Biomedical subjects

I S Dunn

Publications and source records attributed to I S Dunn.

At least 19 recordsLinked to original sources

PubMed2001

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Antigens, Neoplasm↗

PubMed2001

Tracking membrane and secretory immunoglobulin alpha heavy chain mRNA variation during B-cell differentiation by real-time quantitative polymerase chain reaction.

Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B-cell plasmacytoid differentiation. Ratios of alpha heavy chain mRNA secreted (alphas) to membrane (alpham) form were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; TaqMan) using an IgA plasma cell line (NCI-H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI-H929 cells showed the highest alphas: alpham ratio as expected, alphas mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL-2 and IL-10 resulted in a further progression towards the alphas form, correlating with increased human plasma cell antigen-1 (HPC1) mRNA levels. However, alpha mRNA processing and HPC1 expression were independently regulated, as IFN-gamma treatment suppressed HPC1 levels while increasing alphas: alpham ratios. Cytokine-mediated increases in the alphas: alpham ratio resulted from strongly enhanced levels of alphas with relatively constant alpham values. Differentiation-related changes in mRNA processing can thus be tracked by automated quantitative PCR.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2000

Quantifiable analysis of human immunoglobulin heavy chain class-switch recombination to all isotypes.

Somatic recombinational events, including the immunoglobulin heavy chain class-switch, are a normal feature of B-cell maturation. To enable comprehensive and sensitive class-switch analysis in ex vivo human B cells, we have developed multiple digestion-circularization PCR (DC-PCR) techniques for quantifiable detection of switching to all immunoglobulin isotypes. This technology was validated by extensive sequencing of PCR products, tests with control non-lymphoid cells and B-cell lines of known isotypic specificities, and by demonstrating DC-PCR selectivity in a model system. With tonsillar B-cell DNA, switching to gamma 3, gamma 1, alpha1, gamma 2, gamma 4 and alpha2 isotypes was reproducibly detectable among different individuals. Levels of epsilon switching were relatively low and usually required higher total amounts of template DNAs for detection. Quantitation of alpha1 class switching in a panel of human tonsillar whole B cells was performed by the internal-competitor approach, and showed a pattern consistent with previous studies on IgA+ tonsillar cells. We demonstrate that these assays can rapidly show germline status or specific switch rearrangements in B lymphoid cell lines.

B-Lymphocytes↗

PubMed1997

Expression and localization of the retinoblastoma gene during radiation-induced apoptosis in neonatal rat kidney.

An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppressor gene (Rb), or its protein product (pRb), and localization of radiation-induced apoptosis. The rat kidney has two distinct zones of differentiation at birth-an outer nephrogenic zone, in which cells are undifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apoptosis are induced in the nephrogenic zone, with little effect on the differentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) and irradiated renal tissue for Rb mRNA transcript levels and localization (Northern blot and in situ hybridization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel electrophoresis for apoptosis), and cells in S-phase ([3H]thymidine uptake and autoradiography). Northern blots showed no detectable alteration in Rb transcript levels between control and irradiated tissues, whereas Western blots indicated increased expression of pRb in protein extracted from irradiated kidney compared with controls. ISH confirmed that Rb transcripts were not substantially altered in the nephrogenic and differentiated zones in control versus irradiated renal tissue. Immunolocalization of pRb demonstrated little effect in the differentiated zone, but in the nephrogenic zone pRb expression was increased, especially the S-shaped prenephrons, and was also found in many, but not all, apoptotic cells in this zone. The results link radiation-induced apoptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation.

Animals↗

PubMed1997

Tumor selectivity and transcriptional activation by azelaic bishydroxamic acid in human melanocytic cells.

