Abstract␣ 2 adrenergic agonists are used in the anesthetic management of the surgical patient for their sedative/hypnotic properties although the ␣ 2 adrenoceptor subtype responsible for these anesthetic effects is not known. Using a gene-targeting strategy, it is possible to specifically reduce the expression of the individual adrenoceptors expressed in the central nervous system and to thereby determine their role in hypnotic action.Stably transfected cell lines (PC 124D for rat ␣ 2A ; NIH3T3 for rat ␣ 2C adrenoceptors) were exposed to 5 M antisense oligodeoxynucleotides (ODNs) for ␣ 2A and ␣ 2C adrenergic receptor subtypes for 3 d. Individual receptor subtype expression, as determined by radiolabeled ligand binding, was selectively decreased only by the appropriate antisense ODNs and not by the "scrambled" ODNs. These antisense ODNs were then administered three times, on alternate days, into the locus coeruleus of chronically cannulated rats and their hypnotic response to dexmedetomidine (an ␣ 2 agonist) was determined.Only the ␣ 2A antisense ODNs significantly change the hypnotic response causing both an increase in latency to, and a decrease in duration of, the loss of righting reflex following dexmedetomidine; hypnotic response had normalized 8 d after stopping the ODNs. Therefore, the ␣ 2A adrenoceptor subtype is responsible for the hypnotic response to dexmedetomidine in the locus coeruleus of the rat. ( J. Clin. Invest. 1996. 98:1076-1080.)

Early treatment of HIV-1 infection is well tolerated and results in rapid and sustained virological suppression. Preservation of CD4+ cell counts may be achieved with early therapy, independent of seroconversion status. Protease inhibitor-based and nonnucleoside reverse-transcriptase inhibitor-based regimens show comparable performance in tolerability, time to virological suppression, and CD4+ cell count when used as a first regimen.

Histone deacetylases (HDACs) are aberrantly expressed, and inhibitors of HDACs induce apoptosis in lymphoplasmacytic cells (LPCs) in Waldenström macroglobulinemia (WM). The molecular profile by which these agents induce apoptosis in WM LPCs remains to be delineated. We examined the activity of the histone deacetylase inhibitor, vorinostat, and dissected its pro-apoptotic pathways in WM LPCs. Vorinostat induced apoptosis in WM cells through activating specific caspases at varying times. Inhibitors of apoptosis (IAPs) were down-regulated after vorinostat treatment. Cellular stress induced in vorinostat-treated WM cells was reflected by changes in the mitogen activated protein kinase (MAPK) pathways. Activated phospho-p38 MAPK was up-regulated at 12 h, while phospho-extracellular signal-regulated kinase (Erk) abruptly decreased at 24 h. Bortezomib did not augment vorinostat induced primary WM cell killing as reported in other B-cell disorders. These studies support that stress induced apoptosis in vorinostat-treated WM LPCs is mediated through disrupting the activity of the Erk and p38 MAPK pathways.

4785 Waldenstrom's Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration of CD19+ cells and production of a monoclonal IgM protein. Despite advances in treatment, WM remains incurable. As part of these efforts we sought to define the role of HDAC-inhibitors in WM. Gene expression profiling of bone marrow CD19+ cells from 30 WM patients and 10 healthy donors showed over-expression of HDAC4, HDAC9, and Sirt5 in WM patients. Evaluation of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA or Vorinostat), Trichostatin A (TSA), LBH-589 (Panobinostat), and sirtinol demonstrated dose dependent killing of BCWM.1 cells with IC50 of 3.5 uM, 70 nM, 0.8 uM, and 30 uM, respectively, whilst the combination of these agents with bortezomib resulted in at least additive tumor cell killing. TSA is more potent than bortezomib in inducing apoptosis in primary WM tumor cells in patients with prior treatment. TSA and bortezomib showed synergistic effect in 25% of the patients samples tested. We also observed that TSA and bortezomib-induced apoptosis of BCWM.1 cells depended on the activation of a similar set of caspases. Conversely, changes in cell cycle regulators were distinctly different between TSA and bortezomib treated BCWM.1 cells. The results of these studies demonstrate over-expression of distinct members of HDAC in WM cells, and provide a framework for the examination of HDAC-inhibitors as monotherapy, as well as combination therapy with bortezomib in the treatment of WM. Disclosures: No relevant conflicts of interest to declare.

e14582 Background: HDAC inhibitors (HDAC-I) have emerged as a promising class of therapeutics for the treatment of lymphoplasmacytic malignancies, particularly in combination strategies. The basic mechanisms by which these agents modulate malignant cell growth remain to be clarified. In recent studies, we showed that SAHA, TSA, and Sirtinol have significant preclinical activity in Waldenstrom's macroglobulinemia (WM). As part of our effort to identify potential molecular targets for these agents, we performed gene expression profiling (GEP) using WM cells treated with these agents. Methods: We implemented the Bootstrap re-sampling technique on five BCWM1 GEP datasets to characterize the range of gene expression observed. Individual BCWM1 cells were treated with SAHA, TSA and Sirtinol at their established IC50 dosages for 6 and 24 hours prior to gene expression profiling. The genes which changed by two-fold, and fell outside of the 90% range of expression after drug treatment were identified. Results: SAHA upregulated TNF-α and downstream proteins including HSP70, TNFSF9, SERPINB2, as well as STAT4- dependent genes. Sirtinol upregulated metalloproteinase (MMP) and phosphodiesterase (PDE) family members through retinoic acid signaling. We also observed that SAHA, TSA and Sirtinol differentially upregulate elements of the MAP kinase pathways, including signaling through Erk, JNK, and p38 MAPK, indicating a global stress response to HDAC inhibition. Conclusions: The above studies suggest that HDAC inhibitors differentially effect growth of WM cells through both global as well as pathway specific alterations in signaling. Importantly, this data provides a platform for studying the mechanistic role of HDACs in WM, and provide a framework for the use of HDAC-I as single and combination. No significant financial relationships to disclose.