Establishment Of Stable Cell Lines Research Articles

A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion

LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-kappaB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kappaB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kappaB signaling, and increasing stability of the LZAP protein.

Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G)

Phenolic acids are found in abundance throughout the plant kingdom. Consumption of wine or other rich sources of phenolic acids, such as the "Mediterranean diet," has been associated with a lower risk of cardiovascular disease. The underlying mechanism(s), however, has remained unclear. Here, we show that many phenolic acids, including those from the hydroxybenzoic and hydroxycinnamic acid classes, can bind and activate GPR109A (HM74a/PUMA-G), the receptor for the antidyslipidemic agent nicotinic acid. In keeping with this activity, treatment with a number of phenolic acids, including cinnamic acid, reduces lipolysis in cultured human adipocytes and in fat pats isolated from wild-type mice but not from mice deficient of GPR109A. Oral administration of cinnamic acid significantly reduces plasma levels of FFA in the wild type but not in mice deficient of GPR109A. Activation of GPR109A by phenolic acids may thus contribute to a cardiovascular benefit of these plant-derived products.

High level virion production and surface antigen expression with 1.5 copies of hepatitis B viral genome

The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2 kb genome, a 5′-end redundant sequence of 1.1 kb to nt715 and a 3′-end redundant sequence of 500 bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24 h and 48 h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log 10 values of viral loads were also determined with the cell lysate having a higher value (10–11 per ml) than that of the supernatant (6–7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro. The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

The Beta3 499–513 Peptide Region is Required for AlphaIIb/Beta3 Active Complex Formation and Fibrinogen Binding

Background: AlphaIIb/beta3 (αIIb/β3) complex is an important integrin that is involved in the final step of platelet aggregation. Peptides derived from either αIIb or β3 have demonstrated to have an effect on the activation of the complex and its ability to bind fibrinogen. We have previously defined a peptide from β3, which inhibits agonists-induced platelet aggregation. Methods: We used standard methodologies for construction of clones and expression of cDNAs, establishment of stable cell lines that contained these cDNAs. Expression of proteins was detected with immunoblots. Flow cytometric analyses were used to verify the presence of the active and inactive complexes with different antibodies. In addition, a fibrinogen- binding assay was used to determine the inhibition of the active complex by the peptide. Results and discussion: A stable cell line of the co-transfected cDNAs of αIIb, β3 wild type and mutants of β3 (scrambled sequence of the peptide region, replacement of C499A and C512A), expressing the inactive complex on CHO cells, has allowed us to examine the important role of the peptide sequence and the cysteine residues within the peptide. The peptide inhibits the active complex formation and thereby inhibits the binding of FITC-PAC-1 in a dose dependent manner by flow cytometric analyses, as well as binding of [3H]-fibrinogen. In addition, creation of a second stable cell line containing wild type αIIb and the mutated region of β3 (residues 499–513) shows that the binding of FITC-PAC-1 and [3H]-fibrinogen on the mutant activated complex was much lower than the wild type activated complex. Our results indicate that the region 499–513 in β3 is one of the important sites for αIIb/β3 active complex formation and the cysteines play an important role in the process.

PAX6 increases glioma cell susceptibility to detachment and oxidative stress

Glioblastoma multiforme (GBM) is an incurable malignant glioma which is very resistant to radiation and alkylating agent-based chemotherapy. Necrosis is a hallmark for GBM and the layer surrounding necrotic area is packed with cells which are now believed to be those migrating out of the necrotic area. Oxidative stress is a condition that GBM cells encounter in the necrotic zone, which is one of the stressful conditions that GBM cells need to resist in order to survive. Our previous studies revealed that low PAX6 expression is a favorable molecular trait for survival acquired by GBM cells because PAX6 could suppress cell invasion and tumorigenicity. Since detachment of cells from GBM is an early event for cell migration and subsequent invasion, we examined whether PAX6 is involved in cell survival after detachment. PAX6 over-expression was achieved in glioma cells transiently (by adenoviral-mediated transient over-expression) or stably (by the establishment of stable cell lines after transfection). The effect of PAX6 over-expression on the survival and growth of glioma cells after detachment from the culture was determined. Our data revealed that GBM cells (with their low PAX6 levels) survived the detachment procedure well. However, PAX6 over-expression attenuated GBM cell recovery of growth after detachment-induced stress. Importantly, intracellular reactive oxygen species (ROS) levels increased following cell detachment and that PAX6 over-expressing cells retained higher level of ROS than control cells. This may be partially responsible for the impaired growth rate after cell detachment. Addition of anti-oxidant improved the cell viability of PAX6 over-expressing cells, but did not restore their ability to proliferate. To survive, GBM cells must resist oxidative stress in the necrotic zone as well as the intracellular ROS generated during detachment. Since PAX6 over-expression in low PAX6-expressing glioma cells attenuated cell survival and growth after detachment, these results suggest that a reduced PAX6 expression may be a molecular trait that gives glioma cells a real selection advantage over other cell types to survive in stressful conditions, thus resulting in expansion of their population.

