5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma.

BACKGROUND: Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80-85 %) or multiglandular disease (~15 %) causing hypersecretion of parathyroid hormone (PTH) and generally hypercalcemia. Parathyroid cancer is rare (<1-5 %). The epigenetic mark 5-hydroxymethylcytosine (5hmC) is reduced in various cancers, and this may involve reduced expression of the ten-eleven translocation 1 (TET1) enzyme. Here, we have performed novel experiments to determine the 5hmC level and TET1 protein expression in 43 parathyroid adenomas (PAs) and 17 parathyroid carcinomas (PCs) from patients who had local invasion or metastases and to address a potential growth regulatory role of TET1. RESULTS: The global 5hmC level was determined by a semi-quantitative DNA immune-dot blot assay in a smaller number of tumors. The global 5hmC level was reduced in nine PCs and 15 PAs compared to four normal tissue samples (p < 0.05), and it was most severely reduced in the PCs. By immunohistochemistry, all 17 PCs stained negatively for 5hmC and TET1 showed negative or variably heterogeneous staining for the majority. All 43 PAs displayed positive 5hmC staining, and a similar aberrant staining pattern of 5hmC and TET1 was seen in about half of the PAs. Western blotting analysis of two PCs and nine PAs showed variable TET1 protein expression levels. A significantly higher tumor weight was associated to PAs displaying a more severe aberrant staining pattern of 5hmC and TET1. Overexpression of TET1 in a colony forming assay inhibited parathyroid tumor cell growth. CONCLUSIONS: 5hmC can discriminate between PAs and PCs. Whether 5hmC represents a novel marker for malignancy warrants further analysis in additional parathyroid tumor cohorts. The results support a growth regulatory role of TET1 in parathyroid tissue.

Diagnosis of parathyroid carcinoma using immunohistochemical staining against hTERT.

The differential diagnosis of parathyroid carcinoma from benign adenoma is often difficult when its typical clinicopathological features are absent, even with the aid of various molecular markers. We recently demonstrated that telomerase activation through hTERT expression is a unique characteristic that is limited to parathyroid carcinoma and not seen in benign tumors. In the present study, we investigated hTERT expression in parathyroid tumors using immunohistochemistry in an attempt to determine its clinical utility. There was no evidence of immunoreactivity in the 4 normal parathyroid glands and the 18 typical adenomas. In contrast, one atypical adenoma stained positively and homogeneously, and the disease recurred three times clinically. All of the 6 carcinomas demonstrated a clear positive nuclear staining of hTERT. Every hTERT-positive tumor showed a high Ki-67 index, i.e., greater than 4%. Nucleolin, an hTERT-binding protein, was abundantly and homogeneously expressed in all specimens examined independent of the pathological diagnosis and hTERT or Ki-67 expression. Therefore, it is possible that immunostaining with an anti-hTERT antigen (NCL-L-hTERT) individually may distinguish parathyroid carcinoma from benign tumors.

Loss of expression for the Wnt pathway components adenomatous polyposis coli and glycogen synthase kinase 3-beta in parathyroid carcinomas.

The development of parathyroid carcinoma has been associated with inactivating mutations of the Hyperparathyroidism type 2 (HRPT2) gene encoding parafibromin, a member of the human RNA Polymerase II-Associated Factor Complex (hPAF) and functionally linked to the Wingless type (Wnt) pathway. In this study, we characterized the expression of Wnt pathway molecules in parathyroid benign and malignant tumors. Tumors were investigated by immunohistochemistry supplemented with Western blot analyses using monoclonal antibodies. The study comprised 13 tumors from 12 cases of unequivocal parathyroid carcinoma, 18 cases of parathyroid adenoma, as well as non-tumorous parathyroid tissue. Adenomatous polyposis coli (APC) was uniformly expressed in non-tumorous parathyroid tissue and adenomas, but absent in carcinomas from 9 of 12 patients (75%). Expression of glycogen synthase kinase 3-beta (GSK3-beta) was lost in 4/12 carcinomas and in 1/18 adenomas. The loss of APC and GSK3-beta did not lead to augmentation of the Wnt target protein cyclin D1 or the Wnt oncoprotein beta-catenin. Active beta-catenin showed cytoplasmic and nuclear expression in all non-tumorous tissues and tumors. Loss of APC immunoreactivity was significantly associated with parathyroid carcinoma as compared to adenomas (p<0.001), giving a high specificity (100%) and sensitivity (75%) for the detection of parathyroid malignancy. The results suggest the involvement of Wnt-pathway members APC and GSK3-beta in parathyroid carcinoma development. In addition, APC immunohistochemistry could become a useful tool for improved recognition of parathyroid carcinoma together with immunohistochemistry for parafibromin and proliferation index. Furthermore, the involvement of APC related pathways in the disease development opens possibilities to explore therapeutic routes complementary to surgery.

