Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells.

We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackie-adenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systemic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

Vitamin D receptor genotypes in primary hyperparathyroidism.

Vitamin D and parathyroid hormone (PTH) constitute the main regulators of systemic calcium homeostasis. As well as its calcaemic effects, active vitamin D3(1,25(OH)2D3) has a direct regulatory role on parathyroid cells. Active vitamin D3 acts via its receptor (VDR), and binding of the ligand-receptor complex to specific promoter regions of the PTH gene inhibits transcription. Active vitamin D3 constitutes a principal regulator of parathyroid cell growth, and polymorphism in the VDR gene has recently been related to bone mineral density and suggested as predisposing to osteoporosis. Impaired effects of active vitamin D3 may contribute to the relatively enhanced secretion and cell proliferation seen in hyperparathyroidism (HPT). Indeed, VDR dysfunction, of essentially unknown character, has been demonstrated in the pathological parathyroid tissue of primary HPT as well as HPT secondary to uraemia. Consistent with the essential role of active vitamin D3 in parathyroid regulation, the VDR gene polymorphism was studied in 90 postmenopausal women with primary hyperparathyroidism. The VDR genotype bb was found in 60.0% of HPT patients and in 33.3% of the postmenopausal female controls (P < 0.001). As the b allele has been linked to decreased transcriptional activity or messenger RNA stability, reduced VDR expression may impede regulatory actions of vitamin D and may contribute to parathyroid tumorigenesis in these patients.

25-hydroxyvitamin D3 1alpha-hydroxylase and vitamin D receptor expression in papillary thyroid carcinoma.

Vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-alpha-hydroxylase expression have recently been shown to be upregulated in several tumors and thought to represent an important endogenous response to tumor progression. Little is known about the expression of these proteins in thyroid carcinoma, although previous reports have documented evidence of the biological effect of vitamin D in thyroid cells. Using paraffin-embedded and frozen sections of papillary thyroid carcinoma, we utilized real-time quantitative RT-PCR and immunohistochemistry to characterize the expression of VDR and 1-alpha-hydroxylase in thyroid follicular cells, with special emphasis on papillary thyroid carcinoma (PTC). VDR and 1-alpha-hydroxylase expression were increased in PTC compared with normal thyroid tissue and especially high in areas of lymphocyte infiltration. Expression of VDR and 1-alpha-hydroxylase in PTC may be compatible with an overall favorable prognosis for this tumor type and may constitute important prerequisites for using vitamin D and/or vitamin D analogs in the treatment of PTC.

Expression of platelet-derived growth factor beta-receptors on stromal tissue cells in human carcinoid tumors.

Carcinoid tumors of the midgut type are slowly growing neoplasms which often present clinically and histologically pronounced fibrosis around the tumors. Cryosections from 41 neuroendocrine tumors (31 midgut carcinoid tumors, 8 endocrine pancreatic carcinomas, 1 parathyroid carcinoma, and 1 pheochromocytoma) and 22 nonneuroendocrine carcinomas were examined for the presence of platelet-derived growth factor (PDGF) beta-receptor by immunohistochemistry using the monoclonal antibody PDGFR-B2. Twenty midgut carcinoid tumor tissues (66%) and 4 endocrine pancreatic carcinomas (50%) and the parathyroid carcinoma stained positively with the antibody. In contrast, only 2 nonneuroendocrine tumor tissues (10%) were stained, and the staining in these cases was weak. The immunoreaction in the carcinoid tumors was observed in connective tissue cells adjacent to tumor cell clusters but not in the tumor cells themselves. The degree of positive PDGF beta-receptor expression in the carcinoid tissues seems to correlate positively with the presence of macrophages as determined by the monoclonal antibody anti-Leu-M5, but not with other infiltrated lymphocytes identified with the monoclonal antibody anti-Leu-4, or with anti-HLA-DR antibodies. Stromal cells adjacent to tumor cells, including small capillaries, stained more strongly than the stromal cells which were distant from tumor cell clusters. Furthermore, carcinoid tumor metastases from lymph nodes as well as from liver showed stronger immunoreactivity in the stromal cells with the PDGF beta-receptor antibody than the corresponding primary tumors. Our data suggest that carcinoid tumor cells may directly or indirectly induce expression of PDGF beta-receptor on adjacent stromal cells in the tumor tissue, which may contribute to the fibrosis that is often seen around carcinoid tumors.

Mutation of the multiple endocrine neoplasia type 1 gene in nonfamilial, malignant tumors of the endocrine pancreas.

Endocrine pancreatic tumors are rare neoplasms that occur sporadically or as part of a multiple endocrine neoplasia type 1 (MEN1) syndrome. Germ-line mutations of the MEN1 gene, located at 11q13, have been demonstrated in MEN1 kindreds, and loss of heterozygosity (LOH) on 11q13 together with somatic MEN1 mutations have been detected in 20% of nonfamilial parathyroid tumors. Here, we examine 11 non-MEN1 malignant tumors of the endocrine pancreas, 9 nonfunctioning tumors, and 2 glucagonomas. LOH of at least one informative locus on 11q13 was found in 70% of the tumors. Three tumors displayed somatic mutations of the MEN1 gene together with LOH on 11q13, whereas the corresponding germ-line DNA was normal. These findings support the hypothesis that MEN1 gene mutations contribute to the tumorigenesis of nonfamilial, malignant endocrine pancreatic tumors.

Human biodistribution of [111In]diethylenetriaminepentaacetic acid-(DTPA)-D-[Phe1]-octreotide and peroperative detection of endocrine tumors.

Requisites for preoperative and intraoperative tumor localization with [111In]diethylenetriaminepentaacetic acid-D-[Phe1]-octreotide scanning were explored in 23 patients with endocrine tumors (15 carcinoids, 4 insulinomas, and single cases of gastrinoma, medullary thyroid carcinoma, aldosteronoma, and paraganglioma). The patients were subjected to Octreoscan single photon emission computed tomographic examination prior to surgery and well counter investigation of nuclide uptake in tumors and normal tissues sampled at surgery. Somatostatin receptor-positive tumors demonstrated efficient nuclide accumulation with mean tumor:blood radioactivity ratios of 180-370 (for carcinoids and insulinoma), compared with tissue:blood ratios of 302 for spleen, 42 for liver, and < 10-15 in other normal tissues (pancreas, small intestine, and mesenteric fat). Inefficient preoperative visualization of lesions was related to inconspicuous size, as for primary intestinal carcinoids, tiny liver metastases, and a single small insulinoma. High background activity, pronounced tumor fibrosis, and meager accumulation of tracer also interfered with visualization. Tumor deposits in organs with low background activity (such as carcinoid mesenteric metastases and endocrine pancreatic tumors) were generally most readily detected. Intraoperative investigations with hand-held gamma detector probes were disturbed by obvious high background activity. These investigations revealed two preoperatively unrecognized primary intestinal carcinoids, which, however, were both palpable during surgery. These studies, therefore, had little impact on the surgical strategy.

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma.

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.

Dimethyl sulfoxide increases cytoplasmic Ca2+ concentration and inhibits parathyroid hormone release in normal bovine and pathological human parathyroid cells.

The acute effects of dimethyl sulfoxide (DMSO) on parathyroid hormone (PTH) release and the cytoplasmic Ca2+ concentration (Ca2+i) were studied in dispersed bovine cells and cells isolated from human parathyroid adenomas. At extracellular Ca2+ concentrations in the 0.5-3.0 mM range, but not at less than 25 nM, addition of 2% DMSO caused a rapid rise of Ca2+i. This effect corresponded to an inhibition of PTH release and there was a strong negative correlation between Ca2+i and secretion. The actions of DMSO on Ca2+i and PTH release were less pronounced in the pathological human cells. The data are consistent with a DMSO effect on the Ca2+-sensor function of the parathyroid cell, possibly mediated by an altered plasma membrane fluidity.

Kinetic evidence for cytoplasmic calcium as an inhibitory messenger in parathyroid hormone release.

A sudden change of extracellular Ca2+ from 0.5 to 3.0 mM resulted in a transient rise of the cytoplasmic Ca2+ concentration (Ca2+i) followed by a sustained increase in parathyroid cells loaded with the Ca2+-indicator fura-2. The initial transient could be eliminated by increasing the Ca2+ buffering capacity of the cytoplasm. Under such conditions the rise of Ca2+i exhibited kinetics reminiscent of those for 45Ca uptake and cell depolarization. Addition of 0.5 mM Mn2+ mimicked the effect of raising the extracellular Ca2+ concentration, since there was an initial Ca2+i transient followed by a slower entry of Mn2+ into the cells. This reaction pattern was different from that of pancreatic alpha 2-cells in which there was no substantial influx of Mn2+ before depolarization with arginine. When measuring the kinetics of parathyroid hormone (PTH) release it was apparent that Ca2+ inhibition of secretion followed Ca2+i and thus became substantially delayed after eliminating the initial transient. The results support the concept of a depolarizing Ca2+ permeability in the parathyroid cell membrane which can be activated by external Ca2+, and indicate that Ca2+i is an inhibitory messenger of importance for the physiological regulation of PTH release.

Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism.

