Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

No description available.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), is a key calcium (Ca++) regulatory hormone. It is also associated with functions unrelated to Ca++ homeostasis. Here, special attention is paid towards the anticancer properties of 1,25D. 1,25D strongly inhibits the growth of well-differentiated head and neck squamous cell carcinoma (HNSCC) derived cell lines. However, advanced, less differentiated, HNSCC cell lines (e.g. SCC4) are partially resistant to 1,25D. Resistance to nuclear receptor (NR) agonists is a common event that occurs in other NR-related treatments. For example, some leukemias develop resistance to the usually effective retinoic acid (RA) treatment. However, treating RA-resistant cells with HDAC inhibitors (HDACi) sensitizes them to RA. Thus, this study aims to investigate how treatment with TSA, an HDACi, would affect the response of SCC4 cell lines to 1,25D. We found that TSA had a variety of effects on 1,25D-regulated gene expression. Combined treatment with 1,25D and TSA increased the expression of cell-cycle regulating proteins, but also enhanced the downregulation of key target genes. Given the potential of the 1,25D/HDACi combination in combating cancers, two chimeric compounds, each containing parts of 1,25D and an HDACi, were synthesized in collaboration with Dr. James Gleason (Dept. of Chemistry, McGill). These 1,25D analogs have the HDACi-like structure replacing the 1,25D side chain. Both compounds proved to be agonists of the vitamin D receptor. Moreover, the TSA-substituted compound, called triciferol, effectively induced a-tubulin as well as histones acetylation. This study underlines the potential of combining 1,25D and TSA in cancer treatment, and reveals that bi-functional 1,25D analogs can be produced with potentially enhanced therapeutic activity.

Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required. Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment. Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results - Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring. Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5- methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with Ki values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively. A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > pfluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin- 4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor. The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log Ki (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 μM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities. Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy. Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

