Involvement of the cGMP pathway in mediating the insulin-inhibitory effect of melatonin in pancreatic beta-cells.

Recent investigations have demonstrated an influence of melatonin on insulin secretion in pancreatic beta-cells. The effects are receptor-mediated via two parallel signaling pathways. The aim of this study was to examine the relevance of a second melatonin receptor (MT2) as well as the involvement of a third signaling cascade in mediating melatonin effects, i.e. the cyclic guanosine monophosphate (cGMP) pathway. Our results demonstrate that the insulin-inhibiting effect of melatonin could be partly reversed by preincubation with the unspecific melatonin receptor antagonist luzindole as well as by the MT2-receptor-specific antagonist 4P-PDOT (4-phenyl-2-propionamidotetraline). As melatonin is known to modulate cGMP concentration via the MT2 receptor, these data indicate transmission of the melatonin effects via the cGMP transduction cascade. Molecular investigations established the presence of different types of guanylate cyclases, cGMP-specific phosphodiesterases and cyclic nucleotide-gated channels in rat insulinoma beta-cells (INS1). Moreover, variations in mRNA expression were found when comparing day and night values as well as different states of glucose metabolism. Incubation experiments provided evidence that 3-isobutyl-1-methylxanthine (IBMX)-stimulated cGMP concentrations were significantly decreased in INS1 cells exposed to melatonin for 1 hr in a dose- and time-dependent manner. This effect could also be reversed by application of luzindole and 4P-PDOT. Stimulation with 8-Br-cGMP resulted in significantly increased insulin production. In conclusion, it could be demonstrated that the melatonin receptor subtype MT2 as well as the cGMP signaling pathway are involved in mediating the insulin-inhibiting effect of melatonin.

WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors.

BACKGROUND: It is difficult to predict the biologic behavior of pancreatic endocrine tumors in absence of metastases or invasion into adjacent organs. The World Health Organization (WHO) has proposed in 2004 size, angioinvasion, mitotic activity, and MIB1 proliferation index as prognostic criteria. Our aim was to test retrospectively the predictive value of these 2004 WHO criteria and of CK19, CD99, COX2, and p27 immunohistochemistry in a large series of patients with long-term follow-up. DESIGN: The histology of 216 pancreatic endocrine tumor specimens was reviewed and the tumors were reclassified according to the 2004 WHO classification. The prognostic value of the WHO classification and the histopathologic criteria necrosis and nodular fibrosis was tested in 113 patients. A tissue microarray was constructed for immunohistochemical staining. The staining results were scored quantitatively for MIB1 and semiquantitatively for CK19, COX2, p27, and CD99. The prognostic value of these markers was tested in 93 patients. RESULTS: The stratification of the patients into 4 risk groups according to the 2004 WHO classification was reliable with regard to both time span to relapse and tumor-specific death. In a multivariate analysis, the CK19 status was shown to be independent of the WHO criteria. By contrast, the prognostic significance of COX2, p27, and CD99 could not be confirmed. CONCLUSIONS: The 2004 WHO classification with 4 risk groups is very reliable for predicting both disease-free survival and the time span until tumor-specific death. CK19 staining is a potential additional prognostic marker independent from the WHO criteria for pancreatic endocrine tumors.

The effects of cell density and device arrangement on the behavior of macroencapsulated beta-cells.

Over the last several decades, considerable research has focused on the development of cell encapsulation technology to treat a number of diseases, especially type 1 diabetes. One of the key advantages of cell encapsulation is that it permits the use of xenogenic tissue, particularly animal-derived cell lines. This is an attractive idea, because it circumvents the issue of a limited human organ supply. Furthermore, as opposed to whole islets, cell lines have a better proliferative capacity and can easily be amplified in culture to provide an endless supply of uniform cells. We have previously described a macroencapsulation device for the immunoisolation of insulin-secreting 1-cells. The aim of this work was to optimize the viability and insulin secretion of cells encapsulated within this device. Specifically, the effects of cell packing density and device membrane configuration were investigated. The results indicated that cell density plays an important role in the secretory capacity of the cells, with higher cell density leading to increased insulin secretion. Increasing the transport area of the capsule by modifying the membrane configuration also led to an improvement in the insulin output of the device.

