INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells.

AIMS/HYPOTHESIS: Using a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin(+) cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin(+) cells and identify key differences and similarities between them and primary beta cells. METHODS: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS (GFP/w)) and an untargeted hESC line (HES2). INS (GFP/w) allowed efficient identification and purification of GFP-producing (INS:GFP(+)) cells. Insulin(+) cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology. RESULTS: Immunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable K(ATP) and voltage-gated Ca(2+) channel activity, and reduced glucose-induced cytosolic Ca(2+) uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP(+) cells and adult human islets; however, INS:GFP(+) cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis. CONCLUSIONS/INTERPRETATION: INS:GFP(+) cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP(+) cells collectively resemble immature endocrine cells. However, insulin(+) cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.

Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.

Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells.

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.

Rapid assays for evaluating the drug sensitivity of tumor cells.

Assays that assess the ability of cells to incorporate labeled precursors into acid-precipitable material in the presence of adriamycin, daunorubicin, puromycin, vinblastine, melphalan, or methotrexate were investigated as an approach to the detection of resistant cells in human tumor samples. Each assay was evaluated with suitable drug-resistant Chinese hamster ovary cell lines and normal human fibroblasts to determine whether the assays reflected the drug sensitivity of these lines. Moreover, the ability to detect the presence of drug-resistance cells in a mixed population was evaluated. Validated assays were then used to measure the drug sensitivity of cell samples from pleural and peritoneal effusions of patients, mainly with carcinoma of the breast or ovary. Though the responsiveness of the majority of the samples in these assays was similar to that of a human fetal lung fibroblast line, 37 of 142 samples displayed responses consistent with the presence of a significant proportion of drug-resistant cells. Of these 37 nonresponsive samples, 12 displayed nonresponsiveness to three drugs.

Complete cDNA sequences encoding the Chinese hamster P-glycoprotein gene family.

The only function of the transport protein P-glycoprotein (Pgp) that has been identified to date in mammals is its ability to mediate multidrug resistance (MDR) in tumour cell lines. Rodents have three P-glycoprotein (pgp) genes (termed pgp or mdr 1, 2 and 3), and humans have two (mdr1 and mdr3/mdr2). Pgp isoforms differ in their drug transport capabilities: Pgp1 and Pgp2 can mediate MDR, while Pgp3 apparently cannot. The expression of the gene family members is tissue-specific, suggesting that they have unique physiological roles. We report in this paper the complete cDNA sequences for each of the three pgp genes in Chinese hamster. A comparison of the Chinese hamster cDNA sequences with those isolated from human and mouse confirms the identification of the gene family member homologues across these species. An analysis of mammalian Pgp sequences identifies conserved sequences which, it may be speculated, are important for Pgp activity. Previously, three different mdr3 (pgp3 homologous) transcripts, products of alternative splicing, have been reported in humans. Unexpectedly, we find no evidence for a similar alternative splicing event in Chinese hamster: it appears that the expression of pgp3 (mdr3) is different between rodents and humans.

Cloning of 11 alpha-tubulin gene sequences from the genome of Chinese hamster ovary cells.

We have analyzed the complex tubulin gene family in clonal lines of Chinese hamster ovary cells. There are approximately 16 alpha-tubulin genes and a similar multiplicity of beta-tubulin genes. The alpha-tubulin genes are not closely linked to each other nor to the beta-tubulin genes. A genomic library has been constructed in the vector lambda charon 4A containing insert sizes of 13-20 kilobases. The library has been screened with both inter- and intra-species alpha-tubulin probes. Eleven alpha-tubulin clones with different restriction patterns have been isolated and characterized. At least seven of these clones contain the complete gene coding region. One clone appears to represent the transcribed alpha-tubulin gene II. The sequence of an intron from this gene is compared with that from an equivalent gene in the rat.

Mutants of Chinese hamster ovary (CHO) cells with altered colcemid-binding affinity.

