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Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.

Sixteen consecutive patients in one unit were studied during long-term treatment of Paget's disease of bone with salmon calcitonin. Eleven patients developed detectable antibody titres at some time during treatment. In one patient with a high antibody titre evidence of resistance to treatment emerged two years after the development of antibodies, but no other patient showed evidence of resistance. The clinical and biochemical response could be maintained in the absence of an acute calcium-lowering effect of calcitonin. Although antibodies often develop during treatment with heterologous calcitonin, they are only rarely the cause of clinical resistance.

1. The production of 1,25-dihydroxycholecalciferol (1,25-DHCC) from tritiated 25-hydroxycholecalciferol (25-HCC) was assessed in renal tubules prepared from vitamin D-deficient chicks. 2. Cyclic AMP and dibutyryl cyclic AMP enhanced the production of 1,25-DHCC. 3. Bovine parathyroid hormone (BPTH) and its synthetic analogue either had no effect or inhibited the production of 1,25-DHCC when calcium was present in the extracellular medium, but enhanced the conversion when calcium was absent. BPTH increased adenylate cyclase activity and cyclic AMP content of the renal tubule cells. 4. Synthetic salmon calcitonin enhanced the production of 1,25-DHCC, whereas human calcitonin had no effect at the concentrations tested. Salmon calcitonin did not increase chick renal adenylate cyclase activity or cyclic AMP levels. 5. A short period of incubation in media containing strontium gluconate (5 mmol/l) significantly increased production of 1,25-DHCC, whereas no effect was seen after more prolonged exposure to strontium. Calcium depletion decreased production of 1,25-DHCC, but when some calcium was present, variation in extracellular concentration of calcium did not affect conversion. Variation in extracellular phosphate concentration did not affect conversion. 6. A small quantity of 1,25-DHCC added to the medium inhibited the conversion of 25-HCC into 1,25-DHCC. 7. The production of 1,25-DHCC is subject to complex control, and intracellular concentrations of calcium, cyclic AMP and 1,25-DHCC may all be important regulatory influences.

Platelets of different densities are probably produced from megakaryocytes of different ploidy. Thromboxane B2 (TxB2) production was studied in density dependent subfractions of the total platelet population.Platelets were isolated by a new technique using velocity sedimentation into a polyvinylpyrrolidone-coated colloidal silica (Percoll) gradient followed by equilibrium centrifugation through a continuous linear Percoll gradient. Fractionation of the second gradient yielded density-dependent platelet subpopulations which were functionally competent and showed minimal leukocyte and plasma protein contamination. By pooling fractions and adding buffer, suspensions containing similar concentrations of platelets representative of low, intermediate and high density subpopulations were prepared, each containing 20–30% of the total population. Aliquots of each suspension were stirred in an aggregometer and thrombin or arachidonic acid (AA) were added to induce aggregation. After three minutes the reaction was arrested by addition of ice cold ethanol and TxB2 measured by radioimmunoassay.When 0.5 mM AA (final concentration) was the aggregating agent, for every 108 platelet the least dense subpopulations produced 10.2 ng TxB2, and the most dense subpopulations 22.4 ng TxB2,(means of four experiments), whilst the intermediate density subpopulations gave intermediate values. However, thrombin at 0.5 or 1.0 units/ml produced similar amounts of TxB2 in all subpopulations. In a second series of experiments, AA-stimulated serotonin secretion in density dependent platelet subpopulations correlated closely with platelet number.These observations imply functional differences between platelet subpopulations.

We have demonstrated that a variety of cultured breast cancer cell lines, human breast cancers, non-malignant breast tumours and normal rabbit breast tissue contain specific, high-affinity, low-capacity receptors for 1,25-(OH)2D3. Considered with the ability of normal breast tissue to transport calcium from blood into breast milk, which has recently been shown to be stimulated by 1,25-(OH)2D3, the presence of this receptor could explain the high incidence of hypercalcaemia in this common disease and has important implications for the management of metastatic breast cancer. Finally these findings suggest that breast is a normal target organ for 1,25-(OH)2D3 action.

