Identification of Ras-regulated genes by representational difference analysis.

In conclusion, RDA provides a fast, technically simple, and inexpensive way to characterize genes aberrantly expressed due to Ras transformation. The identification and characterization of these genes may provide insight not only into the mechanism by which Ras causes transformation, but also may identify novel targets for rational drug design and development of anticancer drugs.

The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter.

An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene. We show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21(WAF1/Cip1) due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/Cip1) through a specific Sp1-like cis-element in the p21(WAF1/Cip1) proximal promoter. The same element is also required by p53 to activate the p21(WAF1/Cip1) promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21(WAF1/Cip1) promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21(WAF1/Cip1) promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21(WAF1/Cip1) is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21(WAF1/Cip1) in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21(WAF1/Cip1) and may be part of a novel pathway by which cellular responses to stress are modulated.

Understanding Ras: 'it ain't over 'til it's over'.

Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras.

The gut-enriched Krüppel-like factor suppresses the activity of the CYP1A1 promoter in an Sp1-dependent fashion.

The gut-enriched Krüppel-like factor (GKLF) is a newly identified zinc finger-containing transcription factor. Recent studies indicate that GKLF binds to a core DNA sequence of 5'-(G/A)(G/A)GG(C/T)G(C/T)-3', which is found in an endogenous cis element, the basic transcription element (BTE) of the cytochrome P-450IA1 (CYP1A1) promoter. The present study characterizes the ability of GKLF to regulate CYP1A1 expression. By electrophoretic mobility gel shift assay (EMSA) and methylation interference assay, GKLF was found to bind BTE in a manner similar to several other transcription factors known to interact with BTE including Sp1 and BTEB. Cotransfection studies in Chinese hamster ovary cells showed that GKLF inhibited the CYP1A1 promoter in a dose- and BTE-dependent manner. The same experiments also revealed that BTE was responsible for a significant portion of the CYP1A1 promoter activity. EMSA of nuclear extracts from Chinese hamster ovary cells showed that Sp1 and Sp3 were two major proteins that interacted with BTE. Additional cotransfection studies showed that GKLF inhibited Sp1-mediated activation of the CYP1A1 promoter. In contrast, GKLF enhanced Sp3-dependent suppression of the same promoter. Moreover, the ability of GKLF to inhibit Sp1-dependent transactivation was in part due to physical interaction of the two proteins. These findings indicate that GKLF is a negative regulator of the CYP1A1 promoter in a BTE-dependent fashion and that this inhibitory effect is in part mediated by physical interaction with Sp1.

Size-dependent increase in prostanoid levels in adenomas of patients with familial adenomatous polyposis.

Recent studies indicate that nonsteroidal anti-inflammatory drugs have a chemopreventive effect against colorectal neoplasia. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenases, principal enzymes that mediate the formation of prostanoids. To determine whether prostanoids are involved in the pathogenesis of colorectal adenomas, we compared the levels of five major stable metabolic products of the cyclooxygenase pathway in the normal-appearing mucosa and in adenomas of patients with familial adenomatosis polyposis. Of 12 patients tested, 6 had elevated levels of at least one prostanoid in the adenomas. More importantly, the relative levels of three prostanoids [prostaglandin (PG)D2, PGE2, and 6-keto-PGF1alpha] were elevated in adenomas compared to normal-appearing mucosa from the same patients, and the resulting ratios were correlated with the size of the adenoma. These results suggest a role for prostanoids in progression of colorectal polyposis in familial adenomatosis polyposis patients.

Identification of the DNA sequence that interacts with the gut-enriched Krüppel-like factor.

The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Krüppel proteins, including the lung Krüppel-like factor (LKLF), the erythroid Krüppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. In the present study we determined the DNA binding sequence of GKLF using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis a DNA with the sequence 5'-G/AG/AGGC/TGC/T-3' was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the beta-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence was capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. Our results establish GKLF as a sequence-specific transcription factor likely involved in regulation of expression of endogenous genes.

Two potent nuclear localization signals in the gut-enriched Krüppel-like factor define a subfamily of closely related Krüppel proteins.

