Discordance of p53 status in matched primary tumours and metastases in head and neck squamous cell carcinoma patients.

To study the use of p53 as a diagnostic tool in head and neck squamous cell carcinoma (HNSCC), we analysed 15 primary tumours (PT) and matched lymph node metastases (LNM) for overexpression and mutations of p53. The primary goal was to study whether differentiation between primary and metastatic disease through their p53 status would be possible. Immunohistochemistry for p53 protein (antibody BP 53-12-1) was performed. Mutations of the p53 gene were detected by exon-specific amplification of DNA (exons 4-9), followed by exon analysis using denaturing gradient gel electrophoresis (DGGE). Mutant exons were sequenced. p53 overexpression was detected in seven (47%) of the PT and in seven (47%) of the LNM. 6 patients (40%) exhibited p53 protein overexpression in both PT and LNM. 2 patients had a different p53 protein expression in each sample. Mutations in the p53 gene were detected in 6 patients (40%) in the PT and in 7 patients (47%) in the LNM. In 2 patients (13%), the same mutation was found in the PT and in the LNM. 9 patients (60%) had a different mutation in each sample. We conclude that a poor correlation exists between p53 protein overexpression and p53 gene mutation in HNSCC. Also, a poor correlation for both detection techniques exists, when PT and LNM are compared. The p53 status may seem to differ between PT and LNM because of polyclonality in the PT. More sensitive detection techniques could be promising.

Radioresistance and p53 status of T2 laryngeal carcinoma. Analysis by immunohistochemistry and denaturing gradient gel electrophoresis.

BACKGROUND: Animal experiments and tumor cell line studies have shown that p53 alterations can cause radioresistance. This has not yet been demonstrated in patient groups. METHODS: We report p53 status in 20 patients with T2 laryngeal carcinoma and recurrent disease after curative therapy. The control group consisted of 16 patients with T2 laryngeal carcinoma without recurrent disease. The p53 gene (exons 5 to 9) was analyzed by Denaturing Gradient Gel Electrophoresis (DGGE). Expression of p53 in biopsy material was visualized by immunohistochemistry (monoclonal antibody BP 53-12-1). RESULTS: The group with recurrent disease showed a mutation in 9 cases (45%) and overexpression in 14 cases (70%). In 17 cases (85%) either mutation or overexpression was found. The control group showed a mutation in 7 cases (44%) and overexpression in 14 cases (88%). In 14 cases (88%) either mutation or overexpression was found. Adding up both groups a discordance of 50% was found between both detection techniques. The same mutated exon was found in 6 patients (66%) in both primary and recurrent tumors. CONCLUSIONS: A discordance between immunohistochemistry and DGGE exists in 50% of the cases. Assuming that both p53 mutation and p53 overexpression are indicative of a disturbed p53 checkpoint system, 31 cases (86%) in both groups show an alteration of the p53 system. No significant difference in p53 status in patients with or without recurrent disease exists. Analysis of the p53 status is not of prognostic significance for irradiation as primary treatment.

Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma.

Mutations in the K-ras oncogene and in the p53 tumor suppressor gene are commonly identified in sporadic cases of pancreatic adenocarcinoma. Although these genes might serve as useful markers for early diagnosis of pancreatic carcinoma in patients at risk for the development of this disease, familial pancreatic carcinomas have not been studied for these mutations. We recently had the opportunity to examine a pancreas prophylactically removed from a patient with a strong family history of pancreatic carcinoma. This gave us the unique opportunity to study the early events in the development of familial adenocarcinoma of the pancreas. Histopathological examination of the pancreas revealed multifocal papillary and nonpapillary mucinous duct hyperplasia. Seven of these foci were microdissected and analyzed for K-ras and p53 mutations. The K-ras mutations were detected by combined mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. Five of the seven duct lesions harbored activating point mutations in codon 12 of K-ras; a G to A transition was found in four and a G to C transversion in one. In contrast, these lesions did not harbor detectable p53 mutations as determined by denaturing gradient gel electrophoresis of exons 5 to 8, nor was there overexpression of the p53 protein as determined by immunohistochemistry. These findings suggest that mutations in K-ras represent an early event in the pathogenesis of pancreatic carcinoma. In addition, monitoring of patients with a strong family history of pancreatic carcinoma for K-ras mutations may identify patients at risk for the development of invasive carcinoma.

K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.

We examined 82 surgically resected or biopsied, formalin-fixed, paraffin-embedded primary adenocarcinomas of the pancreas for the presence of activating point mutations in codon 12 of the K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. This combination of mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and allele-specific oligonucleotide hybridization results in a rapid and sensitive characterization of the mutations in codon 12 of K-ras. Sixty-eight (83%) of the 82 carcinomas examined harbored a point mutation. Of the 68 mutations, 33 (49%) were guanine to adenine transitions, 27 (39%) were guanine to thymine transversions, and eight (12%) were guanine to cytosine transversions. Mutations were found in carcinomas of the head (61 of 75, 81%) as well as in carcinomas of the body or tail (seven of seven, 100%) of the pancreas. The overall prevalence of K-ras point mutations in adenocarcinomas of the pancreas obtained from patients who smoked cigarettes at some point during their lives (88%; 86% in current smokers and 89% in ex-smokers) was greater than that seen in pancreatic adenocarcinomas from patients who never smoked cigarettes (68%, P = 0.046). The presence of K-ras point mutations did not correlate with tumor ploidy, tumor proliferating index, or patient survival. These results demonstrate that primer-mediated, mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis combined with allele-specific oligonucleotide hybridization can be used to detect and characterize mutations in codon 12 of the K-ras oncogene in formalin-fixed, paraffin-embedded tissues, and the results confirm that activating point mutations in codon 12 of the K-ras oncogene occur frequently in adenocarcinomas of the pancreas.