A reduction in voluntary feed intake is a major factor in the lost productivity of grazing lambs infected by gastrointestinal parasites yet the mechanisms involved are poorly understood. Potential pathways involved in parasite-induced feed intake depression were investigated in lambs with minimal previous exposure to parasites and artificially infected by the small intestinal parasite Trichostrongylus colubriformis. Six in vivo experiments were conducted on lambs housed in individual pens or metabolism crates with similar feeding and experimental procedures. In Experiment 1 (Chapter 4) the effect of T. colubriformis infection on short term feed intake in lambs and of some pharmacological agents on feed intake depression were investigated. Prior to and for the duration of infection, lambs were fed once per day and feed intake recorded at regular intervals over the day (8 h). Following the onset of feed intake depression in the infected group (9 weeks after commencing dosing), all animals were treated with an analgesic (codeine phosphate per os), an anti-inflammatory agent (indomethacin per os), a CCK antagonist (L364-718 by subcutaneous injection) or saline (control) in a replicated Latin square design (n = 8). Although the pattern of feed consumption was similar in infected and non-infected lambs, average daily intake was reduced 32 % and short term intake (recorded at 10 minute intervals for the first hour of feeding, 15 minute intervals for the second hour and hourly for the next 6 hours of feeding) reduced 40 % by infection. This identified the key component by which intake was depressed and enabled the use of a short term intake model and short duration of action compounds to identify the pathways involved in intake depression in this sequence of experiments. None of the pharmacological treatments increased intake in the infected group. These results suggest a reduction in the rate of consumption due to reduced hunger signals, rather than change of meal eating patterns, is the major cause of feed intake depression. Specific conclusions about the pathways investigated using the pharmacological agents could not be obtained. Experiment 2 (Chapter 5) was designed to investigate the roles of pain and osmolality on feed intake depression. Digesta samples collected prior to and during parasite infection and before and after feeding had similar osmolalities (240-260 mosmol/l) which indicated that feeding or infection had no effect on osmolality of digesta. Following the onset of feed intake depression in infected animals, all animals were treated in a Latin square design (n = 4) with no treatment, saline, local anaesthetic (xylocaine) or analgesic (codeine phosphate) solution 15 minutes before feeding, by slow injection into the duodenum. There was no effect of these treatments on food intake. In the second part of the experiment, hyperosmotic solutions (mannitol and NaCI) markedly depressed short term intake in non-infected animals, suggesting a role for osmoreceptors in intake regulation. However these effects were not blocked by local anaesthetic so the depressed intake may have resulted from generalised malaise rather than from specific osmoreceptor effects. In Experiment 3 (Chapter 6) the role of peripheral CCK on intake depression was examined by a dose-response study utilising the CCK antagonist, loxiglumide. Intravenous injection of 5, 10 or 20 mg/kg LW of loxiglumide to infected lambs 10-15 minutes before feeding (n = 6) had no effect on feed intake at any of the dose levels. In experiment 4 (Chapter 7) loxiglumide was infused intravenously for 10 minutes (30 mg/kg/h) before feeding and for the first 2 h (10 mg/kg/h) after feed was offered to minimise any effect of the rate of clearance of loxiglumide on the lack of feed intake response. As well, the rate of marker disappearance from the abomasum was recorded in both infected and non-infected animals. Continuous infusion of loxiglumide did not attenuate parasite induced intake depression nor did it have any effect on abomasal emptying. Abomasal volume was reduced by infection (66.3 vs 162 ml) as was the fractional outflow rate (2.2 vs 2.8 ml/min) but these differences were accounted for by the lower level of feed intake in the infected animals. In Experiment 5 (Chapter 8) brotizolam, a benzodiazepine appetite stimulant, thought to act on the hypothalamus, was administered in a dose-response study to infected and non-infected animals (n = 4) immediately prior to feeding or following termination of the first meal (45 minutes after feeding) and the feed intake response recorded. Brotizolam elevated both the short term (0-0.75 h), daily (22 h) intake and all time intervals in the first 5 h after feeding in infected and non-infected animals when administered after the first meal but when administered prior to feeding elevated intake only over the first 6 h of feeding. In both cases the magnitude of the response was greater in infected animals than in non-infected animals. Brotizolam appeared to increase the rate of eating without having a major impact on meal eating patterns when administered before feeding. Where administration was after the first meal, the effect was due to an "extra" meal being consumed. These findings showed that infected animals can respond to central stimulators of intake although the mechanism of the response is not known. Opioids were implicated in intake depression as the rate of intake rather than meal patterns appeared to be the major parameter depressed under parasitism. This was examined in experiment 6 (Chapter 9) where animals (n = 6) were fasted for 26 h or not fasted, then treated with saline (control), brotizolam (intake stimulant) or naloxone (opioid antagonist) immediately prior to feeding. Fasting stimulated feed intake in the short term (100 % increase in 75 min) and over the day (12 % increase) in both infected and non-infected animals. Following fasting, infected animals ate a similar amount of feed to the non-infected, fasted animals and more than the non-infected, non-fasted animals. The signals resulting from a one day fast were sufficient in the short term to override parasite induced mechanisms causing feed intake depression. Naloxone suppressed the intake stimulatory effects of a 26 h fast in both infected and non-infected animals, which supports a role for endogenous opioids as hunger signals. Where animals were not fasted, naloxone reduced intake only in the non-infected animals which suggested endogenous opioid levels may be lower in infected animals than in non-infected animals. In the final experiment (Experiment 7, Chapter 10) the role of central hunger and satiety mechanism were investigated. Infected and non-infected animals (n = 6) were treated with naloxone or saline by intravenous injection, or saline and met-enkephalinamide (an opioid analogue) by intracerebral infusion, or naloxone and the opioid analogue simultaneously to investigate the role of central opioids in feed intake depression. To determine the role of CCK induced satiety signals on feed intake at a central level, loxiglumide and CCK were infused separately and in combination for 30 minutes prior to feeding and for the first 60 minutes of feed on offer, into a lateral cerebral ventricle of the brain of infected and control animals (n = 6). The opioid analogue tended to increase intake in infected animals but the effect was not significant probably because the dose used was too low to elicit a response in sheep. Naloxone depressed intake only in the infected animals, which conflicted with the results of Experiment 4. As a consequence these results were inconclusive because of the single low dose of opioid analogue used and the conflicting naloxone responses. CCK alone depressed intake by 39-52 % only in infected animals and this effect of the 90 minute infusion was evident over the 8 h short term recording period. Loxiglumide attenuated the feed intake depressive effects of CCK in the infected animals to the extent that intake was elevated above control levels. Loxiglumide alone was an intake stimulant in both infected and non-infected animals. Intake was increased over the entire 8 h but mostly in the second hour when intake was increased by 188 % in infected animals and by 16 % in the non-infected animals and resulted in almost continuous eating. These results showed loxiglumide will temporarily block the effect of parasite infection on feed intake in sheep when administered centrally and the fact that it blocked the effects of exogenous CCK on intake indicated that the effect is mediated via CCK receptors. In conclusion GIT parasite infection reduced both short term and daily feed intake apparently by a change in rate of intake rather than any alteration in meal patterns. It was further suggested that anyone of a number of potential peripheral pathways, including changes to osmolality, gut emptying, pain and inflammation of the gut, alone is not involved in anorexia in sofar as the compounds used could block these factors and the results support the idea that intake depression is mediated via a central mechanism. Intake in infected animals responded to a much greater extent when fasting, i.c.v. loxiglumide or brotizolam were employed. Feed intake thus appears to be regulated through the same mechanisms in infected and non-infected animals. The results from compounds affecting the central mechanism suggest central CCK receptors are important in parasite induced anorexia, possibly by changing the onset of satiety or by interacting with endogenous opioids to reduce the rate of feed intake. Secondly reduced endogenous opioids may be causing the reduction in the rate of feed consumption alone or as a result of other interactions. It was concluded that intake in parasitised animals could be increased to that of control animals by employing procedures and compounds thought to act on the hypothalamus.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 109-141). Also available online.

