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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

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone,doxorubicin,and daunorubicin,reduces daunorubicin accumulation and retention,and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells. View Publication

Abnormalities in mucin-type glycoprotein expression have been documented in a variety of cancers,identifying these molecules as targets for immunologically based therapies and prognostic/diagnostic assays. We examined the expression of the membrane-bound MUC1 mucin in normal,histologically atypical,and neoplastic lung to determine its potential contribution to lung carcinogenesis. In vivo,intense MUC1 immunoreactivity was present in normal type II pneumocytes as well as in a range of atypical lesions derived from type II cells and textgreater60% of primary and metastatic non-small cell lung cancers. Expression was not associated with altered survival,although it was highly correlated with the adenocarcinoma histology. A carcinogenesis model using 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-exposed hamsters revealed that MUC1 mRNA increased prior to the histological appearance of tumors. In vitro studies using MUC1 expressing non-small cell lung cancer cell lines revealed that differentiation away from a type II cell lineage was associated with dramatic down-regulation of MUC1. We propose that MUC1 is a powerful new marker for the type II pneumocyte cell lineage that allows us to follow the type II pneumocyte lineage during the process of lung carcinogenesis. View Publication

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure,but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2,Q105,into threonine and alanine,and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins,and the crystal structure of a pyridinyl imidazole,SB203580,bound to an ERK2 pentamutant,I103L,Q105T,D106H,E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore,we identified 5-iodotubercidin as a potent ERK2 inhibitor,which may help reveal the role of ERK2 in cell proliferation. View Publication

The oncogene p3k,coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137),strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD,myosin heavy chain,creatine kinase,and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis. View Publication

Tankyrase,a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP),was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1),a negative regulator of telomere length maintenance. Like ankyrins,tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro,with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro,suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation. View Publication

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However,the importance of this event for monocyte function is not clear. To address this issue,we used the novel MAP/ERK kinase (MEK) inhibitor,U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK,Jun NH2-terminal kinase (JNK),and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However,the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound,confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function,we measured production of the cytokines IL-1,IL-8,and TNF,as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1,IL-8,TNF,or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS. View Publication

Human blastocyst-derived,pluripotent cell lines are described that have normal karyotypes,express high levels of telomerase activity,and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months,these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers,including gut epithelium (endoderm); cartilage,bone,smooth muscle,and striated muscle (mesoderm); and neural epithelium,embryonic ganglia,and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology,drug discovery,and transplantation medicine. View Publication

Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS,PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol,a lignan,was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K1 of 26 +/- 2 microM but enhanced the PGHS-2 activity nearly twofold. Additionally,resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC50 = 15 microM) better than that of PGHS-2 (IC50 = textgreater 200 microM). Inhibition of the perxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG2 from arachidonic acid. A plausible relationship between these observation and the anticarcinogenic activity of resveratrol is discussed. 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