Douglas W. Burton

Immunoreactive calcitonin in the intermediate lobe of the pituitary gland

Abstract Immunoreactive-calcitonin was observed in all cells of the intermediate lobe of the rat pituitary gland. It may thus be a fragment of the ≥31, 000 daltons precursor molecule of adrenocorticotropin and β-lipotropin.

PTH/PTHrP and Vitamin D Control Antimicrobial Peptide Expression and Susceptibility to Bacterial Skin Infection

Vitamin D and parathyroid hormone work together to maximize innate immune resistance to skin infections. A Sunny Solution to Infectious Disease A role for vitamin D in immune defense has been hypothesized on the basis of observations made over several decades that increased vitamin D intake, often through increased sunlight exposure, provides a therapeutic effect in fighting infectious diseases. However, a critical role for vitamin D status in immune function has not been established by controlled clinical trials. The current study by Muehleisen et al. sought to determine whether parathyroid hormone (PTH), or PTH-related peptide (PTHrP), might also act together with vitamin D in immune defense against bacterial skin infections and thus confound interpretations based solely on vitamin D status. The authors found that human skin keratinocytes showed increased expression of PTHrP in response to bacterial products. PTH or PTHrP was then observed to induce expression of cathelicidin antimicrobial peptide by keratinocytes in culture. When PTH was administered to mice, it enhanced their resistance to skin infection by group A Streptococcus. If vitamin D was absent from the diet of normal mice, they responded with an increase in PTH production and an increase in antimicrobial peptide expression. However, mice lacking the capacity to convert vitamin D to 1,25-vitamin D failed to induce cathelicidin in response to restriction of dietary vitamin D and became much more susceptible to invasive bacterial infection. Because 1,25-dihydroxyvitamin D was found necessary for keratinocytes to express the PTH/PTHrP receptor, this increase in susceptibility to infection may reflect the inability of PTH or PTHrP to induce cathelicidin in a setting of low vitamin D. These findings show that PTH/PTHrP is immunologically active, can boost innate immunity, and may compensate for low vitamin D status. Understanding this system provides a new target for studying the immunological functions of vitamin D. The production of antimicrobial peptides is essential for protection against a wide variety of microbial pathogens and plays an important role in the pathogenesis of several diseases. The mechanisms responsible for expression of antimicrobial peptides are incompletely understood, but a role for vitamin D as a transcriptional inducer of the antimicrobial peptide cathelicidin has been proposed. We show that 1,25-dihydroxyvitamin D3 (1,25-D3) acts together with parathyroid hormone (PTH), or the shared amino-terminal domain of PTH-related peptide (PTHrP), to synergistically increase cathelicidin and immune defense. Administration of PTH to mouse skin decreased susceptibility to skin infection by group A Streptococcus. Mice on dietary vitamin D3 restriction that responded with an elevation in PTH have an increased risk of infection if they lack 1,25-D3. These results identify PTH/PTHrP as a variable that serves to compensate for inadequate vitamin D during activation of antimicrobial peptide production.

Gene therapy of pancreatic cancer with green fluorescent protein and tumor necrosis factor-related apoptosis-inducing ligand fusion gene expression driven by a human telomerase reverse transcriptase promoter

AbstractBackground: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in malignant cells but not in normal cells. Ad/g-TRAIL, an adenoviral vector in which expression of green fluorescent protein (GFP) and TRAIL is driven by a human telomerase reverse transcriptase promoter, has shown promise as a targeted antitumor agent. Methods: To investigate the effects of TRAIL gene therapy on pancreatic cancer, BxPC-3, MIA-PaCa-2, Panc-1, and ASPC-1 cells were treated with Ad/g-TRAIL. Transfection and protein expression were determined by using immunoblotting and identification of GFP with fluorescent microscopy and flow cytometry. Cell viability was determined by proliferation assay. Cell-cycle analysis and quantification of caspase-3 were used to identify apoptosis. The in vivo efficacy of Ad/g-TRAIL was characterized in a novel red fluorescent protein murine model of MIA-PaCa-2 pancreatic cancer. Results: Cells treated with Ad/g-TRAIL expressed GFP and exhibited apoptotic morphology within 2 days of treatment. Treatment with this vector in vitro resulted in less cell viability, increased caspase-3 activity, and a greater apoptotic fraction than treatment with controls. In vivo, treatment with Ad/g-TRAIL significantly suppressed tumor growth. Conclusions:TRAIL gene therapy induces apoptosis of pancreatic tumor cells both in vitro and in vivo and is a promising therapy in the treatment of pancreatic cancer.

Demonstration of chromogranin a in human neuroendocrine cell lines by immunohistology and immunoassay

We have used immunohistology and radioimmunoassay procedures to study Chromogranin A (CgA) in human neuroendocrine tumor cell lines, especially small cell lung cancers (SCLC). By immunohistology, CgA could be detected in 11 of 18 classical SCLC cell lines, in a medullary thyroid carcinoma (MTC) cell line, and in only one of 13 variant‐ or non‐SCLC cell lines. By radioimmunoassay, CgA could be detected in the cells and culture media of all of the classical SCLC cell lines tested. Many of the classical SCLC cell lines also produced calcitonin (CT). These studies demonstrate that CgA production is a common feature of SCLC cell lines, especially those with neuroendocrine characteristics.

