ABSTRACT
Therapies designed to reduce androgen production or receptor activation are effective in limiting prostate tumor growth. However, prolonged treatment with anti-androgen therapies results in the progression of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of disrupting androgen receptor signaling in promoting NED of prostate carcinoma cells and whether it is accompanied by an increase in T-type Ca2+ channel expression. The effect of disrupting androgen signaling was assessed in LNCaP and 22Rv1 prostate cancer cells following treatment with the androgen receptor blocker, bicalutamide, or hormone-depleted media. Treatment of LNCaP cells with bicalutamide or hormone-depleted media for 4-10 d evoked considerable morphological and biochemical changes consistent with NED including the development of long neurite-like processes and the expression of the neuronal marker, tubulin IIIß. PCR analysis of bicalutamide-stimulated cells revealed no significant changes in Cav3.2 mRNA. However, stimulation of LNCaP cells with bicalutamide or hormone-depleted media for 10 d evoked a significant increase in Cav3.2 protein expression and the appearance of functional T-type Ca2+ channels. Inhibition of T-type Ca2+ channel function with various pharmacological blockers disrupted the morphological differentiation of LNCaP cells. Bicalutamide-evoked expression of functional T-type Ca2+ channels in LNCaP cells promoted chemoresistance to docetaxel. These findings indicate that disruption of androgen receptor signaling in prostate cancer cells evokes increased expression of functional T-type Ca2+ channels, which may result in significant morphological and biochemical changes.
ABSTRACT
Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1-3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.
