Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma.

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational "hot spots" in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single-strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13-16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the "hot spots" domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.

Novel somatic MEN1 gene alterations in sporadic primary hyperparathyroidism and correlation with clinical characteristics.

Primary hyperparathyroidism (pHPT) is a common endocrine disease that in more than 95% of cases is sporadic and only in some cases is caused by inherited disorders, isolated or as part of multiple endocrine neoplasia (MEN1 and 2). Somatic mutations of MEN1 gene have also been described in sporadic parathyroid tumors. In our study, we examined the presence of alterations in MEN1 gene in a series of 39 patients who had undergone surgery for sporadic pHPT (35 with parathyroid adenoma or hyperplasia, 4 with a carcinoma). A genotype-phenotype correlation was also analysed. After DNA extraction from paraffin-embedded tissues, we amplified by PCR and sequenced the exons 2-10 of the MEN1 gene. Somatic MEN1 mutations were detected in 6 of the 35 patients with a benign parathyroid lesion examined (17.1%), whereas no alterations were found in the carcinomas. Four novel MEN1 gene mutations were identified as follows: one frameshift mutation (222insT, exon 2), one frameshift deletion (912delTA, exon 5), one in-frame deletion (835del18, exon 4) and one missense mutation (P291A, exon 6). In addition, one missense mutation (L89R, exon 2) and one nonsense mutation (Q536X, exon 10) were previously reported. Moreover, two polymorphisms were also found: one allele carried a R171Q polymorphism (1/39 tumors), while a D418D polymorphism (GAC/GAT) was found in 15 and 8 tumors in hetero (CT) and homozygosity (TT), respectively. In no case (mutations and/or polymorphisms) did we find a genotype-phenotype correlation. In conclusion, our data demonstrate the presence of somatic alterations of the MEN1 tumor suppressor gene in about one fifth of benign sporadic parathyroid tumors. The absence of a genotype-phenotype correlation, however, suggests the involvement of other genetic/epigenetic factors for the full expression of the disease.

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

Thyrotropin receptor mutations and thyroid hyperfunctioning adenomas ten years after their first discovery: unresolved questions.

Ten years after the first description of activating mutations in the thyroid stimulating hormone receptor (TSHR) gene in sporadic autonomous hyperfunctioning thyroid adenomas, there is general agreement in assigning a major pathogenic role of this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristic of these tumours. From the beginning, however, the pathophysiological and clinical relevance of somatic TSHR mutations has been debated and some arguments still exist against a fully causative role of these mutations and the practical value of detecting these mutations for the diagnosis, treatment and prognosis of thyroid hot nodules. Some major issues will be examined herein, including (a) the frequency of TSHR alterations in various reports showing that the genetic abnormality underlying the pathogenesis of a substantial subset of thyroid tumours has yet to be identified; (b) the limitations of the present experimental models, which suggest greater caution in the interpretation of in vitro results; (c) the still unresolved question of absence of genotype-phenotype correlation. Clarification of these issues may hopefully provide new and useful tools for improving the clinical management of this disease.

Expression pattern of the pendrin and sodium/iodide symporter genes in human thyroid carcinoma cell lines and human thyroid tumors.

OBJECTIVE: In the present study we analyzed the pattern of pendrin (PDS) and sodium/iodide symporter (NIS) gene expression in some thyroid carcinoma cell lines and a series of thyroid tumoral tissues. METHODS: Total RNA was extracted from all cell lines and from 53 tissues, and gene expression was examined by RT-PCR. Semiquantitative 'multiplex' RT-PCR was used to assess variations in PDS gene expression among various thyroid pathologies. Pendrin expression was determined in the thyroid cell lines by Western blot analysis. RESULTS: PDS mRNA was expressed in all the cells investigated; conversely, NIS mRNA was detectable only in the B-CPAP cells. Pendrin protein was expressed in B-CPAP and WRO cell lines, reduced in FRO and absent in ARO cells. PDS gene expression was not detected in 5 of 25 differentiated thyroid carcinomas (DTC) while NIS gene was not expressed in six carcinomas. A concordance expression of both PDS and NIS transcripts was found in 20 DTC. In contrast, 2 neoplastic thyroid tissues carrying undetectable PDS mRNA maintained NIS transcript, and 3 thyroid carcinomas negative for NIS mRNA retained the expression of PDS gene. A semiquantitative analysis showed that the mean PDS mRNA levels were significantly decreased in DTC tissues. CONCLUSIONS: Our data demonstrate that pendrin expression: (i) is present in the more differentiated thyroid carcinoma cell lines studied; (ii) is reduced or absent in DTC tissues; (iii) may not correlate with the NIS expression. These alterations may contribute to the loss of iodine concentration ability detected in thyroid tumors.