There is sufficient impetus from the clinical nature of osteogenic sarcoma to stimulate basic studies of the effects of hormones on tumor growth and differentiation. This can probably best be done first by the use of in vitro studies to determine precisely the effects of certain hormones on tumor cell growth and biochemical function. Such investigations would hopefully indicate the direction of in vivo work. The differentiated transplantable tumor described in this paper is clearly hormone-responsive, and offers a means of investigating the effects of other hormones, including growth hormone, androgens, estrogens and glucocorticoids, on specialized function of the osteogenic sarcoma cells, and on the growth of the tumor.

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.

A technological and radiological assessment has been made of the management options for irradiated graphite wastes from the decommissioning of Magnox and Advanced Gas-cooled Reactors. The main contribution to the radionuclide inventory of irradiated graphite is from activation of the graphite and its stable impurities. Three different packaging methods for graphite have been described; each could be used for either sea or land disposal, and each is logistically feasible and could be achieved at reasonable cost. Leaching tests have been carried out on irradiated graphite, under a variety of conditions including those of the deep ocean bed; the different conditions had little effect on the observed leach rates of radiologically significant radionuclides. Radiological assessments were made of four generic options for disposal of packaged graphite: on the deep ocean bed, in deep geologic repositories at two different types of site, and by shallow land burial. Incineration of graphite was also considered, though this option presents logistical problems additional to those of direct disposal. With appropriate precautions during the lifetime of the 60Co content of the graphite, any of the options considered could give acceptably low doses to individuals, and all the options would merit further investigation in site-specific contexts.

Hormonal control of cAMP production in the osteoclast has not been investigated in detail because this bone-resorbing cell has been difficult to isolate. We have used osteoclasts freshly isolated by disaggregation from neonatal rat long bones and settled onto coverslips (100-150 cells per coverslip) to examine the effects of calcitonin and prostaglandin E2 on osteoclast cAMP levels and cytoplasmic spreading. Salmon, eel, and human calcitonin (CT), and various analogs, stimulated cAMP production in a dose-dependent manner with relative potencies as seen in other response systems. Forskolin (10(-7) M) increased the sensitivity and amplitude of the response. Pretreatment with pertussis toxin (200 ng/ml for 3 h) had no effect suggesting that CT does not act through Ni, the inhibitory guanine nucleotide regulatory unit of adenylate cyclase. CT treatment was associated with rapid and dose-dependent induction of a persistent activated state of adenylate cyclase and homologous desensitization, the former being a particular feature of CT action previously observed in nonosteoclastic cells. Quantitative histomorphometry demonstrated a sensitive, dose dependent, and prolonged (greater than 2 h) reduction in osteoclast plan area after exposure to salmon CT. Although prostaglandin E2 also stimulated cAMP production and resulted in cell contraction in osteoclasts this was not associated with persistent activation of adenylate cyclase nor with prolonged contraction. Persistent activation of adenylate cyclase may be an important mechanism in CT inhibition of osteoclast function.

Physiologic regulation of bone cell function is achieved by the combined actions of some circulating hormones and several locally produced cytokines. Interactions among these agents are important, and the regulated generation of proteolytic activities is likely to play a major role in determining net effects on bone cell function. It is likely that the most important local factors in regulating osteoclast formation and activation have yet to be chemically characterized.

Mixed bone cell cultures obtained by sequential collagenase-trypsin digestion of newborn chick, rat, and mouse calvaria responded to calcitonin gene-related peptide (CGRP) with a dose-dependent increase in cyclic AMP formation. The amplitude of response to CGRP in each species was less than that to parathyroid hormone (PTH). The CGRP effect was not the result of an action as a weak calcitonin agonist, since in most instances a calcitonin effect was not observed. Only in early digests of mouse calvarial cells were consistent stimulatory effects of calcitonin on cyclic AMP noted, and these were always considerably less in amplitude than those to CGRP. It is concluded that chick, rat, and mouse bones contain cells in osteoblast-rich populations that respond specifically to CGRP with a rise in cyclic AMP.

