Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin

<p>Abstract</p> <p>Background</p> <p>Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA). This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing.</p> <p>Results</p> <p>The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 μg) injected intrathecally produce thermal hyperalgesia. At higher doses (10 μg) peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons.</p> <p>Conclusion</p> <p>The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury.</p

A retinoid-resistant acute promyelocytic leukemia subclone expresses a dominant negative PML-RARa mutation

The unique t(15;17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor alpha (RAR alpha) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RAR alpha associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RAR alpha protein. In contrast to mutations in RAR alpha found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RAR alpha remains wild-type. In vitro expression of a cloned PML-RAR alpha with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RAR alpha protein retains the ability to heterodimerize with RXR alpha and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RAR alpha engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RAR alpha selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXR alpha and RAR beta and the RA-induced loss of PML-RAR alpha protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RAR alpha-mediated pathway that is independent from gene expression induced or repressed by PML-RAR alpha. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically

Estrogen receptor expression activates the transcriptional and growth-inhibitory response to retinoids without enhanced retinoic acid receptor a expression

Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alfa protein in acute promyelocytic leukemia cells

BACKGROUND: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. METHODS: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RAR alpha fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As2O3 treatment. RESULTS AND CONCLUSIONS: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid-resistant APL cells at concentrations that are achievable in patients. As2O3 induces loss of the PML/RAR alpha fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells

Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells

Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients