Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR.

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.

The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux.

Cytochrome P450 3A4 is an important mediator of drug catabolism that can be regulated by the steroid and xenobiotic receptor (SXR). We show here that SXR also regulates drug efflux by activating expression of the gene MDR1, which encodes the protein P-glycoprotein (ABCB1). Paclitaxel (Taxol), a commonly used chemotherapeutic agent, activated SXR and enhanced P-glycoprotein-mediated drug clearance. In contrast, docetaxel (Taxotere), a closely related antineoplastic agent, did not activate SXR and displayed superior pharmacokinetic properties. Docetaxel's silent properties reflect its inability to displace transcriptional corepressors from SXR. We also found that ET-743, a potent antineoplastic agent, suppressed MDR1 transcription by acting as an inhibitor of SXR. These findings demonstrate how the molecular activities of SXR can be manipulated to control drug clearance.

Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer.

It has recently been shown that the neurological mutant mouse staggerer (sg) harbors a deletion within the Rora gene that encodes the orphan nuclear receptor ROR alpha. This deletion removes an exon encoding part of the ligand binding domain of the putative receptor, generating an ROR alpha truncated protein (ROR alpha(sg)). It is unknown whether sg acts as a null or highly hypomorphic allele. To address this question, we have generated a null mutation of Rora by targeted disruption of its DNA binding domain in ES cells. The Rora-/- mice are viable but display tremor, body imbalance, small size and die between 3-4 weeks, similar to the sg mouse. Histological examination of the cerebellum of Rora-/- and sg mice showed similar defects, including small size and fewer ectopically localized Purkinje cells. Northern blot analysis of cerebellar RNA showed that ROR alpha transcripts are still expressed in the Rora-/- and sg mutants, although with altered mobilities. However, the cerebellum of the Rora-/- mutant does not express the ROR alpha protein. Attempts to complement the defect of the Rora-/- with sg failed, demonstrating conclusively that the sg defects are caused by the absence of functional ROR alpha.

Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors.

ROR alpha1 and RVR are orphan members of the superfamily of nuclear hormone receptors which constitutively activate and repress, respectively, gene transcription by binding to a common DNA sequence. In an attempt to understand the physiological functions of these two transcription factors, we aimed to identify target genes. We have identified a consensus binding site for ROR alpha1 and RVR in the first intron of the N-myc gene that we designated N-myc RORE (ROR response element). Unlike most of the intronic sequence, the region encompassing the N-myc RORE is highly conserved between human and mouse, underscoring its importance. Our studies revealed that ROR alpha1 and RVR specifically bind to the human and mouse N-myc ROREs and transactivate and transrepress, respectively, reporter constructs containing the ROREs. Moreover, Northern blot analysis demonstrated a direct modulation of an exogenously introduced N-myc gene by ROR alpha1 and RVR in COS-1 cells. This effect is mediated through the N-myc RORE, since mutation of this site abolished the regulatory effects of both receptors. While transfection of ROR alpha1 in P19 embryonic carcinoma cells had no effect on the levels of endogenous N-myc mRNA, RVR down-regulated its expression. The regulatory function of the N-myc RORE was further demonstrated by the rat embryonic fibroblast (REF) transformation assay. Mutation of the RORE increased the oncogenic potential of the N-myc gene in the REF assay. The foci were more numerous and significantly larger with the mutated than with the wild-type N-myc gene, regardless of ROR alpha1 or RVR expression. Moreover, concomitant expression of ROR alpha1 and wild-type N-myc resulted in a twofold increase in the number of transformed foci. In contrast, RVR expression resulted in the formation of foci that could be established as permanent clones with a very low frequency compared to foci transformed in its absence. These observations show that ablation of the RORE results in a more oncogenic form of N-myc and suggest that deregulation of the activity of the ROR alpha1 and RVR could contribute to the initiation and progression of certain neoplasias.

Effect on leukemia cell numbers of in vivo administration of immunotherapeutic agents is age-dependent.