Azelaic bishydroxamic acid (ABHA), a potent differentiating agent for lymphoid cells, was selectively toxic for 5 human tumor cell lines and transformed human melanocytes and keratinocytes (dose for 37% survival, D37, 30-100 microg/mL) compared with normal cells (melanocytes, fibroblasts; D37 > 300 microg/mL). Dendritic morphology was the only indicator found for increased differentiation, markers for the pigmentation pathway being unchanged or inhibited by ABHA. In contrast to hexamethylene bisacetamide and azelaic acid, ABHA significantly increased the HIV LTR, SV40 and c-fos promoter activities during a 24 hr treatment. Metallothionein promoter activity was enhanced by 5 hr treatment with ABHA in a sensitive melanoma cell line (MM96L) but was inhibited in a more resistant line (HeLa); c-fos promoter activity was inhibited in HeLa during this time. Transcription from a p53 binding response element was inhibited in MM96L by a 24 hr ABHA treatment but enhanced in HeLa. ABHA may represent a structural prototype for designing more potent and selective anti-melanoma agents.

Acetamides↗

PubMed1997

Biphasic response of the metallothionein promoter to ultraviolet radiation in human melanoma cells.

Because metallothionein (MT) is elevated and may be protective in UV-irradiated skin, we have studied the effects of UV and other agents on MT transcription using the sheep MT 1A promoter, linked to the beta-galactosidase gene and stably transfected into human cell lines. beta-galactosidase reporter activity was inducible by adding Zn2+ ions to the medium (100 microM for 2-4 h). Two differentiating agents, butyric acid and azelaic bishydroxamic acid (ABHA), significantly increased the response to Zn2+ in a melanoma cell line (MM96L-gal). UVB (280-315 nm) had two distinct, time-dependent effects. During the first 4 h after irradiation, high doses of UVB inhibited induction by Zn2+, an effect that was made more acute by simultaneous exposure to the differentiating agents. These changes in reporter activity were not due to alterations in Zn2+ transport into the cell. The UVB-depressed MT response subsequently recovered and by 24 h was double the control, yet remained sensitive to ABHA. Reporter activity in transfected HeLa cells differed from that in MM96L, being depressed 4 and 24 h after UVB and insensitive to ABHA at both times. Galactosidase reporter activity driven by non-MT promoters was not affected by these treatments. Dependence of MT transcriptional activity on UV-related DNA damage could be inferred because equitoxic UVC (254 nm) affected the response to Zn2+ in a similar fashion, whereas UVA, cisplatin and a methylating agent had no effect. The MT response was partly dependent on the PKC signal transduction pathway because it was inhibited by phorbol ester in HeLa, and by bisindolyl maleimide in HeLa and MM96L. The biphasic MT transcriptional response may model a signal transduction pathway that gives an early, depressed response to acute UV damage, with exacerbation by concurrent differentiation stimuli, but switches to a positive, cell-specific and potentially protective response at later times.

Animals↗

PubMed1996

In vitro alpha-complementation of beta-galactosidase on a bacteriophage surface.

Surface display of large multimeric non-secreted proteins is advantageous on the bacteriophage lambda compared with the widely used filamentous phage systems. A model system, the alpha-complementation of beta-galactosidase, was used for both further general characterization of protein-protein interactions on the lambda tail tube surface and for specifically probing the structure of the phage-displayed beta-galactosidase tetramer. In this complementation system, dimeric enzymatically inactive N-terminal deletion mutants of beta-galactosidase (alpha-acceptors) interact with peptides whose sequences span the region of the deletion (alpha-peptides) with the subsequent formation of tetramers and restoration of activity. The lambda phage could tolerate incorporation into their tail tubes of a limited number of copies of V protein (gpV) subunits C-terminally modified with an active alpha-peptide. Purified alpha-peptide phage showed specific in vitro alpha-complementation with an alpha-acceptor extract; the features of this reaction suggested that each complemented monomer can directly associate with an alpha-peptide displayed within the same tail tube structure. In contrast to the alpha-peptide, attempts to surface display an alpha-acceptor protein in a similar manner were unsuccessful. The implications of this work for surface-display cDNA libraries are discussed.

Bacteriophage lambda↗

PubMed1996

Total modification of the bacteriophage lambda tail tube major subunit protein with foreign peptides.

