Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines.

There is a lack of understanding of the casual mechanisms behind the observation that some breast adenocarcinomas have identical morphology and comparatively different cellular growth behavior. This is exemplified by a differential response to radiation, chemotherapy, and other biological intervention therapies. Elevated concentrations of the free radical nitric oxide (NO), coupled with the up-regulated enzyme nitric oxide synthase (NOS) which produces NO, are activities which impact tumor growth. Previously, we adapted four human breast cancer cell lines: BT-20, Hs578T, T-47D, and MCF-7 to elevated concentrations of nitric oxide (or high NO [HNO]). This was accomplished by exposing the cell lines to increasing levels of an NO donor over time. Significantly, the HNO cell lines grew faster than did each respective ("PARENT") cell line even in the absence of NO donor-supplemented media. This was evident despite each "parent" being morphologically equivalent to the HNO adapted cell line. Herein, we characterize the HNO cells and their biological attributes against those of the parent cells. Pairs of HNO/parent cell lines were then analyzed using a number of key cellular activity criteria including: cell cycle distribution, DNA ploidy, response to DNA damage, UV radiation response, X-ray radiation response, and the expression of significant cellular enzymes. Other key enzyme activities studied were NOS, p53, and glutathione S-transferase-pi (GST-pi) expression. HNO cells were typified by a far more aggressive pattern of growth and resistance to various treatments than the corresponding parent cells. This was evidenced by a higher S-phase percentage, variable radioresistance, and up-regulated GST-pi and p53. Taken collectively, this data provides evidence that cancer cells subjected to HNO concentrations become resistant to free radicals such as NO via up-regulated cellular defense mechanisms, including p53 and GST-pi. The adaptation to NO may explain how tumor cells acquire a more aggressive tumor phenotype.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.

Alendronate/interleukin-1beta cotreatment increases interleukin-6 in bone and UMR-106 cells: dose dependence and relationship to the antiresorptive effect of alendronate.

Aminobisphosphonates inhibit bone resorption but have been shown to elicit acute-phase-like elevations in interleukin-6 (IL-6) in bone in vitro. The current studies were carried out to determine the relationship between the antiresorptive effects of the aminobisphosphonate alendronate and its effects on IL-6. Resorption was elicited in cultured 19-day fetal rat limb bones by 72 h treatment with interleukin-1beta (IL-1beta). Bone mass was quantitated at the end of the culture period to assess resorption. IL-6 was determined by bioassay (7TD1 cell proliferation). IL-1beta (18 and 180 pM) stimulated bone resorption and increased IL-6. Alendronate (70 microM) inhibited the IL-1beta-stimulated resorption. Alendronate alone did not affect IL-6 production by the bones. The IL-6 production from bones stimulated with 18 pM IL-1beta was not significantly affected by alendronate, but the IL-6 production from bones stimulated with 180 pM IL-1beta plus alendronate (21 and 70 microM) was higher than with IL-1beta alone. Indomethacin (1 mM) inhibited the IL-6 increase elicited by 180 pM IL-1beta and the enhanced IL-6 production elicited by cotreatment with IL-1beta and alendronate. Since bone cultures contain multiple cell types, further experiments were carried out to determine whether alendronate could increase IL-1beta-stimulated IL-6 production in an osteoblast cell line, UMR-106. Alendronate alone did not affect IL-6 in UMR-106 cells. Alendronate (70 microM) in combination with IL-1beta (180, 1.8, or 8 nM), or 7 microM alendronate, in combination with 8 nM IL-1beta, significantly increased IL-6 in 48 h cell cultures. The results from the bone organ cultures show that alendronate can enhance IL-6 production elicited by higher concentrations of the cytokine IL-1beta in bone, but that this effect on IL-6 does not prevent the inhibitory actions of alendronate on bone resorption. The results with the UMR106 cells indicate that one cellular site at which this enhancement of IL-6 production can occur is the osteoblast.

Serum interleukin-6 and bone metabolism in patients with thyroid function disorders.