On the basis of our previous findings that erythroleukemia-bearing mice of different ages responded positively to immunotherapy [indomethacin +/- recombinant interleukin (rIL-2)] in vivo [stimulated natural killer (NK) cells and increased life span], we aimed, in the present study, to determine if changes in these parameters could be correlated to change in erythroleukemia cell numbers. Infant (< 3 weeks old), young adult (5-8 weeks) and aged (> 10 months) DBA/2 mice were injected with erythroleukemia cells. Some mice remained untreated for the 10-day duration of the tumor-bearing period, while others were treated with either indomethacin alone for the 10 days from tumor inoculation, rIL-2 alone for the last 4 days of the 10-day tumor-bearing period, or with both indomethacin and rIL-2 as above. In all cases, both treated and untreated mice were killed at 10 days after tumor inoculation. Cytospot preparations from single cell suspensions of spleen and bone marrow, in all cases, were stained with MacNeal's tetrachrome hematologic stain and the relative and absolute number of erythroleukemia cells in these organs were determined using light microscopy. The results show that indomethacin- and/or rIL-2 treated leukemic infant and young adult mice have significantly lower numbers of erythroleukemia cells in both the spleen and bone marrow relative to untreated, leukemic mice of corresponding age. Leukemic aged mice, however, show no change in erythroleukemia cell numbers in either organ, regardless of treatment, paralleling our observations of a lack of immunotherapeutic value of these agents on NK cell production/function in aged mice.

Effectiveness of immunotherapy in aged leukemic mice.

We previously showed that immunotherapy using indomethacin combined with rIL-2 in vivo was very effective in stimulating natural killer (NK) cells and in increasing the life span of young adult mice bearing a tumor of hemopoietic origin. The aim of the present study was to test the efficacy and universality, with respect to age, of this treatment in tumor-bearing mice. DBA/2 mice (10-16 months old) were injected with 5 x 10(6) erythroleukemia cells and remained either: (i) untreated (control); (ii) treated with indomethacin (5 micrograms/ml drinking water) for 9 days from tumor onset; (iii) treated with rIL-2 (24 x 10(3) U/injection) twice a day for the last 4 days of the 9-day tumor-bearing period, or (iv) treated with both indomethacin and rIL-2 concomitantly. Some mice from each group (above) were killed after 9 days of tumor growth, while the others were allowed to survive. Spleen and bone marrow cells were collected from the mice of each group and NK (ASGM-1+) cells were quantitated using an immunoperoxidase technique combined with light microscopy. NK cell-mediated activity was assessed using a standard chromium release assay. The results show that although NK cell numbers increase in the presence of the growing tumor, neither indomethacin alone, rIL-2 alone, nor the combination could further increase the numbers of these cells. Furthermore, indomethacin and/or rIL-2 could not induce NK cell-mediated activity in such mice. Moreover, tumor-bearing aged mice treated as above did not have a significantly longer life span than untreated (control) tumor-bearing mice. The present results indicate an age-dependent resistance to a form of immunotherapy already proven very effective in young adult mice. Furthermore, the results of this and our previous studies suggest that immunotherapy, which may be highly effective in one age group, should not be presumed effective throughout life.

Suppression of natural killer cell activity in infant mice occurs after target cell binding.

Natural killer (NK) cell functional activity is absent in mice < 3 weeks of age. However, the mechanism(s) responsible for such inactivity is (are) still unknown. Some evidence suggests basic immaturity of the infant-source NK cells while others suggest suppression of NK cell-mediated activity by other endogenous cells/factors. In the present study, infant-source, spleen-derived, NK cells were highly enriched using anti-NK 1.1 monoclonal antibody coupled to immunomagnetic DYNAbeads. The NK cell characteristics of the isolated cells were tested using three assays: (i) target-binding capacity, (ii) lytic potency as measured in a single cell assay using propidium iodide to label NK cell-lysed dead targets and (iii) proliferative capacity (3H-thymidine incorporation). The results demonstrated that enriched populations of infant NK cells have a greater capacity to bind target cells than unseparated infant or young adult spleen cells. Nevertheless, both unseparated spleen cells and isolated NK cells had a similar 3H-thymidine uptake. The NK cell-mediated lytic capacity of unseparated infant spleen cells was negligible, with 0.5% of YAC-1 targets being killed. However, the lytic capacity of isolated infant-source NK cells was 12% and could be further augmented to 21% after exposure to recombinant interleukin-2. These results suggest that (a) NK cells are present and functional in infant mice, but are suppressed by some endogenous cells/factors, (b) such suppression acts at a postbinding event of NK lysis and (c) such suppression is released when NK cells in infant spleen are removed from that environment.

