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Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study,a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline,MCF-7 AdVp3000. Levels were lower in earlier steps of selection,and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells,but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter,which is overexpressed in mitoxantrone-resistant cells. View Publication

The histologic distinction between epithelial peritoneal mesothelioma and papillary serous carcinoma diffusely involving the peritoneum may be difficult. Although some investigators have indicated that immunohistochemistry can facilitate this differential diagnosis. only a few studies using a limited number of markers have been published. In this study,the immunoreactivity of keratin 5/6,vimentin,epithelial membrane antigen,thrombomodulin,calretinin,MOC-31,Ber-EP4,carcinoembryonic antigen,TAG-72 (B72.3),CD15 (Leu-M1),placental alkaline phosphatase,CA19-9,CA-125,HBME-1,44-3A6,and S-100 protein was investigated in 35 epithelial peritoneal mesotheliomas,and 45 papillary serous carcinomas [30 ovarian (10 primary and 20 metastatic to the peritoneum) and 15 papillary serous carcinomas of the peritoneum]. After analyzing the results,it is concluded that calretinin,thrombomodulin,and keratin 5/6 are the best positive markers for differentiating epithelial malignant mesotheliomas from papillary serous carcinomas diffusely involving the peritoneum. The best diagnostic discriminators among the antibodies considered to be negative markers for mesothelioma are MOC-31,B72.3,Ber-EP4,CA19-9,and Leu-M1. Immunostaining for carcinoembryonic antigen,placental alkaline phosphatase,epithelial membrane antigen,vimentin,HBME-1,44-3A6,CA-125,or S-100 have little or no diagnostic utility in establishing the differential diagnosis between these conditions. The results of this study also confirm previous observations indicating that both papillary serous carcinomas of the peritoneum and serous carcinomas of the ovary have a similar phenotype and,therefore,immunohistochemical studies are not useful in separating these entities. View Publication

MUC1 mucin is expressed by normal and malignant epithelial cells and is thought to function through cell-cell interactions and transmembrane signal transduction events. Secreted cancer-associated MUC1 is immunosuppressive and inhibits human T-cell proliferation. We report here that newly synthesized MUC1 is expressed on the surface of mitogen-activated human T cells and is also found in soluble form in the supernatants from cultures of mitogen-activated human T cells. After removal of the mitogenic stimulus from the T-cell cultures,MUC1 expression is downregulated. The addition of anti-MUC1 monoclonal antibody to mitogen-activated cultures partially inhibits the T-cell proliferative response. These data suggest that MUC1 serves an immunodulatory function for human T lymphocytes. View Publication

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening,potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics. View Publication

The CD34 molecule belongs to the mucin membrane molecule family and is expressed on virtually all normal haematopoietic progenitor cells (HPC). Due to its heavy glycosylation,several different epitopes exist on the molecule. Based on the sensitivity of the glycosylated molecule to degradation with a glycoprotease from Pasteurella haemolytica and neuraminidase,three classes of epitopes have been identified. The class I and II epitopes are probably related to the glycosylated part of the molecule while class III epitopes are core protein related. It has been known for some time that CD34 class I epitopes are absent on CD34 molecules expressed on high endothelial venules. Here we review recent observations that expression of both class I and II epitopes,but not class III epitopes,is impaired on mature myeloid CD34-pos. HPC while no diverse class epitope expression was observed on immature HPC. In addition,cells from patients with CD34-pos. acute myeloid leukaemia of FAB classification M4-M5,i.e.,leukaemic blast cells of relatively mature morphologic phenotype,also express less class I and II epitopes than class III epitopes. It therefore seems that HPC maturation and class I and II epitope deprivation are concomitant events and that CD34 class I and II epitopes are lost prior to downregulation of the CD34 molecule per se. The biological significance of this observation is discussed as well as the need to carefully select CD34-specific monoclonal antibodies for research and clinical purposes. View Publication

Ubiquinone and alpha-lipoic acid are natural constituents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of alpha-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme complexes involved in the citric acid cycle while UQ recycles through semi- and divalently reduced ubiquinones in the respiratory chain. We have proved the validity of the concept about the antioxidant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation,the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product alpha-lipoic acid. Our experiments demonstrate the existence of an dihydrolipoic acid driven recycling of UQ to the antioxidative-active UQH2. The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites. View Publication

Using radioligand binding assays,we determined the equilibrium dissociation constants (KD's) for 37 antidepressants,three of their metabolites (desmethylcitalopram,desmethylsertraline,and norfluoxetine),some mood stabilizers,and assorted other compounds (some antiepileptics,Ca2+ channel antagonists,benzodiazepines,psychostimulants,antihistamines,and monoamines) for the human serotonin,norepinephrine,and dopamine transporters. Among the compounds that we tested,mazindol was the most potent at the human norepinephrine and dopamine transporters with KD's of 0.45 +/- 0.03 nM and 8.1 +/- 0.4 nM,respectively. Sertraline (KD = 25 +/- 2 nM) and nomifensine (56 +/- 3 nM) were the two most potent antidepressants at the human dopamine transporter. We showed significant correlations for antidepressant affinities at binding to serotonin (R = 0.93),norepinephrine (R = 0.97),and dopamine (R = 0.87) transporters in comparison to their respective values for inhibiting uptake of monoamines into rat brain synaptosomes. These data are useful in predicting some possible adverse effects and drug-drug interactions of antidepressants and related compounds. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication

The entry of a cell into mitosis is regulated by an elaborate network of kinases and phosphatases that control both for the timing of cell division and the complete reorganization of the cellular architecture. The mitotic cyclin/Cdks form part of large multiprotein complexes whose other components are only now beginning to be identified. The continuing identification of proteins that contribute to these complexes and changes in the composition of these complexes are likely to give a more integrated view of how mitotic cyclin/Cdk complexes are regulated and how they function-not only to induce mitosis,but also to aid further mitotic progression. Furthermore,assigning specific G2/M functions to distinct mitotic cyclin/Cdk complexes will require the identification of differences in substrate specificities between the mitotic cyclin/Cdk complexes,perhaps in parallel with specific cyclin knockouts in mice. Such investigations will be complicated by potential functional overlap between mitotic cyclin/Cdk complexes in vitro and in vivo. Although cyclin/Cdk1 is thought to be the major kinase that initiates the onset of mitosis,a more complete understanding of how cells move from G2 to a mitotic state will require further identification of kinases operating upstream,downstream and in parallel with Cdk1,their substrates and their relationship with one another during the G2/M transition. View Publication