To determine the possible involvement of interleukin-6 (IL-6) in the bone loss of hyperthyroidism, relationships between thyroid status, biochemical and densitometric parameters of bone metabolism, and IL-6 were studied in female subjects. Patients with hyperthyroidism caused by either toxic nodular goiter or Graves' disease had significantly higher serum IL-6 concentrations than normal controls. Within the control group, serum IL-6 was higher in postmenopausal than in premenopausal women, but this influence of menopausal status was not seen in the hyperthyroid patients. The production of IL-6 by blood mononuclear cells was higher in cells from the hyperthyroid women. Bone turnover was increased in the hyperthyroid patients based on serum osteocalcin and urinary deoxypyridinoline excretion, and the hyperthyroid group also had reduced radius bone mineral content (BMC). A subgroup of hyperthyroid patients who had the lowest BMC (values more than 1 SD below normal age-matched controls) also had serum IL-6 concentrations significantly greater than those of hyperthyroid patients showing less reduction of BMC. The correlations observed in this study support the possibility that IL-6 plays a role in mediating the bone loss that results from excess thyroid hormone.

Circadian rhythm of in vitro bone-resorbing activity in human serum.

Calciotropic hormones as well as biochemical parameters of bone formation and resorption show circadian rhythms. In a previous study in the rat, we observed a circadian rhythm in serum bone-resorbing activity (SBRA). In the present study, we investigated whether there was a circadian rhythm of SBRA in human serum. For this purpose, we studied 10 healthy premenopausal women and 5 healthy men. Blood was collected every 2 h, and urine samples were collected during 4-h periods for 24 h. For the determination of SBRA, media were prepared by reconstituting serum samples with Dulbecco's Modified Eagle's Medium at a ratio of 20% serum and 80% Dulbecco's Modified Eagle's Medium. Limb bones were dissected from 19-day old fetal rats prelabelled with 45Ca and were cultured for 72 h in the presence of the sera. Bone resorption was assessed from the 45Ca released into the culture medium and from that retained in the bone and was expressed as percentage 45Ca release. Serum calcium, phosphorus, PTH, cortisol, and urinary pyridinium cross-links were also determined. SBRA in human serum followed a circadian rhythm with a peak at about 0300 h and a nadir at 0700 h. There was no significant difference between the rhythm of SBRA of women and men. At concurrent time points, SBRA and serum PTH were positively correlated (r = 0.629; P < 0.01), and SBRA and serum cortisol were negatively correlated (r = 0.797; P < 0.01). To further investigate the possible contribution of these hormones to SBRA, either neutralizing anti-PTH antibody or RU-486 (mifepristone), a glucocorticoid receptor antagonist, was added to the serum samples of 6 subjects. Neutralizing the effect of PTH did not change the pattern of SBRA rhythm. The addition of RU-486 had a significant effect on the rhythm of SBRA, reducing the peak and nadir amplitudes. Thus we conclude that cortisol plays a major role in the rhythm of SBRA present in human serum; however, the influence of other factors cannot be excluded. Cortisol may be an important determinant of the circadian rhythm of bone resorption in vivo.

Triiodothyronine potentiates the stimulatory effects of interleukin-1 beta on bone resorption and medium interleukin-6 content in fetal rat limb bone cultures.

It has been demonstrated that thyroid hormones stimulate osteoclasts indirectly and that this effect is mediated by products of other cell types present in bone. To determine if interleukin-6 (IL-6) could be a mediator of thyroid hormone action, we investigated the effect of 3,5,3'-triiodothyronine (T3) on bone resorption (45Ca release) and on the IL-6 concentration in medium from cultured 19-day-old fetal rat limb bones. T3 alone increased 45Ca release significantly only at a fairly high concentration (10(-6)M) under the conditions used. T3 alone, over a 10(-11)-10(-6) M concentration range, failed to elicit a detectable effect on the medium IL-6 content. However, T3 potentiated the stimulatory effect of interleukin-1 beta (IL-1 beta) on IL-6 production in a dose-dependent manner. T3, 10(-8) M, also significantly increased IL-1 beta-stimulated calcium release. Inhibition of IL-1 beta with 1 muM interleukin-1 receptor antagonist (IL-1ra) abrogated the potentiating effects of T3 on IL-1 beta-stimulated IL-6 production and blocked the combined effect of T3 and IL-1 beta on 45Ca release. One micromolar indomethacin significantly, but not completely, inhibited the effect of IL-1 beta, as well as the combined effect of IL-1 beta and T3 on resorption and IL-6 production, indicating the involvement of prostaglandins in these actions. Consistent with this, 1 microM prostaglandin E1 (PGE1) significantly increased both the IL-6 production and the calcium release. By potentiating the effect of IL-1 beta, T3 increased bone resorption at much lower concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin receptors, second messengers, and actions in bone.

