Hidehiro Oka

Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Transforming Growth Factor-β1-Induced Apoptosis in a Human Pituitary Adenoma Cell Line

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues based on its ability to stimulate cAMP production in cultured anterior pituitary cells. Recent studies have suggested a functional role for PACAP in the apoptosis of brain cells. However, the role of PACAP in regulating apoptosis in human pituitary adenomas has not previously been examined. Analysis of the cultured human pituitary adenoma cell line HP75, which expresses all three major PACAP receptors, showed that both PACAP-38 and PACAP-27 inhibited TGF-beta1-induced apoptosis. Treatment with the PACAP receptor antagonists PACAP 6-38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe(6), Leu(17))-VIP (PACAP type II receptor antagonist) blocked the effects of PACAP-38 on the inhibition of transforming growth factor-beta1 (TGF-beta1)-induced apoptosis, confirming the specificity of the role of PACAP. Treatment with forskolin but not phorbol 12-myristate 13-acetate (PMA) also inhibited TGF-beta1-induced apoptosis. TGF-beta1 treatment was associated with an increase in mitogen-activated protein kinase (MAP kinase) when analyzed by Western blotting, but PACAP inhibition of TGF-beta1-induced apoptosis was not associated with activation of MAP kinase. Immunocytochemical analysis of the cell cycle cyclin-dependent kinase inhibitor p27 showed that treatment with TGF-beta1, forskolin, PMA, and PACAP increased p27 expression in cultured HP75 cells. These results indicate that PACAP is a highly specific inhibitor of TGF-beta1-induced apoptosis in the HP75 human pituitary adenoma cell line and that PACAP, TGF-beta1, forskolin, and PMA all stimulate expression of the TGF-beta-regulated cell cycle protein p27 in the HP75 human pituitary adenoma cell line. The HP75 cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.

Pituitary Adenylate-Cyclase-Activating Polypeptide (PACAP) Binding Sites and PACAP/Vasoactive Intestinal Polypeptide Receptor Expression in Human Pituitary Adenomas

Pituitary adenylate-cyclase-activating polypeptide (PACAP) stimulates release of several anterior pituitary hormones by interacting with PACAP receptors on pituitary cells. To learn more about the distribution and possible regulatory roles of PACAP and its receptors in human pituitary adenomas, we investigated the expression of vasoactive intestinal polypeptide (VIP) and PACAP binding sites using receptor autoradiography, PACAP and PACAP/VIP receptor (PVR) mRNAs by reverse transcription polymerase chain reaction (RT-PCR), conventional in situ hybridization, and catalyzed reporter deposition in situ hybridization (CARD-ISH) analyses. PACAP mRNA was expressed in normal human hypothalamus, which was used as a positive control, but not in pituitary adenomas. Receptor autoradiography showed PACAP types I and II binding sites in all groups of pituitary adenomas, except prolactinomas. The highest levels were present in gonadotroph and null cell adenomas. PVR-2 mRNA was expressed in normal pituitaries and in all groups of pituitary adenomas by RT-PCR, whereas PVR-1 and -3 mRNAs were expressed in all groups of pituitary adenomas, except for most prolactinomas. Conventional in situ hybridization studies with digoxigenin-labeled probes demonstrated weak staining for PVR-1, -2, and -3 mRNAs in most tissues. The CARD-ISH technique, which increased the sensitivity of the in situ hybridization method, also revealed PVR-2 mRNA expression in all adenomas, whereas PVR-1 and -3 mRNAs were detected in nearly all adenomas except for prolactinomas. The presence of PACAP mRNA in the hypothalamus, but not in normal anterior pituitary or in pituitary adenomas, and the differential expression of PVRs in adenomas indicate a selective regulatory endocrine and paracrine role of PACAP in normal and neoplastic anterior pituitary cells.

Intraoperative Photodynamic Diagnosis of Brain Tumors Using 5-Aminolevulinic Acid

Glioma is a type of invasive brain tumor, and the properties of the center region of the tumor differ from those of normal brain tissue. Accordingly, the macroscopic differences from normal brain tissue or differences in terms of properties such as hardness allow one to recognize that the glioma is, in fact, tumor tissue. The difference from normal brain tissue in the periphery of the tumor, i.e., the tumor’s region of infiltration, does not show glioma to be as evident, making it difficult to make any objective determination as to whether there is any tumor infiltration. The margin between the normal brain tissue and tumor tissue is not clearly discernible in the band of infiltration around the malignant brain tissue, often preventing the gross-total resection (GTR) of a malignant brain tumor. Researches have shown that GTR of a malignant brain tumor has a significant impact on patient survival (Filippini et al., 2006; Gorlia et al., 2006). Accordingly, any surgery on malignant glioma is performed for the purpose of GTR, and objectively determining the region of infiltration to perform GTR on malignant glioma is necessary to significantly affect patient survival. Photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) has enabled the objective assessment of tumor infiltration whenever surgery on glioma is to be performed (Stummer et al., 1998a, 1998b). When 5-ALA is administered, protoporphyrin IX (PpIX), which is a metabolite of 5-ALA, accumulates to a greater extent in the tumor cells than in normal brain cells. The PpIX used in the procedure reacts to light within the UV range, and emits a red fluorescent hue. Irradiation of tumor-infiltrated regions with UV-range light results in the regions appearing fluorescent red if tumor cells containing an accumulation of PpIX have infiltrated. This effectively allows for an objective assessment of tumor infiltration. Resection utilizing the fluorescence of the glioma after administration of 5-ALA has been shown to assist in the visualization of the tumor tissue during surgery. As a result, the use of 5-ALA is currently being praised as a new approach that allows for a significant increase in the potential to successfully perform GTR of malignant brain tumors (Stummer et al., 1998a, 1998b, 2006, 2011). This chapter will cover the principles of PDD using 5-ALA and provide commentary on the usefulness of this procedure for brain tumors. This will be followed by further commentary on the potential for PDD in regard to brain tumors, and finally a synopsis on the current limitations of and perspectives on this promising technique.