Decline in natural killer cell-mediated immunosurveillance in aging mice--a consequence of reduced cell production and tumor binding capacity.

The mechanism(s) responsible for the functional deficiencies in aging mice of natural killer (NK) cells, the lymphocytes active in early neoplasia development, are unknown. The aim of the present study was to examine the effect of indomethacin, a prostaglandin synthetase inhibitor, and recombinant interleukin-2 (rIL-2), an NK cell stimulant, on the NK cell numbers and function of aging mice. This combination is highly effective in inducing proliferation and activation of the NK cells in young adult mice. Ten- to 13-month-old DBA/2 mice received either indomethacin daily for 9 days in their drinking water, or rIL-2 twice a day for 4 days, or both agents combined. Untreated aging mice had 50% fewer splenic NK cells than did young adult (5-8 weeks) DBA/2 mice, although bone marrow contained NK cell numbers similar to that of young adult mice. Neither indomethacin alone, rIL-2 alone nor the combination of both could induce an increase in NK cell numbers or function in aging mice. Radioautography combined with immunoperoxidase labelling techniques revealed that the production of new NK cells from the bone marrow of aging mice was significantly reduced relative to that of young adult mice. This was reflected in low numbers of newly formed NK cells accumulating in the spleens of aging mice. The target binding capacity, a necessary precytolytic event, of aging mouse NK cells was also reduced compared to that of young adult mice. The results suggest that the functional deficiencies of aging mouse NK cells are a consequence of multiple factors including an absolute loss of NK cells resulting from a reduced production of such cells in the bone marrow and a decreased capacity of NK cells to bind their tumor targets.

Stimulation of natural killer cell numbers but not function in leukemic infant mice: a system primed in infancy allows survival in adulthood.

Infant mice (< 3-4 weeks) demonstrate no detectable natural killer (NK)-cell-mediated immunity. The aim of the present work was to assess, quantitatively and functionally, the possibility that prostaglandin E2, (PGE2), an NK cell inhibitor, may be responsible for the absence of NK-cell-mediated activity in normal and/or erythroleukemia-bearing infant DBA/2 mice prior to the normal age-related onset of NK-cell-mediated lytic capacity. Infants (7 days after birth) were exposed daily to indomethacin via intraperitoneal injection for 10 days and/or recombinant interleukin-2 (rIL-2) daily for 4 days. Significant increases in the number of NK cells in both the spleen and bone marrow were found after 10 days of indomethacin or 4 days of rIL-2 in normal mice. The spleens but not the bone marrow of infants treated with indomethacin from tumor onset (7 days after birth) contained significantly more NK cells 10 days later than did control (tumor+vehicle) infants. Infants treated with rIL-2 during the last 4 days of tumor bearing, i.e., days 13-16 after birth, contained significantly more NK cells in both their spleen and bone marrow, while combined administration of rIL-2 and indomethacin to tumor-bearing, but not normal, infants resulted in a more than additive increase in the NK cell numbers in both organs relative to control (tumor+vehicle or vehicle alone). However, in neither normal nor tumor-bearing infants, could indomethacin, rIL-2, or a combination of both, induce the development of NK-cell-mediated functional (lytic) activity in spite of the generation, in nearly all instances, of high levels of NK cells in the presence of these agents. The observations collectively suggest that the lack of functional (lytic) reactivity of infant-source NK cells, in the presence of agents which potently enhance adult-source NK cells, reflects (1) the innate immaturity of infant NK lineage cells, or (2) the presence in infant NK-cell-containing organs of a function-suppressive mechanism which is indomethacin insensitive, i.e., not PGE2-mediated. The significantly prolonged survival, and even cure, of infant leukemic mice treated with indomethacin and/or rIL-2 may result from the agent-mediated elevated levels of precursor NK cells coming under the influence of some age-related, as yet unidentified, endogenous factor imbuing them with functional capacity.