The gene for human chromogranin A (CgA) is located on chromosome 14.

Chromogranin A (CgA) is a protein that is present in most neuroendocrine tissues and is co-secreted with their resident hormones. We have assigned the CgA gene to human chromosome 14 by hybridization of a CgA cDNA probe cloned from a cDNA library of human medullary thyroid carcinoma cells to spots of individual human chromosomes flow-sorted onto nitrocellulose filters. Southern analysis of human genomic DNA with the same probe revealed only 1-3 restriction bands. These studies indicate that the CgA gene is probably single copy and not a member of a dispersed, multigene family. The CgA gene is not co-localized with the genes of any of the CgA-associated hormones.

Chromogranin A is present in and released by fish endocrine tissue

Chromogranin A (CgA) is a protein that is present in many mammalian endocrine cells and co-secreted with their resident hormones. We have demonstrated the presence of CgA by immunohistology in the ultimobranchial glands and corpuscles of Stannius of rainbow trout. CgA was also detected by radioimmunoassay in the medium of incubated coho salmon ultimobranchial glands. Our observations demonstrate the presence of CgA in endocrine glands of evolutionarily divergent species. These observations are consistent with the hypothesis that CgA participates in the secretory process of a wide variety of hormones.

Parathyroid hormone‐related protein regulates the growth of orthotopic human lung tumors in athymic mice

Parathyroid hormone‐related protein (PTHrP) has growth regulatory effects for many malignant cells and may influence the progression of carcinomas of the breast, prostate, and lung. In the current study, the authors investigated the in vivo and in vitro effects of PTHrP neutralizing antibody and PTHrP treatment on the growth of BEN cells, a human lung squamous cell carcinoma line that expresses PTHrP and its receptor.

Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells

We have studied the pattern of chromogranin A (CgA)-related species in different human endocrine cells that produce CgA and also express the calcitonin gene. Antibodies against CgA peptides that span its linear sequence were used in Western analysis of cell lines derived from medullary thyroid carcinoma (MTC), small cell lung cancers (SCLC), epidermoid cell lung cancer (ECLC) and a pulmonary carcinoid tumor (CRND). Each of the cell lines demonstrated a distinct pattern of CgA-related species. Gel filtration studies also revealed multiple and different forms of immunoreactive CgA in the cell lines. Although proteolysis may contribute to our results, these observations suggest that native CgA is processed to smaller species in a tissue-specific pattern by different endocrine cells. More conclusive studies, however, are necessary to establish that cell processing leads to the specific CgA moieties that we have observed.

Hypercalcemia due to Parathyroid Hormone-Related Protein Secretion by Melanoma

About 1–2% of melanoma patients develop hypercalcemia. We report hypercalcemia without bone metastasis in a 46-year-old woman with advanced melanoma. The hypercalcemia was associated with elevated serum parathyroid hormone-related protein (PTHrP) levels. An even higher concentration (10 times the serum level) in pleural effusion caused by pleural metastases implied that the source of the increased circulating PTHrP was the melanoma. Immunohistochemical staining of paraffin sections, performed using a monoclonal antibody (9H7) against the peptide sequence 109–141 of human PTHrP, detected PTHrP in the cytoplasm and nucleoli of melanoma cells in an autopsy specimen but not in specimens from this patient prior to onset of hypercalcemia. Considering the evidence, it is very likely that PTHrP production by melanoma caused hypercalcemia in this patient.

GSK3 and PKB/Akt are associated with integrin‐mediated regulation of PTHrP, IL‐6 and IL‐8 expression in FG pancreatic cancer cells

We have demonstrated recently that PTHrP is upregulated in pancreatic adenocarcinoma and that the ECM exerts regulatory control, at least in part, over PTHrP expression. In our present study, we examined the potential signaling interactions between these 2 pathways. Our results demonstrate that, under serum‐free conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn is mediated by the α5β1 integrin, whereas adhesion to Type I collagen is mediated by the α2β1 integrin. α5β1 integrin‐mediated adhesion to Fn results in a phenotype that includes a reduction in cell proliferation, increased E‐cadherin localization in cell–cell contacts, increased β‐catenin localization throughout the cell, inhibition of haptokinetic cell migration, and increased expression of PTHrP, IL‐6 and IL‐8 relative to α2β1 integrin‐mediated adhesion on Type I collagen. A phosphoprotein immunoblotting screen of FG pancreatic cancer cells grown on either Fn or Type I collagen indicates that GSK3 and PKB/Akt are differentially phosphorylated on these 2 substrates. These results implicate GSK3 and PKB/Akt in the integrin‐mediated regulation of PTHrP, IL‐6 and IL‐8 in pancreatic cancer.