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Background & Aims: Neonatal intestinal obstruction (meconium ileus [MI]) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and nongenetic modifiers to the development of this major complication of CF. Methods: A total of 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Results: Specific mutations in the CF transmembrane conductance regulator ( CFTR ), the gene responsible for CF, correlated with MI, indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings ( P = 1 × 10 −5 ), showing that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome ( P = 8 × 10 −4 ). Unlike MI, concordance analysis indicated that the risk for development of distal intestinal obstruction syndrome in CF patients is caused primarily by nongenetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene on chromosome 19 in a region previously linked to MI. Conclusions: The CFTR gene along with 2 or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, whereas intestinal obstruction in older CF patients is caused primarily by nongenetic factors.

Background The importance of the IGF system in HPT has been previously demonstrated. Additionally, the role of vitamin A in HPT has been reported. Retinoic acid (RA), a derivative of vitamin A, is a ligand for the IGF II receptor (IGF2R). We have evaluated the interactions of RA with the IGF system in a primary parathyroid cell culture model. Materials and Methods Primary cell cultures were prepared from nine patients. Following adhesion, the cells were transferred to serum-free medium and dosed once with growth factors ± RA for 96 hours. Proliferation was assessed by measuring tritiated thymidine incorporation. Results Compared with the control group (100%), both IGF I and II increased DNA synthesis significantly. Retinoic acid significantly reduced the basal DNA synthesis to 82.2% ± 4.2% compared with control (P < 0.05). Retinoic acid ×10−5 M completely abrogated the proliferative actions of IGF II (70.2% ± 9.7%, P < 0.05) but had no significant effect on the IGF I response (P > 0.05). To evaluate the role of IGF2R or IGFBPs in mediating the actions of RA, the IGF II analogs [Leu27]IGF II (10–20-fold reduced IGF I receptor affinity) and des(1–6) IGF II (lower IGFBP binding affinity) were used. The IGF II inhibitory effect of RA was enhanced in the presence of analogs [Leu27]IGF II (P = 0.052) but not with des(1–6)IGF II (P > 0.05), compared with wild-type IGF II. Conclusions These data implicate a novel antiproliferative role for RA in enhancing the pericellular clearance of IGF II via the IGF2R preventing ligand activation of the IGF I receptor. This may have broader implications for RA effects in other tumors.

Effects of extracellular calcium ([Ca(2+)](ext)) on parathyroid cells are mainly due to the activation of a plasma membrane calcium receptor (CaR) coupled with release of intracellular calcium. In addition, high [Ca(2+)](ext) activates the sphingomyelin pathway in bovine parathyroid cells, generating ceramides and sphingosine. This study explored the direct effects of synthetic ceramides on [Ca(2+)](i) in human parathyroid cells. Cells from five parathyroid adenomas removed from patients with primary hyperparathyroidism were dispersed and maintained in primary culture. Intracellular calcium concentration ([Ca(2+)](i)) [Ca(2+)](i) was monitored using standard quantitative fluorescence microscopy in Fura-2/AM-loaded cells. Laser scanning microscopy was used to monitor the intracellular distribution of a fluorescent ceramide analogue (BODIPY-C5). After addition of 10 microM C2-ceramide (N-acetyl-d-erythro-sphingosine), [Ca(2+)](i) increased rapidly (30-60 s) to a peak three times above basal levels in 70% of cells (37/55 cells in four experiments). This effect appeared to be due to release of Ca(2+) from intracellular stores rather than Ca(2+) entry from the extracellular medium. C2-responsive cells had a smaller [Ca(2+)](i) response to subsequent stimulation with the CaR agonist-neomycin (1 mM). These responses were specific to C2 since C6-ceramide (N-hexanoyl-d-erythro-sphingosine) did not affect basal [Ca(2+)](i) nor the responses to an increase in [Ca(2+)](ext) and to neomycin. C5-BODIPY generated intense perinuclear fluorescence, suggesting targeting of the ceramides to the Golgi apparatus. These data demonstrate that endogenous generation of ceramides has the potential to modulate changes in [Ca(2+)](i) and secretion in response to [Ca(2+)](ext) in human parathyroid cells.

Cells from 22 renal cell carcinomas (RCC) were established in culture. Sensitivity of the tumour cells to doxorubicin alone and in combination with racemic verapamil, which reverses multidrug resistance, was tested using a [75Se]selenomethionine uptake assay to measure protein synthesis. The effect of verapamil was expressed as a potentiation index: LD50doxorubicin/LD50doxorubicin + verapamil. The potentiation index in 15 of these carcinomas was determined for cells within the first 14 days of culture. At 3.3 mumol/l concentration of verapamil, of the tumours sensitive to doxorubicin alone (LD500.75 microgram/ml) five of seven showed a potentiation index of2. For the less sensitive tumours the analogous proportion was seven of eight. Tumour cell expression of glycoprotein P-170, associated with multidrug resistance, was estimated using the monoclonal antibody C-219. Initial expression levels were unrelated to the action of verapamil. In five tumours the proportion of cells expressing P-170 declined as the period of culture increased. This was not associated with any consistent change in the LD50 for doxorubicin or in potentiation of doxorubicin sensitivity by verapamil. Cell cloning associated with prolonged cell growth in vitro could mimic tumour cell cloning which accompanies the formation of metastases. Thus reduced expression of P-170 on prolonged cell growth in vitro may be a pointer to the efficacy of combination therapy in the treatment of patients with metastatic renal cell carcinoma.

