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Expression of the POMC gene in the adrenal gland and adrenocortical tumors causing

hypercortisolism in dogsWritten by J. Burghard

December 2009Supervisors:

S. GalacH.S. Kooistra

J.A. Mol

Department: Clinical Sciences of Companion animals, Faculty of Veterinary medicine, Utrecht University

Expression of the POMC gene in the adrenal gland and adrenocortical tumors causing hypercortisolism in dogs1

AbstractIntroduction: Hypercortisolism the most common endocrine disease in dogs. Chronic elevated plasma levels of cortisol result in clinical signs such as polyuria and polydipsia, polyphagia and alopecia. One of the causes for cortisol expression is an adrenocortical tumor (AT). So far, it was believed that AT secrete cortisol independently from the pituitary and ACTH. However, human research has shown that there is expression of adrenocorticotropic hormone (ACTH) and its precursor POMC in the tumors. In this research, the expression of POMC in ATs in dogs was studied in 26 carcinomas and 12 adenomas. For comparison also 15 normal adrenal cortexes served as control. Materials and methods: RT-PCR was performed on all the adrenal tissue samples. PCR products have been sequenced. After this, a qPCR was performed. Finally immunohistochemistry was done.Results: In the PCR, POMC expression was found in different samples, in both ATs and normal adrenal tissue. qPCR gave no reliable results. Results of the immunohistochemistry seemed to match with the results of the PCR, except for the normal tissue slides.


Contents

1. Abstract 22. Introduction

- Hypothalamic-Pituitary-Adrenal axis 4- Hypercortisolism 5

3. Materials and methods- Primers 9- cDNA 9- RT-PCR 10- qPCR 10- Immunohistochemistry 11

4. Results- RT-PCR 12- qPCR 12- Immunohistochemistry 13

5. Discussion 146. Acknowledgements 157. References 168. Appendices

1: Sample names 192: Protocol Immunohistochemistry 213: Results RT-PCR 22


2. IntroductionHypothalamic-Pituitary-Adrenal axisAdrenal cortisol secretion is regulated by the hypothalamus-pituitary-adrenal axis. In the hypothalamus corticotrophin-releasing hormone (CRH) is produced and secreted in the portal vessels of the pituitary gland. CRH-secretion is increased by stress and hypoglycaemia, plasma cortisol provides the negative feedback [1]. In the pituitary gland, the CRH stimulates corticotrophs, which account for 15% of the pituitary cells. They produce POMC, a precursor molecule which is cleaved in to multiple products (fig 1). One of them is adrenocorticotropic hormone (ACTH), a 39-amino acid peptide [2,3]. ACTH in the dog is secreted in pulses, ranging from six to twelve per 24 h period. ACTH is secreted into the blood and transported to the adrenal glands by the circulation [4]. The adrenal gland consists of a cortex and medulla. The cortex consists of 3 different zones: The outer zona glomerulosa that produces mineralocorticoids, the middle zona fasciculata which produces glucocorticoids (cortisol and corticosterone) and the inner zona reticularis which produces androgens and, to a minor degree, cortisol [2]. Negative feedback of pituitary corticotrophin-secretion is regulated by plasma cortisol [1]. Cortisol acts on the metabolism of carbohydrates, proteins and lipids. By doing so the plasma glucose levels are increased. Gluconeogenesis is increased by stimulation of the enzymes of the gluconeogenic pathway. For gluconeogenesis amino acids are needed. Cortisol provides these by stimulating the protein catabolism. Another action is reducing uptake of glucose by muscle and fat tissue. All of the above can lead to hyperglycemia, which can lead to insuline resistance. In order to still have a energy supply for the muscles, lipogenesis is decreased and lipolysis is increased, leading to a higher level of glycerol and free fatty acids. Cortisol also suppresses inflammatory and immunological responses, leading to destruction of tissue and fibroplasia. At high levels, cortisol can reduce the resistance to, for instance, bacteria and viruses.