Endothelins are a class of peptides that are produced by and elicit responses in many tissues. A growing literature documents the presence and effects of endothelins in bone. Both endothelinA and endothelinB receptors have been demonstrated in osteoblastic cells by ligand binding. Major signal transduction pathways for endothelin in bone cells appear to be stimulation of phospholipid turnover, by activation of A, C and D phospholipases, stimulation of calcium flux from intracellular and extracellular stores and activation of tyrosine kinases. Endothelins also modulate calcium signaling elicited by other agents in osteoblastic cells. The parathyroid hormone-stimulated calcium transient in UMR-106 cells is enhanced by endothelins, acting through an endothelinB receptor, whereas the parathyroid hormone-stimulated increase in cyclic AMP is inhibited by endothelins. Phenotypic responses to endothelin-1 include changes in alkaline phosphatase activity, stimulation of osteocalcin and osteopontin message, stimulation of collagen and noncollagenous protein synthesis, inhibition of osteoclast motility and stimulation of prostaglandin-dependent resorption. Endothelin-1 also enhances the interleukin-1-induced increase in interleukin-6. Endothelins can also potentially affect calcium metabolism through their actions to inhibit the secretion of parathyroid hormone.

[Zn-content of erythrocytes in overt and subclinical hyperthyroidism]. / Vörösvérsejt Zn-tartalom vizsgálata manifeszt és szubklinikus hyperthyreosisban.

Assaying the markers in blood reflecting thyroid hormone effect at peripheral tissue level previous data revealing a significantly decreased red-blood cell Zn-content in overt hyperthyroidism compared to euthyroid controls could be confirmed (p < 0.001). In patients with thyrotoxicosis during Metothyrin treatment the Zn-concentration of red-blood-cells normalised about 8 weeks later than the abnormally elevated serum thyroxine and triiodothyronine levels. On the basis of the present findings it is assumed that the Zn-concentration of red-blood cells reflects to a certain degree the circulating levels of thyroid hormones 2 to 3 months prior to the performed examination. The measurement of red-blood cell Zn-content does not seem to be a reliable method for the detection of a possible "tissue"-thyrotoxicosis in cases of subclinical hyperthyroidism.

[Changes in osteocalcin serum levels in children with growth hormone deficiency during substitution therapy]. / A szérum osteocalcin-szint változása növekedési hormon-hiányos gyermekek szubsztitúciós kezelése során.

UNLABELLED: Osteocalcin (OC), a specific marker of osteoblast activity, was measured in 21 short, growth hormone deficient (GHD) children before and during 2 years of growth hormone (GH) therapy. Anthropometric and serum measurements were performed in every three-month during the first year (15 patients) and after the second year (16 patients). Mean OC concentration was significantly lower in GHD children compared to normal value (11.9 +/- 2.1 ng/ml (n = 15) and 11.4 +/- 2.5 ng/ml (n = 16) vs. 17.5 +/- 4.9 ng/ml). During the GH treatment serum OC increased continuously: 15.5 +/- 2.4 - 20.5 +/- 8.2 - 26.1 +/- 8.6 - 25.1 +/- 9.8 ng/ml (n = 15) and 24.9 +/- 9.1 ng/ml (n = 16) then decreased (16.6 +/- 9.7 ng/ml). OC level measured in the 9th and 12th months was markedly higher than in normal children (p = 0.01 and p < 0.001). IN CONCLUSION: 1. Serum OC is low in short statured GHD children. 2. In the first 9-12 months of GH therapy OC raises continuously exceeding the normal mean value. 3. During the second year of GH treatment OC decreases to the normal level. 4. OC concentration measured does not correlate with change of other parameters observed (growth velocity, bone maturation, height for age). 5. Although OC is a sensitive marker of bone formation, it has no prognostic value concerning the growth.