Telomerase activation and expression of its catalytic subunits in benign and malignant tumors of the parathyroid.

PURPOSE: Telomerase activity (TA) has been extensively studied in tumors of many organs, but not the parathyroid gland. Therefore, we investigated TA in parathyroid tumors, and examined the mRNA expression of its catalytic subunits. METHODS: We examined 17 single adenomas, one hyperplastic parathyroid gland, and one metastatic parathyroid cancer and quantified TA by fluorescence-based TRAP analysis. We also studied the expression of telomerase catalytic subunits in nine adenomas and one cancer by reverse transcription - polymerase chain reaction (RT-PCR) analysis. RESULTS: Telomerase activity was not detected in any of the adenomas or the hyperplastic gland; however, the metastatic cancer was highly positive for TA. Both human telomerase RNA ( hTR) and human telomerase-associated pretein (hTEP-1) mRNA were detected universally in every specimen tested. Conversely, human telomerase reverse transcriptase (hTERT) mRNA was not detected by the conventional electrophoresis-based technique. Faint expression of hTERT mRNA was detected by real-time RT-PCR only in the sample of parathyroid cancer. DISCUSSION: We hypothesize that TA plays a role in the malignant transformation of parathyroid disease and suggest that hTERT mRNA expression could be the key step for TA as in other malignancies. Telomerase activity and hTERT may be useful molecular targets of parathyroid cancer.

Demonstration of human telomerase reverse transcriptase (hTERT) in human parathyroid tumours by in situ hybridization with a new oligonucleotide probe.

BACKGROUND AND OBJECTIVE: Telomerase activity is present in most malignant tumours and might provide a mechanism for unlimited replication of neoplastic cells. Expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is associated with telomerase activity, and it is overexpressed in most parathyroid carcinomas. We have therefore studied hTERT expression in parathyroid tumours. METHODS: In the present study, we assayed telomerase activity by the telomeric repeat amplification protocol (TRAP) and used in situ hybridization (ISH) to study hTERT mRNA expression in four parathyroid metastatic cancers, six adenomas and two parathyroid hyperplasia tissue specimens. RESULTS: Telomerase activity was detected by the TRAP assay in all of the parathyroid cancers (100%) but in none of the eight parathyroid benign lesions. hTERT gene expression was detected in the nuclei of the parathyroid cancer cells in all of the lesions (100%) but in none of the eight parathyroid benign lesions. CONCLUSIONS: Our results demonstrate a correlation between telomerase activity and hTERT gene expression measured by ISH (P < 0.001) in parathyroid tumours. We succeeded in very clearly and sensitively demonstrating hTERT mRNA in parathyroid tissues by using a new probe.

Consistent decrease in telomere length in parathyroid tumors but alteration in telomerase activity limited to malignancies: preliminary report.

Telomerase is known to be activated and telomere length altered in various types of malignant and benign tumors, but whether this is also the case for parathyroid lesions has hitherto been unclear. We therefore investigated telomerase activity and telomere length in 3 parathyroid metastatic cancers, 6 adenomas, 2 cases of parathyroid hyperplasia, and 16 samples of normal parathyroid tissue. Telomerase activity, assayed by the telomeric repeat amplification protocol, was detected in all of the parathyroid cancers (100%), in none of the 8 parathyroid benign lesions, and in only 1 of the 16 normal parathyroid samples (8.3%). Telomere length, determined by the terminal restriction fragment assay, was reduced in the tumor tissues with a mean telomere length of 8.23 +/- 0.86 kbp compared with the 12.61 +/- 0.81 kbp for the 16 age-matched subjects (p = 0.002). The results indicate that telomerase activity and telomere length may reflect the biologic behavior of individual parathyroid lesions.