Fig. 1: Cleavage of pro-opiomelanocortin (POMC) in ACTH and MSH, which are secreted simultaneously.ACTH: adrenocorticotropic hormone, MSH: melanocyte stimulating hormone, CLIP: corticotrophin-like intermediate lobe peptide, END: endorphin, LPH: lipoprotein, J peptide: joining peptide. (Ettinger et. al. 2005)

Hypercortisolism/Cushing’s syndromeIntroductionCushing’s syndrome or hypercortisolism is one of the most common endocrinological disorders in dogs. It is characterised by a chronic excess of plasma cortisol levels. Estimated incidence is 1 to 2 cases/1000 dogs/ year [5]. The median age in which Cushing’s syndrome occurs is 10 years. Some breeds seem to have a predisposition. In, among others, Poodles, Dachshunds, Beagles, Labrador retrievers and German Shepherds have a higher incidence of hypercortisolism then other breeds. Typical clinical signs in dogs are polyuria, polydipsia, polyphagia, panting, abdominal enlargement, endocrine alopecia, mild muscle weakness and lethargy [6, 7, 8]. There are three forms of this disease: a pituitary or ACTH dependent form, an ACTH independent form and an iatrogenic form. The first form, the pituitary dependent form, also called Cushing’s disease or the ACTH-dependent form. It is caused by a tumor in the pituitary that leads to an overproduction and excretion of ACTH. This tumor is usually located in the anterior lobe [6, 7, 9]. Because ACTH stimulates cortisol production in the adrenal gland, this results in hypercortisolism. In patients with hypercortisolism, this is the most common form. It occurs in about 80-85% of the patients [4, 6, 7].In ACTH-independent hypercortisolism, elevated cortisol secretion is a result of an adrenocortical tumor. This tumor produces cortisol independent from ACTH and suppresses ACTH secretion by negative feedback. The tumor can be either an adenoma or carcinoma and is usually unilateral. About 15-20% of the patients with Cushing’s syndrome have an adrenocortical tumor. Adenomas and carcinomas occur at the same incidence [7]. An andrenocortical tumour is considered carcinoma when the tumor size exceeds 2 cm in diameter and when there is histological evidence of invasion of neoplastic cells into blood vessels, peripheral fibrosis, capsular invasion, a trabecular growth pattern, hemorrhage, necrosis, and single cell necrosis. Typical histological characteristics for adenomas are hematopoiesis, fibrin thrombi, and cytoplasmic vacuolization [11]. They can metastasize to the lungs and liver. [5, 6, 7, 10].The third form, the iatrogenic form is caused by long-term or excessive administration of glucocorticoid drugs to the dog, usually by a veterinarian.Ectopic ACTH secretion has been described in dogs, but is rare [12].

DiagnosisFor diagnosing one of the three forms above, there are several endocrinological tests available. There are different screening tests, which can only indicate if hypercortisolism is present or not. The first and least invasive is measuring the urinary corticoid creatinine ratio (UCCR). For this test the owner collects morning urine samples on two consecutive days, the UCCR in these samples is averaged. This way the UCCR gives an indication of cortisol secretion over a period of time and is adjusted for fluctuations in plasma levels caused by the pulsatile release. Secondly, the sensitivity of the pituitary-adrenocortical system to suppression is tested with the low-dose dexamethasone suppression test. Dexamethasone is a glucocorticoid, which normally acts as a suppressor of the ACTH secretion from the pituitary. In dogs with hypercortisolism, the pituitary is very resistant to the


glucocorticoid feedback. Therefore in these dogs, after intravenously administrating dexamethason, there should be no decline in the plasma cortisol levels, while there should be a decline in healthy dogs. This test can have a false positive result due to stress. This can be avoided by combining UCCR measuring and administrating the dexamethason orally, which can be done by the owner at home [4, 6-9, 13,].