Four human cancer cell lines (MCF 7, T 47D, ZR 75 and BEN), each of which possess calcitonin-responsive adenylate cyclase, have been investigated. Pre-treatment of cells with calcitonin resulted in two effects on their adenylate cyclase. First, the ability of adenylate cyclase to respond to further stimulation by calcitonin was reduced. Secondly, membranes from calcitonin-preincubated cells had increased activity of adenylate cyclase compared with control membranes, the activated state not only surviving washing and cell-disrupting steps but persisting for some hours after the removal of hormone from the incubation medium. This phenomenon has therefore been termed "persistent activation" and may be peculiar to the action of calcitonin. It is dose dependent and is seen with calcitonins of various origins (sCT, hCT, pCT and [Asu1-7] eCT). When pre-bound hormone was removed from the cell surface by acid washing, adenylate cyclase activities in preincubated membranes fell towards control activities and responsiveness of the enzyme to a second challenge with calcitonin returned. Inhibitors of protein synthesis prevented full recovery from desensitization but had no effect on the return towards control of residual enzyme activity in preincubated membranes. These results are consistent with continued receptor occupancy leading to a persistent activation of adenylate cyclase until internalization of the receptor has occurred.

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [3H]leucine. Insulin, at contrations from 10(-12) M to 10(-6) M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serum-free medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH1C1, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.

A human lung cancer cell line (BEN cells) was found to have a calcitonin-responsive adenylate cyclase. Various calcitonins and synthetic analogs stimulated adenylate cyclase activity withe same relative potency as they show in lowering blood calcium in the rat. Preincubation of the cells with calcitonin, followed by washing, led to loss of subsequent adenylate cyclase response to hormone. This was a dose-dependent phenomenon. The binding of [125I]salmon calcitonin to freshly subcultured cells was studied. The ability of calcitonins and analogs to compete for binding paralleled their efficacies in stimulating adenylate cyclase. Binding was saturable, reversible, and consisted of a single class of noninteracting sites with a mean Kd of 10.75 X 10(-10) M, K of 0.93 X 10(9)/M, and mean receptor number of 2.71 X 10(4)/cell. It is not known whether the calcitonin receptor is inappropriate to the cell of origin of the tumor. The BEN cells provide a means of isolating and studying the properties of the calcitonin receptor and of evaluating the significance for the tumor of a hormone-responsive adenylate cyclase.

The amino-terminal fragment of a tumoral parathyroid hormone-related peptide (PTHrP (1–34)) produced by a human squamous cell carcinoma of the lung was recently synthetized. In the present work its effect on the amiloride-sensitive sodium transport, taken as an estimate of the Na+/H+ exchanger activity of cultured opossum kidney (OK) epithelia was compared to that of synthetic bovine parathyroid hormone (bPTH (1–34)). Both PTHrP (1–34) and bPTH (1–34) inhibited the initial rate of amiloride-sensitive22Na transport. Half maximal inhibitory activity was obtained at about 10−11 M for both PTHrP (1–34) and bPTH (1–34). In conclusion, tumoral PTHrP (1–34) appears to be as effective as bPTH (1–34) for inhibiting the amiloride-sensitive Na transport, and presumably for decreasing the activity of the Na+/H+ exchanger present in the apical membrane of kidney epithelial cells.

In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.

The possibility of a relationship between cyclic AMP formation and metabolic processes in tumours has been investigated. Changes in basal activity and hormone-responsiveness of adenylate cyclase were demonstrated in plasma membranes and intact cells from pre-cancerous liver of rats fed a diet containing the carcinogen 3'-methyl-4-dimethylaminoazobenzene. Basal adenylate cyclase activity in hyperplastic parathyroid gland membranes was 200% higher than that in parathyroid adenoma membranes, corresponding with their relative rates of parathyroid hormone secretion in vitro. Membrane adenylate cyclase activity in hypernephromas was consistently 100--300% higher than in adjacent human renal cortex. Furthermore the adenylate cyclase activity of the tumour membranes was not influenced by a wide range of hormones which were effective stimulants in 'normal' renal cortex membranes. Conversion of 25-hydroxycholecalciferol to 1,25-dihydrocholecalciferol could not be demonstrated in either hypernephroma or adjacent renal cortical tissue. However, three of the four hypernephromas tested secreted a bone-resorbing factor. Cyclic AMP formation was increased by salmon, human and porcine calcitonins in both plasma membranes and intact cells from a poorly differentiated epidermoid cell carcinoma which was itself secreting calcitonin in culture. This phenomenon might be related to a feedback regulation of calcitonin production in this cell line. The observations are consistent with the concept of a relationship between cyclic AMP formation and certain metabolic functions (e.g. hormone production) in tumour cells.

