Androgen receptor signaling regulates T-type Ca2+ channel expression and neuroendocrine differentiation in prostate cancer cells.

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.

Posttranscriptional regulation of T-type Ca(2+) channel expression by interleukin-6 in prostate cancer cells.

BACKGROUND: At early stages, the growth of prostate cancers is androgen dependent. At later stages, however, the growth of prostate cancers becomes androgen independent, which leads to an increase in mortality. The switch to an androgen-refractory state is associated with neuroendocrine differentiation (NED) of prostate cancer cells. Several factors including interleukin-6 (IL-6) and increased cAMP production promote NED of prostate cancer cells. In this work we investigated whether IL-6 evoked NED of LNCaP cells results in a significant change in T-type Ca(2+) channel expression in comparison to non-stimulated LNCaP cells. METHODS: T-type Ca(2+) channel subunit Cav3.2 expression was studied using PCR analysis, western blot and whole cell recordings. Tubulin IIIß expression and neurite-like morphology was assessed to investigate the role of T-type Ca(2+) channels in the differentiation of prostate cancer cells. RESULTS: Treatment of LNCaP cells with IL-6 for 4days evokes considerable morphological and biochemical changes consistent with NED. Transcripts of the T-type Ca(2+) channel subunit Cav3.2 but not Cav3.1 or Cav3.3 are detected in IL-6 stimulated cells. Real time PCR analysis of IL-6 stimulated cells indicates no significant change in Cav3.2 mRNA expression in comparison to non-stimulated cells. LNCaP cells stimulated with IL-6 show a threefold increase in T-type Ca(2+) channel subunit Cav3.2 protein expression, suggesting that channel expression is upregulated by a posttranscriptional mechanism. Electrophysiological recordings reveal that increased Cav3.2 protein expression following IL-6 stimulation of LNCaP cells does not result in increased expression of functional channels in the membrane. Functional expression of Cav3.2 channels in LNCaP cells is facilitated by co-stimulation with IL-6 and the cAMP-stimulating agent, forskolin (FSK). Inhibition of T-type Ca(2+) channel activity in IL-6 stimulated LNCaP cells prevents the development of morphological characteristics consistent with NED. CONCLUSIONS: These results indicate that the functional expression of T-type Ca(2+) channels is regulated by the interplay of multiple factors in LNCaP cells.

Regulation of T-type calcium channel expression by sodium butyrate in prostate cancer cells.

Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2 T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100µM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells.

Identification of two spliced leader RNA transcripts from Perkinsus marinus.

Spliced leader (SL) variants are present in a number of mRNAs from Perkinsus marinus. Three different SLs of 22 nucleotides (nt) in length were previously reported, with a consensus sequence of (DCCGUAGCCAUYUUGGCUCAAG). A truncated 21 nt SL, with an (A) at nt-1 and a (U) deletion at nt-13, has also been reported. Here, we report an additional 21 nt SL variant with (G) at nt-1. Using cDNA analysis, a full-length SL RNA transcript was identified for both 21 nt SLs (SL2[A] and SL2[G]). This transcript is 81 nt in length and contains a conserved transcription termination sequence present in closely related dinoflagellates.

Catalytically active monomer of class mu glutathione transferase from rat.

Although rat glutathione transferase M1-1 is crystallized as a homodimer (GST M1-1), we have generated monomers (GST M1) of the enzyme by adding potassium bromide to buffer solutions containing the wild-type enzyme and by introducing point mutations in the electrostatic region of the subunit interface. The wild-type enzyme was evaluated in 0.05 M MES (pH 6.5) containing up to 3 M KBr. We report that the addition of KBr greatly influences the monomer-dimer equilibrium of the wild-type enzyme and that at 3 M KBr GST M1 has a specific activity close to that of GST M1-1. Since the effect of KBr is likely due to charge screening at the subunit interface, the influence on the monomer-dimer equilibrium exerted by the amino acid residues in the electrostatic region of the interface (Arg77, Asp97, Glu100, Asn101) was investigated. Mutations introduced at positions 97, 100, and 101 promote monomerization, resulting in enzymes that exhibit a decreased weight average molecular weight in comparison to that of the wild-type enzyme. However, only mutations at position 97 result in enzymes that have catalytic activity in the monomeric form. The mutations introduced at positions 100 or 101 result in enzymes whose activity can be accounted for by the amount of dimeric enzyme present. Our results indicate that the electrostatic region of the interface is important in the monomer-dimer equilibrium of glutathione transferase and that, although GST M1-1 may be more active than GST M1, the dimer is not required for catalytic function.

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1.

The "mu loop," an 11-residue loop spanning amino acid residues 33-43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35-44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the "Deletion Enzyme." Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4 degrees C and 10 degrees C, but it rapidly loses activity at 25 degrees C, unlike the wild-type enzyme. In the temperature range of 4 degrees C through 25 degrees C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates.

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1.

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.