The gut-enriched Krüppel-like factor (GKLF) is a newly identified transcription factor that contains three C2H2 Krüppel-type zinc fingers. Previous immunocytochemical studies indicate that GKLF is exclusively localized to the nucleus. To identify the nuclear localization signal (NLS) within GKLF, cDNA constructs with various deletions in the coding region of GKLF were generated and analyzed by indirect immunofluorescence in transfected COS-1 cells. In addition, constructs fusing regions representing putative NLSs of GKLF to green fluorescent protein (GFP) were generated and examined by fluorescence microscopy in similarly transfected cells. The results indicate that GKLF contains two potent, independent NLSs: one within the zinc fingers and the other in a cluster of basic amino acids (called 5' basic region) immediately preceding the first zinc finger. In comparison, putative NLSs within the zinc fingers and the 5' basic region of a related Krüppel protein, zif268/Egr-1, are relatively less efficient in their ability to translocate GFP into the nucleus. A search in the protein sequence data base revealed that despite the existence of numerous Krüppel proteins, only two, the lung Krüppel-like factor (LKLF) and the erythroid Krüppel-like factor (EKLF), exhibit similar NLSs to those of GKLF. These findings indicate that GKLF, LKLF, and EKLF are members of a subfamily of closely related Krüppel proteins.

Colonic epithelium-enriched protein A4 is a proteolipid that exhibits ion channel characteristics.

Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.

Expression of the gut-enriched Krüppel-like factor gene during development and intestinal tumorigenesis.

We examined the expression of GKLF (gut-enriched Krüppel-like factor), a recently identified zinc finger-containing transcription factor, in mice during development using the ribonuclease protection assay. In the adult, the level of GKLF transcript is abundant throughout the gastrointestinal tract. Between embryonic days 10 and 19 (E10 and E19) of development, the initial level of whole embryo GKLF transcript is low but begins to rise on E13 and peaks on E17. In the newborn, GKLF transcript level is higher in the colon than in the small intestine although the levels in both organs rise with increasing age. Expression of GKLF was also examined in the intestinal tract of the Min mouse, a model of intestinal tumorigenesis. The level of GKLF transcript is significantly decreased in the intestine of Min mice during a period of tumor formation when compared to age-matched control littermates. Our findings indicate that GKLF expression correlates with certain periods of gut development and is down-regulated during intestinal tumorigenesis, suggesting that GKLF may play a role in gut development and/or tumor formation.

Identification and characterization of a gene encoding a gut-enriched Krüppel-like factor expressed during growth arrest.

A cDNA clone, named gut-enriched Krüppel-like factor (GKLF), was isolated from an NIH 3T3 library using a probe encoding the zinc finger region of the immediate-early transcription factor zif/268. The deduced GKLF amino acid sequence contains three tandem zinc fingers that are related to members of the Krüppel family of transcription factors. By indirect immunofluorescence, GKLF is localized to the cell nucleus. In cultured fibroblasts, GKLF mRNA is found in high levels in growth-arrested cells and is nearly undetectable in cells that are in the exponential phase of proliferation. The growth-arresting nature of GKLF is demonstrated by an inhibition of DNA synthesis in cells transfected with a GKLF-expressing plasmid construct. In the mouse, GKLF mRNA is present in select tissues and is most abundant in the colon, followed by the testis, lung, and small intestine. In situ hybridization experiments indicate that GKLF mRNA is enriched in epithelial cells located in the middle to upper crypt region of the colonic mucosa. Taken together, these results suggest that GKLF is potentially a negative regulator of cell growth in tissues such as the gut mucosa, where cell proliferation is intimately coupled to growth arrest and differentiation.

Galactose consumption, metabolism, and follicle-stimulating hormone concentrations in women of late reproductive age.

