(epsilon)-DNA adduct-forming chemicals were developed and applied to humans and animal models. The aims were to identify hitherto unknown sources and mechanisms of exogenous and endogenous DNA damage, to examine the effect of drug polymorphism on BP adduct levels, and to develop QSAR between tumorigenic potency, heritable genetic damage and structural elements of alkylating carcinogens (Vogel and Nivard (1994) Mutation Res., 395, 13-32). (i) BP-DNA adducts: An HPLC/fluorimetry assay suitable for measuring (+)-anti-BP-diol-epoxide (BPDE) adducts in human tissues and white blood cells (WBC) was developed (Alexandrov et al. (1992) Cancer Res., 52, 6248-6253). In smokers, a positive correlation was found between pulmonary CYP1A1-related catalytic activity (AHH) and the level of lung BPDE-DNA adducts. In coke oven workers, an enhancing effect of smoking on BPDE-adduct levels in WBC was demonstrated (Rojas et al. (1995) Carcinogenesis, 16, 1373-1376). (ii) 3-Alkyladenines (3-alkAde): Alkylating carcinogens form 3-alkAde adducts in DNA which depurinate to yield 3-alkAde in urine, for which a detection method was developed (Friesen et al. (1991) Chem. Res. Toxicol., 4, 102-106; Prevost et al. (1990) Carcinogenesis, 11, 1747-1751), using immunoaffinity purification and GC-MS analysis. The usefulness of 3-alkAde analysis for the determination of the whole-body dose of alkylating agents derived from exogenous and endogenous sources was demonstrated. (iii) Etheno-DNA adduct-forming agents: Etheno(epsilon)-DNA base adducts (epsilon A, epsilon dC, epsilon dG) are promutagenic DNA lesions that are formed by occupational (vinyl halides) and environmental (urethane) carcinogens. An ultrasensitive detection method was developed (Nair et al. (1995) Carcinogenesis, 16, 613-617), based on immunoaffinity purification and 32P-postlabelling of epsilon-nucleoside 3'-monophosphates. Liver DNA from unexposed rats, mice and from human samples contained background levels of epsilon dA and epsilon dC (Bartsch et al. (1994) Drug. Metab. Rev., 26, 349-371). As formation of epsilon dA and epsilon dC adducts by lipid peroxidation products was demonstrated (El Ghissassi et al. (1995) Chem. Res. Toxicol., 8, 278-283), they may serve as markers for oxidative stress. Results from testing this hypothesis are presented.     

The role of telomerase for pancreatic carcinogenesis in human and hamster

Telomerase activity and terminal restriction flagment (TRF) length were investigated in human and hamster pancreatic duct adenocarcinomas. In the hamster primary and transplantable pancreatic carcinomas and cell lines, telomerase activity increased 86 to 215.7 times relative to the levels in normal spleen and pancreas, and reduction of TRF length was observed. In 38 human pancreatic ductal carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymph node involvement and distant metastasis. These results suggest that telomerase play an important role for pancreatic duct carcinogenesis.     

Estrogen receptors and cathepsin D in human thyroid tissue

BACKGROUND: To investigate the significance of estrogen receptors (ER) in the pathogenesis of thyroid dysplasia, the authors analyzed, by analogy with breast cancers, ER and three estrogen-regulated proteins: progesterone receptor (PR), cathepsin D, and pS2 protein, in cytosols of 42 human thyroid tissues. METHODS: ER and PR were measured by an immunoenzymatic assay and cathepsin D and pS2 by an immunoradiometric assay. Tissue specimens included 7 normal tissues, 6 benign nodules, 8 toxic adenomas, 7 from patients with Graves disease, and 14 carcinomas. RESULTS: ER was present at very low concentrations, with no statistical difference between neoplastic and nonneoplastic tissues. The mean levels of cathepsin D, expressed as pmol/mg protein minus thyroglobulin, were higher in the 14 carcinomas (P = 0.0003), the 7 specimens from patients with Graves disease (P = 0.006), and the 8 toxic adenomas (P = 0.04) than in the 7 normal thyroid tissues. A significant difference also was observed between the carcinomas (P = 0.003) and six benign nodules. Compared to TNM parameters, cathepsin D concentrations correlated with tumor size: higher cathepsin D levels were found in pT4 than in pT2 and pT3 carcinomas. All the tissues tested were negative for PR and pS2 protein. CONCLUSIONS: The results clearly indicate a significant difference between neoplastic and normal thyroid tissue in terms of the amount of cathepsin D, but not that of ER. This suggests that cathepsin D probably is not regulated by estrogen but simply is a marker of protease activity during invasion by thyroid carcinomas.     

