Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.     

Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.     

Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models

BACKGROUND: Some anaerobic and facultatively anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in tumors. In this study, we exploited attenuated Salmonella choleraesuis as a tumoricidal agent and a vector to deliver the endostatin gene for tumor-targeted gene therapy. METHODS: Attenuated S. choleraesuis carrying a eukaryotic expression plasmid encoding reporter gene was used to evaluate its abilities of tumor targeting and gene delivery in three syngeneic murine tumor models. Furthermore, S. choleraesuis carrying the endostatin expression vector was administered intraperitoneally into tumor-bearing mice, and its antitumor effect was evaluated. RESULTS: Systemically administered S. choleraesuis preferentially accumulated within tumors for at least 10 days, forming tumor-to-normal tissue ratios exceeding 1000-10,000 : 1. Transgene expression via S. choleraesuis-mediated gene transfer also persisted for at least 10 days. Host immune responses and tumor hypoxia may influence tumor-targeting potential of S. choleraesuis. When systemically administered into mice bearing melanomas or bladder tumors, S. choleraesuis carrying the endostatin expression vector significantly inhibited tumor growth by 40-70% and prolonged survival of the mice. Furthermore, immunohistochemical studies in the tumors revealed decreased intratumoral microvessel density, reduced expression of vascular endothelial growth factor (VEGF), and increased infiltration of CD8(+) T cells. CONCLUSIONS: These results suggest that tumor-targeted gene therapy using S. choleraesuis carrying the endostatin expression vector, which exerts tumoricidal and antiangiogenic activities, represents a promising strategy for the treatment of solid tumors.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

The role of intrahepatic gammadelta-T cells for liver injury induced by Salmonella infection in mouse.

Liver injury was induced after infection with Salmonella choleraesuis 31N-1. In T-cell receptor-delta knockout mice, serum alanine transferase level was significantly decreased in comparison with normal control mice after Salmonella infection. On the contrary, in vivo administration of anti-gammadelta T-cell receptor monoclonal antibody (UC7-13D5) to stimulate gammadelta-T cells in infected mice significantly increased serum alanine transferase level but decreased bacterial growth compared with infected mice given control antibody (UC8; hamster IgG). These data suggest that gammadelta-T cells have effector activities not only for protection but also for liver injury during Salmonella infection.     

The mitogenic action of insulin-like growth factor I in normal human mammary epithelial cells requires the epidermal growth factor receptor tyrosine kinase

The signals used by insulin-like growth factor I (IGF-I) to stimulate proliferation in human mammary epithelial cells have been investigated. IGF-I caused the activation of both ERKs and Akt. Activation of ERKs was slower and more transient than that of Akt. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, prevented activation of ERKs but not Akt by IGF-I. Inhibition of the EGFR with function-blocking monoclonal antibodies also specifically blocked IGF-I-induced ERK activation. These effects occurred in primary mammary epithelial cells and in two cell lines derived from normal mammary epithelium but not in mammary fibroblasts or IGF-I-responsive breast carcinoma cell lines. Although IGF-I stimulated the proliferation of both normal and carcinoma cell lines, ZD1839 blocked this only in the normal line. ZD1839 had no effect on IGF-I receptor (IGF-IR) autophosphorylation in intact cells. IGF-I-induced ERK activation was insensitive to a broad spectrum matrix-metalloproteinase inhibitor and to CRM-197, an inhibitor of the EGFR ligand heparin-bound epidermal growth factor. EGFR was detectable within IGF-IR immunoprecipitates from normal mammary epithelial cells. Treatment of cells with IGF-I led to an increase in the amount of tyrosine-phosphorylated EGFR within these complexes. ZD1839 had no effect on complex formation but completely abolished their associated EGFR tyrosine phosphorylation. These findings indicate that IGF-I utilizes a novel EGFR-dependent signaling pathway involving the formation of a complex between the IGF-IR and the EGFR to activate the ERK pathway and to stimulate proliferation in normal human mammary epithelial cells. This form of regulation may be lost during malignant progression.     

