A balance of histone acetylation and deacetylation governs the regulation of genes that are involved in the dif- ferentiation and stratification of the mammalian epidermis. Class II HDACs (HDAC4, 5, 6, 7, 9, 10) frequently undergo nucleocytoplasmic flux resulting in gene derepression. Of the Class I HDACs (HDAC1, 2, 3, 8), HDAC2 has only been described in a nuclear setting. Here we report that a specific in vivo subpopulation of epidermal keratinocytes undergoing apoptotic-like terminal differentiation demonstrate complete cytoplasmic sequestration of HDAC2, robust Keratin-10 ex- pression, and canonical nuclear fragmentation. Paralleling our in vivo findings, proteosomal degradation of total cellular HDAC2 enhanced Keratin-10 expression in undifferentiated HFK cells. Forced HDAC2 nuclear overexpression and re- tention results in a partial differentiation block as measured by reduced Keratin-10 expression and delayed chromatin fragmentation. We offer a preliminary model whereby cytoplasmic sequestration of the HDAC2 transcriptional corepres- sor contributes, in part, to the process of mammalian epidermal differentiation. (words 150)

1289 Reversible acetylation of core histone proteins is currently the best-understood mechanism for regulating higher order DNA structure. Histone acetyltransferases (HAT) and histone deacetylases (HDACs) are key enzymes involved in the regulation of DNA based processes. Loss of radiation and androgen sensitivity are two themes associated with increased rates of prostate cancer morbidity and mortality. Remodeling of local chromatin structure has been shown to be a central factor governing both gene expression and cellular radiation sensitivity. HDAC mediated gene repression has generally been associated with chromatin condensation and may additionally serve to protect chromatin from the damaging effects of ionizing radiation. Thus, inhibition of HDAC activity and subsequent DNA decondensation may sensitize chromatin to radiation induced damage. We propose that HDAC activity likely plays a role in the acquisition and/or maintenance of radioresistant prostate cancer proliferation. Preliminary evidence has revealed that the class I HDACs 1,2, 3, 8 as well as the class II HDACs 4,5,6,7 and 9 are all expressed in our prostate cancer model system, namely the androgen-dependent cell line LNCaP and the hormone-refractory isogenic cell line C4-2. Although displaying distinct differences in radiosensitivity, the relative HDAC expression levels appear to be equivalent between LNCaP and C4-2 cells. Addition of the broad spectrum HDAC inhibitor TSA resulted in radiosensitization of C4-2 cells. We suggest that the preferential inhibition of specific HDAC isoform(s) activity and/or localization may play a role in modulating the radiation response. We propose that inhibition of HDAC activity can be therapeutically exploited to modulate radiation sensitivity. Thus, we will explore the hypothesis that targeted inhibition of specific HDAC isoforms will enhance the radiosensitivity (and possibly the hormone responsiveness) of prostate cancer cells.

Progression of prostate cancer ultimately results in a disease that is refractory to hormone ablation therapy but nevertheless continues to require the androgen receptor. Progression to hormone refractory disease is often correlated with overexpression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. Many of these growth factor receptors engage the Ras/mitogen-activated protein (MAP) kinase pathway as part of their signaling activities. This raises the possibility that chronic activation of Ras/MAP kinase signaling could cause or contribute to the progression of prostate cancer. We have demonstrated previously that MAP kinase activation correlates with the progression to advanced hormone refractory disease in patient samples. Here we demonstrate that stable expression of Ras effector-loop mutants that activate the Ras/MAP kinase pathway is sufficient to reduce the androgen requirement of LNCaP prostate cancer cells for growth, prostate-specific antigen expression, and tumorigenicity. We propose that chronic activation of endogenous c-Ras by autocrine and paracrine growth factor stimulation sensitizes the androgen receptor transcriptional complex to subphysiological levels of androgen. This provides a common mechanism for prostate cancer progression driven by diverse agonists.