OBJECTIVE: To test the hypothesis that high galactose consumption and low activity of galactose-1-phosphate uridyl transferase (transferase) is associated with early ovarian senescence among nongalactosemic women. DESIGN: Cross-sectional study. Data collection consisted of a self-administered questionnaire with sections on diet (food frequency data to measure galactose consumption), reproductive, and medical histories. One blood sample was collected to measure FSH and transferase activity; FSH was used as a measure of ovarian senescence. Among women who were having menstrual periods at least every 8 weeks, the blood sample was drawn in the early follicular phase (days 2 to 4) of a menstrual cycle. PARTICIPANTS: Two hundred ninety-five women volunteers ages 38 to 49 years who had not had a hysterectomy or oophorectomy were recruited through posters and advertisements. MAIN OUTCOME MEASURE: Serum FSH concentrations. RESULTS: Controlling for age, smoking, and body mass, transferase activity and FSH were unrelated. However, FSH levels were 29% higher (95% confidence intervals, 9% to 52%) among women who reported consuming > or = 6 g galactose/d. CONCLUSION: These data do not support the hypothesis that low transferase activity represents a genetic predisposition for early ovarian senescence, as measured by FSH levels in women ages 38 to 49 years. However, the hypothesized positive association between galactose consumption and FSH was supported.

Collision between law and ethics: consent for treatment with adolescents.

The dilemmas between legal obligations and ethical responsibilities can often create problems in clinical work. The treatment of minors, and particularly adolescents, can present special issues to the clinician that are becoming increasingly frequent and difficult. The issue of informed consent for treatment of adolescents raises serious questions for the clinical practitioner who is faced with both legal and ethical dilemmas in making decisions about treatment. There are an increasing number of cases where adolescents may seek treatment yet are in circumstances that preclude parental consent. This paper uses case material to illustrate some of the legal, ethical, and treatment considerations in the situation of adolescent treatment where parental consent is problematic.

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

Signal transduction in human neutrophil leucocytes: effects of external Na+ and Ca2+ on cell polarity.

Stimulation of neutrophil leucocytes with chemotactic factors is known to result in membrane permeability changes, as evidenced by fluxes of Na+ and K+ across the cell membrane together with an increased uptake of Ca2+ from the medium. These fluxes have been implicated in the transduction mechanisms of various responses, including locomotion and subsequent chemotaxis. We have previously reported that exposure of unstimulated, round neutrophils held in suspension, to the chemotactic peptide fMet-Leu-Phe confers morphological polarity on the neutrophils by stimulating waves of contraction, which are also intimately connected with locomotion on an appropriate substratum. As the acquisition of polarity is the important first step in the chemotactic response we have investigated the effects of modifying the external ionic environment and of various ion channel blockers on the polarizing response of neutrophils held in suspension. Removal and chelation of both Ca2+ and Mg2+ from the external medium did not inhibit the acquisition of polarity and a variety of inorganic Ca2+ channel blockers together with the organic Ca2+ antagonists, verapamil and D600, were ineffective in inhibiting the response. Replacement of Na+ in the external medium with choline inhibited the polarizing response completely but tetrodotoxin, which blocks fast Na+ channels, and amiloride, which inhibits Na+/K+ exchange, had no effect. Inhibition of the Na+/K+-ATPase with ouabain and also tetraethylammonium ions, which block potassium channels, had no inhibiting effect on polarization. These results indicate that while Ca2+ and Mg2+ are not required in the external medium, Na+ is essential, and therefore Na+/K+ fluxes across the cell membrane play a role in initiating locomotion.

An alternative health care setting for children with cancer: a residential summer camp.

Increasing concern for the psychosocial needs of children with cancer has paralleled increasing survival rates. This paper discusses the way in which a summer camp program can help to meet many social needs of children with cancer. Also discussed is the proposal of a campsite to be operated year-round for families of children with cancer, and the way in which such a program can help to meet many needs of these families.

Neutrophil leucocyte chemotaxis: a simplified assay for measuring polarizing responses to chemotactic factors.

A method is described which greatly simplifies the screening of compounds which are potentially chemotactic for neutrophil leucocytes. Neutrophils isolated from blood by a standardised procedure are greater than 95% spherical in morphology. Addition of chemotactic factors in isotropic, non-gradient, concentrations induces the spherical shape to become polarized. The degree of polarity depends on the concentration of the factor used, as does the percentage of cells which become polarised. All compounds which induce a good response in assays measuring cell accumulation or orientation in gradients induced a consistent polarizing response in non-gradient conditions with the cells held in suspension. The advantage of this simplified assay over methods currently in use are discussed.

Behaviour of neutrophil leucocytes in uniform concentrations of chemotactic factors: contraction waves, cell polarity and persistence.