Generation and characterization of islet cell tumor in pTet-on/pTRE-SV40Tag double-transgenic mice model.

A line of double-transgenic mice that develop neoplasms arising primarily in the pancreas was established. In these mice, the oncogene SV40 T antigen (Tag) was detected in the pancreas with and without the control of Tet-on system. The transgenic mice that developed pancreatic tumors as early as 20 weeks of age showed hypoglycemia on a blood glucose test. Pathological and immunohistochemical characterizations demonstrated that the tumors belonged to neuroendocrine neoplasms arising from pancreatic islets. A change in IGFs/IGF-1R signaling pathway was detected using real-time PCR analysis. A potential association between the IGFs/IGF-1R system and SV40Tag was studied to further explain the cancerogenesis of the double-transgenic mice by Western blot analysis and immunoprecipitation experiments. The results suggest that a Tag transgenic mice model could be used to study the molecular mechanism of the tumorigenesis of islets.

Immunocytochemical identification of low-affinity NTS2 neurotensin receptors in parietal cells of human gastric mucosa.

The biological effects of neurotensin (NT) are mediated by two distinct G protein-coupled receptors, NTS(1) and NTS(2). Although it is well established that neurotensin inhibits gastric acid secretion in man, the plasma membrane receptor mediating these effects has not been visualized yet. We developed and characterized a novel antipeptide antibody to the carboxy-terminal region of the human NTS(2) receptor. The cellular and subcellular distribution of NTS(2) receptors was evaluated in various human gastrointestinal tissues. Specificity of the antiserum was demonstrated by (1) detection of a broadband migrating at M(r) 90 000-100 000 in Western blots of membranes from NTS(2)-expressing tissues; (2) cell-surface staining of NTS(2)-transfected cells; (3) translocation of NTS(2) receptor immunostaining after agonist exposure; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with its immunizing peptide. In the gastrointestinal tract, NTS(2) receptor immunoreactivity was highly abundant in parietal cells of the gastric mucosa, in neuroendocrine cells of the stomach small and large intestine, and in cells of the exocrine pancreas. NTS(2) receptors were clearly located in the plasma membrane and uniformly present on nearly all target cells. The presence of NTS(2) receptors was rarely detected in human tumors. This is the first localization of NTS(2) receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The abundant expression of low-affinity NTS(2) receptors on the plasma membrane of human parietal cells provides a morphological substrate for the direct inhibition of gastric acid secretion observed after i.v. administration of neurotensin.

Ryanodine receptor-operated activation of TRP-like channels can trigger critical Ca2+ signaling events in pancreatic beta-cells.

There is little information available concerning the link between the ryanodine (RY) receptors and the downstream Ca(2+) signaling events in beta-cells. In fura-2 loaded INS-1E cells, activation of RY receptors by 9-methyl 5,7-dibromoeudistomin D (MBED) caused a rapid rise of [Ca(2+)]i followed by a plateau and repetitive [Ca(2+)]i spikes on the plateau. The [Ca(2+)]i plateau was abolished by omission of extracellular Ca(2+) and by SKF 96365. In the presence of SKF 96365, MBED produced a transient increase of [Ca(2+)]i, which was abolished by thapsigargin. Activation of RY receptors caused Ca(2+) entry even when the ER Ca(2+) pool was depleted by thapsigargin. The [Ca(2+)]i plateau was not inhibited by nimodipine or ruthenium red, but was inhibited by membrane depolarization, La(3+), Gd(3+), niflumic acid, and 2-aminoethoxydiphenyl borate, agents that inhibit the transient receptor potential channels. The [Ca(2+)]i spikes were inhibited by nimodipine and ryanodine, indicating that they were due to Ca(2+) influx through the voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR). Activation of RY receptors depolarized membrane potential as measured by patch clamp. Thus, activation of RY receptors leads to coherent changes in Ca(2+) signaling, which includes activation of TRP-like channels, membrane depolarization, activation of the voltage-gated Ca(2+) channels and CICR.

Functional MR microimaging of pancreatic beta-cell activation.