Strong promoter activity of human and rat islet amyloid polypeptide/amylin gene constructs in mouse beta cells (beta TC 3).

Islet amyloid polypeptide (IAPP)('amylin') is co-produced with insulin in pancreatic beta cells and is the formative polypeptide of pancreatic amyloid in patients with type 2 (non-insulin-dependent) diabetes mellitus. Islet amyloid and type 2 diabetes occur in man, but not in rat. To study transcription regulation of IAPP gene expression in man and rat, luciferase reporter constructs containing different portions of the upstream region of both IAPP genes were expressed in transfected cells. Both the human and the rat IAPP gene constructs revealed higher promoter activity in beta cells (particularly in beta TC3 cells) than in non-beta cells. In both IAPP genes potential transcription elements, with homology to insulin gene transcription elements, were identified. beta TC3 cells provide a good model system in which to study regulation of human and rat IAPP gene expression.

No constant relationship between islet amyloid polypeptide (IAPP) and insulin expression in insulinomas.

The relationship between insulin and islet amyloid polypeptide (IAPP) expression was studied at the level of RNA and peptide in 3 patients operated on for insulinoma. Two patients had a solitary tumour; one patient suffered from the MEN-I syndrome. In the 3 tumours, as in normal pancreas, the same 2 IAPP-specific mRNA species, approximately 1600 and 2100 nucleotides, respectively, were detected. In 2 patients, the IAPP mRNA concentration of the tumour was lower than the insulin mRNA concentration; in the third patient, however, the specific IAPP hybridization signal was at least equal to that for insulin. Amyloid deposits were found in one solitary tumour and in the tumour from the MEN-I patient, both staining strongly positive with anti-IAPP antibodies; cytoplasmatic IAPP was weak. In conclusion, at least in some insulinomas, no constant relationship exists between insulin and IAPP expression.

Islet amyloid polypeptide (IAPP) is synthesized in the islets of Langerhans. Detection of IAPP polypeptide and IAPP mRNA by combined in situ hybridization and immunohistochemistry in rat pancreas.

We investigated the localization of IAPP mRNA by means of in situ hybridization in tissue sections of rat pancreas. A 35S-labeled, IAPP-specific DNA probe--hybridized specifically in the islets of Langerhans. This localization was confirmed by immunohistochemical localization of insulin and IAPP polypeptides on adjacent tissue sections. Moreover, combined in situ hybridization of IAPP mRNA and immunohistochemistry of insulin and IAPP polypeptide on the same section, using insulin as specific marker shows the presence of IAPP mRNA in the islets of Langerhans.

Amylin-induced in vivo insulin resistance in conscious rats: the liver is more sensitive to amylin than peripheral tissues.

UNLABELLED: Amylin is a polypeptide of 37 amino acids, predominantly synthesized in pancreatic Beta cells. The peptide was suggested to be dysregulated in Type 2 (non-insulin-dependent) diabetes mellitus and it antagonized certain actions of insulin in vitro in rat muscle. This led to speculation that amylin is involved in the pathogenesis of Type 2 diabetes. We have examined the in vivo effects of rat amylin, amidated at the carboxy-terminus, on insulin-mediated carbohydrate metabolism in conscious rats, using the hyperinsulinaemic (+/- 1 nmol/l) euglycaemic (6 mmol/l) clamp technique combined with [3-3H]-glucose infusion. Basal plasma amylin levels were less than or equal to 75 pmol/l. Applied amylin levels of 220 +/- 75 pmol/l (infusion rate of 12.5 pmol/min) antagonized only the insulin action on liver, resulting in a 100% increase of hepatic glucose output. Amylin levels of 4750 +/- 750 pmol/l (infusion rate of 125 pmol/min) induced a 250% increase of insulin-inhibited hepatic glucose output and, in addition, a 30% decrease of insulin-stimulated peripheral glucose up-take. Amylin did not affect: 1) the metabolic clearance rate of insulin, 2) the levels of plasma glucagon, epinephrine, norepinephrine, and corticosterone, 3) in vitro insulin binding and insulin-stimulated receptor autophosphorylation. This suggests that amylin antagonizes insulin action via binding to a yet unknown receptor. IN CONCLUSION: amylin causes in vivo insulin resistance and the liver seems the predominant organ regulated by this hormone. The in vivo effects of amylin mimic the pathophysiological abnormalities of insulin action in Type 2 diabetes.