The next step to determine the actual cause of hypercortisolism is a high dose dexamethasone suppression test. In dogs with ACTH-dependent hypercortisolism, higher doses of dexamethasone usually overcome the resistance of the pituitary to glucocorticoid feedback. In dogs with ACTH-independent hypercortisolism the pituitary gland is chronically suppressed by the cortisol form the adrenal tumour, therefore, no matter how high the dose; it will not suppress plasma cortisol levels. Therefore a decrease of >50% of plasma cortisol confirms ACTH-dependent hypercortisolism. Often the test for diagnosing hypercortisolism is combined into one test with differentiating between the forms by using UCCR measurement together with oral dexamethason administration. When the high-dose dexamethason suppression test gives a decrease of <50% the hypercortisolism can still be ACTH-dependent, due to an extremely resistant pituitary tumour. To differentiate further measuring plasma ACTH is done. In patients with an adrenocortical tumor ACTH is usually suppressed. If interpretation of the ACTH values is uncertain the next steps are to perform a CRH-stimulation test and visualization of the adrenals and pituitary by computed tomography (CT) or nuclear magnetic resonance imaging (MRI)[4, 6-9, 13]. Measuring the plasma - MSH is also possible, high values are seen especially with tumors of the pars intermedia of the pituitary gland [4].Visualization of an adrenocortical tumor can, besides CT and MRI, also by ultrasonography, which is also used for examining the liver to search for metastases. To exclude metastases in the lungs thoracic radiographs or a CT scan should be made.


TreatmentWith all the different causes of hypercortisolism, there are also different ways of treating them. For a dog with ACTH independent hypercortisolism, the first choice is adrenalectomy, with the condition that there are no metastases. Because the non tumorous adrenocortical tissue is atrophic after the longstanding glucocorticoid excess, substitution of glucocorticoids with is needed. Substitution with hydrocortisone is started during the surgery and administered until oral medication with cortisone acetate is possible. This is gradually reduced and then stopped six to eight weeks after surgery [4, 6, 12, 13]. Adrenocortical tumours can also be treated medically. The adrenocorticolytic drug o,p’-DDD is used if tumour tissue cannot be completely removed during surgery, in case the disease recurs after an adrenalectomy is performed and also in cases of metastases. The aim is complete destruction of all adrenocortical cells. The downside of this is that the dog needs lifelong substitution therapy; on the upside are the chances of recurrence much lower.The third option is to use the competitive inhibitor of 3- β-hydroxysteroid dehydrogenase/isomerase system, named trilostane, which suppresses cortisol synthesis. Trilostane is used is neither adrenalectomy of o,p’DDD treatment are an option. It is also used as an palliative treatment in case of metastases. With this therapy also lifelong treatment is needed, because trilostane does not affect the tumour itself [4, 6, 12, 14].Dogs that have pituitary dependent hypercortisolism have two more options for treatment: microsurgical transsphenoidal hypophysectomy and radiation therapy. Both of these treatments can only be performed by a specialist. First a computed tomography (CT) scan is necessary for locating the tumour and assessing its size.In using hypophysectomy the cause of hypercortisolism in removed, however lifelong substitution therapy for thyroxine with L-thyroxine is necessary. In 83% of the patients, the plasma cortisol levels are normalised after surgery. In 10% of the dogs, the hypercorticolism comes back [12]. Radiation is mostly used as a way to reduce tumor size and should always be combined with one of the medical treatments [6-9].

PrognosisFor patients with a pituitary tumor the life expectancy is less then one year if there is no treatment. With treatment most animals can continue satisfactorily for several years [4]. Dogs with an adrenocortical tumor have a mean survival time of 36 months, after adrenalectomy. This time is dependent on the presence of metastases, if they are present, prognosis is poor. A dog then usually dies within one year. [7]


ResearchSo far,hypercortisolism in ATs has been explained by autonomic cortisol secretion independently from the pituitary and ACTH. However, human studies have shown that ectopic ACTH secretion is possible in different kind of tumors, for instance in breast and prostate cancer. These studies have demonstrated that these tumors also express POMC [16-23]. And recently Lefebvre et al [24,25] studied the POMC and ACTH expression in cortisol-secreting ATs. They reported that both ACTH and POMC were expressed in functional ATs.[24,25]. As mentioned before, in physiological circumstances ACTH is produced by the pituitary gland by cleavage of POMC. After being excreted in to the blood it is transported to the adrenal gland by the circulation. The POMC found in the studies by Lefebvre et al. [24,25] can not originate from the circulation. Because of the feedback-mechanism of the adrenal gland to the pituitary, the pituitary is suppressed by the large amounts of cortisol excreted by the tumor [6].Because of the differences between human Cushing’s syndrome and the disease in dogs, subject of this study was to determine if POMC and ACTH are expressed in ATs This will be done by using PCR to determine the possible expression of POMC and in case of positive results, the expression level by qPCR will be measured. Secondly immunohistochemistry of the ACTH will be performed to evaluate if the expression of the gene also leads to protein expression. If there is expression of the POMC gene it would explain why sometimes measurements of plasma ACTH levels in dogs with ATs are higher than expected. Besides that, it could also mean new possibilities of treatment for dogs. If removing the tumour is difficult because of its position or if the owner chooses not to do surgery, there can be targeted on blocking ACTH or its receptor, in stead of suppressing or destroying the adrenal cortex with drugs.