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain beta-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image beta-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated beta-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 microM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and beta-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and beta-cell mass.

Cytologic diagnosis of pancreatic endocrine tumors by endoscopic ultrasound-guided fine-needle aspiration: a review.

Precise localization and diagnosis of pancreatic endocrine tumors (PETs) is important, because pancreatic PETs have different clinical and biological behavior and treatment modalities than do exocrine pancreatic tumors. In contrast to the much more common exocrine adenocarcinomas, cytologic studies of PET are relatively rare and many cytopathologists lack experience with the cytomorphologic features of these tumors.During the last 10 yr, endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) has matured into an accurate, highly sensitive, and cost-effective modality for the preoperative localization of pancreatic PETs. This has resulted in an increased number of PETs first sampled as cytology specimens. This manuscript focuses on the cytomorphologic features most suggestive of pancreatic PETs, differential diagnosis, and diagnostic pitfalls of PETs. The technical development of EUS-guided FNA and the ancillary studies for pancreatic PETs are also reviewed. The data summarized in this review indicate that EUS-FNA is a valuable method in the recognition of pancreatic PETs and in most cases cytopathologists could reach a correct diagnosis of these tumors, including their hormone producing capability on aspirated cytologic material.

Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas.

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.

XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor.

The cis-acting sequences that mediate insulin gene expression exclusively in pancreatic islet beta-cells are localized within the 5'-flanking region between nucleotides -340 and -91. We have identified an evolutionarily conserved, A+T-rich element at -201/-196 basepairs in the rat insulin II gene that is essential for efficient expression in beta-cells. Affinity-purified antibody to the XIHbox 8 protein super-shifted the major beta-cell-activator factor complex binding to the -201/-196 element. XIHbox 8 is a Xenopus endoderm-specific homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and developing pancreas. Antibody to XIHbox 8 specifically interacts with a 47-kilodalton protein present in this DNA complex. Immunohistochemical studies revealed XIHbox 8-like proteins within the nucleus of almost all mouse islet beta-cells and a subset of islet alpha- and beta-cells. These results are consistent with the proposal that an XIHbox 8-related homeoprotein of 47 kilodalton is required for expression of the mammalian insulin gene in beta-cells. Experiments conducted with antiserum raised to somatostatin transcription factor-1 (STF-1), a recently isolated mammalian XIHbox 8-related homeoprotein, indicate that the STF-1 protein is the mammalian homolog of Xenopus XIHbox 8.

Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid.

There is a significant correlation between the occurrence of pancreatic islet amyloid and beta-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.

Different Raman spectral patterns of primary rat pancreatic ß cells and insulinoma cells.

As a noninvasive and label-free analytical technique, Raman spectroscopy has been widely used to study the difference between malignant cells and normal cells. Insulinomas are functional ß-cell tumors of pancreatic islet cells. They exhibit many structural and immunohistochemical features in common with normal pancreatic ß cells; thus, they are typically difficult to distinguish under the microscope, especially in vivo. We investigated insulinoma and primary rat pancreatic ß-cell populations using Raman spectroscopy. The details of the optical heterogeneity between these two populations were determined based on different Raman regions primarily involving nucleic acid and protein contents, which are the most distinct cellular contents in these two types of cells. Using principal component analysis­linear discriminant analysis, these two cell types can be readily separated. The results of this work indicate that Raman spectroscopy is a promising tool for the noninvasive and label-free differentiation of insulinoma cells and normal pancreatic ß cells.

Laparoscope resection of retroperitoneal ectopic insulinoma: a rare case.

Ectopic insulinoma is a very rare and dormant tumor. Here we report the case of a 79-year-old female who presented with repeated episodes of hypoglycemia and was diagnosed with insulinoma based on laboratory and imaging examinations. Computed tomography and positron emission tomography revealed a tumor in the retroperitoneum under and left of the hepatoduodenal ligament, which was resected successfully using a laparoscopic approach. Pathologic results revealed an ectopic insulinoma, which was confirmed immunohistochemically. Ectopic insulinomas are accompanied by hypoglycemia that can be misdiagnosed as drug- or disease-induced. These tumors are difficult to diagnose and locate, particularly in atypical cases or for very small tumors. Synthetic or targeted examinations, including low blood glucose, elevated insulin, proinsulin, and C-peptide levels, 48-h fasting tests, and relevant imaging methods should be considered for suspected cases of insulinoma. Surgery is the treatment of choice for patients with insulinoma, and laparoscopic resection is a feasible and effective method for select ectopic insulinoma cases.