The gene encoding proopiomelanocortin in the dog.

The regulation of the synthesis of ACTH in the dog is of interest for studies of the physiology of the pituitary-adrenocortical axis as well as for studies of the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite this broad interest the nucleotide sequence encoding ACTH and its precursor proopiomelanocortin (POMC) is not known, nor is it clear whether there are differences in POMC mRNA from the anterior lobe or the intermediate lobe of the normal pituitary or from pituitary tumours causing ACTH excess. Following the preparation of a cDNA library from the canine intermediate lobe of the pituitary gland, the part of the mRNA that is translated into the proopiomelanocortin prohormone was amplified using a polymerase chain reaction. Sequence analysis revealed the highest homology with the porcine mRNA sequence. Translation in a single reading frame revealed highly homologous areas in the amino-terminal, carboxy-terminal, and ACTH part of the prohormone, whereas a high diversity was noticed at the sequence preceding ACTH and the beginning of beta-lipotropin. Northern blot analysis disclosed the presence of a POMC mRNA of approximately 1300 nucleotides. There were no size differences between the anterior lobe, intermediate lobe, and pituitary tumour derived POMC mRNA. The highest expression levels of POMC mRNA as related to the expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase were found in the intermediate lobe of the canine pituitary gland. It is concluded that excessive production of ACTH by pituitary tumours is not caused by relatively high expression levels or alterations in the size of mRNA.

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.

Islet amyloid polypeptide: identification and chromosomal localization of the human gene.

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction.

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.

Two juxtaposed tyrosyl-OH groups participate in phi X174 gene A protein catalysed cleavage and ligation of DNA.

Bacteriophage phi X174 encoded gene A protein is an enzyme required for initiation and termination of successive rounds of rolling circle phi X DNA replication. This enzyme catalyses cleavage and ligation of a phosphodiester bond between nucleotide residues G and A at the phi X origin. The cleavage reaction which occurs during initiation involves formation of a free GOH residue at one end and a covalent bond between tyrosine-OH of the gene A protein and 5' phosphate of the A residue, at the other end of the cleavage site. During termination the covalently bound gene A protein cleaves the phosphodiester bond between G and A at the regenerated origin and ligates the 3' and 5' ends of the displaced genome-length viral DNA to form a circle. Since tyrosyl-OH mediated rearrangements of phosphodiester bonds in DNA may also apply to other enzymes involved in replication or recombination such as topoisomerases we have studied this interesting mechanism in greater detail. Analysis of 32P-labelled gene A protein-DNA complex by tryptic digestion followed by sequencing of 32P-containing peptides showed that two tyrosyl residues in the repeating sequence tyr-val-ala-lys-tyr-val-asn-lys participate in phosphodiester bond cleavage. Either one of these tyrosyl residues can function as the acceptor of the DNA chain. In an alpha-helix the side chains of these tyrosyl residues are in juxtaposition. An enzymatic mechanism is proposed in which these two tyrosyl-OH groups participate in an alternating manner in successive cleavage and ligations which occur during phosphodiester bond rearrangements of DNA.

Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein.

Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region. During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein. Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage. In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur. A model for termination of rolling circle replication which is based on these findings is presented. Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo. The functional significance of this cleavage in vivo is discussed.

The bond in the bacteriophage phi X174 gene A protein--DNA complex is a tyrosyl-5'-phosphate ester.

The bacteriophage phi X174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin. It leaves a free 3'-OH at nucleotide 4305 (G) of the phi X DNA sequence and binds covalently to the DNA. The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG[32P]ATATTAATAAC. This octadecamer, which corresponds to nucleotides 4299-4316 of phi X viral DNA, is cleaved by gene A protein. Gene A protein is bound to the labelled phosphate via a tyrosyl residue, indicating that binding occurs to the nucleotide corresponding to 4306 (A) of the phi X viral DNA strand.

Gene A protein of bacteriophage phi X174 is a highly specific single-strand nuclease and binds via a tyrosyl residue to DNA after cleavage.

The sequence specificity of the endonuclease activity of gene A protein and A* protein was studied using synthetic oligonucleotides containing (part of) the sequence of the origin of phi X RF DNA replication and single-stranded (ss) DNA fragments of phi X and G4. From a comparison of the sequences that are cleaved a consensus sequence for cleavage of ssDNA by gene A protein has been deduced. This consensus sequence occurs in ssDNA of both phi X and G4 at the origin and at one additional site. This is surprising since the rolling circle mechanism demands that gene A protein cleaves at the origin only. However, it could be shown that in the presence of SSB protein the ssDNAs of phi X and G4 are only cleaved at the origin, which is probably due to a strong gene A protein binding site, the key sequence, which forms part of the 30 b.p. origin region of phi X and related bacteriophages. Gene A protein and A* protein bind covalently to the DNA at the 5'-end of the cleavage site. Using a uniquely, internally 32p-labelled oligonucleotide as a substrate, it was shown that gene A protein and A* protein are bound via a tyrosyl residue to the 5'-phosphate of the phosphodiester bond which is cleaved.

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.