3. Materials and MethodsPrimersThe predicted sequence of the POMC gene, available on http://www.ncbi.nlm.nih.gov/ GI: 73979845 was used for developing a primer pair. The primers where made using the Primer Select module of the program DNA Star Lasergene (DNAstar, Inc., Madison, USA) for developing a primer pair. The following primers were used:Forward: 5’-CCTGCTGCTGGCCTTGCTGCTTCA-3’Reverse: 5’-CGCCCGCCGCCACCTCTTCTTCCT-3’ The primers where compared to the total DNA of the dog, to see if no other products could be formed. This was done using the Basic Local Alignment Search Tool (BLAST) at the website www.ncbi.nlm.nih.gov.

cDNAThe primers were used on cDNA made in an earlier student research project. For a few samples new cDNA was made. For deriving this cDNA the following steps where done:

1. TissuesATs were derived from dogs with hypercorticism, which have been presented at the Department of Clinical Sciences of companion animals at the Faculty of Veterinary medicine in Utrecht. After adrenalectomy the ATs were classified by a single pathologist and separated in two groups: adenomas and carcinomas. Classification was based on the extent of differentiation of the tumor cells, encapsulation, tumor growth pattern invasiveness, infiltration in surrounding tissues, presence of metastases and mitotic figures. Also a number of normal adrenals were used, derived from healthy dogs. Samples are listed in appendix 1.

2. RNA isolationThe RNeasy Mini Kit (Qiagen), was used to derive RNA from the frozen tissue samples, according to the protocol ‘Purification of total RNA from animal tissues’ in the RNeasy Mini Handbook (4th edition, Qiagen, April 2009). Homogenizing was done with a rotor-stator homogenizer. A DNA-ase-step was performed during RNA purification according to an additional protocol in the RNeasy Mini Handbook: the ‘optional on-column DNase digestion with the RNase-free DNase set’. After RNA isolation, the concentrations of the RNA were measured with a UV-Vis spectrophotometer (Nanodrop ND1000®, Thermo Fisher Scientific).

3. cDNAThe amount of RNA, needed for generating cDNA, was calculated out of the RNA concentrations. In total 1,5 μg RNA per sample was used. The cDNA was made using iScript (Biorad) and the PCR protocol on the right was used [26].


Table 1: cDNA protocolTemperature Time25°C 5 min42°C 30 min85°C 5 min4°C ‘forever’

http://www.ncbi.nlm.nih.gov/

RT-PCRFirst a RT-PCR was done to see if POMC is expressed in the adrenal cortex. The choice to start with RT-PCR, instead of directly starting with qPCR, was made because of the high levels of G and C nucleotides in the POMC gene. Therefore it was uncertain if the qPCR would work.The primers, together with the cDNA, MilliQ, a HF buffer, dNTP’s, Phusion Taq and MilliQ were inserted in to a C1000 thermal cycler (Biorad Laboratories, Inc). The following protocol was used:

The PCR product was loaded on a 1% agarose gel, together with a 100kb ladder for size comparison. After electrophoresis the gel was viewed with a Biorad Geldoc 2000 using the Quantity One software. All adrenal samples where also compared to a positive control, for which pituitary cDNA was used.