Telomerase in endocrine and endocrine-dependent tumors.

Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.

Telomerase is not activated in human hyperplastic and adenomatous parathyroid tissue.

BACKGROUND: Telomerase is a specific enzyme that appears to have a key role in cellular senescence and the progression of neoplastic tissue. High telomerase activity has been found in several cancers, but not in most normal and benign tissue. Little is known about the influence of telomerase on the abnormal growth associated with hyperparathyroidism. OBJECTIVE: To analyse telomerase activity in parathyroid tissue obtained from 29 patients undergoing surgery for primary hyperparathyroidism. DESIGN: Tissue for telomerase activity measurements was collected from six hyperplastic, 20 adenomatous and 22 normal parathyroid glands. METHODS: The highly sensitive PCR-based telomeric repeat amplification protocol, TRAP, combined with ELISA, was used to detect telomerase activity in tissue extracts containing 3.0 microg protein. RESULT: Telomerase was not activated in any of the analysed tissue by 3 microg protein. Reassay of 12 samples containing 6.0 microg protein verified these negative TRAP results. CONCLUSION: Our findings indicate that telomerase is not a part of the mechanism promoting parathyroid proliferation and the underlying conditions remain to be determined.

Expression of matrix metalloproteinase gelatinase A messenger ribonucleic acid in parathyroid carcinomas.

BACKGROUND: The incidence of parathyroid cancer in patients with hyperparathyroidism is less than 1%. However, these few cases cause diagnostic problems in the absence of clear-cut invasion of adjacent organs or metastasis. New markers are needed to increase diagnostic accuracy. METHODS: Thirty-one parathyroid tumors from patients with primary hyperparathyroidism were collected worldwide. Eighteen tumors were classified as unequivocal cancers, whereas 13 tumors were considered equivocal because of a lack of infiltrative growth or evidence of recurrence. Paraffin sections were hybridized with a 35S-labeled riboprobe complementary to gelatinase A mRNA, dipped in photographic emulsion, developed, counterstained, and then evaluated by light- and dark-field microscopy. RESULTS: Fourteen of the 18 unequivocal parathyroid cancers expressed gelatinase A, as compared with the equivocal tumors, of which only 4 of 13 showed expression. The strongest hybridization signal was seen in stromal cells at the tumor border, most likely fibroblasts and macrophages. No expression was detected in tumor cells. CONCLUSIONS: Invasive growth of many tumors is facilitated by proteolytic enzymes, such as gelatinase A. The presence of gelatinase A mRNA in parathyroid tumors strengthens the suspicion of malignancy but cannot be used as a definitive marker of malignancy.

Telomerase repeat amplification protocol (TRAP): A new molecular marker for parathyroid carcinoma.

Telomerase results to be active in human germ, stem cells, several malignant cell tumors and in immortalized cell lines. In order to investigate if molecular mechanisms other than Rb gene inactivation can be helpful to diagnose malignancy of parathyroid tumors, we decided to investigate the presence of active telomerase in homogenates from different pathological parathyroid tissues (hyperplastic, adenomatous, carcinomatous, and normal) and primary cell cultures. The TRAP assay was performed to detect this activity in histologically characterized normal, hyperplastic, adenomatous, and carcinomatous human parathyroid tissues, primary cell lines, and one metastatic tissue from parathyroid carcinoma. Only malignant parathyroid glands and the metastatic tissue were TRAP positive. Our findings suggest that telomerase expression could represent an important molecular mechanism underlying the acquisition and progression of an aggressive phenotype of epithelial parathyroid cells and it may help to predict their malignant potential. The TRAP assay is easy to perform and it could become an additional tool to be included in the harmamentarium for the molecular diagnosis of parathyroid carcinoma.

Thyroid-stimulating hormone activates phospholipase C in normal and neoplastic thyroid tissue.