Immunolocalization of secretogranin II and insulin in a nerve growth factor-differentiated insulinoma cell line.

RINm5F, a rat insulin-secreting pancreatic cell line, responds to nerve growth factor (NGF) by extending neurite-like processes. Secretogranin II (SgII), a marker of neuroendocrine secretory organelles, has recently been found to be a good marker of neuronal differentiation in both human neuroblastoma and rat pheochromocytoma cells. The present paper reports the results obtained from immunocytochemical studies, which show that NGF increases the expression of SgII-immunolabeled organelles in RINm5F cells. We also demonstrate that NGF increases the expression of insulin and that SgII and insulin are predominantly, but not always, colocalized. These results suggest that this insulinoma cell line may be a good model for studying the role of SgII in neuroendocrine secretion mechanisms.

Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats.

We have identified two rat insulinoma cDNAs that code for proteins homologous to the Kex2 dibasic protease of yeast and the mammalian furin gene product. A 5.0-kilobase (kb) cDNA, termed BDP, coding for a 752-amino acid protein and a 2.5-kb cDNA coding for a 636-amino acid protein, which was found to be the rat equivalent of the human insulinoma PC2 protein, were isolated. The proteins encoded by these clones contain a specific N-terminal signal sequence, indicating that both enter the secretory pathway. Neither protein contains a C-terminal transmembrane domain as is found in kex2 and furin, suggesting that the proteins may be soluble. Both proteins contain regions surrounding the active site residues which show amino acid identities to both kex2 (43% for BDP and 41% for RPC2) and furin (57% for BDP and 53% for RPC2). Probes specific for the mRNAs of each protein were used to localize the expression of each protein in endocrine and neuroendocrine tissues.

Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.

Presence of somatostatin receptors negatively coupled to adenylate cyclase in ectopic growth hormone-releasing hormone- and alpha-subunit-secreting tumors from acromegalic patients responsive to octreotide.

The functional study of SRIH receptors was performed in ectopic GHRH-secreting tumors from two patients with acromegaly; patient 1 presented with multiple endocrine neoplasia type 1 with GHRH- and insulin-secreting pancreatic tumors, and patient 2 presented with a multihormone-secreting carcinoid tumor (including GHRH and alpha-subunit secretion, as demonstrated by clinical and immunohistochemical studies). In both cases, plasma GH levels were responsive to octreotide. In patient 2, plasma GHRH and alpha-subunit levels were responsive to octreotide. In vitro perifusion studies of a tumor fragment from patient 1 also showed inhibition of GHRH secretion by SRIH. A high density of specific SRIH-binding sites was visualized by autoradiography in GHRH tumors from both patients. SRIH specific binding was much higher in the GHRH tumors (6.6-8.4 fmol/surface unit) than in the insulinoma (1.9 fmol/surface unit). The binding inhibition constant (IC50) was in the nanomolar range (0.9-3 nmol/L) in the GHRH tumors. SRIH-14 inhibited forskolin-stimulated adenylate cyclase in the GHRH tumors from both patients, but not in the insulinoma. The functional SRIH receptors negatively coupled to adenylate cyclase present in ectopic GHRH-secreting tumors mediate the inhibitory effect of octreotide on GHRH secretion and on previously underrecognized ectopic alpha-subunit secretion from carcinoid tumors.

Cloning of a cDNA encoding rat bone marrow stromal cell antigen 1 (BST-1) from the islets of Langerhans.

We have isolated the rat bone marrow stromal cell antigen 1-encoding cDNA (BST-1) from a pancreatic islet cDNA library. The cDNA encodes a 319-amino-acid (aa) protein whose aa sequence shows homology with mammalian CD38 (33%), Aplysia ADP-ribosyl cyclases (33%), as well as mouse (86%) and human (72%) BST-1.

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells.

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.

Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.