Steve Huang

Memory of Extracellular Adenosine A2A Purinergic Receptor-mediated Signaling in Murine T Cells

Accumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, “signaling” mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The “memory” of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2Areceptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivounder adenosine deaminase deficiency and hypoxic conditions in,e.g., solid tumors.

Protein patterns and proteins that identify subtypes of glioblastoma multiforme.

Glioblastoma multiforme (GBM) has been subdivided into two types based on clinical and genetic findings: primary tumors, which arise de novo, and secondary tumors, which progress from lower grade gliomas to GBMs. To analyse this dichotomy at the protein level, we employed selective tissue microdissection to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins. Protein patterns were analysed in a blinded manner from the clinical and genetic data. 2-DGE clearly identified two distinct populations of tumors. 2-DGE was reproducible and reliable, as multiple samples analysed from the same patient gave identical results. In addition, we isolated and sequenced 11 proteins that were uniquely expressed in either the primary or the secondary GBMs, but not both. We demonstrate that specific proteomic patterns can be reproducibly identified by two-dimensional gel electrophoresis from limited numbers of selectively procured, microdissected tumor cells and that two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. Proteins that are expressed distinctively may have important implications for the diagnosis, prognosis, and treatment of patients with GBM.

Multiple Leiomyomas of the Esophagus, Lung, and Uterus in Multiple Endocrine Neoplasia Type 1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant hereditary disorder characterized by multiple parathyroid, pancreatic, duodenal, and pituitary neuroendocrine tumors. Nonendocrine mesenchymal tumors, such as lipomas, collagenomas, and angiofibromas have also been reported. MEN1-associated neuroendocrine and some mesenchymal tumors have documented MEN1 gene alterations on chromosome 11q13. To test whether the MEN1 gene is involved in the pathogenesis of multiple smooth muscle tumors, we examined the 11q13 loss of heterozygosity (LOH) and clonality patterns in 15 leiomyomata of the esophagus, lung, and uterus from five patients with MEN1. Forty sporadic uterine leiomyomata were also studied for 11q13 LOH. LOH analysis was performed using four polymorphic DNA markers at the MEN1 gene locus; D11S480, PYGM, D11S449, and INT-2. 11q13 LOH was detected in 10 of 12 (83%) MEN1-associated esophageal and uterine smooth muscle tumors. In contrast, LOH at the MEN1 gene locus was demonstrated only in 2 of 40 (5%) sporadic uterine tumors. LOH at 11q13 was not documented in three lung smooth muscle tumors from a single patient with MEN1. Ten tumors from two female patients were additionally assessed for clonality by X-chromosome inactivation analysis. The results demonstrated different clonality patterns in multiple tumors in the same organ in each individual patient. The data indicate that leiomyomata of the esophagus and uterus in MEN1 patients arise as independent clones, develop through MEN1 gene alterations, and are an integral part of MEN1. However, the MEN1 gene is not a significant contributor to the tumorigenesis of sporadic uterine leiomyomata.

Somatic point mutation of the wild-type allele detected in tumors of patients with VHL germline deletion

The majority of patients with Von Hippel-Lindau (VHL) disease are affected by a VHL germline mutation involving one copy of the VHL gene. Loss of heterozygosity of the second VHL allele can be consistently demonstrated in tumor tissue from these patients, suggesting that allelic deletion is a very early or even initiating event for tumorigenesis. Approximately 20% of VHL disease patients, however, exhibit germline deletion of one entire copy or at least a substantial part of the VHL gene. To investigate the nature of the ‘second genetic hit’ in this patient population, we analysed two renal cell carcinomas and one CNS hemangioblastoma from three unrelated patients for genetic changes of the second copy of the VHL gene. All three tumors showed retention of one VHL allele by FISH. Single-strand conformation polymorphism and mutation analysis of microdissected tumor DNA revealed somatic point mutations of the wild-type VHL copies in each of the three tumors. The results indicate that the ‘two hit model’ is equally applicable to patients with VHL germline mutation and VHL germline deletion. In contrast to tumors from patients with VHL germline mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion.

Allelic imbalance of the mutant and wild-type RET allele in MEN 2a-associated medullary thyroid carcinoma

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.

Somatic VHL gene deletion and point mutation in MEN 2A-associated pheochromocytoma.

Multiple endocrine neoplasia type 2 (MEN 2) is an inherited cancer syndrome that includes pheochromocytoma. Germline mutations in RET are responsible for MEN 2 but the precise pathogenetic mechanisms of tumorigenesis are unknown. We have recently identified possible mechanisms of tumor formation in patients with MEN 2A-related pheochromocytoma. Two of nine tumors investigated, however, did not reveal either of these mechanisms. In the present study, we therefore searched for other possible mechanisms underlying the pathogenesis of MEN 2A-related pheochromocytoma. Hereditary pheochromocytoma also occurs in patients with von Hippel-Lindau (VHL) disease, a syndrome consisting of tumors caused by inactivation of the VHL tumor suppressor gene. A subset of sporadic pheochromocytomas have somatic mutations in RET or VHL, suggesting that both genes contribute to pheochromocytoma pathogenesis in a subset of tumors. It is unknown, however, whether VHL gene alterations would be associated with tumorigenesis in hereditary, MEN 2-related pheochromocytoma. We therefore investigated four pheochromocytomas from patients with MEN 2A and RET germline mutations for the presence of allelic deletion and/or somatic mutation of the VHL gene. LOH analysis using the polymorphic markers D3S1038 and D3S1110 that map to the VHL gene locus 3p25/26, revealed evidence for somatic VHL gene deletion in all four MEN 2A-related pheochromocytomas. Mutation analysis of the VHL gene showed frameshift mutations in two tumors and a splice acceptor mutation in one tumor. The remaining tumor did show LOH but not mutation of the VHL gene. These results suggest that somatic genetic alterations of the VHL gene may play a role in the tumorigenesis of some MEN 2A-related pheochromocytomas.