Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter.

The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with anti-Rb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both effects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene.

Identification by RNA-based arbitrarily primed PCR of the involvement of cytochrome c oxidase in the development of resistance to methotrexate.

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.

Tips and traps in brain MRI: applications to vascular disorders.

The French Society of Radiology's guide to good use of medical imaging examinations recommends MRI as the first-line examination for exploring cerebrovascular events or disorders. This paper will discuss the main traps in the images when stroke is suspected and provide the technical tips or knowledge necessary for an optimal radiological report.

Effects of nitric oxide synthesis inhibition on mesenteric perfusion in young pigs.

We tested the hypothesis that nitric oxide (NO) modulates postprandial hyperemia in young pigs. To test this hypothesis, we studied five groups of 3-wk-old pigs: group 1: milk fed, N(G)-monomethyl-L-arginine (L-NMMA) and L-arginine (L-Arg) treated (n = 10); group 2: milk fed, placebo treated (n = 8); group 3: water fed, L-NMMA and L-Arg treated (n = 6); group 4: water fed, placebo treated (n = 4); and group 5: fasted sham fed, L-NMMA and L-Arg treated (n = 6). After catheter placement and electromagnetic flow probe instrumentation of the mesenteric artery, systemic blood pressure and mesenteric artery blood flow were measured during preprandial baseline, postprandial, and postprandial intra-arterial L-NMMA- and L-Arg-infused study periods. The same measurements were made in the milk- and water-fed placebo-treated groups except that diluent replaced the L-NMMA and L-Arg infusions. In the milk- and water-fed placebo-treated groups, a significant (analysis of variance, P < 0.05), postprandial hyperemia was observed. The change in blood flow was greater (P < 0.05) in the milk-fed group than in the water-fed group. Inhibition of NO synthesis with L-NMMA diminished (P < 0.05) the hyperemic responses to both water and milk feeding and resulted in a decrease (P < 0.05) in mesenteric artery perfusion in the fasted sham-fed state. We conclude that, in young pigs, NO is a modulator of mesenteric vascular tone in both the postprandial and fasted states.

Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.

[Isolation of mycobacteria in patients with cystic fibrosis: a prospective study]. / Aislamiento de micobacterias en pacientes con fibrosis quística: estudio prospectivo.

OBJECTIVE: A prospective study to assess the incidence of mycobacterial infection in patients with cystic fibrosis in our geographical area was performed. PATIENTS AND METHODS: A monitored follow-up was carried out in 91 patients over a period of 20 months, during which time 522 respiratory samples were obtained. These were processed by standard techniques of decontamination with sodiumlaurylsulphate, cultured on Löwenstein-Jensen medium and identified by biochemical and cultural characteristics and hybridization by specific probes. At the same time, the clinical reports of the patients with positive culture were reviewed. RESULTS: Positive cultures of mycobacteria were obtained from 4 patients. Environmental mycobacteria were isolated in three of them (M. xenopi, M. fortuitum and M. avium) and M. chelonei and later M. tuberculosis in the forth. None of the isolations of environmental mycobacteria were associated with deterioration of pulmonary function, while the isolation of M. tuberculosis in one of the patients coincided with an episode of decompensation in respiratory function. None of the patients presented sensitivity of the tuberculin skin test. CONCLUSIONS: It is advisable to investigate the mycobacteria in the presence of exacerbation of the respiratory process, above all taking into account the high incidence of tuberculosis in our geographical area. The isolation of environmental mycobacteria was not associated with pulmonary deterioration, but they represent a potential danger as opportunist pathogens, affecting patients of which many are candidates for lung transplants.

Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1.

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-acting elements for the transcription factors Sp1 and E2F. Given the ability of Sp1 to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell-growth-phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Sp1-binding or E2F-binding sites in the dhfr minimal promoter.

Effects of anti-sense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.

The effect of incubations with anti-sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'-untranslated region, the translational start, the splice sites and branch point of intron I and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation-start site, at a range of concentrations between 1 and 4 microM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co-transfected DHFR minigenes. Cell incubation with ATNL caused a time-dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti-sense oligonucleotides using DHFR as the target.

Purine enzyme profile in human colon-carcinoma cell lines and differential sensitivity to deoxycoformycin and 2'-deoxyadenosine in combination.

Different cell lines, 2 from human colon carcinoma (LoVo and HT29) and 1 from Chinese hamster ovary (CHO K-I), were examined to assess the effect of deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA), and 2'-deoxyadenosine (dAdo) on their growth. When used alone, neither dCF or dAdo were cytotoxic for the 3 cell lines, while their combination caused inhibition of cell growth, with the following sensitivity: CHO K-I > LoVo > HT29. We studied the pattern of enzymatic activities involved in the metabolism of dAdo in the 3 cell lines. The phosphorylation of dAdo by adenosine kinase appears to play a central role in the toxicity of the deoxynucleoside in combination with dCF. In fact, CHO K-I cells, which are the most sensitive, possess the highest level of this enzyme. Moreover, the cytotoxic effect was almost completely reversed in the 3 cell lines when inhibitors of adenosine kinase, such as 5'-amino-5'-deoxyadenosine and iodotubercidine, were added to the culture medium together with dCF and dAdo. In addition, baby hamster kidney (BHK) adenosine-kinase-deficient (AK-) cells were highly resistant to this treatment. Uptake inhibition of dAdo using dipyridamole also caused reversal of the toxicity. The AMP and deoxyAMP dephosphorylating activities, much lower in the CHO K-I cells, also appear to play a central role in the toxicity of dAdo when adenosine deaminase is inhibited. However, our data suggest that other factors may modulate the toxic effect, such as S-adenosyl-homocysteine-hydrolase inhibition by dAdo at high concentrations.

Expression profiles of a human pancreatic cancer cell line upon induction of apoptosis search for modulators in cancer therapy.

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.