Bone mineral density in patients with endogenous subclinical hyperthyroidism: is this thyroid status a risk factor for osteoporosis?

OBJECTIVE: The aim of the present study was to elucidate whether endogenous subclinical hyperthyroidism due to a solitary autonomously functioning thyroid nodule affects bone metabolism and is a risk factor for osteoporosis. DESIGN: In a cross-sectional study measurements of bone mineral density were performed in premenopausal and post-menopausal women. Patients were categorized into non-toxic nodular goitre (n = 32), subclinical hyperthyroid (n = 37) and toxic solitary autonomous thyroid nodule (n = 22) subgroups and the results were compared with those of sex and age-matched control reference population (n = 68). MEASUREMENTS: Lumbar spine and femoral neck bone mineral densities were measured by dual energy X-ray absorptiometry. Single-photon absorptiometry was applied to the measurement of bone mineral content in the midshaft of the radius. RESULTS: In the non-toxic nodular goitre group, bone densities for all the scanned sites did not differ from the sex and age-matched reference population. At the L2-4 scanning site a significant decrease in the bone mineral density could be observed only in the toxic nodular goitre group and this decrease was more marked in the postmenopausal (P < 0.001) than in the premenopausal females (P < 0.05). At the femoral neck and midshaft radius the mean densitometric values were slightly, but significantly, lower only in the post-menopausal subclinical hyperthyroid group compared with the reference population (P < 0.01). The bone mineral density of the femoral neck, as well as the bone mineral content of the midshaft radius, was significantly decreased in both the premenopausal and post-menopausal patients with a toxic solitary nodule. CONCLUSION: This study indicates that the bone mineral density of the lumbar spine, femoral neck and the midshaft of the radius are not significantly decreased in premenopausal patients with endogenous subclinical hyperthyroidism resulting from a solitary autonomously functioning thyroid nodule. Conversely, findings hint at the possibility that long-lasting endogenous subclinical hyperthyroidism may be a contributing factor to the development of osteoporosis in some post-menopausal women, mostly at sites where cortical bone preponderates.

T-cell receptor V gene use in autoimmune thyroid disease: direct assessment by thyroid aspiration.

We have examined the hTcR V gene family use of T-cells present in the aspiration thyroid biopsy specimens of patients with hyperthyroid Graves' disease (n = 8) and Hashimoto's autoimmune thyroiditis (n = 5). Nine of the 13 specimens had cytologically identified thyroid follicular cells, and 12 of the 13 contained human thyroglobulin-specific mRNA, confirming successful sampling. Of 18 hTcR V alpha and 19 hTCR V beta gene families tested for in the individual aspirates, a mean +/- SEM of 6.8 +/- 0.9 V alpha and 9.6 +/- 1.4 V beta gene families were present in the Graves' aspirates, while 12.2 +/- 1.7 and 16.8 +/- 0.4 V alpha and V beta gene families were present in the aspirates of patients with Hashimoto's thyroiditis. These samples, which offer a window onto the natural history of autoimmune thyroid disease, demonstrate significant hTcR V alpha and beta gene restriction in hyperthyroid Graves' disease, but much less restriction of both V alpha and V beta gene families in Hashimoto's disease. Such data extend our earlier information based only on examination of highly selected surgical specimens of patients with autoimmune thyroid disease to the much more typical patient. We conclude that hTcR V gene restriction of varying degrees is present in the majority of patients with autoimmune thyroid disease, but appears to be more easily detected in Graves', rather that Hashimoto's, disease.