Growth hormone-releasing hormone receptor (GHRH-R) mRNA expression in human pituitary adenomas: A study by catalyzed reporter deposition-In situ hybridization (CARD-ISH)

Growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene transcription, synthesis, and secretion of growth hormone via growth hormone-releasing hormone receptor (GHRH-R). In a previous study using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blotting, GHRH-R mRNA was detected in all types of pituitary adenomas. On the other hand, in a recentin situ hybridization (ISH) study of 16 adenomas, including GH (n=8), PRL (n=1), ACTH (n=2), gonadotroph (n=4), and null-cell (n=1) adenomas, GHRH-R mRNA expression was observed only in GH adenomas and one gonadotroph adenoma. Thus, the extent of GHRH-R mRNA in pituitary adenomas is not clear. To clarify these different findings, we investigated the expression of GHRH-R mRNA in various types of human pituitary adenomas, including GH (n=7), PRL (n=4), ACTH (n=3), gonadotroph (n=8), and null-cell adenomas (n=13) using conventional ISH and the CARD-ISH technique, which is more sensitive than previous nonisotopic ISH techniques. By conventional ISH, GHRH-R was found in 71% of GH, 50% of PRL, 67% of ACTH, 12% of gonadotroph, and 8% of null-cell adenomas. By catalyzed reporter deposition-in situ hybridization (CARD-ISH), GHRH-R mRNA was expressed in 100% of GH, 50% of PRL, 67% of ACTH, 62% of gonadotroph, and 70% of null-cell adenomas. Hybridization with the control sense probe was consistently negative. These results show that GHRH-R mRNA is expressed in all types of adenomas with GH adenomas showing the strongest signal for GHRH-R mRNA, suggesting that GHRH-R has a role mainly in the function of GH adenomas, but may also play a role in tumor growth and/or function of other types of adenomas. These experiments also show that CARD-ISH can be used to detect low copy numbers of specific mRNAs because of the increased sensitivity of this technique.

Porphyrin Synthesis from 5-Aminolevulinic Acid in Patients with Glioma

The molecule 5-aminolevulinic acid (5-ALA) is a substrate for a heme synthesized in cells us‐ ing succinyl CoA and glycine. Initially, 5-ALA is converted to porphobilinogen (PBG), and the metabolism progresses by the action of PBG deaminase, and its product is incorporated into the mitochondria via uroporphyrinogen and coproporphyrinogen by an enzymatic reaction and transformed into protoporphyrin IX (PPIX). This PPIX is then converted to a heme by the action of ferrochelatase. These metabolic reactions are conducted in the liver and erythroblasts of normal human subjects [16]. It is thought that a larger amount of 5-ALA is incorporated by rapidly proliferating tumor cells as compared to normal cells, due to the active synthesis of heme [5, 20]. In the metabolic pathway of 5-ALA, it is reported that the rate limiting enzyme in normal cells is different from that in tumor cells [16]. PBG deaminase is the rate limiting en‐ zyme in normal cells [3]. Large amounts of uroporphyrinogen, coproporphyrinogen, and PPIX are produced in tumor cells compared to normal cells because metabolism after the PBG is pro‐ moted by high PBG deaminase activity and low ferrochelatase activity [15]. The PPIX accumu‐ lates in tumor cells and produces a red fluorescent response to ultraviolet light. Intraoperative photodynamic diagnosis using this method is used for patients with glioma [21, 24, 26]. There are numerous reports concerning the PPIX concentration in the tumor tissue; however, re‐ ports concerning plasma titers and urinary excretion of uroporphyrinogen and copropor‐ phyrinogen after 5-ALA administration are rare [25]. In this study, 5-ALA was administered to healthy adult volunteers, and changes in serum and urinary porphyrins were measured. Uri‐ nary excretion of porphyrins in volunteers and patients with brain tumors after the 5-ALA ad‐ ministration were compared and examined.

Repeated neuroendoscopic palliative surgery in elderly patients with predominantly cystic craniopharyngioma in the third ventricle: three case reports

Abstract Objective: The management of craniopharyngioma in the elderly remains controversial. We sought to better characterize the outcomes of elderly patients treated with neuroendoscopic palliative surgery (NPS). Methods: We report the cases of three patients older than 80 years, whose cystic craniopharyngioma was treated by repeated NPS. Results: One male and two female patients, ranging in age from 80 to 83 years (mean=81.3 years) had predominantly cystic lesions in the third ventricle and a suprasellar enhanced mass. Two patients underwent two NPS procedures and the other received three. The operating time ranged from 40 to 140 min (mean=93.4 min), and the interval between operations ranged from 2 to 24 months (mean=11.3 months). The length of postoperative hospitalization ranged from 4 to 28 days (mean=11.1 days). There were no perioperative complications, and all patients experienced symptom improvement immediately after the operation. Conclusion: Repeated NPS can be an effective alternative treatment for cystic craniopharyngioma in the third ventricle, especially in elderly patients.