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.

The effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast-rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti-cyclic AMP and FITC-labelled goat anti-rabbit IgG. Treatment of rat osteo-clasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium-regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.

Parathyroid hormone related protein (PTHrP) has been demonstrated in the tumour cells of squamous cell carcinomas originating in a variety of organs, in undifferentiated small cell tumours of the bronchus, and in carcinoma of the kidney. The protein hormone is thought to produce the hypercalcaemia which may complicate some of these malignancies. By using an antibody raised in rabbits against the N-terminal portion of the molecule, the polypeptide may be demonstrated in cells of the prickle cell layer of normal skin and in the cells of hair follicles. Skin tumours showing squamous or hair follicle differentiation are shown to contain the protein antigen, while basal cell carcinomas and tumours with sweat gland differentiation do not. The hormone may be primordial in origin and the progenitor of parathyroid hormone.

The methods for establishing osteoblast-rich rat calvarial cell cultures have been described, together with methods for the use of clonal osteogenic sarcoma cells of osteoblast phenotype. The latter clonal lines are useful for several purposes, but all the precautions and quality control measures necessary in the study of clonal lines must be observed. Some of the techniques for studying biochemical responses to hormones in these cells have also been detailed, but clearly others are applicable, including studies of the synthesis of matrix constituents. Osteoclast-like cells have not been considered in this chapter, because osteoclast culture methods have not yet been developed to the degree of purity and reproducibility necessary for this type of biochemical approach.

A protein responsible for the biochemical syndrome similar to primary hyperparathyroidism associated with certain tumors has been recently characterized and its effects at the level of bone and kidney reported. However, the relative role of tubular reabsorption of calcium (Ca) and bone resorption in the pathogenesis of hypercalcemia induced by this factor is still debated. We investigated the effects of a synthetic amino-terminal fragment of parathyroid hormone-related protein [PTHrP-(1-34)] administered chronically by intraperitoneal osmotic minipumps in thyroparathyroidectomized (TPTX) rats. Clearance studies performed on day 6 of treatment after a 24 h fast revealed an increase in renal tubular reabsorption of Ca and a decrease in renal tubular reabsorption of phosphate (Pi), accompanied by an increase in cAMP excretion. PTHrP-(1-34) (90 pmol/h) stimulated bone resorption as evaluated by an increment in fasting urinary Ca excretion. Although the bone resorption inhibitor aminopropylidene diphosphonate fully corrected urinary Ca excretion and reduced plasma Ca from 3.04 +/- 0.07 to 2.44 +/- 0.21 mM (p less than 0.05), this latter value remained considerably higher than in TPTX control rats (1.54 +/- 0.12 mM, p less than 0.01). In contrast, when the agent WR-2721, which is known to decrease the renal tubular reabsorption of Ca by a PTH-independent mechanism, was given, a further drop in plasma Ca and an increase in urinary Ca excretion were observed. These findings are similar to those found in animals implanted with the hypercalcemic Leydig cell tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructural and biochemical properties of four clonal osteogenic sarcoma lines, UMR 104, 105, 106, and 108, have been compared with uncloned osteogenic sarcoma cells and normal osteoblast-rich cells derived from newborn rat calvaria. High alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone were used as biochemical markers of osteoblastic cells. Cloning enriched both of these parameters above those of the parent tumor and far higher than that seen in normal cells, suggesting enrichment of the osteoblast phenotype. Both of these properties have been retained through many passages in culture. Morphologically, the clonal lines have also retained the "blast"-like appearance of the uncloned osteogenic sarcoma cells and consist mainly of flat, relatively featureless cells. Many cells with mitotic figures were observed, indicating continuous cell division taking place in the malignant cells. Each clonal line gave rise to characteristic tumors when reinjected into rats. It is concluded that the clonal osteogenic sarcoma lines are highly differentiated tumor lines which have conserved the differentiated properties of the mature osteoblast, making them a suitable model for the study of the effects of hormones on the growth of a differentiated tumor, as well as for the study of hormonal regulation of the osteoblast.