SequencingSequencing of the PCR-products was done with the ABI PRISM® 3130 xl Genetic Analyzer (Applied Biosystems) to verify if the right product was formed. The resulting sequence was compared with the sequence of the POMC-gene using the Basic Local Alignment Search Tool (BLAST) at the website www.ncbi.nlm.nih.gov. The following samples where used:

Table 3: Samples used for sequencingSample Tissue Number of samples for sequencingB Normal 4: forward and reverse primer, in duplicateG Normal 4: forward and reverse primer, in duplicateN Normal 4: forward and reverse primer, in duplicate6 Carcinoma 2: forward and reverse primerVIII Adenoma 2: forward and reverse primer

qPCRBecause of the results of the RT-PCR, the first set of primers where not suitable to use in qPCR. Therefore a second primer pair was used, also used in research in POMC expression in pituitary adenomas by Teshima et al [27, 28]. Primers:Forward: 5’-GGCCTCTGTGGAAGTGAGTG -3’Reverse: 5’-ACGCCAGCAGGTTACTTTCC-3’These primers where first used in a RT-PCR with the Sybr green PCR master mix, to see if product is formed with this mix. The same protocol as before was used, only at 60°C.Besides the primers used by Teshima also a combination of the forward primer from the first set and the reverse primer of Teshima’s pair was used. With these primers, besides the RT-PCR, a gradient from 55-65°C was performed using the qPCR machine (Biorad labratories Inc).


Table 2: RT-PCR protocol98 °C, 30 seconds45 cycles of 98 °C, 10 seconds

55 °C, 10 seconds72°C, 10 seconds

72 °C, 5 minutes

http://www.ncbi.nlm.nih.gov/

ImmunohistochemistryImmunohistochemistry was performed on a total of 37 slides.The following samples where used:

Slides where chosen on results from the RT-PCR and availability, not for all the samples used for PCR a slide was available. The protocol used for immunohistochemistry is found in Appendix 2. For staining a monoclonal mouse anti- ACTH (1-24) antibody was used, manufactured by the Department of Infectious Diseases and Immunology, of the Faculty of Veterinary Medicine at the Utrecht University. The slides are made from paraffin embedded blocks, on Superfrost and Silan coated glass.


Table 4 : samples used for immunohistochemistryCarcinoma Adenoma Normal tissue

1 I A5 II B6 III C7 IV D9 V F

10 VI G11 VII H12 VIII K13 IX L15 M1617202122232425

ResultsRT-PCRThe RT-PCR showed:Normal adrenals: 8 bands at the correct height (300bp) in 15 samples (53,3%).Carcinomas: 13 bands in 25 samples (52%)Adenomas: 5 bands of 10 samples (50 %)This PCR also showed a lot of byproducts, smaller than 100bp in size. Also bands with a size of 500bp are seen here. Pictures of remaining gels are included in appendix 3.

Figure 4: gel after 45 cycles

SequencingResults of sequencing:Table 6: sequencing resultsSample Tissue ResultPositive control Pituitary POMCB Normal POMCG Normal POMCN Normal POMC6 Carcinoma POMCVIII Adenoma Not useful for determination

qPCRWith the primers used by Teshima et al, the negative (water) controls keep turning out positive, even after ordering a complete new set, using filter tips and wearing gloves. Sequencing the product showed that is was POMC. With the combination of the first and second primer sets, the RT-PCR results of the negative controls where good (negative), but in the qPCR they where positive again. Therefore there are no results of the qPCR available.


Table 5: samples shown in fig 4.Line in gel Sample nr Tissue Result1 100bp ladder2 Pos control Pituitary +3 D Normal +4 7 Carcinoma +5 8 Carcinoma -6 9 Carcinoma -7 10 Carcinoma +8 11 Carcinoma +9 12 Carcinoma +10 13 Carcinoma +

ImmunohistochemistryImmunohistochemistry showed coloring in total of 19 tissue slides of both adenomas (5) and carcinomas (14). The adenomas showed more colored cells per slide. Interesting to see is that adenoma and carcinoma samples that where positive earlier in the PCR are now also positive and PCR-negative samples are here also negative. Another remarkable result is that none of the normal tissue slides showed coloring.