When raising the extracellular Ca2+ concentration stepwise from 0.5 to 3.0 mM, bovine parathyroid cells reacted with initial transient and sustained elevations of the cytoplasmic Ca2+ concentration (Ca2+i), as well as more than 50% inhibition of parathyroid hormone (PTH) release. Human parathyroid adenoma cells and bovine cells cultured for 1 day or exposed to a low concentration of a monoclonal antiparathyroid antibody exhibited right-shifted dependencies of PTH release and Ca2+i on extracellular Ca2+ and reduced Ca2+i transients. The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) further right-shifted the dose response relationship for Ca2+ regulated Ca2+i of the adenoma cells, whereas the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) tended to normalize it, without affecting Ca2+i of normal bovine cells. In cells from an oxyphil adenoma and a parathyroid carcinoma as well as in bovine cells cultured 4 days or exposed to a high concentration of the antiparathyroid antibody, there were no Ca2+i transients, very small increases in steady-state Ca2+i and nonsuppressible PTH release. The results suggest that reduced availability of a putative Ca2+-receptor and increased protein kinase C activity may be important factors in the decreased Ca2+ sensitivity of abnormal parathyroid cells.

Paradoxical effects of K+ and D-600 on parathyroid hormone secretion and cytoplasmic Ca2+ in normal bovine and pathological human parathyroid cells.

The effects of K+ and the Ca2+ channel blocker D-600 on parathyroid hormone (PTH) release and cytoplasmic Ca2+ activity (Ca2+i) were measured at different Ca2+ concentrations in dispersed parathyroid cells from normal cattle and from patients with hyperparathyroidism. When the extracellular Ca2+ concentration was raised within the 0.5-3.0 mM range Ca2+i increased and PTH secretion was inhibited. There was also a stimulatory effect of Ca2+ on secretion as indicated by a parallel decrease of Ca2+i and PTH release when extracellular Ca2+ was reduced to less than 25 nM. Addition of 30-50 mM K+ stimulated PTH release and lowered Ca2+i. The effect of K+ was less pronounced in the human cells with a decreased suppressability of PTH release. The Ca2+ channel blocker D-600 had no effect on Ca2+i and PTH release in the absence of extracellular Ca2+. However, at 0.5-1.0 mM Ca2+, D-600 increased Ca2+i and inhibited PTH release, whereas the opposite effects were obtained at 3.0 mM Ca2+. The transition from inhibition to stimulation occurred at a higher Ca2+ concentration in the human cells and the right-shift in the dose-effect relationship for Ca2+-inhibited PTH release tended to be normalized by D-600. It is suggested that K+ stimulates PTH release by increasing the intracellular sequestration of Ca2+ and that the reduced response in the parathyroid human cells is due to the fact that Ca2+i already is lowered. D-600 appears to have both Ca2+ agonistic and antagonistic actions in facilitating and inhibiting Ca2+ influx into the parathyroid cells at low and high concentrations of extracellular Ca2+, respectively. D-600 and related drugs are considered potentially important for the treatment of hyperparathyroidism.

Longitudinal studies of mild primary hyperparathyroidism.

In 1969, a health survey was offered to all inhabitants of a town district in Sweden. A clinical examination was carried out, and among other variables, a measurement was made of serum calcium. The same procedure was repeated in 1971. From these two investigations a cohort of 176 individuals (1.1%) with sustained hypercalcemia was identified who could be followed during the subsequent 15 years. Comparisons were made with an age- and sex-matched control group from the same health survey. Survival was significantly lower in the hypercalcemic cohort than in the control group. This reduction was related to the degree of hypercalcemia and apparently mainly due to diseases of the circulatory organs. There was no marked deterioration of renal function, and although there was in some patients a moderate progression of the hypercalcemia, none developed a hypercalcemic crisis during 15 years of follow-up. In consecutively referred patients with primary hyperparathyroidism, psychiatric disturbances of mainly a depressive character were found upon detailed analysis within a majority of the patients, and parathyroid surgery resulted in a clear improvement in mental health.

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells.

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.

Hyperparathyroidism is associated with reduced expression of a parathyroid calcium receptor mechanism defined by monoclonal antiparathyroid antibodies.

Parathyroid tissue from patients with hyperparathyroidism (HPT) exhibited reduced immunohistochemical reactivity with monoclonal antiparathyroid antibodies, previously shown to stain intensely the surface of normal human parathyroid cells and to interfere with a receptor mechanism of these cells which is involved in the sensing and gating of Ca2+. Parathyroid hormone (PTH) release and cytoplasmic Ca2+ concentrations (Ca2+i) of dispersed cells from the pathological parathyroid glands had right-shifted dependencies on extracellular Ca2+, and exposure to the antibodies rendered both Ca2+i and PTH release almost completely insensitive to changes in ambient Ca2+. The results suggest that reduced expression of a parathyroid calcium receptor mechanism may be an important cause for the aberrant PTH release in HPT.