Clones of CHO cells stably resistant to colcemid have been isolated in the presence of the nonionic detergent Tween 80 after mutagen treatment. Successive single-step selections for increasing resistance were performed resulting in lines after three selection steps about 10 fold more resistant to colcemid than the parental cells. Three observations indicate that these colcemid-resistant (CMR) mutants are different from the colchicine-resistant permeability mutants isolated previously. First, their relative resistance to colcemid was not diminished in the presence of detergent which promoted increased drug permeability. Second, the CMR clones displayed limited cross-resistances only to tubulin-binding compounds. Third, the binding affinity of labeled colcemid by cytoplasmic extracts from CMR clones was reduced, and the reduction was greater in the more resistant clones. No reduction in binding of labeled colcemid was observed in the membrane-altered colchicine-resistant mutants. All these observations are consistent with the CMR clones being tubulin-altered mutants. In further support of this conclusion, we observed that tubulin purified from a CMR mutant still possessed reduced colcemid-binding affinity compared with that from parental cells.

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

We have examined mutant lines of Chinese hamster ovary cells that have increased resistance to the antimicrotubule drug Colcemid. Analysis of the functional properties of purified microtubule protein indicates that increased tolerance to the drug in vivo is reflected in altered properties of microtubules and tubulin in vitro. In this study, we have examined one series of related mutants and have found different microtubule alterations associated with each selection step. These changes include decreased Colcemid-binding affinity, an altered electrophoretic pattern of tubulin subcomponents, increased resistance to Colcemid inhibition of polymerization in vitro and, in one case, a decreased critical concentration for microtubule assembly. Characterized mutants of the class described here will be useful for probing the regulation of microtubule assembly in vivo.

CDw150(SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive properties of this virus.

Natural isolates of measles virus readily infect several lymphocyte cell lines. These viruses appear to use a receptor other than CD46, the molecule to which most laboratory strains of virus bind. Methods used to identify and characterize this lymphocyte receptor for measles virus are described in this study. A binding assay with a soluble form of measles virus H protein demonstrated that B-cell lines, activated with Epstein-Barr virus, or T cells, transformed with human T-cell leukemia virus, exhibit this receptor on their cell surfaces. On the other hand, resting lymphocytes, monocytes, or immature leukocytes either failed to express or possessed reduced levels of this receptor. A cDNA library derived from B95-8 marmoset B-cell lines was used to identify this receptor through expression cloning. This molecule was shown to be CDw150, which is also known as the signaling lymphocytic activation molecule (SLAM). When the lymphocyte receptor was expressed in Chinese hamster ovary (CHOP) or human embryonic kidney (293T) cells, these cells became susceptible to lymphotropic as well as laboratory strains of measles virus. Binding assays confirmed that either lymphotropic or laboratory strains of measles virus could adhere to human or marmoset CDw150, but interaction with the mouse homolog was weak. These infections were independent of the presence of CD46 on the host cell surface. Interaction of measles virus with CDw150(SLAM) could explain the immunosuppressive properties of this virus.

Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding.

CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis.

Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance.

Increased expression of P-glycoprotein, a plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (170K), occurs in a wide variety of cell lines that exhibit pleiotropic resistance to unrelated drugs. The presence of P-glycoprotein in human cancers refractory to chemotherapy suggests that tumour cells with multidrug resistance can arise during malignant progression. We have discovered striking homology between P-glycoprotein and the HlyB protein, a 66K Escherichia coli membrane protein required for the export of haemolysin (protein of Mr 107K). P-glycoprotein can be viewed as a tandem duplication of the HlyB protein. The hydropathy profiles of the two proteins are similar and reveal an extensive transmembrane region resembling those found in pore-forming plasma membrane proteins. The C-terminal region of P-glycoprotein and the HlyB protein contain sequences homologous to the nucleotide-binding domains of a group of closely related bacterial ATP-binding proteins. We propose a model for multidrug resistance in which P-glycoprotein functions as an energy-dependent export pump to reduce intracellular levels of anticancer drugs.

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway.

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells.

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.