The steroid/thyroid hormone receptor superfamily is a large class of ligand-dependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development, homeostasis, and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basal level via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors, particularly the vitamin D, estrogen, and retinoid receptors, and modulates their transcriptional activity. It was discovered that these receptors interact with different α-helical LXXLL motifs of RAC3 in vitro. Mutation of specific motifs differentially impairs the ability of RAC3 to enhance transcription by the receptors in vivo. In addition, the intrinsic transcriptional activation function of RAC3 was also characterized. Here, a single LXXLL motif, NR box v, was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent, the coactivator pocket of RARα overlaps with the corepressor pocket, with the exception of helix 12, which is required for coactivator, but not corepressor binding. Recruitment of RAC3 or SMRT also correlates directly with the ability of RARα to activate or repress transcription, respectively. Intriguingly, it was discovered that the AF-2 domain of RXRα inhibited cofactor binding to RXRα heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXRα cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXRα is the primary docking point for cofactors at RXRα heterodimeric complexes. Taken together, this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.

The synapse is the primary locus of cell-cell communication in the nervous system. The elaboration of a functional synapse requires both a specialized structure and an efficient communication system. For my thesis work, I studied proteins implicated in each of these functions: the structural molecules dystroglycan and dystrophin, and the signaling elements Insulin Receptor Substrate p58/53 and insulin receptor. The α/β-dystroglycan complex, believed to be the heart of cellmatrix adhesion in muscle and other tissues, provides a link between dystrophin, a cytoskeletal protein at the base of the muscle cell's Dystrophin Associated Protein Complex, and the extracellular matrix. In addition, dystrophin is found at central synapses, tightly associated with the postsynaptic density. The absence of dystrophin and the secondary loss of its associated proteins causes the genetic disease Duchenne Muscular Dystrophy. DMD affects both muscle and brain, causing a severe muscular dystrophy and lower IQs than control groups. In the first portion of my thesis work, I sought to determine the role of dystroglycan, dystrophin's peripheral partner, at central synapses. I probed Northern blots of brain regions to delineate the distribution of brain β-dystroglycan mRNA and to uncover any β-dystroglycan-related transcripts in brain. Then, using subcellular brain fractions, and cultured hippocampal neurons, I determined that whereas α-dystroglycan is associated with central synapses, β-dystroglycan is not. This discovery is surprising, and differs from the finding that dystrophin and α- and β-dystroglycan colocalize at the presynaptic membrane of retinal photoreceptors. In the course of the above mentioned work, using the anti-β-dystroglycan antiserum Ab98, I discovered a pair of proteins that were tightly associated with the postsynaptic density. These polypeptides of 58 kDa and 53 kDa (p58/53) were highly enriched in postsynaptic density (PSD) fractions from rat cerebral cortex, hippocampus, and cerebellum. In pursuit of a potential synapse-specific dystroglycan relative, I purified p58 and p53 by a combination of hydrophobic interaction chromatography and two-dimensional gel electrophoresis. Mass spectroscopy and peptide microsequencing revealed that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Whereas IRSp58/53 has no significant homology to β-dystroglycan other than the one span of peptides that confers its antibody cross-reactivity, its localization to the PSD newly implicates insulin signaling at synapses. Analysis of IRSp58/53 mass profiles, peptides, and mRNA indicated that IRSp58 and IRSp53 are the product of the same coding sequence. Immunolocalization showed that IRSp58/53 is expressed in the synapserich molecular layer of the cerebellum. Immunostaining of cultured hippocampal neurons showed that both IRSp58/53 and insulin receptor are highly concentrated at synapses. Like IRSp58/53, insulin receptors are a component of the PSD fraction. Together, these data suggest that the synapse is a specialized site for insulin signaling in the brain.

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.