Establishment of stable cell lines of Drosophila germ-line stem cells

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam(-) GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma.

Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays.

We have developed a series of fluorescent splicing reporter minigenes for the establishment of cell-based screens to identify splicing regulatory proteins. A key technical advance in the application of these reporters was the use of two different fluorescent proteins: EGFP and monomeric Red Fluorescent Protein (mRFP). Through establishment of stable cell lines expressing such dual color fluorescent reporters, these minigenes can be used to perform enhanced screens for splicing regulatory proteins. As an example of such applications we generated fluorescent minigenes that can be used to determine the splicing of mutually exclusive FGFR2 exons IIIb and IIIc by flow cytometry. One minigene contained a coding sequence for EGFP whose translation was dependent on splicing of exon IIIb, whereas a second minigene required exon IIIc splicing for translation of an mRFP coding sequence. Stable incorporation of both minigenes into cells that express endogenous FGFR2-IIIb or FGFR2-IIIc resulted in EGFP or mRFP fluorescence, respectively. Cells stably transfected with both minigenes were used to screen a panel of cDNAs encoding known splicing regulatory proteins, and several were identified that induced a switch in splicing that could be detected specifically by an increase in green, but not red, fluorescence. We further demonstrated additional minigenes that can be used in dual color fluorescent screens for identification of splicing regulatory proteins that function through specific intronic splicing enhancer elements (ISEs). The methods and minigene designs described here should be adaptable for broader applications in identification of factors and mechanisms involved in alternative splicing of numerous other gene transcripts.

Dual Function for a Unique Site within the β2I Domain of Integrin αMβ2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.

Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons

Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.

Generation and characterization of a cell line with inducible expression of Ca(v)3.2 (T-type) channels.

Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.

SnRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN

Splicing snRNPs (small nuclear ribonucleoproteins) are essential sub-units of the spliceosome. Here we report the establishment of stable cell lines expressing fluorescently tagged SmB, a core snRNP protein. Analysis of these stable cell lines has allowed us to characterize the nuclear pathway that leads to snRNP accumulation in nuclear speckles and has identified a limiting nucleolar step in the pathway that can be saturated by overexpression of Sm proteins. After nuclear import, newly assembled snRNPs accumulate first in a subset of Cajal bodies that contain both p80-coilin and the survival of motor neurons protein (SMN) and not in bodies that contain p80-coilin but lack SMN. Treatment of cells with leptomycin B (LMB) inhibits both the accumulation of snRNPs in nuclear bodies and their subsequent accumulation in speckles. The formation of Cajal bodies is enhanced by Sm protein expression and the assembly of new snRNPs. Formation of heterokaryons between HeLa cell lines expressing Sm proteins and primary cells that usually lack Cajal bodies results in the detection of Cajal bodies in primary cell nuclei. Transient over-expression of exogenous SmB alone is sufficient to induce correspondingly transient Cajal body formation in primary cells. These data indicate that the level of snRNP protein expression and snRNP assembly, rather than the expression levels of p80-coilin or SMN, may be a key trigger for Cajal body formation.

Characterization of Ligand Binding of a Soluble Human Insulin-like Growth Factor I Receptor Variant Suggests a Ligand-induced Conformational Change

Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted alpha-beta'-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 x 10(8) M-1 (25 degrees C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 degrees C) and in two different buffer systems at 25 degrees C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)2) receptor complex in solution are deduced to 0.96 x 10(8) M-1 (25 degrees C). The determined heat capacity change for the process is large and negative, -0.51 kcal (K mol)-1. Further, the entropy change (DeltaS) at 25 degrees C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding.

Comparison between cytomegalovirus promoter and elongation factor-1α promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein

The establishment of stable cell lines expressing the hepatitis C virus (HCV) core protein may be important for studies of HCV pathogenesis. Human and mouse cell lines were generated expressing the HCV core protein using expression vectors driven by either the cytomegalovirus (CMV) or elongation factor-1αa (EF-1α) promoters. Following transient transfection, HCV core protein was expressed in all cell lines. However, stable human hepatocellular carcinoma (HCC) and murine myeloma cell lines expressing the HCV core protein were only established using constructs driven by the EF-1α promoter. In contrast, stable expression of the hepatitis B virus (HBV) middle envelope protein (MHBs) was obtained successfully in these cell lines using an expression vector driven by the CMV promoter. Inhibitory activity of the first 69 amino acids of the HCV core protein on the CMV promoter was found by using chimeric MHBs/HCV core protein constructs. Growth of cloned cell lines expressing the HCV core protein was slower than that of nonexpressing cell lines. However, morphological changes and cell death were not observed in the stable cell lines expressing HCV core protein. These results indicate that the HCV core protein was not directly cytotoxic to HCC and myeloma cell lines but that specific promoter elements are required to establish stable expression of the nucleocapsid structural protein.