Multiple allelic deletions and intratumoral genetic heterogeneity in men1 pancreatic tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome with multiple tumors of the endocrine pancreas, the parathyroid, the pituitary, and other tissues. The MEN1 gene at 11q13 is homozygously mutated in the majority of MEN1 tumors. Here we present a genome-wide loss of heterozygosity (LOH) screening of 23 pancreatic lesions, one duodenal tumor, and one thymic carcinoid from 13 MEN1 patients. Multiple allelic deletions were found. Fractional allelic loss varied from 6-75%, mean 31%. All pancreatic tumors displayed LOH on chromosome 11, whereas the frequency of losses for chromosomes 3, 6, 8, 10, 18, and 21 was over 30%. Different lesions from individual patients had discrepant patterns of LOH. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells, whereas other chromosomal loci were deleted in portions of the analyzed tumor. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. Fractional allelic loss did not correlate to malignancy or to tumor size. Our findings indicate that MEN1 pancreatic tumors fail to maintain DNA integrity and demonstrate signs of chromosomal instability.

Vitamin D receptor (VDR) and parathyroid hormone messenger ribonucleic acid levels correspond to polymorphic VDR alleles in human parathyroid tumors.

Calcitriol, via its receptor (VDR) is a main regulator of PTH secretion and parathyroid cell proliferation. Recently, marked overrepresentation of the polymorphic VDR alleles b, a, and T was found in patients with primary hyperparathyroidism (pHPT), which suggests pathogenic importance in the disease. Using the ribonuclease protection assay, relative VDR and PTH messenger ribonucleic acid (mRNA) levels of parathyroid adenomas from 42 patients with sporadic pHPT were related to these VDR polymorphisms. The tumors of patients homozygous for the b, a, or T alleles demonstrated significantly lower VDR and higher PTH mRNA levels than those exhibiting the BB, AA, or tt genotypes (P < 0.0001-0.02), whereas heterozygotes had intermediate values. A similar discrepancy was found when comparing the baT and non-baT haplotypes (0.042 +/- 0.005 vs. 0.064 +/- 0.004 for VDR; 34.4 +/- 3.7 vs. 21.6 +/- 2.2 for PTH; both P < 0.005). The lower VDR mRNA levels associated with the b, a, and T alleles may affect the calcitriol-mediated control of parathyroid function and thereby contribute to the development of sporadic pHPT.

Vitamin D receptor polymorphisms correlate to parathyroid cell function in primary hyperparathyroidism.

Calcitriol acts via its receptor (VDR) and inhibits PTH secretion and parathyroid cell proliferation. Increased prevalence of the polymorphic VDR alleles b, a, and T has been demonstrated in sporadic primary hyperparathyroidism. Sixty-two patients with primary hyperparathyroidism due to parathyroid adenoma (mean age, 69.5 +/- 1.4 yr) were genotyped for these VDR polymorphisms. Dispersed cells of the adenomas were exposed to increasing concentrations of extracellular Ca2+ and analyzed for PTH release and cytoplasmic Ca2+ concentrations. Ca2+-mediated PTH inhibition exhibited higher ED50 and less suppression in the cells of patients who were homozygous for the b, a, and T alleles (P < 0.05-0.10). When analyzing haplotypes, the patients with baT demonstrated a ED50 of 1.81 +/- 0.15 vs. 1.29 +/- 0.10 for BAt (P < 0.05). As VDR alleles were unrelated to parathyroid intracellular Ca2+, influences of polymorphic VDR alleles on PTH secretion seem to involve mechanisms other than the Ca2+-sensing protein of the parathyroid cell surface.

Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism.

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20-58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.

The vitamin D receptor (VDR) start codon polymorphism in primary hyperparathyroidism and parathyroid VDR messenger ribonucleic acid levels.

Vitamin D regulates parathyroid cell proliferation and secretion of PTH. Increased prevalence of the polymorphic vitamin D receptor (VDR) alleles b, a, and T has been reported in sporadic primary hyperparathyroidism (PHPT), suggesting that these genetic variants may predispose to the disease. Recently, another polymorphism in the VDR gene was related to bone mineral density, and this VDR-FokI polymorphism causes different lengths of the VDR, implying possible functional consequences. The VDR-FokI polymorphism was studied in 182 postmenopausal women with sporadic PHPT and in matched controls. No significant differences in distribution of the VDR-FokI genotypes could be detected between the groups, although there was a tendency toward overrepresentation of the F allele in the PHPT patients (P = 0.05). There were no significant associations with age, serum calcium, serum PTH, bone mineral density, or parathyroid tumor weight. The VDR genotypes were unrelated to VDR and PTH messenger ribonucleic acid levels in the parathyroid adenomas of 42 PHPT patients. In 23 PHPT patients, the Ca2+-PTH set-points were determined in vivo and were unrelated to the VDR alleles. We suggest that the VDR-FokI polymorphism has at most a minor pathogenic importance in the development of PHPT.