Homeostatic regulation of intestinal villous epithelia by B lymphocytes

The epithelial cell of the small intestine is one of the most rapidly regenerating cells in the body. However, the cellular mechanism and biological significance underlying this rapid regeneration remain elusive. In this study we examined the intestinal epithelia of mutant mice that lack B and/or T cells and those of normal littermates. The absence of B cells in Ig mu-chain mutant mice or B and T cells in recombination-activating gene (RAG)-2(-/-) as well as SCID mutant mice was associated with a marked acceleration of epithelial cell turnover and an up-regulation of the expression of MHC class II molecules. No such effects were observed in T cell-deficient TCR-delta and -beta double-mutant mice. As far as the goblet cells of villous epithelium are concerned, absolute numbers of them remained the same among these mutant mice that have no B and/or T cells. Alymphoplasia (aly/aly) mutant mice that lacked Peyer's patches and Ig-producing cells in the lamina propria, but harbored a large number of intestinal mucosal T cells, also displayed a significant acceleration of epithelial cell turnover and, to some extent, up-regulated expression of MHC class II molecules. Notably, the accelerated epithelial cell turnover was not observed and returned to normalcy in the Ig mu-chain mutant mice that had been given antibiotic-containing water. These findings indicate that B cells down-regulate the generation and differentiation of intestinal epithelial cells in the normal wild-type condition and suggest that enteric microorganisms are implicated in the accelerated generation of epithelial cells in mice that have no B cells.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation

The function of the E5 protein of human papillomaviruses (HPV) is not well characterized, and controversies exist about its role in the viral life cycle. To determine the function of E5 within the life cycle of HPV type 31 (HPV31) we first constructed HPV31 mutant genomes that contained an altered AUG initiation codon or stop codons in E5. Cell lines were established which harbored transfected wild-type or E5 mutant HPV31 genomes. These cell lines all maintained episomal copies of HPV31 and revealed similar phenotypes with respect to growth rate, early gene expression, and viral copy number in undifferentiated monolayer cultures. Following epithelial differentiation, genome amplification and differentiation-dependent late gene expression were observed in mutant cell lines, but at a rate significantly reduced from that observed in cells containing the wild-type genomes. Organotypic raft cultures indicated that E5 does not effect the expression of differentiation markers but does reduce expression of late viral proteins. Western analysis and immunofluorescence staining for cyclins during epithelial differentiation revealed a decreased expression of cyclin A and B in E5 mutant cells compared to HPV wild-type cells. Using a replating assay, a significant reduction in colony-forming ability was detected in the absence of E5 expression when cells containing wild-type or E5 mutant HPV genomes were allowed to proliferate following 24 h in suspension-induced differentiation. This suggests that HPV E5 modifies the differentiation-induced cell cycle exit and supports the ability of HPV31-positive keratinocytes to retain proliferative competence. In these studies, E5 was found to have little effect on the levels of the epidermal growth factor receptor (EGFR) or on its phosphorylation status. This indicates that EGFR is not a target of E5 action. Our results propose a role for high risk HPV E5 in modulation of late viral functions through activation of proliferative capacity in differentiated cells. We suspect that the primary target of E5 is a membrane protein or receptor that then acts to alter the levels or activities of cell cycle regulators.     

Loss of Androgen Receptor-Dependent Growth Suppression by Prostate Cancer Cells Can Occur Independently from Acquiring Oncogenic Addiction to Androgen Receptor Signaling

The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein.     

Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells

Intramacrophage survival appears to be a pathogenic trait common to Salmonellae and definition of the metabolic requirements of Salmonella within macrophages might provide opportunities for novel therapeutic interventions. We show that loss of PurG function in Salmonella enterica serovar Typhimurium SL1344 leads to death of the bacterium in RAW264.7 cells, which was due to unavailability of purine nucleotides but not thiamine in the phagosome of RAW264.7 cells. Phagosomal escape of purG mutant restored growth, suggesting that the phagosomal environment, but not the cytosol, is toxic to Salmonella purine auxotrophs. NADPH oxidase inhibition restored the growth of purG mutant in RAW264.7 cells, implying that the Salmonella -containing vacuole acquires reactive oxygen species (ROS) that are lethal to purine auxotrophs. Under purine limiting conditions, purG mutant was unable to repair the damage caused by hydrogen peroxide or UV irradiation, suggesting that ROS-mediated DNA damage may have been responsible for the attenuated phenotype of purG mutant in RAW264.7 cells and in mice. These studies highlight the possibility of utilizing the Salmonella purine nucleotide biosynthetic pathway as a prospective therapeutic target and also underline the importance of metabolic pathways in assembling a comprehensive understanding of the host–pathogen interactions inside phagocytic cells.     

