Positive tetracycline control of expression of p15INK4B from an Epstein-Barr autonomous plasmid in a human melanoma cell line.

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

Inflammatory nevus comedonicus in children.

More than 100 years has passed since the first report of a nevus comedonicus. The earliest reports emphasized the inflammatory aspect of the nevus comedonicus as being the most significant problem. In the past 30 years, publications have ignored the inflammatory aspect of nevus comedonicus while emphasizing a variety of associated malformations. In this review, we describe five prepubertal children with prominent and persistent inflammatory changes limited to areas within a nevus comedonicus. In our experience, inflammation can be severe and resistant to treatment. Ultimately, surgical removal of the involved skin was required in two children.

Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: selective enhancement of collagenase gene expression in human dermal fibroblasts in culture.

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E2 (PGE2) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01-1.0 microgram/ml) of PGE2 led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE2 had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE2 did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE2 by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-alpha, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.

Effects of secretin and caerulein on pancreatic digestive enzymes in cultured rat acinar cells.

Caerulein is proposed to regulate the synthesis of pancreatic proteases and amylase. Similarly, secretin is implicated in the regulation of pancreatic lipase synthesis. Evidence of these regulations is predominantly from in vivo studies. We therefore examined the effects of caerulein and secretin directly on acinar cells to eliminate possible interactions with other regulatory factors. Cellular and media enzyme activities and relative synthesis were measured after 24 h of hormonal treatment. Cells were incubated with [14C]-amino acids and then subjected to two-dimensional gel electrophoresis to separate individual acinar proteins for subsequent determination of incorporated radioactivity and relative synthesis. In general, all enzyme activities decreased (33%; p < 0.02) over time in culture and medium enzyme activities increased (370%; p < 0.00001) in all treatment groups. Caerulein further decreased cellular content of all enzymes (p < 0.002) and increased media amylase and lipase activities (p < 0.02). Caerulein, however, significantly increased the relative synthesis of trypsin (28%) and tended to increase that of chymotrypsin (25%; p < 0.06), which supports its proposed role in protease regulation. Secretin, on the other hand, did not significantly affect the cellular or medium activities or the relative synthesis of any pancreatic enzyme evaluated. Therefore, this study does not support the proposed role of secretin in lipase regulation.

Acceleration of asynchronous enzyme transport in the rat exocrine pancreas following hormonal stimulation in vivo.

We have measured the rate of secretion in vitro of individual rat exocrine pancreatic enzymes and proenzymes from cells in pancreatic lobules of control animals and animals subjected to 24 h optimal hormonal prestimulation with cerulein, in vivo. With the controls secreted proteins were first detectable at 20 to 25 min of chase after a 5-min pulse label, and the amount of total secretion increased slowly thereafter. With stimulated lobules secretion was first detectable at 10 to 15 min postpulse. Comparison of the rates of secretion from control and experimental lobules showed that 24 h prestimulation in vivo resulted in a fivefold increase in the rate of secretion. Measurement of the rates of secretion of the individual enzymes and proenzymes showed that they were all increased to the same extent. In both situations the serine endoproteases, proelastase 2, chymotrypsinogen 1, and trypsinogen 1 and 2, were the most rapidly secreted proteins, while amylase, the procarboxypeptidases and prophospholipase A2 were amongst the slowest. The difference in the rates of secretion of proelastase 2 and prophospholipase A2, the two extremes, was three-fold before and after prestimulation, and their halftimes of secretion from the hormonally prestimulated lobules were 34 min and 190 min, respectively. Both electron microscopic autoradiography and immunocytochemistry showed that in the hormonally prestimulated cells the secreted proenzymes and enzymes followed the normal regulated exocytotic pathway and were transported between the Golgi apparatus and the apical plasma membrane in dense cored secretory granules.

Isolation and sequence of the canine pancreatic phospholipase A2 gene.

