Immunocytochemical detection of p53 in human thyroid carcinomas is associated with mutation and immortalization of cell lines.

Mutations in the tumor suppressor gene p53 are the most-common mutations found in human cancers. In thyroid cancers, p53 mutations generally are found only in poorly differentiated and undifferentiated tumors and in cell lines. To determine the prevalence of p53 mutations in thyroid neoplasms and thyroid cell lines, we screened 58 thyroid tissues and 3 thyroid cell lines, p53 primers bracketing exons 4, 5/6, 7, and 8 were used to amplify genomic DNA using the PCR. Mutations were screened by denaturing gradient gel electrophoresis and confirmed by sequencing. The two papillary thyroid cancer cell lines and the follicular thyroid carcinoma cell line (positive control) had transitions (CGT->CAT) in exon 8, codon 273, resulting in the replacement of arginine with histidine. No normal thyroid tissues or primary tumors from which the cell lines were derived demonstrated exon 8 mutations, using this technique. p53 immunocytochemistry demonstrated a progression of p53 immunopositivity between synchronous and metachronous neoplasms, paralleling the neoplastic progression from a benign adenoma to primary carcinoma, regional, and distant metastasis and ultimately, the cell lines, where intense immunopositivity is noted. In addition, fluorescence in situ hybridization, using probes specific for the p53 locus, revealed the presence of 3 homologues of p53 in the follicular cell line and 2 homologues in the papillary and Hürthle cell lines. These results suggest that a point mutation present in a small number of original tumor cells and amplification of the mutant allele may be responsible for immortalizing well-differentiated thyroid cancer cells into cell lines.

Desensitization of adenylate cyclase in Chinese hamster ovary cells transfected with human thyroid-stimulating hormone receptor.

Desensitization or decreased response to the same (homologous) or other stimuli (heterologous) is a well known process. Homologous desensitization to TSH has been demonstrated in normal thyroid tissue. Chinese hamster ovary cells (CHO) transfected with normal human TSH receptor (hTSHR) DNA, in contrast, have been reported not to desensitize. The purpose of our investigation was to determine whether CHO cells transfected with hTSHR desensitize in response to TSH and postreceptor stimulation. CHO cells were stably transfected with plasmid DNA containing hTSHR; nontransfected CHO cells served as the control. TSH (10 mU/ml), 5'-beta,gamma-imido-triphosphate [Gpp(NH)p; 0.1 mM], sodium fluoride (NaF; 10 mM), forskolin (10 microM), and (Bu)2cAMP (100 microM) were used to determine whether homologous or postreceptor heterologous desensitization of adenylate cyclase activity occurred in CHO-transfected cells. Intracellular cAMP accumulation was determined by RIA. Cells were incubated with TSH (to stimulate TSH receptor), Gpp(NH)p, NaF (to stimulate G-protein), forskolin (to stimulate adenylate cyclase activity), and (Bu)2cAMP (nonmetabolized cAMP analog). A second incubation was carried out with TSH (10 mU/ml). Maximal desensitization to either TSH or postreceptor stimulation was observed at 2 h. When transfected CHO cells were preexposed to TSH (10 mU/ml) for 4 h, even the smallest dose of TSH (0.001 mU/ml) caused desensitization. All substances that increased the intracellular cAMP concentration, such as TSH, Gpp(NH)p, NaF, forskolin, and (Bu)2cAMP, caused desensitization. The decrease in the cAMP response to TSH added in the second incubation was 63% less than the initial response to TSH or to postreceptor stimulation (P = 0.0001). In conclusion, desensitization of hTSHR-transfected CHO cells occurs in response to both receptor and postreceptor stimulation that increase cAMP levels. Because hTSHR transfected CHO cells desensitize, no specific thyroid factor(s) other than increased levels of cAMP is required.

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.

Double parathyroid adenomas. Clinical and biochemical characteristics before and after parathyroidectomy.