The essential component of any hypothesis of random or directed cell movement is the mechanism of cell polarity. In this paper we describe the polar behaviour of human neutrophil leucocytes in uniform concentrations of chemotactic factors both in suspension and while moving across surfaces. Neutrophils exposed to uniform concentrations of chemotactic factors in suspension around the dissociation constant (Kd) for the receptor rapidly become distinctly bipolar; neutrophils exposed to supraoptimal uniform concentrations (100-fold greater than Kd) of chemotactic factors in suspension, although morphologically active, never reached the same degree of polarity as cells in optimal concentrations. These differences in polarity were shown to be the direct result of equatorial contraction waves stimulated on the cell surface by interaction with chemotactic factors. In optimal concentrations of chemotactic factors, contraction waves were initiated from one region of the cell, whereas in supraoptimal concentrations of chemotactic factors contraction waves emanated from all areas of the cell surface. Asymmetry in the distribution of surface receptors for Fc and C3b were observed in neutrophils polarized in uniform concentrations of chemotactic factor. In neutrophils, motile but not well polarized (in 10(-6) M-N-formylmethionyl-leucyl-phenylalanine (fMLP), receptors were uniformly distributed. In neutrophils polarized in concentrations of fMLP near the Kd for the receptor (10(-8) M) receptors for C3b and Fc were localized in the anterior region of the moving cell. The link between contraction waves, cell polarity and receptor redistribution and their initiation by chemotactic peptides is discussed in the context of neutrophil locomotion and response to chemical signals.

Contraction waves in lymphocyte locomotion.

In this paper we propose that the constriction ring, a prominent feature of moving leucocytes, is a major source of locomotive force. Analysis of time-lapse films of lymphocytes in suspension and moving through three-dimensional collagen gels, demonstrated that the constriction ring was the morphological manifestation of a wave of circular contraction that moved antero-posteriorly. In lymphocytes in suspension the wave moved, although the cells could not. Analysis of lymphocytes moving through a collagen gel revealed that the waves remained stationary with respect to the external environment while the cell appeared to move forward through them. Passage of a single equatorial contraction wave resulted in cell lengthening: a shortening of the region posterior to the constriction was observed in cells moving through collagen gels, but not in lymphocytes held in suspension, suggesting that attachment of cells to the collagen network was necessary for longitudinal contraction. Lymphocyte attachment to collagen gels was mediated through the rapid extension of bleb-like structures into the collagen network. Transmission electron microscopy (TEM) failed to demonstrate any organized structure at the constriction ring. NBD-Phallacidin staining of lymphocytes together with TEM demonstrated that F-actin was distributed evenly throughout the length of the cell. Cell polarity was clearly recognizable by the distribution of coated vesicles, microvilli, and all organelles to the rear, and Thy 1-2 to the front, of motile cells, but polarity could be reversed by the passage of a single contraction wave starting at the rear of the cell, without prior redistribution of these structures.

Invasion of collagen gels by mouse lymphoid cells.

Small mouse lymphocytes from lymph nodes rapidly invaded three-dimensional collagen gels (in the absence of any added chemical attractant). In short-term assays (2-8 hr) this property was restricted to 20-25% of the cell population. Invasion was an active process involving cell locomotion. Time-lapse cinematography revealed that movement was erratic with frequent changes in cell speed. Tracks of cell paths within collagen gels demonstrated that lymphocytes made narrow angles of turn and thus showed a 'persistent random-walk' similar to other cell types moving on plane substrata. Analysis of lymphocyte movement within aligned collagen gels demonstrated that locomotion was biased in the axis of fibre alignment, i.e. lymphocytes showed contact guidance. Separated B lymphocytes invaded collagen gels at a slower rate than unseparated lymph node cells, as also did T cells purified by filtration through nylon wool columns. This latter anomaly implied that nylon wool filtration selectively depleted cells with invasive characteristics from a heterogeneous lymphocyte population. A comparison of Peyer's patch and lymph node lymphocytes showed that both populations invaded at the same rate but the latter cell type did this in greater numbers. This difference may reflect the different proportions of B and T lymphocytes in the two tissues. Lymphocytes from oxazolone-stimulated lymph nodes showed greatly increased movement into collagen matrices compared to unstimulated control lymph node lymphocytes. This increase was demonstrated to be a property of the blast cell population by separating the cells on Percoll gradients into lymphoblast-enriched and -depleted populations.