Population dynamics of natural killer cells and T lymphocytes in murine spleen and bone marrow during the development of erythroleukemia: the effect of indomethacin.

Quantitative and functional methods were used to assess changes in NK (ASGM-1+, nonblast, lymphoid) cells and cytotoxic/suppressor T (Lyt-2+) cells in the spleen and bone marrow (BM) of normal DBA/2 mice and/or mice bearing early (7 days)- and late (14 days)-stage erythroleukemia +/- concomitant oral administration of indomethacin. In normal mice, indomethacin increased ASGM-1+ cells in the spleen and BM by 7 days of continuous drug administration relative to control, while the numbers of Lyt-2+ cells in both organs remained unaltered. The presence of a tumor in untreated mice resulted, by 14 days development, in elevated numbers of ASGM-1+ cells in the spleen and BM. Lyt-2+ cells in the spleen increased transiently (7 days) in tumor-bearing (TB) mice and returned to normal levels by 14 days TB. No difference in either early- or late-stage TB was observed in the numbers of Lyt-2+ cells in the BM. In spleen and BM of TB mice, indomethacin increased ASGM-1+ cells by 7 days TB. ASGM-1+ cells returned to untreated levels by 14 days TB in both organs in spite of continued drug treatment. The numbers of Lyt-2+ cells in both the spleen and BM of 7 days indomethacin-treated TB mice were reduced relative to those of untreated TB mice. By 14 days TB, drug treatment had no effect on Lyt-2+ cell numbers in the spleen or BM. Spontaneous cytolytic activity of the spleen and BM did not always parallel changes in the numbers of ASGM-1+ cells, indicating the presence of late, immature, prelytic NK cells among the ASGM-1+ cell numbers. The results demonstrate that the numbers of NK and cytotoxic/suppressor T cells are markedly but differentially affected during tumor growth and/or time of exposure to indomethacin in an organ-specific manner.

Population dynamics of natural killer cells in the spleen and bone marrow of normal and leukemic mice during in vivo exposure to interleukin-2.

By quantitative and functional methods, changes were assessed in NK(ASGM-1+) cell numbers and NK cell-mediated lytic function of the spleen and bone marrow of mice bearing a tumor of hemopoietic origin (FLV-induced erythroleukemia) for 9 days +/- simultaneous administration of indomethacin (10 micrograms/ml drinking water) +/- rIL-2 (3x/day, 12 x 10(3) Units/injection) during the last 4 days of tumor-bearing. Recombinant IL-2 alone during the last 4 days of tumor-bearing increased both the NK(ASGM-1+) cell numbers (p less than 0.001) and the functional activity (24-fold) of the spleen. In the bone marrow, however, no change in the numbers of NK(ASGM-1+) cells was observed relative to untreated tumor-bearing mice, but the NK cell-mediated lytic activity of that organ was augmented 30-fold. The continuous presence of indomethacin from the onset of tumor-bearing prior to rIL-2 treatment during the last 4 days of tumor-bearing, further boosted both the already high, rIL-2 driven numbers of NK(ASGM-1+) cells in the spleen (p less than 0.01), as well as splenic NK cell lytic function (2-fold). In the bone marrow, continuous presence of indomethacin prior to and during the terminal 4 days of co-administration with rIL-2 increased 3-fold the numbers of NK(ASGM-1+) cells relative to that of the bone marrow of tumor-bearing mice given rIL-2 alone, and resulted in lytic activity of that organ which was 140% of that of the rIL-2 treated, tumor-bearing mice. The results indicate that under the combined influence of indomethacin and rIL-2, the production of NK(ASGM-1+) cells was augmented in the bone marrow of tumor-bearing mice, export of immature NK(ASGM-1+) cells from the bone marrow was increased, and import of immature NK(ASGM-1+) cells by the spleen was increased. The increased NK(ASGM-1+) cell numbers in each organ was reflected in increased lytic function.