Background Genetic mutations and upregulation of growth factors are implicated in the pathogenesis of hyperparathyroidism. The aim of this study was to evaluate the role of Wilms' tumour suppressor gene (WT-1) and the insulin-like growth factor (IGF) axis in hyperparathyroidism. Methods The expression of WT-1 and IGF components was examined by immunohistochemistry, reverse transcriptase–polymerase chain reaction and western immunoblotting in a panel of parathyroid specimens from both primary and secondary hyperparathyroidism. A human parathyroid cell culture model was established to examine the parathyroid response to IGF stimulation. Results There was a significantly lower level of WT-1 expression in parathyroid tumours than in normal parathyroid glands. Most tumours expressed IGF-I and IGF-II receptors and responded to IGF stimulation. Only IGF-I was present in normal parathyroid glands, whereas IGF-II was expressed exclusively in parathyroid tumours. Conclusion Abnormal expression of WT-1 and the IGF axis may play a role in the pathogenesis of hyperparathyroidism.

Background. Primary hyperparathyroidism (HPT) predominately affects perimenopausal women, leading to speculations that an estrogen imbalance may be liable. We have previously demonstrated the importance of the insulin-like growth factor (IGF) axis in HPT. Because the antiestrogen tamoxifen has been shown to modulate the IGF axis, we examined the interactions of selective estrogen receptor modulators (SERMs) and IGF in HPT. Methods. Estrogen receptors were evaluated by Western immunoligand blotting. Sixteen parathyroid glands from 19 patients were included. After adhesion, the cells were treated with IGF (I or II) ± estrogen ± SERMs (tamoxifen, ICI 182,780) for 96 hours in serum-free media. Proliferation was assessed by measuring tritiated thymidine incorporation. Results. Both primary and secondary HPT express estrogen receptors α and β. Primary and secondary HPT had comparable responses to SERMs, they were analyzed together. Compared with control (100%), IGFs (I and II) induced a significant increase in DNA synthesis. Estradiol at 10−8 and 10−7 mol/L (physiologic range) had no significant effects on IGF (I and II, P > .05). Both tamoxifen and ICI 182,780 inhibited basal DNA synthesis (P < .05) and abolished the effects of both IGF I and II (P < .05). Conclusions. SERMs are capable of reducing basal and IGF-stimulated DNA synthesis. This reduction in proliferation has implications for cancer biology and therapeutic potential for SERMs in HPT.

In addition to modulating insulin-like growth factors action, it is now clear that insulin-like growth factor-binding protein-3 also has intrinsic effects on cell growth and survival. We have compared the effects of insulin-like growth factor-binding protein-3 and transforming growth factor-beta on cell proliferation and death of Hs578T cells and the normal breast epithelial cell line, MCF-10A. The growth of MCF-10A cells was inhibited at low concentrations of insulin-like growth factor-binding protein-3 but stimulated at high concentrations. These differential effects were unaffected in the presence of an insulin-like growth factor-I receptor antagonist. A synthetic peptide corresponding to the serine phosphorylation domain of insulin-like growth factor-binding protein-3 (that does not bind to insulin-like growth factors) also mimicked these differential actions. The growth of both cell lines was significantly inhibited by transforming growth factor-beta, this was associated with a 14-fold increase of insulin-like growth factor-binding protein-3 secreted by the Hs578T cells but a five-fold decrease of insulin-like growth factor-binding protein-3 secreted by MCF-10A cells. Replacement doses of exogenous insulin-like growth factor-binding protein-3 overcame the transforming growth factor-beta-induced growth inhibition in the MCF-10A cells. Cell death induced by ceramide was significantly reduced by insulin-like growth factor-binding protein-3 in the MCF-10A cells and depleting insulin-like growth factor-binding protein-3 with transforming growth factor-beta in these cells consequently increased their susceptibility to ceramide. In contrast, insulin-like growth factor-binding protein-3 enhanced apoptosis induced by ceramide in the Hs578T cells but transforming growth factor-beta treated Hs578T cells were resistant to apoptosis. The addition of anti-sense mRNA to insulin-like growth factor-binding protein-3 significantly abrogated this effect of transforming growth factor-beta. These data indicate that insulin-like growth factor-binding protein-3 has intrinsic activity capable of inhibiting or enhancing the growth and survival of breast epithelial cells depending on the cell line and exposure to other cytokines. British Journal of Cancer (2002) 86, 1963–1969. doi:10.1038/sj.bjc.6600355 www.bjcancer.com © 2002 Cancer Research UK

Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using fura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca(2+)](ext)), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca(2+)](ext). Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2 microM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca(2+)](ext) known to stimulate secretion. A high [Ca(2+)](ext) and lanthanum (La(3+)) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5-30 min) in the presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment. These data suggest that FM1-43 fluorescence is determined by: (i) changes in cell membrane surface area associated with secretion-associated events, (ii) displacement/quenching by extracellular cations and (iii) endocytosis of the dye. In parathyroid cells, a rise in FM1-43 fluorescence occurred during incubation in a high (inhibitory) [Ca(2+)](ext) if the cytoplasmic calcium concentration ([Ca(2+)](i)) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10-50 microM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) [Ca(2+)](ext) if [Ca(2+)](i) was increased by addition of the calcium ionophore A23187 (10-25 microM). These data suggest that [Ca(2+)](i), rather than the absolute value of [Ca(2+)](ext), is the main modulator of secretion from parathyroid cells.

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.

Of patients presenting with renal cell carcinoma, some 25 % have metastatic disease and 74 % of these will die of the disease within one year. Mitozantrone has been used to treat patients with metastatic renal carcinoma [1, 2] but with minimal success. It is questionable, however, whether useful plasma levels of mitozantrone were achieved in one of these studies.

Summary— Ten renal carcinomas were established in culture and their sensitivity to mitozantrone ± verapamil, as measured by (75 Se) selenomethionine incorporation, was studied. The 5 tumours with spread beyond the kidney were those in which sensitivity to mitozantrone was enhanced by verapamil. With 4 of the 10 tumours and 1 further carcinoma, similar experiments were performed using doxorubicin. This drug was more active, its anti-tumour action being related to concentration. Verapamil was more effective in potentiation of doxorubicin and this action increased in proportion to the concentration of verapamil added.

Background: Primary HPT predominately affects females and has a peak incidence at the perimenopausal period. This has led to speculations that an estrogen imbalance may have a role in this condition. We have previously demonstrated the importance of the insulin-like growth factor (IGF) axis in HPT. The SERM, tamoxifen (tam) has been shown to modulate the IGF axis. We propose to examine the interactions of SERMs and IGF in a primary parathyroid cell culture model. Methods: Estrogen (E2) receptors were evaluated by Western immuno-ligand blotting. Sixteen parathyroid glands from 14 patients were included. Following adhesion, the cells were transferred to serum free media and dosed once with IGF (I or II) ± estrogen ± SERMs (Tam), faslodex (fas, ICI 182780)) for 96 h. Proliferation was assessed by measuring tritiated thymidine incorporation. Results: Both primary (1) and secondary (2) HPT express α and β ERs. 1HPT and 2HPT had comparable responses and were analysed together. Compared with control (100%), IGFs (I & II) induced a significant increase in DNA synthesis. E2 at 10−8 and 10−7m (physiological range) inhibited DNA synthesis but had no significant effects on IGF (I & II) (P > 0.05). Tam inhibited basal DNA synthesis (P < 0.05) and abolished the effects of both IGF I and II (P < 0.05). To clarify whether these effects can be ascribed to Tam's ER antagonist property, Fas (a pure ER antagonist) was examined. Fas at 10−7m inhibited both IGF I and II (P < 0.05). Conclusions: Tam and Fas are capable of reducing basal and IGF stimulated DNA synthesis. There are obvious implications for cancer biology as well as therapeutic potential for SERMs in HPT.

Mouse mammary carcinoma cells were exposed in vitro to increasing concentrations of doxorubicin hydrochloride [adriamycin (ADR)] or 5-fluorouracil (5-FU). Uptake of [75Se]selenomethionine (75SeM) in a methionine-deficient medium measured the resulting inhibition of protein synthesis by the tumour cells. This was compared with the ability of the 75SeM labelled tumour cells to localize in mouse lungs and to form pulmonary tumours following intravenous (i.v.) injection into isogenic hosts. These parameters were also related to the ability of the drugs to inhibit pulmonary tumour formation in vivo when injected into mice which had received tumour cells i.v. Results from five different tumours were pooled for analysis. At the highest drug concentration (10 micrograms/ml ADR, 100 micrograms/ml 5-FU) inhibition of protein synthesis was significantly related to the in vivo action of the drugs in limiting formation of pulmonary tumors (P less than 0.02 using the rank difference coefficient). There was also a direct relationship between pulmonary localization of tumour cells following exposure to drugs, their ability to form tumour nodules (P less than 0.025) and the in vivo action of the drugs in inhibiting tumour formation (P less than 0.05). Thus inhibition of protein synthesis in vitro and pulmonary localization following i.v. injection may be of value in predicting the in vivo effect of cytotoxic drugs.