Showing papers on "Cell culture published in 1996"

Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours

Abstract: Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.

Sheep cloned by nuclear transfer from a cultured cell line

Abstract: Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.

Telomerase activity in human germline and embryonic tissues and cells

Abstract: Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16-20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes

Abstract: We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts

Abstract: Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Transfecting Mammalian Cells: Optimization of Critical Parameters Affecting Calcium-Phosphate Precipitate Formation

Abstract: DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation

Telomerase activation by the E6 gene product of human papillomavirus type 16.

Abstract: Activation of telomerase, a ribonucleoprotein complex that synthesizes telomere repeat sequences, is linked to cell immortalization and is characteristic of most cell lines and tumours. Here we show that expression of the human papillomavirus type 16 (HPV-16) E6 protein activates telomerase in early-passage human keratinocytes and mammary epithelial cells. This activation was observed in cells pre-crisis, that is, before they became immortal, and occurred within one passage of retroviral infection with vectors expressing HPV-16 E6. Studies using HPV-16 E6 mutants showed that there was no correlation between the ability of the mutants to activate telomerase and their ability to target p53 for degradation, suggesting that telomerase activation by HPV-16 E6 is p53 independent. Keratinocytes expressing wild-type HPV-16 E6 have an extended lifespan, but do not become immortal, indicating that telomerase activation and E6-mediate degradation of p53 are insufficient for their immortalization. These results show that telomerase activation is an intrinsic, but insufficient, component of transformation by HPV.

Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA

Abstract: We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells.

Abstract: Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Expression of galectin-3 modulates T-cell growth and apoptosis

Abstract: Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.

A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.

Abstract: We constructed gfph, a synthetic version of the jellyfish Aequorea victoria green fluorescent protein (gfp) cDNA that is adapted for high-level expression in mammalian cells, especially those of human origin A total of 92 base substitutions were made in 88 codons in order to change the codon usage within the gfp10 coding sequence so that it was more appropriate for expression in mammalian cells We also describe a series of versatile recombinant adeno-associated virus and adenovirus vectors for delivery and expression of genes into mammalian cells and, using these vectors, demonstrate the efficient transduction and expression of the gfph gene in the human cell line 293 and also in vivo, within neurosensory cells of guinea pig eye Cells infected with recombinant adeno-associated virus-GFPH can be readily sorted by fluorescence-activated cell sorting, suggesting that the newly designed gfph gene could be widely used as a reporter in many gene delivery technologies, including human gene therapy

TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.

Abstract: Metastasis of epithelial tumor cells can be associated with the acquisition of fibroblastoid features and the ability to invade stroma and blood vessels. Using matched in vivo and in vitro culture systems employing fully polarized, mammary epithelial cells, we report here that TGF-beta1 brings about these changes in Ras-transformed cells but not in normal cells. When grown in collagen gels in the absence of TGF-beta, both normal and Ras-transformed mammary epithelial cells form organ-like structures in which the cells maintain their epithelial characteristics. Under these conditions, treatment of normal cells with TGF-beta results in growth arrest. The same treatment renders Ras-transformed epithelial cells fibroblastoid, invasive, and resistant to growth inhibition by TGF-beta. After this epithelial-fibroblastoid conversion, the Ras-transformed cells start to secrete TGF-beta themselves, leading to autocrine maintenance of the invasive phenotype and recruitment of additional cells to become fibroblastoid and invasive. More important, this cooperation of activated Ha-Ras with TGF-beta1 is operative during in vivo tumorigenesis and, as in wound healing processes, is dependent on epithelial-stromal interactions.

Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.

Abstract: The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors.

Abstract: Currently, the preferred host for the production of early region-1 (E1)-deleted recombinant adenoviruses (rAdV) is cell line 293, which was generated by transformation of human embryonic kidney cells by sheared adenovirus 5 (Ad5) DNA. To develop alternative hosts for the production of rAdV, we generated adenovirus-transformed human cell lines by transformation of human embryonic retinoblasts (HER) with a plasmid containing base pairs 79–5789 of the Ad5 genome. One of the established HER cell lines, which we called 911, exhibited favorable growth characteristics and was chosen for further study. This cell line is demonstrated to have several characteristics in common with the well-known 293 cell line: The 911 cell line is highly transfectable, and exhibits similar frequencies of homologous recombination. However, it has additional characteristics that make it a useful alternative for 293. The 911 cells perform particularly well in plaque assays. Upon infection with E1-deleted adenoviruses, plaques...