Desensitization in normal and neoplastic human thyroid cell lines

Because some papillary thyroid cancers continue to grow when thyroid-stimulating hormone (TSH) levels are suppressed, we questioned whether desensitization (i.e., a decreased cAMP response to repeat stimulation with TSH) occurs in normal and neoplastic thyroid tissue. If desensitization does occur, is it similar or different in these human thyroid cells? Normal and papillary thyroid cancer cells from the same patient were cultured as we have previously described. Normal and neoplastic thyroid tissues responded to TSH (0.01-10.0 mU/ml) by increasing cAMP production and growth in a dose-dependent manner. In normal cells there was an 11-fold mean increase in cAMP production at 4 hours, and all thyroid cultures responded. In neoplastic cells cAMP production increased from 1.5-fold to 3.0-fold with a mean 2.0-fold increase at 4 hours. In normal thyroid cells the cAMP response to a second TSH stimulus (desensitization) decreased up to 75% (range 25-75%), and desensitization occurred in all normal thyroid cell cultures. In neoplastic thyroid cells, however, the cAMP response to a second TSH stimulus decreased up to 17% (range 0-17%); and desensitization occurred in only two of the five neoplastic thyroid cell cultures. Thus when normal thyroid and neoplastic cells from the same patients were studied, greater desensitization occurred in the normal cells (75% vs. 17%). These studies document that there is greater desensitization in normal tissue than in neoplastic thyroid tissue, which may account for the increased growth of thyroid neoplasms in the presence of ever-changing low levels of TSH.     

Telomerase activity in benign and malignant human thyroid tissues

Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomerase repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity plays a role in carcinogenesis and immortalization. In this work, the status of telomerase activity during the development of human thyroid cancer was determined using telomeric repeat amplification protocol (TRAP) in 14 nodular hyperplasia, 14 adenomas, 23 papillary carcinomas and 11 follicular carcinomas. Positive telomerase activity was detected in 2 of 14 nodular hyperplasias (14%), 4 of 14 adenomas (29%), 12 of 23 papillary carcinomas (52%) and 10 of 11 follicular carcinomas (91%). The cancers that are negative for telomerase activity are mostly in early stage (stage I or II). These results suggest that telomerase reactivation plays a role during the development of thyroid cancer.     

Autofluorescence in normal and malignant human oral tissues and in DMBA-induced hamster buccal pouch carcinogenesis

Light-induced fluorescence spectroscopy was conducted on human oral malignant and normal tissues. Under 330-nm excitation wavelength, significant differences in fluorescence intensity were observed around 380- and 460-nm emission. Furthermore, 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in hamster buccal pouch was investigated to elucidate whether similar alterations of fluorescence spectroscopy occurred during the development of squamous cell carcinoma. Similar to the spectral profiles of human oral malignant and normal tissues, the most intense fluorescence peaks in the pouches occurred at 380 nm and 460 nm emission under 330 nm excitation wavelength. At 380 nm emission, the fluorescence intensity of normal pouch mucosa was stronger than those of DMBA-treated abnormal tissues at different stages of carcinogenesis. However, at 460 nm emission, the fluorescence intensity of DMBA-treated tissues was not only stronger than that of normal pouch mucosa but also shifted to 470 nm. These results suggest that under 330 nm excitation wavelength fluorescence spectroscopy may be useful for the detection of oral malignant lesions.     

Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves' thyroid tissue

Milligram quantities of the human membrane autoantigen thyroid peroxidase (TPO) have been purified to a high degree of homogeneity by a combination of detergent solubilisation, monoclonal antibody affinity, and ion exchange chromatography, from pooled Graves' disease thyroid glands. The purified TPO of greater than 90% purity was enzymatically active as judged by its ability to oxidise guaiacol. Crystals of TPO have been grown from solutions of the protein solubilised in sodium deoxycholate, in the presence of ammonium sulphate. The crystals exhibited birefringence under polarised light, indicative of molecular order. Crystallisation of this large, membrane autoantigen represents the first step in delineating the complete three-dimensional structure of a human autoantigen involved in destructive thyroiditis.     

Manganese superoxide dismutase content and localization in human thyroid tumours

We describe a procedure for detecting and localizing cytomegalovirus DNA sequences based on in situ hybridization at the ultrastructural level. A digoxigenin-labelled probe, identified with an anti-digoxigenin colloidal gold-labelled antiserum, was employed on infected cells embedded in a new acrylic resin (Bioacryl). The silver enhancement method on the same specimen was used to more easily reveal the reaction also at low magnification. The immunolocalization was characterized by high specificity with virtually no background staining and a good maintenance of submicroscopic cell features.     