Establishment of stable cell lines expressing potentially toxic proteins by tetracycline-regulated and epitope-tagging methods.

A tetracycline-regulated expression system is combined with the FLAG-epitope tagging method for conditional expression of potentially toxic proteins in mammalian cells. This strategy allows a controlled expression of exogenous gene products and also provides a unique way of protein purification. Two mammalian expression plasmids containing the FLAG sequence and flanking multiple cloning sites were created for conditional protein expression. The cDNAs encoding human basal transcription factors TBP, TAFII55 and the p62 subunit of TFIIH were individually cloned into these vectors and introduced into a HeLa-derived cell line that constitutively expresses a tetracycline-regulated transactivator (tTA). The established clonal human cell lines express FLAG-tagged basal transcription factors in a manner modulated by the amount of tetracycline in the growth medium. In the absence of tetracycline, tTA binds to the DNA recognition sites of the expression plasmid and induces the expression of tagged proteins. When tetracycline is added back to the growth medium, the induced protein starts to decay. This provides us with an estimation of the in vivo half-lives of TBP and TAFII55, which were assessed to be less than 20 and 6 hours, respectively, in HeLa cells. The level of induced proteins in the absence of tetracycline could be further enhanced by including the antibiotic G418 to presumably boost the production of tTA which in turn activates the expression of tagged proteins.

Growth Hormone Promotes the Association of Transcription Factor STAT5 with the Growth Hormone Receptor

Members of the cytokine/growth hormone (GH)/prolactin receptor superfamily transduce signals by association and activation of JAK tyrosine kinases. For GH receptor (GHR), both JAK2 and the GHR undergo tyrosine phosphorylation upon GH stimulation. Also, GH has recently been shown to activate the transcription factor STAT5 by tyrosine phosphorylation. In the present study, we demonstrate that GH induces rapid tyrosine phosphorylation of different isoforms of STAT5 in mouse L cells stably transfected with a cDNA encoding porcine GHR (pGHR). In this cell system, STAT5 directly interacts with the GHR in a GH-dependent manner. Additionally, GH-induced tyrosine phosphorylation of STAT5 and the interaction of STAT5 with GHR can be observed in mouse 3T3-F442A cells which express endogenous mouse GHR. Interestingly, when cDNAs encoding the two mouse STAT5 homologs (STAT5A and STAT5B) were individually transfected into mouse L cells expressing pGHR, only STAT5A demonstrated the ability to interact with the pGHR and subsequently underwent GH-dependent tyrosine phosphorylation. STAT5B did not. Therefore, the GH-dependent interaction of a particular STAT5 with tyrosine-phosphorylated GHR may play an important role in GH-mediated signal transduction.

Stable high expression of human gamma-aminobutyric acid a receptors composed of alpha and beta subünitS

Multiple classes of pharmacological agents including benzodiazepines, cage convulsants like t-butylbicyclophosphorothionate (TBPS), barbiturates and neuroactive steroids allosterically modulate the y-aminobutyric acidA receptor-chloride ionophore complex (GRC). The function of benzodiazepines requires a GRC comprised of α, β and γ subunits, while TBPS, barbiturates and neuroactive steroids will allosterically modulate GRCs comprised of only a and β subunits. Binary αβ complexes are still hypothesized to be expressed in the mammalian brain particularly during development and could contribute to the pharmacological action of neuroactive steroids and barbiturates. In order to examine binary αβ complexes we report here the establishment of stable cell lines that express high levels of human GABA a receptors comprised of α1β1, α2β1 and a3β1 subunit combinations. The apparent potencies for allosteric modulation of [ 35S]TBPS for most naturally occurring neuroactive steroids for the binary subunit combinations was similar to that of the γ-containing subunit combinations. Also discussed is the usefulness of these cell lines for the biophysical analysis of the GABa a receptor stoichiometry.

Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression.

Establishment of Stable Cell Lines Producing Anti‐Pseudomonas aeruginosa Monoclonal Antibodies and Their Protective Effects for the Infection in Mice

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 micrograms/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype-specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 micrograms MoAbs per 10(6) cells in 24 hr. It is practicable to use these cell lines for large-scale production of anti-P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.