The influence of calcitonin on cell growth was examined in the human breast cancer cell line, T 47D. These cells possess specific high-affinity receptors for calcitonin as well as a sensitive calcitonin-responsive adenylate cyclase. In the T 47D cells, low doses of salmon calcitonin initially stimulated cell growth and the incorporation of [3H]thymidine into acid-insoluble macromolecules. This initial stimulation was followed by an inhibitory effect of calcitonin upon cell proliferation, which occurred during the log phase of growth, was dose dependent, and resulted in prolongation of doubling time from 36 to 90 hr. DNA and protein content correlated well with cell number. By 7 to 9 days of treatment, cell numbers of calcitonin-treated cells reached a mean of 66.5 +/- 3.7% of control (p less than 0.001, n = 8) (range, 51.3 to 82.9%). This biphasic effect of calcitonin on T 47D cells was reproduced by human calcitonin and prostaglandin E2 in the order of potency with which they influence adenylate cyclase. Epidermal growth factor (10(-9)M) and insulin (10(-9)M) stimulated the growth of T 47D cells, but this effect was abolished when either hormone was combined with salmon calcitonin (3 x 10(-10)M). Calcitonin specifically activated type II isoenzyme of cyclic adenosine 3':5'-monophosphate-dependent protein kinase in the T 47D cells. In view of other published data relating activation of this isoenzyme to growth regression in cancer cells, this response to calcitonin may be causally related to the inhibitory effect of the hormone upon cell replication in T 47D cells. The mechanism of the early stimulatory effect of calcitonin upon mitogenesis is not explained, although the possibility of stimulation of activity of type I isoenzyme of cAMP-dependent protein kinase has not been entirely excluded in the present experiments.

Blood platelets have a major role in haemostasis through their aggregation and adherence to the damaged vessel wall. During this process, platelet granular constituents are released. Although the functions of many of these release products are well known, biological functions of some well defined platelet specific proteins have not been determined. This category includes platelet factor 4 (PF4) and β-thromboglobulin (βTG), both small proteins of molecular weight 30,000–36,000 (refs 1, 2) which are stored in the α granule of the platelet and have not been detected in any other tissue. βTG is composed of four identical noncovalently bound subunits and its amino acid sequence is significantly homologous with that of PF4 (ref. 3). Although PF4 possesses a marked ability to neutralise heparin, no physiological or pathophysiological role has been attributed to either protein. The discovery of prostacyclin (PGI2) has led to a reappraisal of the factors regulating haemostasis and especially of the role of the vascular endothelium in preventing platelet adherence and aggregation4–6. The initial action of PGI2 on the platelet is to stimulate adenylate cyclase7–9, and it is the most potent inhibitor of platelet aggregation yet discovered. Also, cultures of arterial and venous endothelial cells have been found capable of producing PGI2 (refs 10,11). We now report results indicating that βTG reduces the production of PGI2-like activity in cultured bovine endothelial cells, which are shown to possess a specific receptor for βTG. The characteristics of this receptor suggest that βTG may act locally at high concentrations to favour platelet aggregation by diminishing PGI2 production.

An osteogenic sarcoma was induced in an inbred strain of the Sprague Dawley rat using seven serial injections of 32P-orthophosphate. The tumor was maintained by transplantation over a 3-year period in the same inbred strain. During this time it retained its bone-like differentiation. Tumor membranes and freshly isolated tumor cells also retained responsiveness to parathyroid hormone and to prostaglandins of adenylate cyclase and cyclic nucleotide formation respectively. The potencies of these agents and their analogues and metabolites were found to be proportional to their efficacies as bone resorbing agents. Thus, the tumor was shown to be a model for the study of hormone-responsiveness for tumor growth and differentiation, and also of the effects of agonists which act on bone-like cells.

