Faculty Bibliography

Type 2 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) is a multi-functional enzyme that possesses 3alpha-, 17beta- and 20alpha-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3alpha-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of Delta(4)-androstene-3,17-dione to testosterone, 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), and 3alpha-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence trans-activation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.

We report a case of an adenomatoid tumor (AT) of an adrenal gland with micronodular adrenal cortical hyperplasia (ACH). A 51-year-old man was found to have newly developed hypertension with clinical evidence of primary aldosteronism. A computerized tomogram of the abdomen revealed a solitary mass in the right adrenal gland. He underwent a right adrenalectomy for a presumptive clinical diagnosis of a solitary aldosterone-producing adrenal cortical adenoma. On histopathologic examination, the adrenal gland demonstrated an AT, diagnosed by the characteristic histological features, immunohistochemical stain results, and electron microscopic findings. The surrounding adrenal cortex showed multiple small hyperplastic cortical nodules. After the adrenalectomy, the patient's blood pressure normalized. Primary AT of the adrenal gland coexisting with micronodular ACH associated with hypertension has not been previously reported.

GLI is the prototype for the Gli-Kruppel gene family characterized by a consensus C2-H2 zinc finger domain and is believed to function as a transcription activator in the vertebrate Sonic hedgehog-Patched signal transduction pathway. Understanding GLI gene regulation may be of importance to understanding causes of human birth defects and cancer. To begin to understand the regulation of this developmentally important gene we have cloned the human GLI gene and functionally characterized its 5' flanking region. The GLI gene is composed of 12 exons and 11 introns and in the zinc finger coding region shares a highly conserved splicing pattern with several other Gli family members in both vertebrates and C. elegans. A major transcription initiation site was identified upstream of the GLI translation start site along with three minor transcription initiation sites. The region surrounding the transcription initiation sites lacks TATA and CCAAT consensus sequences, has a high GC content, includes a CpG island, and contains several GC boxes. A 487bp segment surrounding the transcription initiation sites increased expression of a luciferase reporter gene 15-fold in Tera-1 cells and was defined as the core promoter region of human GLI. In transgenic mice this region directed beta-galactosidase expression to the central nervous system on embryonic days 10.5-12.5 and to sites of endochondral ossification on embryonic days 12.5 and 13.5 in a pattern comparable to the endogenous expression pattern of mouse gli within these tissues. The previously identified gastrointestinal expression of gli was not driven by this region and may require elements outside of the core promoter. Sequence analysis of the 5' flanking region of the mouse gli gene and the full-length mouse gli cDNA demonstrated high homology with human GLI, suggesting conservation of GLI regulation and function.

Three proteins have been identified in mammals, GLI, GLI2, and GLI3, which share a highly conserved zinc finger domain with Drosophila Cubitus interruptus and are believed to function as transcription factors in the vertebrate Sonic hedgehog-Patched signaling pathway. To understand the role GLI plays in the Sonic hedgehog-Patched pathway and mechanisms of GLI-induced transcriptional regulation, we have characterized its transcriptional regulatory properties and contributions of specific domains to transcriptional regulation. We have demonstrated that GLI activates expression of reporter constructs in HeLa cells in a concentration-dependent manner through the GLI consensus binding motif and that a GAL4 binding domain-GLI fusion protein activates reporter expression through the GAL4 DNA binding site. GLI-induced transcriptional activation requires the carboxyl-terminal amino acids 1020-1091, which includes an 18-amino acid region highly similar to the alpha-helical herpes simplex viral protein 16 activation domain, including the consensus recognition element for the human TFIID TATA box-binding protein-associated factor TAFII31 and conservation of all three amino acid residues believed to contact directly chemically complementary residues in TAFII31. The presence of this region in the GLI activation domain provides a mechanism for GLI-induced transcriptional regulation.

We have previously established that a dimer repeat of the complete HPV 16 genome is sufficient to cause multiple organ malignancies, either carcinomas or T-cell lymphomas, in transgenic mice. Here, we report the expression of oncogenes supporting the notion that these tumors arose via multiple oncogenic pathways. In these mice, the transgenic HPV 16 genome cosegregated with the tumor phenotype. E6/E7 expression was observed in both carcinomas and T-cell lymphomas, while E2 expression was observed only in T-cell lymphomas. Some of the T-cell lymphomas revealed E2 expression alone, implying that oncogenic pathways of HPV other than the one involving E6/E7 existed in these transgenic mice. To establish that this is the case, expression of genes downstream from E6/E7 and oncogenes involved in T-cell lymphoma formation were analyzed. p53 mutations were observed in two of five tumors that lacked E6 expression. High levels of c-myc gene expression were observed in five of six tumors with E7 expression, suggesting that a pathway involving E7, inactivation of Rb, and activation of c-myc is important in tumorigenesis of HPV 16 in these transgenic animals. High levels of expression of the c-Pim gene were also noted in two of three c-myc-expressing T-cell lymphomas, suggesting cooperation between these two proto-oncogenes. Activation of Hox-11, Tal2/SCL-2, and Rbtn1/Ttg1 expression, which are highly associated with human T-cell acute lymphoblastic leukemia (T-ALL), was observed in three of three T-cell lymphomas with E2 expression but not E6/E7 expression, showing that pathways to tumor formation not involving E6/E7 exist in these transgenic animals. At least two oncogenic pathways to tumors in HPV 16 transgenic mice exist, one involving E6/E7 and c-myc and the other involving E2 and lymphomagenic oncogenes.

BACKGROUND: GLI is an oncodevelopmental gene in the vertebrate hedgehog/patched signaling pathway that is spatiotemporally regulated during development and is amplified in a subset of human cancers. GLI is the prototype for the Gli-Kruppel family of transcription factors, which includes the Drosophila segment polarity gene ci, the C. elegans sex-determining gene tra-1, and human and mouse GLI3, all of which contain a conserved domain of five C2-H2 zinc fingers. GLI3 mutations have been implicated in the mouse mutant extra toes, as well as in human Greig cephalopolydactaly syndrome and the autosomal dominant form of Pallister-Hall syndrome. As such, GLI and the vertebrate hedgehog/patched signaling pathway appear to play important roles in both normal development and neoplasia. MATERIALS AND METHODS: Since it is not known whether aberrant GLI expression is similarly linked to developmental disorders, we developed gain-of-function transgenic mice which express human GLI ectopically. RESULTS: Affected transgenic mice exhibit a phenotype of failure to thrive, early death, and Hirschsprung-like patches of gastrointestinal dilatation. The colons of affected mice have greatly attenuated smooth muscle layers and abnormal overlying epithelium. The density of myenteric plexuses is reduced in the colonic walls. The severity of the phenotype is related to the level of transgene expression. CONCLUSIONS: The transgenic mouse model supports a role for GLI in gastrointestinal development. As part of the vertebrate hedgehog/patched signaling pathway, GLI is essential to mesoderm and CNS ectoderm development and transgenic GLI expression affects neuronal, muscular, and epithelial cell differentiation in the gut. Expression of human GLI in mice results in impairment of enteric neuronal development and a Hirschsprung-like phenotype.