Biologic markers in blood reflecting thyroid hormone effect at peripheral tissue level in patients receiving levothyroxine replacement for hypothyroidism.

Plasma fibronectin, serum procollagen-III-peptide and sex-hormone binding globulin as not specific markers of thyroid hormone effect at peripheral tissue level were determined and their values were related with serum levels of TSH, free-thyroxine and triiodothyronine during levothyroxine sodium replacement therapy for hypothyroidism. Low levels of biologic markers characteristic of hypothyroidism were normalized in consequence of hormone replacement and a negative correlation between their serum levels and TSH concentration was demonstrated in most subjects. However, in some patients a discrepancy in the response to levothyroxine between the pituitary and other target organs could be revealed. Additional evidence was disclosed that the pituitary thyrotroph sensitizes a minor decrease in serum thyroxine level, which would not be recognized by other target organs. Furthermore, it was revealed that during L-T4 replacement therapy in a large fraction of patients with subnormal serum TSH concentration blood levels of the measured markers often exceeded the upper limit of the normal range indicating the possibility of "tissue"-thyrotoxicosis beside the pituitary in other target organs, too. According to the present study which takes into consideration markers reflecting end-organ responsiveness to thyroid hormones it is recommended to adjust the dose of levothyroxine to maintain serum TSH in the normal range. For patients with subnormal TSH concentration a close follow-up is obligatory and in case od concomitantly raised free-thyroxine level the reduction of the levothyroxine dosage is proposed.

Radioimmunoassay of thyrotropin-releasing hormone in euthyroid subjects and in patients with thyroid diseases.

An episodic pattern of fluctuation in serum TRH level was demonstrated in euthyroid males. It is suggested that these rises in serum TRH-IR are due to its pulsatile hypothalamic release, however, it must be remembered that a high proportion of circulating TRH is derived from extrahypothalamic sources. The conception that the TSH surge during late evening is secondary to an increased TRH release could not be proved in the present study. In the euthyroid and primary hypothyroid groups the mean values of serum TRH-IR did not differ, while its level was slightly, but not significantly lower in hyperthyroid patients. The evaluation of a single TRH determination is impeded by the short half-life, low serum level and its production in extrahypothalamic sites, too.

[Biological markers reflecting peripheral effects of thyroid hormones in autonomous thyroid adenoma]. / Pajzsmirigyhormonok perifériás hatásaira utaló biológiai markerek vizsgálata pajzsmirigy autonom adenomában.

In some patients with functioning thyroid autonomous nodules preclinical hyperthyroidism is detected. It is important to know, whether in this intermediate clinical state beside the suppression of pituitary TSH secretion other target organs are also affected by serum free-thyroxine and free-triiodothyronine levels still within the normal range. Determining some sensitive, but not specific biologic markers reflecting the impact of thyroid hormones at the peripheral tissue level, it was demonstrated that in the group of preclinical hyperthyroidism the mean level of plasma fibronectin exceeded that of the controls (mean +/- S. D.: 583.5 +/- 163.9 vs. 424.2 +/- 84.1 micrograms/ml, p less than 0.001), serum procollagen-III-peptide concentration was already significantly raised, though its value was still within the normal range (mean +/- S. D.: 0.73 +/- 0.17 vs. 0.57 +/- 0.16 U/ml, p less than 0.05), conversely, mean sex-hormone binding globulin level was the same as in euthyroid controls (mean +/- S. D. 47.4 +/- 18.2 vs. 48.3 +/- 16.3 nmol/l). The value of all three parameters was significantly elevated in patients with toxic nodular goiter. Based on the results of this study "tissue"-thyrotoxicosis is suspected in some patients with preclinical hyperthyroidism, which may have therapeutical implications.

Serum bone Gla protein in streak gonad syndrome.