Organization and expression of the human zo-2 gene (tjp-2) in normal and neoplastic tissues.

One of the tight junction components, zonula occludens protein 2 (ZO-2), is expressed as two isoforms, ZO-2A and ZO-2C, in normal epithelia. In pancreatic adenocarcinoma of the ductal type ZO-2A is absent, but none of the common mechanisms of gene inactivation is responsible for lack of ZO-2A expression. In the current study, we report the complete organization of the human zo-2 gene (tjp-2), its alternative splicing, and its expression in normal and neoplastic tissues of several organ sites. In addition to pancreatic adenocarcinoma, ZO-2 was found to be de-regulated in breast adenocarcinoma, but not in colon or prostate adenocarcinoma. The latter are considered to be of acinar rather than ductal type. Thus, our data indicate the importance of zo-2 (tjp-2) gene regulation in ductal cancer development and should help to understand the defects of intercellular interactions, critical for suppressing the malignant phenotype.

zo-2 gene alternative promoters in normal and neoplastic human pancreatic duct cells.

We have observed that 2 forms of zonula occludens 2 (ZO-2) protein, ZO-2A and ZO-2C, are expressed in normal human pancreatic duct cells, but only ZO-2C in pancreatic duct adenocarcinoma. We report here partial organization of the zo-2 gene. Transcription of 2 forms of ZO-2 mRNA is driven by alternative promoters P(A) and P(C). Lack of expression of ZO-2A in neoplastic cells is caused by inactivation of the downstream promoter P(A). Analysis of the promoter P(A) sequence and function in normal and neoplastic cells demonstrated that neither structural changes (mutations) nor a change in the pool of transcription factors is responsible for its inactivation. Although hypermethylation was found in a large number of cancer clones, treatment with 5-aza-2'-deoxycytidine did not fully cause the promoter function to recover. We conclude that the initial down-regulation of zo-2 promoter P(A) activity in pancreatic duct carcinomas is due to the structural or functional alteration(s) in the regulatory elements, localized outside the analyzed promoter region. Methylation of P(A) is responsible for the inactivation of the suppressed promoter at the late stages of tumor development.

Tight junction protein ZO-2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma.

Differential display of hamster mRNA identified a fragment present in normal pancreatic duct cells that is not expressed in pancreatic duct carcinoma cells. Sequence analysis showed an 88% and 82% identity, respectively, to the cDNA of the canine and human tight junction zo-2 gene. Semi-quantitative RT-PCR analysis of human ZO-2 revealed a striking difference in the expression of various regions of the ZO-2 transcript in normal and neoplastic cells and the presence of an abnormality at the 5'-end of mRNA. RACE analysis identified 2 human ZO-2 mRNAs that encode proteins of different lengths, designated as ZO-2A and ZO-2C. The difference between the 2 forms of ZO-2 is the absence of 23 amino acid residues at the N terminus of ZO-2C compared with ZO-2A. Although ZO-2C was expressed in normal pancreatic cells and a majority of neoplastic tissues analyzed, ZO-2A was undetectable except in one case in all of the pancreatic adenocarcinomas analyzed. This suggests the presence of a yet to be identified motif important for cell-growth regulation within the 23-amino acid residue N-terminal peptide of ZO-2A, MPVRGDRGFPPRRELSGWLRAPG.

Hypersensitivity pneumonitis due to humidifier disease: seek and ye shall find.