Two methods have been used to covalently cross-link [125I]-salmon calcitonin to its receptor on a human lung carcinoma cell line, BEN, and the human breast cancer cell lines T47D and MCF 7. The first method was to use a specific photoaffinity derivative of salmon calcitonin and the second employed the chemical cross-linker, disuccinimidyl suberate. In both cases a cross-linked component of approximate molecular weight 80-90,000 on BEN cells was identified by polyacrylamide gel electrophoresis. This is consistent with the size of the cross-linked component found on T47D breast cancer cells using the photoactive salmon calcitonin as described in previous work. Disuccinimidyl suberate was unable to cross-link [125I]-salmon calcitonin either on T47D or MCF cells. However, photoactive salmon calcitonin cross-linked to a component of approximately 80-90,000 Mr on the MCF 7 cells. Thus, whereas the photoactive salmon calcitonin could cross-link a similar receptor component in all cell lines, the ability of disuccinimidyl suberate to do so was apparently cell specific. These data confirm that the calcitonin receptor comprises a component of approximately 85,000 Mr in cell lines examined thus far.

Cyclic AMP-dependent protein kinases prepared from crude cytosols of chick kidney, rat kidney and rat liver were found on dilution to exhibit complex kinetics. Dilution of the cytosols appears to increase the state of activation of the enzymes. This effect was due to the presence of inhibitory agents in the cytosol which had a greater inhibitory effect on the cyclic AMP-dependent than on the cyclic AMP-independent enzyme. Two types of inhibitory activity were found by column chromatography, one resistant to trichloroacetic acid precipitation and boiling but affected by trypsin digestion and the other resistant to boiling and trypsin digestion but precipitated by trichloroacetic acid. Inhibitory activity corresponding to the former characteristics has been described previously but the presence of additional soluble inhibitory agents in the cytosol has not been documented. The complete characterisation of this previously undescribed inhibitory activity requires further investigation. The relevance of such cytosolic inhibitory activity to the interpretation of states of activation of protein kinase enzymes is discussed.

Malignant and benign human breast tumours as well as rabbit breast tissue were examined for specific receptors for 1,25-dihydroxyvitamin D3 using competitive binding studies and sucrose density gradient analysis. Classical high affinity, low capacity receptors for 1,25-dihydroxyvitamin D3 were found in breast and node tissue in seven of ten patients with breast cancer and in all three patients with benign neoplasms. An inflammatory breast mass showed no binding. Similar receptors were found in breast tissue from pregnant and lactating rabbits. Taken with other recent data, these results suggest that 1,25-dihydroxyvitamin D3 may activate calcium transport in the malignant as well as in the normal lactating breast.

A parathyroid hormone-related protein (PTHrP) has been invoked as being responsible for the humoral hypercalcemia of malignancy. Eight of the first 13 amino acids of PTHrP are identical with those in PTH, but there is no other significant homology. The PTHrP gene is located on chromosome 12, whereas that for PTH is on chromosome 11, and the two genes are probably related by a duplication process. Antisera against PTHrP(1-34), which cross-react poorly or not at all with PTH, and antisera against other parts of PTHrP not homologous to PTH were used in immunocytochemistry using a peroxidase-antiperoxidase method, to identify PTHrP in the cytoplasm of cells in a series of unselected parathyroid adenomata. The study was based on our evidence that PTHrP is produced by fetal parathyroid and may be the predominant calcium-regulating hormone in the mammalian fetus. Glands from five patients with parathyroid hyperplasia secondary to chronic renal failure also stained positively for PTHrP. No evidence was obtained for PTHrP in sections from five patients with primary parathyroid chief cell hyperplasia or in a small group of patients with the multiple endocrine neoplasia type 1 or type 2 syndromes.

A photoaffinity derivative of salmon calcitonin has been produced by transglutaminase-mediated incorporation of N-(beta-aminoethyl)-4-azido-2-nitroaniline into the hormone. The derivative, purified by high pressure liquid chromatography, retained the abilities to bind to cultured T47D breast cancer cells and to stimulate adenylate cyclase in these cells. In both these respects it was equipotent with synthetic salmon calcitonin. Photolysis of the 125I-labeled photoactive salmon calcitonin derivative bound to T47D cells was accompanied by specific labeling of only one component (Mr approximately 85,000) as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling was observed only upon photolysis and was inhibited by unlabeled synthetic salmon calcitonin but not by the inactive calcitonin analogue, 8-glycine human calcitonin. Reduction did not alter the apparent molecular weight of the calcitonin receptor complex. No macromolecular forms of calcitonin were produced by photolysis in the absence of T47D cells.