OBJECTIVE: There is considerable debate about whether double parathyroid adenomas are a discrete entity or represent hyperplasia with parathyroid glands of varying sizes. This distinction is important because it impacts on the extent of parathyroid resection and the success of the parathyroid operation. SUMMARY BACKGROUND DATA: Double parathyroid adenomas have been reported to occur in 1.7% to 9% of patients with primary hyperparathyroidism (HPT). It is important for surgeons to differentiate between double adenoma and hyperplasia with glands of varying sizes using gross examination during the initial procedure because microscopic findings of a small biopsy specimen at frozen-section examination may not be diagnostic. METHODS: From 1982 to 1992, 416 unselected patients (309 women and 107 men) with primary HPT without familial HPT or multiple endocrine neoplasia (MEN) were treated by one surgeon at the University of California at San Francisco. Double adenoma occurred in 49 patients, solitary adenoma in 309 patients, and hyperplasia in 58 patients. The authors analyzed the clinical manifestations, the preoperative and postoperative serum levels of calcium, phosphate, and parathyroid hormone (PTH), and the success rate and outcome after parathyroidectomy and compared their results in 49 patients with double adenomas to the results for patients with solitary adenomas or hyperplasia. RESULTS: Ten of the patients with double adenomas (20.4%) were referred for persistent HPT after removal of one abnormal parathyroid gland. The ages of the patients with double adenoma, single adenoma, and hyperplasia were 61 +/- 14, 56 +/- 15, and 58 +/- 7 years, respectively. Fatigue, muscle weakness, and bone pain were common in patients with double adenomas, whereas nephrolithiasis occurred more frequently in patients with solitary adenoma (p = 0.0001). Serum calcium and PTH levels (per cent of upper limit of normal) fell from 11.5 +/- 1.2 mg/dL and 487% to 9.5 +/- 0.8 mg/dL and 61% for patients with double adenomas; from 11.9 +/- 0.9 mg/dL and 378% to 9.3 +/- 1.4 mg/dL and 101% for patients with single adenoma; and from 10.9 +/- 0.5 mg/dL and 418% to 9.1 +/- 0.7 mg/dL and 94% for patients with hyperplasia, respectively. There was no recurrence in the patients with double adenomas with a mean follow-up time of 5.8 years. CONCLUSIONS: Double adenomas are a discrete entity and occur more often in older patients. Patients with double adenomas can be successfully treated by removal of the two abnormal glands.

Increased phospholipase C activity in neoplastic thyroid membrane.

The phospholipase C (PLC)-protein kinase C (PKC) signal transduction pathway appears to be important for cellular growth of many normal and neoplastic tissues. Because alterations in the thyroid-stimulating hormone (TSH) receptor-adenylate cyclase-protein kinase A system in some thyroid tumors do not correlate with tumor size, invasiveness, or metastatic potential, we studied the PLC activity in both normal and neoplastic thyroid tissues from 11 patients. Five of these patients had follicular adenomas and 6 had papillary carcinomas. An 8,000 x g membrane fraction and a 105,000 x g cytosol fraction were prepared from the normal and neoplastic human thyroid tissues. PLC hydrolyzes phosphatidylinositol, 4,5-diphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Phospholipase C activity was determined measuring the hydrolysis of [3H]-PIP2. The activity of PLC in the neoplastic thyroid tissue membrane fraction (20.91 +/- 2.28 nmol PIP2 hydrolyzed/mg protein/120 min) was higher than that in normal thyroid membrane (14.27 +/- 0.82) (p < 0.05). In contrast, PLC activity was similar in the neoplastic (16.12 +/- 0.86 nmol PIP2 hydrolyzed/mg protein/120 min) and normal (16.66 +/- 0.60) cytosol. There was no difference between PLC activity in the membrane fraction from adenomas (21.21 +/- 3.71 nmol PIP2 hydrolyzed/mg protein/120 min) when compared with thyroid carcinomas (20.67 +/- 3.14). Neoplastic thyroid membranes have greater PLC activity than that found in normal thyroid membranes from the same patients. Although PLC activity in benign and malignant thyroid membranes was similar, the increased PLC activity in thyroid neoplasms may be responsible for or contribute to the enhanced growth of some thyroid tumors.