Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes.

In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors. Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.

Synergy between a human c-myc transgene and p53 null genotype in murine thymic lymphomas: contrasting effects of homozygous and heterozygous p53 loss.

Activation of the c-myc oncogene and functional loss of the p53 tumour suppressor gene are among the most frequently recorded genetic lesions in neoplasia but their combined effect has not previously been investigated. By breeding together mice transgenic for human c-myc (CD2-myc) and mice carrying an inactive p53 allele (p53-/-) we found that these genetic lesions act synergistically in vivo. Offspring carrying the CD2-myc transgene and the homozygous p53 null mutation (p53-/-/CD2-myc) were viable but developed thymic lymphomas with dramatically increased frequency and reduced latency compared to both parental groups. The tumour phenotype was similar to that previously recorded for CD2-myc mice (predominantly CD3+, CD4+8+) but tumour clonal complexity and metastasis was significantly greater in the p53-/-/CD2-myc mice. In contrast, no significant increase in tumour incidence was seen in p53+/-/CD2-myc vs p53+/+/CD2-myc mice over a 6 month observation period. However, the loss of wild type p53 in a proportion of tumour cells in p53+/-/CD2-myc lymphomas suggests that wild type allele loss can occur as a late progression step rather than an initiating step in these tumours. We suggest that p53 loss of function may collaborate with the CD2-myc transgene at more than one stage in thymic lymphoma development.

p53 and tumour viruses: catching the guardian off-guard.

Oncogenic viruses have evolved direct and indirect mechanisms to overcome the tumour suppressor p53. Fortunately, tumour development is limited by the narrow cell tropisms of the viruses concerned and the host immune response. However, such viruses are helping to elucidate the p53 response pathway and may play a future role as novel cancer therapeutic agents.

Piperazinylalkyl heterocycles as potential antipsychotic agents.

We recently reported on a series of pyrrole Mannich bases orally active in inhibiting the conditioned avoidance response (CAR) in rats. These compounds exhibit affinity for both D2 and 5-HT1A receptors, and some are noncataleptogenic. Such a profile suggests that they may be potential antipsychotic agents which lack the propensity for causing extrapyramidal side effects and tardive dyskinesias in humans. One of these compounds, 1-[[1-methyl-5-[[4-[2-(1-methylethoxy)phenyl]- 1-piperazinyl]methyl]-1H-pyrrol-2-yl]methyl]-2-piperidinone (RWJ 25730, 1), was chosen for further development but found to be unstable in dilute acid. In order to improve stability, we replaced the pyrrole methylene linkage to the piperazine ring with ethylene, employed ethylene and dicarbonyl as linkers between the lactam and the pyrrole ring, placed electron-withdrawing groups on the pyrrole ring, and substituted acyclic amide for lactam. In addition, we replaced the pyrrole segment with other heterocycles including thiophene, furan, isoxazole, isoxazoline, and pyridine. Generally, replacement of the N-methylpyrrole segment with thiophene, furan, isoxazoline, or pyridine afforded compounds equipotent with 1 in CAR, which were more stable in dilute acid. In the case of side chain or lactam modifications, CAR activity was significantly decreased or abolished, with the exception of 6. For the most part, the modifications to 1 resulted in the decrease or loss of D2 receptor binding. However, within this series, 5-HT1A receptor binding was greatly increased, with thiophene 40 exhibiting an IC50 of 0.07 nM. The CAR activities of pyrroles 6 and 12, thiophene 40, furans 44-47, isoxazolines 49 and 50, and pyridine 54 coupled with their weak or nonexistent D2 binding and strong 5-HT1A binding suggest that they may be acting via a nondopaminergic mechanism or that dopaminergic active metabolites are responsible. Pyrrole 6 and furans 44 and 47 show promise as antipsychotic agents based on their CAR activity, receptor-binding profile, and solution stability.

N-aryl-N'-benzylpiperazines as potential antipsychotic agents.

N1-(2-Alkoxyphenyl)piperazines additionally containing an N4-benzyl group bearing alcohol, amide, imide, or hydantoin functionalities were prepared and evaluated in the conditioned avoidance response (CAR) test predictive of clinical antipsychotic activity and in in vitro receptor-binding assays. Certain of the compounds display high affinity for the D2, 5-HT1A, and alpha 1-adrenergic receptors. Structures bearing acyclic amide, lactam, and imide functionalities display good biological activity, with a preference for the 1,3-disubstituted phenyl ring relative to the 1,4- and 1,2-congeners (7 vs 10 and 12). Every possible position of hydantoin attachment was investigated (e.g., substitution at N1, N3, and C5). The hydantoin involving attachment to N1 (24) was found to have good biological activity, whereas those hydantoins with attachment to N3 or C5 (22, 23, and 25) were inactive. Several of the smaller acetylated derivatives (30 and 33) have fair in vivo activity, which was lost in the case of the larger benzoyl analog 31. Uracil congener 34 had modest affinity for the D2 receptor (65 nM) as well as excellent in vivo activity. Benzylamino compounds display (viz. 27 and 35-38) moderate CAR activity but have surprising receptor affinity, often greater than those of comparable structures bearing a carbonyl (36 vs 7). Benzyl and benzhydryl alcohol compounds 40-48 are more active than amino structures 27 and 35-38 and also exhibit excellent in vivo activity in the CAR test with modest D2 and 5-HT1A receptor binding.