A genomic library has been constructed in EMBL3 lambda phage using high molecular mass DNA isolated from canine spleen. A cDNA clone, shown to code for preprophospholipase A2 which is processed to the prosecretory form prior to release from secretory cells, was used to identify a lambda clone which contains the complete phospholipase A2 gene. Restriction enzyme and DNA sequence analysis indicate that the primary transcriptional unit for the phospholipase gene, approximately 9.0 kb, is organized into four exon sequences. Exon 1 encodes the 5' nontranslated sequence, the ATG initiation codon, and the hydrophobic core of the signal peptide. Exons 2-4 encode regions of the peptide of residues -11 to 43, 43 to 86 and 86 to 124, respectively. The 5' flanking region shows a TATA box at position -29 and multiple CAAT boxes at positions -279, -206, -183 and -159. Regions of the 5' flanking sequence in the canine sequence, from nucleotides -47 to -74 and -91 to -129, show high similarity to similar regions in the human gene. However, an analysis of 400 nucleotides of the 5' flanking sequence in transient expression studies was unable to identify tissue-specific promoter or enhancer sequences. Within 5' nontranslated regions the canine and human genes share a pyrimidine-rich sequence which may be involved in differential regulation of mRNA translation.

Lipase synthesis in the rat pancreas is regulated by secretin.

Conscious rats were infused with optimal doses of secretin (16 clinical units [CU]/kg/h), cerulein (0.25 microgram/kg/h), and both for varying periods of time over 24 h. The presence of zymogen granules in acinar cells and the tissue content of enzymes showed progressive decreases over 3 and 12 h for cerulein and secretin stimulation, respectively. Stimulation with either hormone resulted in a two-fold increase in protein synthesis at 6 and 12 h. Kinetically, the increases observed in protein synthesis were not directly coupled to the observed decreases in tissue content of enzymes. Cerulein stimulation selectively increased the synthesis of anionic trypsinogen forms 1 and 2 and chymotrypsinogen forms 1 (anionic) and 2 (cationic) and decreased the synthesis of amylase. Secretin stimulation selectively promoted the synthesis of lipase and proelastase 2. Lipase synthesis was increased 2.8-, 5.8-, and 4.8-fold at 6, 12, and 24 h, respectively. Proelastase 2 synthesis was increased 2.3-, 3.0-, and 3.4-fold at the same time points. In combination, secretin and cerulein stimulation resulted largely in competitive effects, suggesting that the two hormones exert effects on protein synthesis through different mechanisms. The findings suggest that the anticoordinate changes observed in the synthesis of functional groups of pancreatic enzymes by nutritional substrates in the diet are modulated by specific hormones. Synthesis of the majority of protease zymogens but not cationic trypsinogen or proelastase 2 is modulated by cholecystokinin and its peptide analogs. In contrast, the synthesis of pancreatic lipase and proelastase 2 is regulated by secretin. The findings indicate for the first time that secretin regulates the synthesis of specific proteins in the exocrine pancreas.

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin. I. Changes in enzyme content, pancreatic fine structure and total rate of protein synthesis.

Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 micrograms X kg-1 X h-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU X kg-1 X h-1) and infusion period (1-24 h), except an increased number of coated vesicles in duct cells. Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin. II. Changes in individual rates of enzyme and isoenzyme biosynthesis.

Intravenous infusion of synthetic secretin for periods up to 24 h in conscious rats was combined with in-vitro amino acid incorporation in isolated pancreatic lobules and high-resolution separation of individual enzyme proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis. With this method persistent changes in the biosynthesis of ten enzyme and isoenzyme proteins can be studied as a result of prolonged secretin stimulation. Three major patterns of response were observed: progressive increases in the synthetic rates were found in six out of ten enzyme proteins with most pronounced changes in the synthetic rates of lipase (4.10-fold increase), two forms of proelastase (2.80-fold increase, respectively), the two acidic forms of trypsinogen and chymotrypsinogen (2.60- and 2.40-fold increase, respectively), and of ribonuclease (2.30-fold increase). Only moderate changes (1.30- to 1.90-fold increase) occurred in the synthetic rates of four isoenzymatic forms of procarboxypeptidase and the basic forms of chymotrypsinogen and trypsinogen, respectively. No absolute change in the rate of synthesis was observed in both forms of amylase. These data obtained after secretin stimulation differ significantly from previous results after caerulein stimulation, but it is not clear so far whether this is due to differential effects of the two second messengers released by each of the hormones on the level of transcription or translation.