Preliminary biological evaluation of polyamidoamine (PAMAM) StarburstTM dendrimers

Abstract: PAMAM Starburst™ dendrimers are spherical macromolecules composed of repeating polyamidoamino units. They can be produced in successive “generations,” each with a defined size, molecular weight, and number of terminal amino groups. Because of these well-defined characteristics, PAMAMs are finding utility in a variety of applications, many of which are biological in nature. Little is known, however, about the biological behavior of the PAMAMs, which is critical to their use in vivo. Generation 3 (G3; MW = 5,147; 24 terminal amines), 5 (G5; MW = 21,563; 96 amines), and 7 (G7; MW = 87,227; 384 amines) PAMAMs were studied in V79 cells or in Swiss-Webster, mice for a number of biological properties, including (1) in vitro toxicity, (2) in vivo toxicity, (3) immunogenicity, and (4) biodistribution. Potential biological complications were observed only with G7 at the highest level tested. No evidence of immunogenicity was seen. The biodistribution properties of the Starburst™ dendrimers were rather unusual. G3 showed the highest accumulation in kidney tissue (∼15% ID/g over 48 h); G5 and G7 appeared to preferentially localize in the pancreas (peak levels ∼32% ID/g at 24 h, and ∼20% ID/g at 2 h, respectively). In addition, G7 showed extremely high urinary excretion, with values of 46 and 74% ID/g at 2 and 4 h, respectively. In general, the dendrimers did not exhibit properties that would preclude their use in biological applications. Depending on the situation (desired endpoint, dose, and generation used), however, the biodistribution of biological preparations should be carefully studied. © 1996 John Wiley & Sons, Inc.

Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3.

Abstract: Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of CD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increase. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-beta, PMA caused a strong increase in PKC-delta and a weak increase in PKC-alpha, PKC-epsilon, and PKC-zeta expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.

Replicative senescence: implications for in vivo aging and tumor suppression.

Abstract: Normal cells have limited proliferative potential in culture, a fact that has been the basis of their use as a model for replicative senescence for many years. Recent molecular analyses have identified numerous changes in gene expression that occur as cells become senescent, and the results indicate that multiple levels of control contribute to the irreversible growth arrest. These include repression of growth stimulatory genes, overexpression of growth inhibitory genes, and interference with downstream pathways. Studies with cell types other than fibroblasts will better define the role of cell senescence in the aging process and in tumorigenesis.

Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma

Abstract: Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Abstract: Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells.

Abstract: Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P-glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates.

An intestine-specific homeobox gene regulates proliferation and differentiation.

Abstract: Precise regulation of cellular proliferation, differentiation, and senescence results in the continuous renewal of the intestinal epithelium with maintenance of a highly ordered tissue architecture. Here we show that an intestine-specific homeobox gene, Cdx2, is a transcription factor that regulates both proliferation and differentiation in intestinal epithelial cells. Conditional expression of Cdx2 in IEC-6 cells, an undifferentiated intestinal cell line, led to arrest of proliferation for several days followed by a period of growth resulting in multicellular structures containing a well-formed columnar layer of cells. The columnar cells had multiple morphological characteristics of intestinal epithelial cells. Enterocyte-like cells were polarized with tight junctions, lateral membrane interdigitations, and well-organized microvilli with associated glycocalyx located at the apical pole. Remarkably, there were also cells with a goblet cell-like ultrastructure, suggesting that two of the four intestinal epithelial cell lineages may arise from IEC-6 cells. Molecular evidence for differentiation was shown by demonstrating that cells expressing high levels of Cdx2 expressed sucrase-isomaltase, an enterocyte-specific gene which is a well-defined target for the Cdx2 protein. Taken together, our data suggest that Cdx2 may play a role in directing early processes in intestinal cell morphogenesis and in the maintenance of the differentiated phenotype by supporting transcription of differentiated gene products. We propose that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium.

Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.

Abstract: The goal of our studies was to establish procedures for subculturing normal human tracheobronchial epithelial (NHTBE) cells without compromising their ability to differentiate into mucous and ciliated cells (i.e., differentiation competence) and to study the regulation of airway secretions by epidermal growth factor (EGF) and retinoic acid (RA). Primary NHTBE cells were obtained from a commercial source and subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for differentiation competence in air-liquid interface (ALI) cultures. The apical secretions of cultured NHTBE cells were characterized by immunoblotting, Western blotting, or enzyme-linked immunosorbent assay using a variety of antibodies. They contained mucin-like materials as well as lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI). We found that an EGF concentration of 25 ng/ml, which is commonly used in airway cell cultures, adversely affected growth, mucin production, and morphology of ALI cultures and that RA was essential for mucociliary differentiation. Without RA, the epithelium became squamous and mucin secretions decreased 300- to 900-fold. In contrast, secretion of lysozyme, lactoferrin, and SLPI was significantly increased in RA-depleted cultures. Cells of passage 2 (P-2) through P-4 remained competent to differentiate into mucous and ciliated cells when grown in ALI cultures. However, mucin secretion and ciliagenesis decreased in P-3 and P-4 cell cultures and P-3 but not P-4 cell cultures exhibited bioelectric properties characteristic of airway epithelium. We concluded that P-2 and P-3 NHTBE cell cultures retain many important features of normal airway epithelium. This enables one to conduct many studies of airway cell biology with a greatly expanded (6,000-fold) cell pool.