Conversion of Androgen Receptor Signaling From a Growth Suppressor in Normal Prostate Epithelial Cells to an Oncogene in Prostate Cancer Cells Involves a Gain of Function in c-Myc Regulation

In normal prostate, androgen-dependent androgen receptor (AR) signaling within prostate stromal cells induces their secretion of paracrine factors, termed “andromedins” which stimulate growth of the epithelial cells. The present studies demonstrate that androgen-dependent andromedin-driven growth stimulation is counter-balanced by androgen-induced AR signaling within normal adult prostate epithelial cells resulting in terminal G₀ growth arrest coupled with terminal differentiation into ΔNp63-negative, PSA-expressing secretory luminal cells. This cell autonomous AR-driven terminal differentiation requires DNA-binding of the AR protein, is associated with decreases in c-Myc m-RNA and protein, are coupled with increases in p21, p27, and SKP-2 protein expression, and does not require functional p53. These changes result in down-regulation of Cyclin D₁ protein and RB phosphoryation. shRNA knockdown documents that neither RB, p21, p27 alone or in combination are required for such AR-induced G₀ growth arrest. Transgenic expression of a constitutive vector to prevent c-Myc down-regulation overrides AR-mediated growth arrest in normal prostate epithelial cells, which documents that AR-induced c-Myc down-regulation is critical in terminal growth arrest of normal prostate epithelial cells. In contrast, in prostate cancer cells, androgen-induced AR signaling paradoxically up-regulates c-Myc expression and stimulates growth as documented by inhibition of both of these responses following exposure to the AR antagonist, bicalutamide. These data document that AR signaling is converted from a growth suppressor in normal prostate epithelial cells to an oncogene in prostate cancer cells during prostatic carcinogenesis and that this conversion involves a gain of function for regulation of c-Myc expression.     

Cytoplasmic dynein-dynactin complex is required for spermatid growth but not axoneme assembly in Drosophila

Spermatids derived from a single gonial cell remain interconnected within a cyst and elongate by synchronized growth inside the testis in Drosophila. Cylindrical spectrin-rich elongation cones form at their distal ends during the growth. The mechanism underlying this process is poorly understood. We found that developing sperm tails were abnormally coiled at the growing ends inside the cysts in the Drosophila Dynein light chain 1 (ddlc1) hemizygous mutant testis. A quantitative assay showed that average number of elongation cones was reduced, they were increasingly deformed, and average cyst lengths were shortened in ddlc1 hemizygous testes. These phenotypes were further enhanced by additional partial reduction of Dhc64C and Glued and rescued by Myc-PIN/LC8 expression in the gonial cells in ddlc1 backgrounds. Furthermore, DDLC1, DHC, and GLUED were enriched at the distal ends of growing spermatids. Finally, ultrastructure analysis of ddlc1 testes revealed abnormally formed interspermatid membrane, but the 9 + 2 microtubule organization, the radial spoke structures, and the Dynein arms of the axoneme were normal. Together, these findings suggest that axoneme assembly and spermatid growth involve independent mechanisms in Drosophila and DDLC1 interacts with the Dynein-Dynactin complex at the distal ends of spermatids to maintain the spectrin cytoskeleton assembly and cell growth.     

Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells

An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.     

Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene

Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.     

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues). This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research. Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal and tumor prostate epithelial cells from the same system.     

Prolactin sensitivity of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for alpha-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50-100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports reports had suggested, but rather are generally less sensitive to hormonal stimulants.     

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.     

Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation

Summary A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics of normal epithelial cells. The cells formed a small cyst consisting of keratined squamous epithelium in syngenic hosts. A screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to malignant cells, and suggest that conversion from a state of cell cycle-dependent autocrine expression of one or more members of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy. The work was supported in part by grants CA37589 and DK35310 to Dr. McKeehan, from the National Cancer Institute, Bethesda, MD.