STUDY OBJECTIVES: This study reports a classic case of hypersensitivity pneumonitis (HP) with classic histologic changes in lung tissue and the research used to identify the causative antigens. DESIGN: A patient with clinical, radiographic, pulmonary function abnormalities and a lung biopsy consistent with HP had no identifiable antigen exposure. SETTING: Evaluation of the patient's activities provided no suggestion of antigen exposure. Her home was evaluated. It was found that her humidifier ran continually without being cleaned but water was added periodically. MEASUREMENTS: Serologic analysis demonstrated precipitating antibodies against her humidifier water and ten antigens in the hypersensitivity lung disease serologic panel. CONCLUSION: Removal of the humidifier, cleaning of the house, and a course of prednisone resulted in the return of the patient to a normal state.

Multiple genetic alterations in hamster pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinomas induced in the Syrian golden hamster (SGH) by N-nitrosobis(2-oxopropyl)amine share many similarities with the human disease, including mutations of the K-ras oncogene. In vitro carcinogenesis studies with immortal SGH pancreatic duct cells indicate that neoplastic transformation in this system can occur without mutational inactivation of p53 suppressor gene. In this study we extend the genetic analysis of the in vivo SGH model to increase the number of cases analyzed for the status of K-ras and to determine further the spectrum of alterations involved; we have studied the status of the p53, DCC, and Rb-1 suppressor genes and the status of the mdm2 oncogene, which can involve p53 indirectly. The partial SGH-coding sequence of mdm2 and DCC was determined. K-ras mutation in the second position of codon 12 was present in 17 of 19 (90%) of tumors. Immunohistochemistry and single strand conformation polymorphism analysis showed no evidence of p53 mutation in 21 tumors. RNase protection assays showed overexpression of mdm2 in 5 of 19 (26%) tumors. Semiquantitative reverse transcription-PCR analysis showed a complete or partial loss of DCC expression in 10 of 19 (53%) neoplasms and of Rb-1 (42%) expression in 8 of 19 tumors when compared to matched controls. Deregulation of these genes appears to be significant in SGH pancreatic carcinogenesis as indicated by their frequencies. However, the fact that 6 tumors showed either only a K-ras mutation or the absence of alterations of the 5 genes analyzed indicates that additional as yet unstudied or unknown genes are also involved in SGH pancreatic duct carcinogenesis.

In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations.

Neoplastic transformation of Syrian golden hamster (SGH) pancreatic duct cells was induced by in vitro treatment with the direct-acting carcinogens N-methylnitrosourea (MNU) and N-(2-hydroxypropyl)nitrosourea (HPNU), with subsequent selection by sustained culture in serum- and epidermal growth factor (EGF)-deprived medium. The present study examines the efficacy of serum and EGF deprivation as a selection pressure and the effect of the carcinogen dose, frequency and interval of exposure on tumorigenesis and K-ras mutation. Selection of carcinogen-initiated duct cells by serum and EGF deprivation is highly reproducible and effective, increasing the incidence of tumors from 26 to 93% for MNU or from 0 to 100% for HPNU. SGH pancreatic duct cells exposed to 0.5 mM MNU for 13 weeks (long-treatment schedule) produced K-ras mutations at codon 12 in six of six tumors. However, when cells were exposed to 0.125, 0.25 or 0.5 mM MNU daily for 5 days (short-treatment schedule), mutations of K-ras at codon 13 were identified in four of 16 tumors, the remaining 12 showing no mutations. Duct cells exposed to 0.5 mM HPNU by the short-treatment schedule produced K-ras mutations in codon 13 in six of six tumors, as contrasted to 12 tumors that developed from cells exposed to 0.125 or 0.25 mM HPNU, which all contained K-ras codon 12 mutations. The current experiments demonstrate that K-ras mutation in pancreatic carcinogenesis in vitro by MNU or HPNU can be modified by the nature and dose of the carcinogen as well as the frequency and duration of exposure.

Genomic p53 mutation in a chemically induced hamster pancreatic ductal adenocarcinoma.

