In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors

Summary The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with ³⁵S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors.

The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with 35S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: an in situ hybridization study.

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.     

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.     

In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using ³²P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.     

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.     

Localization of type 7 17beta-hydroxysteroid dehydrogenase in mouse tissues. In situ hybridization studies

The enzyme type 7 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estrone (E1) into estradiol (E2). In order to obtain detailed information about the exact sites of action of type 7 17beta-HSD, we have studied the cellular localization of type 7 17beta-HSD mRNA in mouse tissues using in situ hybridization (ISH). In parallel studies, we also measured the enzyme mRNA levels by quantitative real time (RT)-PCR. In the ovary, strong hybridization signal was restricted to corpus luteum cells. In the female mammary gland, type 7 17beta-HSD mRNA was found to be expressed in stromal cells surrounding the ducts. In the clitoral and preputial glands, specific labeling was observed in the epithelial cells of both acini and small ducts. In the adrenal gland, hybridization signal was observed in the zona fasciculata and reticularis in the cortex. In the liver, hybridization signal was found in all the hepatocytes. In the colon, type 7 17beta-HSD mRNA expression was restricted to epithelial cells of the mucosa. From the results obtained with quantitative real time RT-PCR, it appears, with a very few exceptions, that in tissues exhibiting low mRNA expression no ISH signal could be detected. The present data suggest that E2 can be formed through the action of type 7 17beta-HSD in specific cell types in the ovary and peripheral tissues, in addition to type 1 17beta-HSD, thus providing tissues with an alternative route of formation of E2.     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization.

The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.     

Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization

Apolipoprotein D (apoD) and lecithin:cholesterol acyl transferase (LCAT) are found on high density lipoprotein particles (HDLs) and have been postulated to form part of a complex involved in the transport of cholesterol from peripheral tissues to the liver for excretion. We have examined the sites of synthesis of the mRNAs for these two proteins in the rhesus monkey by in situ hybridization. ApoD mRNA-containing cells were widely distributed throughout peripheral tissues in interstitial and connective tissue fibroblasts often associated with blood vessels or capillaries. ApoD mRNA was also found localized in cells associated with peripheral nerves, neuroglial cells, cells in the subarachnoid space on the surface of the brain including the pial cells, perivascular cells, and scattered neurons in the brain. LCAT demonstrated a much more restricted pattern of synthesis and was found to be synthesized by hepatocytes, the basal cell layer of the epidermis, and in brain cell populations distinct from those that synthesize apoD. In the brain LCAT was synthesized by scattered neurons, neuroglial cells, ependymal cells, as well as a discrete cell layer in the cerebellum. ApoD has been shown to possess extensive homology to retinol binding protein, which has a binding pocket for vitamin A. We propose that apoD may also function to bind cholesterol or its derivatives in compartments not in direct contact with the blood. The findings of both apoD and LCAT synthesis in the brain suggest that they play a significant role in lipid transport in the brain.     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.