Table 7 : samples used for immunohistochemistryCarcinoma samples Adenoma samples Normal adrenal tissue samplesSample number PCR result

IHC result

Sample number PCR result IHC result

Sample number

PCR result IHC result

1 + + I + + A + -5 + + II No result - B + -6 + + III - - C - -7 + + IV - + D + -9 - + V + + F + -

10 + + VI + + G + -11 + + VII No result - H - -12 + + VIII + + K + -13 + + IX + + L - -15 No result - M - -16 - -17 - -20 + +21 + +22 - -23 + +24 + +25 - -

Figure 5 and 6: slide of sample 1 after IHC

Discussion


The results of the RT-PCR and immunohistochemistry from the present study demonstrate the POMC gene expression in ATs (both adenomas and carcinomas) and normal adrenal glands. However, POMC expression was not found in all of the samples. This could be an explanation why in blood test only some dogs with an AT have normal to low ACTH-levels. In the normal adrenals POMC expression was found by RT-QPC, however, the immunohistochemistry was negative. Another expectation was that adenomas would stain more intense than carcinomas, since these are better differentiated than the carcinomas. Adenomas also tend to give rise to higher plasma cortisol levels than carcinoma. As expected, immunohistochemisty of adenomas demonstrated more intense staining than it was present in carcinomas. Based on these results, it can be concluded that although there is gene expression in the normal adrenals, there is no expression of protein. It is possible that the gene is translated, but not converted into protein. This could be because the different cleavers that exist for POMC [29], it may be that the one to make ACTH is not present in the normal adrenals, maybe these cleavers are only activated in tumour, and even then, not in all of them. This could be a subject for further research. Looking at the results of the RT-PCR, there are a lot of byproducts formed. Even at higher temperatures they are still there. The products are smaller than 100bp, but to big to be primer-dimers. They were not sequenced to identify them. A big question still is why the qPCR was not successful and why the water-controls kept turning out positive. It looked like a contamination problem with the primers, but even after ordering a new pair the problem remained. One explanation is a manufacturer’s problem, but it still could be an unnoticed contamination. After all, Teshima et al [26,27] used the same primer sequences.By using a combination of primers the problem seemed solved in the RT-PCR, however in the qPCR the water controls came out positive again, probably because the qPCR is more sensitive. In the future, a different primer pair could be used.

AcknowledgementsExpression of the POMC gene in the adrenal gland and adrenocortical tumors causing hypercortisolism in dogs

14

First I would like to thank Sara Galac for the opportunity to do this project and Hans Kooistra for his help planning and getting this project started. Thanks to Jan Mol for good advices on this project. And last but not least I would like to thank Monique van Wolferen and Adri Slob for showing me around the lab and helping me with the work.

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Appendices

Appendix 1. Sample names


Table 1: Carcinoma samplesSample number Name owner Patient nr RNA conc (ng/μl)

1 Mathot 502937 1079,432 Schaap 805009 992,23 Verkuilen 805151 603,294 Akone 806093 234,35 Snoeij 807065 551,116 Bonebakker 808416 240,51/ 1143,367 Bennekom, van 802715 657,638 Zuiderwijk 803090 529,649 Does, de 311316 430,35

10 Verberk H01.0223B 416,3111 Verweel 228018 411,3312 Waldeck 600227 717,0813 Hazekamp 601184 404,9614 Keizer 602408 653,7415 Neumayer 506709 315,5716 Vermeulen 410807 252,4117 Askeland 608328 527,5418 Bergman 300154 537,5219 Bonefas 900143 645,8820 Bot 803755 464,8421 Doring 228380 382,2822 Fischer 600914 357,6823 Jellema 508028 448,8124 Struyk 216027 397,62/ 3014,1725 Veen, van der 303841 326,7526 Van de Reek 412393 827,9927 Ottens 700948 580,35

Table 3: Normal adrenal tissue samplesSample number Sample description RNA conc (ng/μl)

A BNR GDL rechts normaal 1-6-05 L 1365B Bijnier 11-10-05 GDL 233,73


Table 2: Adenoma samplesSample Number Name owner Patient nr RNA conc (ng/μl)

I Baljet 501795 863,57II Brouwer 221016 697,45III Geest, van 306624 534,06IV Groenedijk 216001 510,5V Harms H01.5333.C 430,94VI Ribbens 609558 785,85VII Rooy 225696 1225,34VIII Wilmes 400056 1291,62IX De Jong 603713 737,45X Van Laar H01.3118.Y 955,5XI Vente 702869 756,25XII Dijkstra 704792 658,43

