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Biomedical subjects

P Friedlander

Publications and source records attributed to P Friedlander.

12 recordsLinked to original sources
PubMed1997

Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.

A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.

Cell Line, Transformed↗
PubMed1996

Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein.

In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters.

Animals↗
PubMed1996

Regulation of mutant p53 temperature-sensitive DNA binding.

We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 --> Ala, Arg-175 --> His, Arg-248 --> Trp, Arg-249 --> Ser, and Arg-273 --> His). While all mutants were defective for binding to DNA at 37 ;C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33 ;C), and several mutants activated transcription from a p53-responsive promoter at 26 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37 degrees C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.

Amino Acid Sequence↗
PubMed1996

A mutant p53 that discriminates between p53-responsive genes cannot induce apoptosis.

Human wild-type (wt) p53 can induce apoptosis in transiently transfected H1299 cells maintained at 37 degrees C, whereas tumor-derived mutant forms of p53 (with the mutation Ala-143, His-175, or Trp-248) fail to do so. At 37 degrees C, p53 with a mutation to Ala at amino acid 143 (p53Ala143) was transcriptionally inactive. However, at 32 degrees C, p53Ala143 strongly activated transcription from several physiologically relevant p53-responsive promoters, to extents similar or greater than that of wt p53. Unexpectedly, p53Ala143 was defective in inducing apoptosis in H1299 cells at 32 degrees C. Concomitantly with the loss of apoptotic activity, p53Ala143 was found to be deficient in its ability to activate transcription from the p53-responsive portions of the Bax and insulin-like growth factor-binding protein 3 gene promoters. It is proposed that there may exist distinct classes of p53-responsive promoters, whose ability to be activated by p53 can be regulated differentially. Such differential regulation may have functional consequences for the effects of p53 on cell fate.

Apoptosis↗
PubMed1993

cDNA clones contain autonomous replication activity.

We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa). Two of three cDNA clones (343, 363) containing 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication. One of these cDNA clones, 343, is enriched by selection for poly(A)+ RNA. In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells. However, six out of ten cDNA clones contained neither 'O'-family or Alu homologous sequences and were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells. cDNA clones, from an oligo-d(T)-primed library of human poly(A)+ enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (from non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.

Animals↗
PubMed1989

Effects of soft contacts of differing thickness on corneal wound healing in rabbits.

This study was undertaken to determine the effects of thin (60 microns) and thick (240 microns) soft contact lenses of equal water content (70%) and power on nonlesioned and lesioned rabbit corneas. In one group of animals, corneas were not lesioned. Thin lenses were placed on left corneas and thick lenses on right corneas. In a second group, lesions were made in both corneas. Left corneas were covered with thin lenses and right corneas with thick lenses. Post-treatment times were 8 hr and 24 hr. At sacrifice, one-half of the cornea was fixed in 4% buffered glutaraldehyde for SEM study. The other half was cut into segments, fixed in a buffered glutaraldehyde-ruthenium red (RR) solution post-osmicated in osmium containing RR and prepared for TEM. At both 8 hr and 24 hr SEM showed cell migration in lesioned corneas covered with thin lenses but not in lesioned corneas covered with thick lenses. At 8 hr, TEM of nonlesioned and lesioned corneas showed no changes in the thickness of the corneal epithelium or the RR staining of the surface. At 24 hr, in nonlesioned corneas covered with thick lenses, the RR staining of microvilli and the height of the corneal epithelium were less than in nonlesioned corneas covered with thin lenses. In lesioned corneas covered with thick lenses, the thickness of the cornea was markedly reduced, the RR staining of microvilli was less and basal cells were more compressed than in lesioned corneas covered with thin lenses. The results of this study indicate that the thickness of a soft contact lens is important in treating corneal trauma.

Animals↗
PubMed1985

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells.

The effects of manno-1-deoxynojirimycin (ManDJN) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) were compared in IEC-6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N-linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H-sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7-9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7-9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP-treated cells were non-glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus-infected MDCK cells [(1984) J. Biol. Chem. 259, 12409-12413].

1-Deoxynojirimycin↗
PubMed1985

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture.

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.

1-Deoxynojirimycin↗
PubMed1978

Isolation of human tumour-specific antigens associated with beta2 microglobulin.

In the present study the tube LAI assay was used to monitor the isolation of the TSA of 4 different types of human cancers. Each tumour antigen was found to be specific for tumours arising in the organ from which the TSA was initially derived and which were histopathologically similar. Immunochemical studies revealed that these molecules co-isolate with normal human HLA antigens and are associated with beta2m. On Sephadex G-150, the majority of the papain-solubilized tumour antigen eluted in the mol. wt range 70,000-150,000. Analysis of this material by SDS-PAGE and 6M guanidine-HC1 column chromatography indicated that the material is composed of smaller subunits with prominent peaks at approximately 40,000, 25,000 and 12,000 mol. wt. Immunoadsorbent affinity chromatography of the solubilized tumour-membrane constituents on AH-Sepharose-linked horse anti-human-beta2m indicated that the tumour antigens, like HLA molecules, contain a beta2m subunit. The specificity of binding of TSA to the immunoadsorbent columns and the immunologically specific abrogation of LAI reactivity were clearly shown. The present study, therefore, indicates that by the isolation of beta2m, human tumour antigens can also be isolated, since human tumour antigens are associated with beta2m. Whether human TSAs may perhaps be modified histocompatibility antigens remains to be answered. Although the change upon malignant transformation in the pattern of the cell-surface proteins expressing the TSA determinant remains obscure, it would appear that for tumours arising within a given organ, a consistent alteration of cell-surface proteins occurs.

Antigens, Neoplasm↗