BACKGROUND: Thyroid-stimulating hormone (TSH) stimulates thyroid growth through two signal transduction pathways: the G protein-adenylate cyclase system and the G protein-phospholipase C (PLC) system. The adenylate cyclase system has been studied extensively, but there is little information available concerning PLC activity in thyroid neoplasms. METHODS: Human thyroid membranes were incubated for 30 minutes at 37 degrees C in the presence or absence of bovine TSH (300 mU/ml). PLC activity was assayed by liberation of inositol phosphates from the enzymatic hydrolysis of tritiated phosphatidylinositol bisphosphate. Fifty-six tissues were assayed (normal, 23; multinodular goiter, 5; follicular adenoma, 9; and differentiated thyroid cancer, 19 [9 low risk and 10 high risk]). RESULTS: TSH significantly increased PLC activity in normal, benign, and most malignant thyroid neoplasms. Although there were no differences in basal or TSH-stimulated PLC activity between the groups of normal thyroid, multinodular goiter, follicular adenoma, or the cancers, one half of the high-risk cancers had an aberrant PLC response. CONCLUSIONS: This is the first demonstration that TSH stimulates PLC activity in normal and neoplastic human thyroid tissue. Aberrant TSH-stimulated PLC activity was present in half of the aggressive thyroid neoplasms.

Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells.

The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients. During 2 weeks culture at different [Ca], the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the [Ca] at 50% suppression was 1.1 mM. At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH. The results indicated that secretion of PA from human parathyroid cells is regulated similarly to that of PTH. The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue. In homogenates, the highest specific activity of PA was in microsomal fractions. The Mr of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA (tPA). Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase. In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA.

Phospholipid/Ca2+-dependent protein kinase activity in human parathyroid adenoma.

We have examined the activities of phospholipid/Ca2+-dependent and cyclic AMP-dependent protein kinases of the parathyroid adenomas and the atrophic glands which were resected from three patients with primary hyperparathyroidism. Phospholipid/Ca2+-dependent protein kinase activity of atrophic parathyroid gland was exclusively present in cytosol fraction (90.7 +/- 12.3%). On the other hand, phospholipid/Ca2+-dependent protein kinase activity of parathyroid adenomas was 66.9 +/- 6.4% in cytosol and 33.1 +/- 6.4% in membrane fraction, suggesting a translocation of the enzyme from the cytosol to the membranes. Cyclic AMP-dependent protein kinase activity appeared to be higher in parathyroid adenoma than in atrophic parathyroid gland in both cytosol and membrane fractions.

Presence of neuron-specific enolase and somatostatin in human parathyroid tissues.

The association of parathyroid abnormalities with apudomas prompted us to examine parathyroid tissues for the presence of neuron-specific enolase and somatostatin. Enolase was present in extracts of 29 out of 29 parathyroid specimens; tissue content was significantly higher in adenoma than in hyperplasia tissues (p less than 0.005). Somatostatin was present in 14 of 33 specimens. Immunoreactive somatostatin measured in tissue extracts' fluids coeluted on Sephacryl chromatography along with synthetic somatostatin-14 in studies of two parathyroid carcinoma specimens. Since neuron-specific enolase has been found only in neural and neuroendocrine cells, our results suggest that human parathyroid glands may contain neuroendocrine elements. The differential content of neuron-specific enolase in adenoma versus hyperplasia specimens may be diagnostically useful in selected cases. The significance of the presence of somatostatin in some but not all parathyroid tumors requires further investigation. Taken together with our prior findings of gastrin and pancreatic polypeptide in some human parathyroid glands, we postulate that human parathyroid tumors contain neural crest elements.

The two-hit theory of neoplasia: implications for the pathogenesis of hyperparathyroidism.