C Bijnier (Vis? YS? Y5? Onduidelijk) 29-09-05 237,28D BNR normaal J 360,41E Bijnier 11-10-05 1483F 27-02-05 BN Boll (krabbel die op een 2 of 3 kan lijken?) 361,9G BN1 630H 0222753 L rig bijnier RE 754I BNL Beagle 5 G 1093,62J 6567 adrenal gland 14-09-05 Vera 604,55K Bijnier 2-11-05 K 292,69L GLD 16(0? Of iets doorgekrast?)5 bijnier links normaal E 518,63M BNR Beagle 5 Q 630,21N BN B (??) C 378,49O BN gezond 3(?) 794,75

Appendix 2: Protocol for Immunohistochemistry

Antibody: monoclonal mouse anti-ACTH (1-24)


1-Deparaffinize sections 5 min Xylene5 min Xylene3 min ethanol 96%3 min ethanol 80%3 min ethanol 70%2 min ethanol 60%2 min ethanol 30%5 min PBS

2- Antigen retrieval 1:100 antigen retrieval solution Vector H-33001.boiled in microwave 2 ½ min at 1000W, 10 min packed in tin foil, 10 min unwrapped cooling down

3-2x 5 min PBS 4-Incubate 5 minutes in H2O2 1% (1.71mL 35% in 60 mL MQ)5-Dip in dH2O6-Incubate 5 minutes in PBSFrom this point on, everything is done in PBS supplemented with 0.1% Tween (PBT) (1 ml Tween and 100 ml PBS 10 X to 1 l MQ)7-Cover with 10% Normal Goat Serum (NGS) in PBT. Incubate 60 min8-Cover slides with primary antibody diluted in 10% NGS/PBT. Incubate overnight (4°C) (anti-ACTH 1:1002) 9-Wash slides 2 X 5 minutes in PBT10-Cover slides with secondary antibody (Envision Goat anti-mouse3) Incubate 45 min.11-Wash slides 2 X 5 minutes in PBT12-Reveal antigen-antibody complexes: DAB (See protocol Vector laboratories)13- Wash slides in MQ water14-Counsterstain with hematoxylin4

15- Wash in running tap water 10 min16- Mount with aqueous mounting medium5

1 Vector labratories Inc, Burlingame. 2 Department of Immunology, Faculty of Veterinary Sciences, Utrecht 3 DakoCytomation Envision+ ® System Labelled Polymer- HRP4 Vectorlab Inc, Hematoxylin OS H-34045 Vectamount H-5000, permanent mounting medium, Vectorlabratories Inc, Burlingame

Appendix 3: RT-PCR results


Figure 1: gel with normal and carcinoma samples


Table 4: samples and results in figure 1Line in gel Sample no. Tissue Result

1 100bp ladder2 Pos control Pituitary +3 A Normal +4 B Normal +5 C Normal -6 E Normal -7 F Normal +8 G Normal +9 H Normal -10 I Normal -11 J Normal -12 K Normal +13 L Normal -14 M Normal -15 N Normal +16 O Normal -17 1 Carcinoma +18 2 Carcinoma -19 3 Carcinoma +20 4 Carcinoma -

Figure 2: gel with carcinoma and adenoma samples


Table 5: samples and results in figure 2Line in gel Sample no. Tissue Result1 100bp ladder2 Pos control Pituitary +3 5 Carcinoma +4 6 Carcinoma +5 16 Carcinoma -6 17 Carcinoma -7 18 Carcinoma -8 19 Carcinoma -9 20 Carcinoma +10 21 Carcinoma +11 22 Carcinoma -12 23 Carcinoma +13 24 Carcinoma +14 25 Carcinoma -15 26 Carcinoma -16 27 Carcinoma -17 I Adenoma +18 III Adenoma -19 V Adenoma +20 VI Adenoma +

Figure 3: gel with remaining adenoma samples


Table 6: samples and results of figure 5Line in gel Sample no. Tissue Result1 100bp ladder2 Pos control Pituitary +3 IV Adenoma -4 VIII Adenoma +5 IX Adenoma +6 X Adenoma -7 XI Adenoma -8 XII Adenoma -

expression of the pomc gene in the adrenal gland and ... · web viewexpression of the pomc gene in...

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