This study investigates possible alterations in exons 5 through 8 of the p53 gene and altered p53 protein expression in the Syrian golden hamster model of pancreatic ductal adenocarcinoma. In the Syrian hamster p53 sequence, 1469 base pairs are presented for the genomic regions surrounding exons 5 through 8, along with the primer sequences specific for the enzymatic amplification of the individual exons. Single-stranded conformation polymorphism was analyzed on the products obtained by enzymatic amplification of hamster genomic DNA extracted from 2 transplantable pancreatic ductal adenocarcinomas, 6 groups of N-methylnitrosourea (MNU)-treated pancreatic duct cells, and 17 MNU-induced pancreatic ductal adenocarcinomas. The two transplantable adenocarcinomas were a well-differentiated ductal adenocarcinoma established from a N-nitrosobis(2-oxopropyl)amine-induced tumor and a poorly differentiated ductal adenocarcinoma established from a spontaneous tumor. An altered mobility indicated a conformational change in the first part of exon 5 in the solid form of the well-differentiated ductal adenocarcinoma. Direct sequencing of the amplified product revealed an A-->C transversion in codon 135, which corresponds to codon 132 in the human p53 gene. A conformational change in exon 7 was observed in 1 of 6 MNU-treated cell samples, and none of the 17 resultant tumors. Direct sequencing confirmed a deletion of one C of the three in codon 263, which generates a frameshift mutation. No conformational change was observed in any other products. Positive staining with PAb240 or DO7 antibodies against human p53 or with an antibody generated in our laboratory against the hamster p53 fusion protein was observed only in the solid form of well-differentiated ductal adenocarcinoma and in rare cells scattered in 4 of 28 MNU-induced tumors analyzed. This study provides a system to analyze p53 gene alterations in the hamster and is the initial report of a specific p53 mutation in a hamster pancreatic ductal adenocarcinoma.

Exploring pathogenetic mechanisms using transgenic animals.

Molecular technologies for the permanent germ-line transformation of animals are now well established and routine. These new strains of animals, called transgenic, offer an unprecedented opportunity to gain a basic understanding of human genetic disorders. In this brief review we discuss the role of transgenic animals in the creation of new models of human disease and their experimental use in biomedical research. Models are now available for the study of the genetic processes involved in the pathogenesis of neoplasia, diabetes, atherosclerosis, and developmental abnormalities. Many others are available and new ones are being produced at a great rate. Principles of gene replacement therapy are amenable to analysis with transgenic animals and the information gained will be important for the development of rational therapy.

K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster.

K-ras oncogene mutation has been shown to be a frequent event in pancreatic ductal adenocarcinomas induced by the carcinogen N-nitroso-bis(2-oxopropyl)amine in the hamster. The present study examines the mutational status of the K-ras oncogene in lesions that precede the appearance of invasive ductal adenocarcinomas. Syrian golden hamsters (80-100 g) received 12 weekly doses of 15 mg/kg N-nitroso-bis(2-oxopropyl)amine and were serially sacrificed at 8, 12, 14, 16, or 24 weeks following the initiation of treatment. Ten microns-thick sections of formalin-fixed paraffin-embedded pancreas were examined for hyperplasia, papillary hyperplasia, carcinoma in situ, and invasive and metastatic ductal carcinoma. Marked lesions of interest were scraped from the slide, subjected to polymerase chain reaction-mediated amplification of the first exon of the K-ras gene, and probed by oligonucleotide-specific hybridization for mutations at codon either 12 or 13. Of 186 samples assayed, K-ras codon 12 mutations were detected in 26% of hyperplasias, 46% of papillary hyperplasias, 76% of carcinoma in situ, 80% of adenocarcinomas, and 43% of lymph node metastases. Codon 12 mutations were exclusively G to A changes at the second position. Codon 13 mutations were only detected in 9 of 168 samples. These results suggest that K-ras activation is an early event in N-nitroso-bis(2-oxopropyl)amine-induced pancreatic carcinogenesis in the hamster.

Stimulation of DNA synthesis in pancreatic duct cells by gastrointestinal hormones: interaction with other growth factors.