Knudson's two-hit or two-mutational-event hypothesis for the initiation of neoplasia suggests that two somatic mutations are necessary for initiation of sporadic neoplasms, and that a genetic plus a somatic mutation are required for hereditary neoplasms. Glucose-6-phosphate dehydrogenase (G6PD) studies of parathyroid tumors of 11 heterozygotes with hyperparathyroidism (HPT), including one with hereditary HPT, have shown both A and B isoenzymes. These findings suggest multicellular development of parathyroid tumors and are compatible with tumors being manifestations of either nonneoplastic hyperplasia or a genetic first mutational event. Parathyroid carcinoma may be the result of a second mutation in cells made susceptible by previous genetic or somatic mutations. Comparison of parathyroid cancer in families with hereditary HPT with sporadic cases from the literature reveals a generally younger onset with hereditary HPT compatible with the two-hit theory. Consideration of these age-of-onset and G6PD findings suggest that hyperparathyroidism may result from either non-neoplastic or mutational-induced processes even though no histologic distinctions have been observed in the hyperplasia associated with these processes.

[Correlation of tumor weight and typical pathologic laboratory parameters in primary and secondary hyperparathyroidism]. / Untersuchungen zur Korrelation von Tumorgewicht und typisch pathologischen Laborparametern beim primären und sekundären Hyperparathyreoidismus.

In a retrospective trial the correlation between tumor weight and typically pathological laboratory findings in serum (parathyroid hormone, calcium, alkaline phosphatase, phosphorous) was investigated in 48 of our patients with primary and 44 with secondary hyperparathyroidism. There was no significant correlation. These laboratory findings therefore do not allow conclusions as to the tumor weight to be drawn.

Human parathyroid adenoma adenylate cyclase: stimulation by histamine that is blocked by cimetidine.

Recent evidence suggests that the histamine receptor blocking agent cimetidine can decrease parathyroid hormone release from human parathyroids. To determine the mechanism for inhibition we examined the ability of histamine 1 X 10(-5) moles/liter to stimulate adenylate cyclase in a particulate membrane preparation from 13 human parathyroid glands. Histamine significantly increased adenylate cyclase activity as compared to control; however, the degree of stimulation was variable among the individual tissue samples. Enzyme stimulation was dose dependent over the concentration range of 1 X 10(-7) to 1 X 10(-4) moles/liter. Cimetidine at 1 X 10(-4) moles/liter completely abolished the histamine mediated increase in activity, but did not block the epinephrine-induced stimulation. The identification of an adenylate cyclase system in certain human parathyroid adenomas that is stimulated by histamine and blocked by cimetidine may offer a basis for the pharmacologic alteration of parathyroid hormone secretion.

Adenylate cyclase activity in human parathyroid tissues: reduced sensitivity to suppression by calcium in parathyroid adenomas as compared with normal glands form normocalcemic subjects or noninvolved glands from hyperparathyroid subjects.

To examine whether alterations in parathyroid adenylate cyclase might be associated with glandular hyperfunction, we compared enzyme activity in membranes from 7 normal glands with activity from 18 abnormal and 5 noninvolved glands from patients with primary hyperparathyroidism. Compared with the normal glands, the specific enzyme activity after full stimulation with guanyl-5'yl imidodiphosphate was significantly decreased in both hyperplastic and noninvolved glands from the hyperparathyroid subjects. While the enzyme activity of all tissues could be suppressed by calcium, a twofold higher calcium concentration was required for comparable suppression of the enzyme from adenomas as compared with normal or noninvolved glands. Alterations in the adenylate cyclase complex of hyperplastic parathyroid glands may explain, in part, the elevated "set point" for calcium homeostasis in primary hyperparathyroidism.

Adenylate cyclase of human parathyroid gland.

Experiments were performed on a particulate fraction from human parathyroid glands. A high activity of adenylate cyclase was detected which was linear with time and protein concentration. The enzyme had an optimum pH in the range of 7-8 and a Km for ATP of 0.44 X 10(-3) M. Ca++ had a profound inhibitory effect; a concentration of 0.5 mM Ca++ reduced enzyme activity by 60%. Maximal enzyme activity was obtained with 5 mM Mg++; higher concentrations of this cation also inhibited enzyme activity. The effect of Mn++ was similar to that of Mg++. Enzyme activity was stimulated by NaF, catecholamines, glucagon, and calcitonin. The effect of catecholamines seems to be mediated through beta-adrenergic receptors.