Osteoporosis is one of the most common complications of streak gonad syndrome (SGS), however its pathogenesis is still unclear. Bone Gla protein (BGP) has been found to be a serum marker of bone turnover in various metabolic disease states. In the present study serum BGP and alkaline phosphatase (AP) were measured in 13 osteoporotic patients with SGS and in 56 healthy women. Mean (+/- SD) serum BGP levels were normal (7.5 +/- 2.0 ng/ml) in seven patients who had been on estrogen-progestin replacement therapy and became significantly elevated (P less than 0.001) 2 and 3 months after discontinuation of the treatment (15.3 +/- 2.3 and 13.2 +/- 1.0 ng/ml, respectively). Mean (+/- SD) serum AP (207 +/- 65 U/l) showed significant increases (P less than 0.05) 2 months after withdrawal of hormonal substitution (287 +/- 74 U/l). Mean (+/- SD) serum BGP (15.4 +/- 3.5) and AP (287 +/- 49) levels were significantly higher (P less than 0.001 and less than 0.05, respectively) in six patients with SGS who had not been on hormonal substitution. These findings are consistent with those obtained in postmenopausal women suffering from "high remodelling osteoporosis" and suggest that bone turnover in osteoporotic patients with SGS is increased and the skeletal loss is a consequence of accelerated bone loss rather than decreased bone formation.

[Decreased serum osteocalcin level in non-alcoholic and alcoholic chronic liver diseases]. / Osteocalcin szérum szint csökkenése nem-alkoholos és alkoholos idült májbetegekben.

Serum level of osteocalcin (OC) is believed to be a specific biochemical parameter of bone formation. Decreased serum OC has been reported in alcohol-intoxicated subjects, in patients with primary biliary cirrhosis and in patients with chronic alcoholic liver disease. The question was, whether lower OC level could be detected in patients with nonalcoholic and non-cholestatic chronic liver disease. The serum OC was measured by RIA developed in our laboratory. Results were compared to age and sex matched controls. Decreased OC level was found in 35 out of 47 (74%) patients with non-alcoholic and non-cholestatic liver disease as chronic persistent hepatitis, chronic active hepatitis, fatty liver and cirrhosis, in 21 out of 26 (80%) patients with alcoholic liver disease and in 8 out of 15 (53%) primary biliary cirrhosis. None of the patients had elevated value. There was no correlation between the decreased OC level and the duration or severity of the liver disease and the laboratory parameters as bilirubin, AST, ALT, alkaline phosphatase, albumin, prothrombin, and serum 25-OH-D3 vitamin level. Decreased OC was found also in the patients without cirrhosis. The possible causes are discussed. Relying upon these findings it is supposed that chronic liver disease by itself can influence the osteoblast activity also by some unknown mechanism.

Biologic blood markers reflecting thyroid hormone effect at peripheral tissue level in patients receiving levothyroxine replacement for hypothyroidism.

Plasma fibronectin, serum procollagen-III-peptide and sex-hormone-binding globulin as non-specific markers of thyroid hormone effect at peripheral tissue level were determined and their values were related with serum levels of TSH, free-thyroxine and triiodothyronine during levothyroxine sodium replacement therapy for hypothyroidism. Low levels of biologic markers characteristic of hypothyroidism were normalized in consequence of hormone replacement and a negative correlation between their serum levels, and TSH concentration was demonstrated in most subjects. However, in some patients a discrepancy in the response to levothyroxine between the pituitary and other target organs was revealed. Additional evidence was disclosed that the pituitary thyrotroph sensitizes a minor decrease in serum thyroxine level, which would not be recognized by other target organs. Furthermore, it was revealed that during L-T4 replacement therapy in a large fraction of patients with subnormal serum TSH concentration blood levels of the measured markers often exceeded the upper limit of the normal range indicating a possibility of "tissue" thyrotoxicosis, besides the pituitary, in other target organs, too. According to the present study, which takes into consideration markers reflecting end-organ responsiveness to thyroid hormones, it is recommended to adjust the dose of levothyroxine to maintain serum TSH in the normal range. For patients with subnormal TSH concentration a close follow-up is obligatory and in case of concomitantly raised free-thyroxine level the reduction of the levothyroxine dosage is proposed.