Pancreatic duct cells of the Syrian hamster were grown as monolayers on thin layers of type I collagen coated onto microporous membranes. The effects of a number of potential trophic factors were tested by their ability to increase [3H]thymidine incorporation into cellular DNA. To measure the effect of growth factors, cells were subjected to a period of growth factor depletion to induce a state of partial quiescence in DNA synthesis. Cells responded with a significant increase in thymidine incorporation after the addition of epidermal growth factor (EGF) alone or a growth factor mixture containing EGF plus insulin, transferrin, selenium, linoleic acid, bovine pituitary extract, triiodothyronine, and dexamethasone. When the serum substitute, Nu Serum IV (5%, vol/vol), was added to this mixture, addition of several gastrointestinal (GI) hormones including secretin, vasoactive intestinal polypeptide (VIP), bombesin, and gastrin caused significant increases in thymidine incorporation at concentrations of 0.01-1 microM. At 1 microM, these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) greater than bombesin (153%) greater than VIP (138%) greater than gastrin (126%). Cholecystokinin octapeptide, a known trophic factor for pancreatic acinar cells, did not cause significant increases in thymidine incorporation in cultured duct cells. These results suggest that pancreatic duct cells possess receptors for a number of GI hormones and respond to the trophic effects of hormones known to stimulate pancreatic growth in vivo.

Prognostic implications of proliferative activity and DNA aneuploidy in Astler-Coller Dukes stage C colonic adenocarcinomas.

Paraffin-embedded surgical specimens from 69 patients who underwent resections of otherwise untreated Dukes stage C adenocarcinoma of the colon were examined for proliferative activity, DNA aneuploidy, DNA index, and proportion of aneuploid cells by flow cytometry. Results were correlated to clinical characteristics of the patients and to overall survival times. DNA aneuploid tumors were identified in 60 cases (87%), diploid tumors in 9 cases (13%). The mean S-phase fraction for all cases was 17.6%, with a standard deviation (SD) of 7.8. In univariate statistical analysis, younger patient age, lower tumor proliferative activity, DNA index less than or equal to 1.2, and presence of only 1-4 lymph nodes with tumor involvement were found to be significant predictors of improved patient survival. In multivariate Cox regression analysis, low tumor proliferative activity, younger patient age, and location of the tumor in the right or transverse colon were found to be significant independent predictors of increased patient survival. When tumor proliferative activity was stratified into statistically defined subgroups, patients with tumors of low proliferative activity (S-phase less than mean - 0.5 SD) had significantly longer survival than patients with tumors of moderate proliferative activity (S-phase value greater than mean - 0.5 SD and less than mean +0.5 SD) or high proliferative activity (S-phase greater than mean +0.5 SD). These results suggest that tumor proliferative activity in Dukes C colon carcinoma may be an important biological factor in determining patient prognosis.

Enhancement of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine-induced tumorigenesis in Sprague-Dawley rats by orotic acid.

The effect of orotic acid (OA) on the carcinogenicity of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) in rats was evaluated. A group of 5 week old Sprague-Dawley male rats were placed on a synthetic 20% protein diet containing 1% OA. A second group was placed on a regular, OA-free diet of similar composition. Approximately 2 weeks later, animals from both groups grown to 100 g were treated with 400 mg/kg HPOP delivered continuously for 14 days via 2002 Alzet osmotic pumps implanted s.c. Rats fed the OA diet were kept under this diet for 13 weeks following initiation of HPOP treatment and subsequently were placed on the regular diet for another 12 weeks, at which time they were killed. In the absence of OA, HPOP-treated rats developed adenomas in the kidney and lungs at incidences of 5 and 33% respectively, while pancreas and liver were unaffected. On the other hand, rats fed the OA diet and treated with HPOP developed renal mesenchymal tumors and pulmonary adenomas at incidences of 70 and 65% respectively. In addition, HPOP induced cystic lesions in the pancreas of animals fed the OA diet. The enhancement of the tumorigenic effectiveness of HPOP was at least partly ascribed to the effect of OA treatment on the rate by which carcinogen-induced alkylation of DNA was repaired in various tissues. Accumulation of N7-methylguanine in kidney, lung and pancreas of rats fed the OA diet was 1.6, 1.9 and 2.4 times higher than in respective organs of animals fed the regular diet. Similarly, concentrations of the premutagenic O6-methylguanine (O6-MeG) were 3.0, 3.1 and 2 times greater in the kidney, lung and pancreas of rats fed the OA diet than in the respective organs of those fed the regular diet. Feeding an OA diet to HPOP-treated rats did not have an effect on either the resistance of the liver to this carcinogen or on the level of O6-MeG accumulation in the DNA of this tissue.