Mechanisms involved in sodium arsenite-induced apoptosis of human neutrophils

Apoptosis is a distinct mechanism by which eukaryotic cells die. Factors governing the induction of polymorphonuclear leukocyte (PMN) apoptosis should be important in understanding resolution of acute inflammation. The mechanisms for induction of PMN apoptosis remain uncertain; however, oxidative stress has been suggested. The aims of this study were to determine whether reactive oxygen intermediates play a role in PMN apoptosis and to investigate inhibition of this process by selective use of antioxidants. PMN were isolated from 10 healthy volunteers. PMN (1 x 10(6) PMN/mL) were cultured in 40, 80, and 160 microM of arsenite for 2, 6, 12, 18, and 24 h. Apoptosis was assessed qualitatively by morphology and gel electrophoresis and quantitatively by CD16 receptor expression and propidium iodide DNA staining. There was a significant (P < 0.05) increase in the rate of apoptosis on incubation with arsenite (80 and 160 microM). To investigate the mechanism of this process, intracellular respiratory burst activity was measured following arsenite culture. We found that arsenite-induced PMN apoptosis correlated with an increase in intracellular respiratory burst. To further investigate the role of oxidative injury in inducing apoptosis, the antioxidants catalase, dimethyl sulfoxide (DMSO), glutathione (GSH), N-acetylcysteine (NAC), and taurine were investigated and we demonstrated that GSH, NAC, and taurine were significantly protective against arsenite-induced apoptosis. However, catalase and DMSO failed to induce protection. This study demonstrates that arsenite induces PMN apoptosis through an oxygen-dependent mechanism that can be prevented through selective antioxidants.     

Iodothyronine content of human thyroid albumin

Stable thyroid hormones (T4 and T3)1) have been demonstrated in pure albumin isolated from normal human thyroid tissue iodinated in vivo. Five samples of albumin were separated from other thyroid proteins by acrylamide gel electrophoresis. After pronase hydrolysis, the content of Thyroid hormones was measured chemically (T4 + T3) as well as by competitive radioactive measurement (T4) and radioimmunoassay (T3). The purity of the albumin and validity of these measurements were confirmed by different techniques. The synthesis of thyroid hormones is not therefore a property unique to Tg and may occur in albumin. However the amount of iodothyronines in the albumin (average 0.004 residue per molecule) is much less than that found in Tg (0.5 residue per molecule). In the albumin as in Tg the number of hormone residues per molecule is proportional to the number of atoms of iodine. At an equivalent iodine concentration, the albumin seems capable of forming the thyroid hormones as well as Tg. The difference between these two proteins, in their capacity to synthesize thyroid hormones, seems to depend on their capacity for iodination. This difference of iodination does not seem to be linked with the number of tyrosyl residues, but might be related to the position of these residues.     

Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin

We report a 21-year-old woman presenting with a slowly progressive tetraparesis, optic nerve atrophy on both sides, and autonomic disturbances since early childhood. The patient has been carefully followed up for 5 years with clinical and ancillary investigations. The results and the time course strongly suggest an underlying degenerative syndrome affecting parts of three major systems: autonomic, motor and visual. Some symptoms resemble familial dysautonomia (FD, Riley-Day syndrome), however, hallmarks of FD, such as absence of fungiform papillae of the tongue, abnormal reaction on intradermal histamine injection, absent tendon reflexes, are missing, and central motor disturbances have not been described in FD. We consider this syndrome a slowly progressive multisystemic degeneration with two unusual hitherto unreported features: the combination of affected systems (autonomic and motor systems, optic nerves), and the early onset.     

Hypermutability in carcinogenesis

The presence of numerous chromosomal changes and point mutations in tumors is well established. At least some of these changes play a role in the development of the tumors. It has been suggested that the number of these genetic changes requires that tumorigenesis involves an increase in mutation rate. However, the presence of numerous changes can also be accounted for by efficient selection. What is required to settle the issue is some measure of nonselected mutations in tumors. In order to determine whether the tumor suppressor TP53 (coding for the protein p53) is hypermutable at some stage of carcinogenesis, the frequency of silent and multiple mutations in this gene has been examined. Silent mutations make up approximately 3% of the total recorded but constitute 9.5% of the mutations found in tumors with multiple mutations. Multiple closely linked mutations are also observed. Such multiple mutations suggest the operation of an error-prone replication process in a subclass of cells. The published data indicate that TP53 is hypermutable at some stage of tumor development. It is not yet clear whether TP53 is unique or whether other genes display a similar pattern of silent and multiple mutations.