Progression of prostate cancer to androgen-refractory disease is correlated with increased expression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. Many of these growth factor receptors engage Ras as part of their normal signaling activities, raising the possibility that activation of endogenous c-Ras could be a common mechanism for prostate cancer progression. Here we demonstrate that inducible expression of a dominant negative form of Ras restores androgen sensitivity to a hormone-refractory prostate cancer cell line. We show that expression of RasN17 in the hormone-refractory C4-2 cell line enhances in vitro sensitivity to the growth-inhibitory action of the antiandrogen Casodex and inhibits anchorage-independent cell growth. Moreover, although induction of RasN17 by itself has no observable effect on the growth of C4-2 xenografts in intact male mice, it restores androgen dependence to the C4-2 xenografts so that they dramatically regress after surgical androgen ablation.

The spatial and temporal expression of subunit 1 of the olfactory cyclic nucleotide-gated channel was investigated using affinity-purified anti-fusion protein antibodies. Immunoreactivity was most prominent in the ciliary layer of the olfactory epithelium, but high protein expression was also seen along the entire length of olfactory receptor neuronal axons to the level of the glomeruli. Electron microscopy showed that the long, thin distal compartments of olfactory cilia labeled more prominently than their thicker proximal segments. This was true as soon as these distal parts began to develop. Using light microscopy, developmental expression of olfactory cyclic nucleotide-gated channel subunit 1 could be detected in discrete populations of olfactory receptor neurons by embryonic day 14. Other signaling molecules are expressed either later (Golf) or only at the level of the epithelial surface and not in axons (adenylyl cyclase type III). Following unilateral lesions of the olfactory bulb, olfactory cyclic nucleotide-gated channel subunit 1 immunoreactivity was present early and throughout developing olfactory receptor neurons; adenylyl cyclase type III immunoreactivity, in contrast, was detectable only later, and again present only in the cilial layer. These results support the hypothesis that this subunit of the olfactory cyclic nucleotide-gated channel may be involved in olfactory axon guidance, in addition to its well-described role in olfactory signal transduction.

We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.

Cyclic nucleotide-gated (cng) channels are important components of signaling systems mediating sensory transduction. In vertebrate photoreceptors, light activates a signaling cascade that causes a decrease in intracellular cGMP concentrations, closing retinal cng channels. Signal transduction in olfactory receptor neurons is believed to proceed via G-protein-mediated elevation of intracellular cAMP in response to odorant binding by 7-helix receptors. cAMP opens the olfactory cng channel, which is highly permeable to Ca2+. Here we demonstrate by in situ hybridization and immunohistochemistry with subunit-specific antibodies that both subunits of the heteromeric rat olfactory cng channel are also widely expressed in the brain. Expression of the retinal rod cng channel, however, can be detected only in the eye. In the adult hippocampus, the olfactory cng channel is expressed on cell bodies and processes of CA1 and CA3 neurons. In cultured embryonic hippocampal neurons, the channel is localized to a subset of growth cones and processes. We recorded conductances with the electrophysiological characteristics of the heteromeric olfactory cng channel in excised inside-out patches from these cultured neurons. We also show that Ca2+ influx into hippocampal neurons in response to cyclic nucleotide elevation can be detected using fura-2 imaging. Cyclic nucleotide elevation has been implicated in several mechanisms of synaptic plasticity in the hippocampus, and these mechanisms also require elevation of intracellular Ca2+. Our results suggest that the "olfactory" cng channel could regulate synaptic efficacy in brain neurons by modulating Ca2+ levels in response to changes in cyclic nucleotide concentrations.

Desensitization of signal transduction may occur through a number of processes, including receptor internalization, receptor uncoupling mediated by receptor phosphorylation, and receptor downregulation. Heterologous desensitization (nonagonist mediated) may proceed via protein kinase A (PKA), protein kinase C (PKC), or other kinases. Homologous or agonist-induced desensitization of the β2-adrenergic receptor occurs through agonist-activated specific receptor phosphorylation that is catalyzed by a specific receptor kinase called the β-adrenergic receptor kinase (β-ARK). Complete quenching of the signal requires the action of β-arrestin (β-ARR). As the putative odorant receptors are members of the G-protein-coupled receptor family, we investigated the possibility that these proteins were involved in olfactory desensitization.