Orally active benzamide antipsychotic agents with affinity for dopamine D2, serotonin 5-HT1A, and adrenergic alpha1 receptors.

New antipsychotic drugs are needed because current therapy is ineffective for many schizophrenics and because treatment is often accompanied by extrapyramidal symptoms and dyskinesias. This paper describes the design, synthesis, and evaluation of a series of related (aminomethyl)benzamides in assays predictive of antipsychotic activity in humans. These compounds had notable affinity for dopamine D2, serotonin 5-HT1A, and alpha1-adrenergic receptors. The arylpiperazine 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoyl]p ipe ridine (mazapertine, 6) was chosen because of its overall profile for evaluation in human clinical trials. The corresponding 4-arylpiperidine derivative 67 was also highly active indicating that the aniline nitrogen of 6 is not required for activity. Other particularly active structures include homopiperidine amide 14 and N-methylcyclohexylamide 31.

Selection for loss of p53 function in T-cell lymphomagenesis is alleviated by Moloney murine leukemia virus infection in myc transgenic mice.

Thymic lymphomas induced by Moloney murine leukemia virus (MMLV) have provided many examples of oncogene activation, but the role of tumor suppressor pathways in these tumors is less clear. These tumors display little evidence of loss of heterozygosity, and MMLV is only weakly synergistic with the Trp53 null genotype, suggesting that viral lymphomagenesis involves mechanisms which do not require mutational loss of Trp53 function. To explore this relationship in greater depth, we infected CD2-myc transgenic mice with MMLV and examined the role of Trp53 in the genesis of these tumors. Most (19 of 27) of the tumors from MMLV-infected, CD2-myc Trp53(+/-) mice retained the wild-type Trp53 allele in vivo while tumors of uninfected CD2-myc Trp53(+/-) mice invariably showed allele loss from a significant fraction of primary tumor cells. The functional integrity of the Trp53 gene in these tumors was indicated by ongoing allele loss or selection for mutational stabilization during in vitro propagation and by the radiosensitivity of selected Trp53(+/-) tumor cell lines. An inverse correlation was noted between retention of the wild-type Trp53 allele and expression of p19(ARF), providing further evidence of negative-feedback control of the latter by p53. However, expression of p19(ARF) does not appear to be counterselected in the absence of p53, and its integrity in Trp53(+/-) tumors was indicated by its transcriptional upregulation on Trp53 wild-type allele loss in vitro in selected tumor cell lines. The role of MMLV was investigated further by analysis of proviral insertion sites in tumors of CD2-myc transgenic mice sorted for Trp53 genotype. A proportion of tumors showed insertions at Runx2, an oncogene which has been shown to collaborate independently with CD2-myc and with the Trp53 null genotype, and at a novel common integration site (ptl-1) on chromosome 8. Genotypic analysis of the panel of tumors suggested that neither of these integrations is functionally redundant with loss of p53, but it appears that the combination of the MMLV oncogenic program with the CD2-myc oncogene relegates p53 loss to a late step in tumor progression or in vitro culture. While the means by which these tumors preempt the p53 tumor suppressor response remains to be established, this study provides further evidence that irreversible inactivation of this pathway is not a prerequisite for tumor development in vivo.

Moloney murine leukemia virus-induced lymphomas in p53-deficient mice: overlapping pathways in tumor development?

The effect of Moloney murine leukemia virus (MoMLV) infection was examined in mice lacking a functional p53 gene. Virus-infected p53-/- mice developed tumors significantly faster than uninfected p53-/- or virus-infected p53+/+ littermates. However, the degree of synergy between MoMLV and the p53 null genotype was weaker than the synergy between either of these and c-myc transgenes. A similar range of T-cell tumor phenotypes was represented in all p53 genotype groups, including p53-/- mice, which developed thymic lymphomas as the most common of several neoplastic diseases. Lack of p53 was associated with higher rates of metastasis and the ready establishment of tumors in tissue culture. Loss of the wild-type allele was a common feature of tumors in p53+/- mice and was complete in tumor cells in vitro, but this appeared to occur by a mechanism other than proviral insertion at the wild-type allele. A lower average MoMLV proviral copy number was observed in tumors of the p53 null and heterozygote groups, suggesting that the absence of a functional p53 gene reduced the number of steps required to complete the malignant phenotype. Mink cell focus-forming virus-like proviruses were detected in tumors of all infected mice but were relatively rare in p53 null mice. Analysis of c-myc, pim-1, and pal-1 showed that these loci were occupied by proviruses in some cases but at similar frequencies in p53 wild-type and null mice. In conclusion, while inactivation of p53 in the germ line predisposes mice to tumors similar in phenotype to those induced by MoMLV, it appears that virus-induced tumors generally occur without p53 loss. We speculate that a bcl-2-like function carried or induced by MoMLV may underlie this p53-independent pathway.

Piperazinyl benzamidines: synthesis and affinity for the delta opioid receptor.

Piperazinyl benzamidines were prepared and found to bind to the rat delta (delta) opioid receptor. The most active compounds had a N,N-diethylcarboxamido group and a N-benzyl piperazine. The most potent among these was N,N-diethyl-4-[4-(phenylmethyl)-1-piperazinyl][2-(trifluoromethyl)phenyl]iminomethyl]benzamide (27) with a 1.22nM K(i) for the rat delta opioid receptor and ca. 1000 x selectivity relative to the mu opioid subtype.