The bacteriophage lambda has been shown previously to tolerate a high multiplicity of peptide additions to the C-terminus of the major tail tube subunit protein (gpV, the product of the V gene). However, it was not clear whether all gpV copies within a functional virion could tolerate such modification. Complementation tests with phage bearing either V gene amber mutations or a precisely deleted V gene were used to test the extent of possible tail tube peptide display. Expression of plasmid-encoded gpV fused C-terminally with certain foreign peptides allowed rescue of such V gene-defective phage to essentially wild-type levels. After extensive purification such phage were shown by sensitive Western blotting to contain only the modified form of gpV. Peptide-modified gpV could also form indefinite tail tube polymeric structures (polytubes).

Amino Acid Sequence↗

PubMed1996

Inhibition of retinoblastoma protein translation by UVB in human melanocytic cells and reduced cell cycle arrest following repeated irradiation.

In human melanocytes and a human melanoma cell line (MM96L), the level of the retinoblastoma gene product (pRB) detected by Western blotting transiently decreased to 55% and 70% of controls respectively 9-12 h after a noncytostatic exposure (75 Jm-2) to UVB (280-315 nm) and to 2% and 14% 48 h after a cytostatic exposure (300 Jm-2). The pRB levels in fibroblasts and HeLa showed minimal loss, and under some conditions increased compared with unirradiated cells. Equitoxic doses of gamma radiation, cisplatin or the antimetabolite deoxyinosine had little effect on pRB levels. UVC (254 nm) was less inhibitory compared with equitoxic UVB. No loss of pRB mRNA was found in MM96L after UVB, nor was pRB protein stability significantly affected. Synthesis of new pRB in MM96L 24 h after UVB was 16% of controls, suggesting that loss of pRB results from a UVB-specific inhibition of translation. Compared with HeLa cells and fibroblasts, MM96L cells exhibited reduced cycle arrest if irradiated when pRB was depleted by a previous UVB exposure. These results suggest a mechanism whereby down-regulation of pRB translation by UVB may play a role in genesis of melanoma.

Cell Cycle↗

PubMed1996

A melanoma cell line sensitive to expression of a fusion protein binding the retinoblastoma protein family pocket domain.

An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta-galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells.

Amino Acid Sequence↗

PubMed1996

Purine deoxynucleoside metabolism in human melanoma cells with a high spontaneous mutation rate.

A human melanoma cell line (MM96L) had a spontaneous mutation rate at the HGPRT locus of approx. 7 times normal. The cells had elevated dATP and dGTP pools, lacked purine nucleoside phosphorylase (PNP) and were sensitive to killing by deoxyadenosine, deoxyinosine and related purines but not to inosine or hypoxanthine. Four other melanoma cell lines exhibited a range of nucleoside sensitivities and dNTP pool sizes. Failure of intact MM96L cells to degrade exogenous deoxyadenosine and deoxyinosine to hypoxanthine was confirmed by NMR of culture medium. Normal melanocytes were PNP+ and were insensitive to deoxyinosine. Comparison of the metabolites of [14C]deoxyinosine from MM96L and a PNP+ cell line of similar doubling time (HeLa) showed that both cell types produced 14C-labelled guanine and adenine nucleotides, with [14C]dATP and [14C]dADP being found in MM96L. This indicates that human sAMP synthetase or a similar enzyme catalyses the conversion of dIMP to dAMP, the resultant elevation of dATP causing base misincorporation and a mutator phenotype.

Alanine↗

PubMed1996

Mammalian cell binding and transfection mediated by surface-modified bacteriophage lambda.

Bacteriophage lambda virions whose tail tube major subunit (V) proteins are modified with a cyclizable Arg-Gly-Asp (RGD) peptide are able to promote the binding of certain mammalian cells to a solid surface. This effect was shown to be specific by peptide competition experiments, and control phage lacking the RGD peptide showed no significant cellular interaction. RGD-modified but not control phage bearing a reporter gene could transfect COS cells at a significant frequency. Phage-mediated transfection therefore benefits when the efficiency of only one step in the multi-stage uptake process is improved.

Animals↗

PubMed1996

Phage display of proteins.

Phage display of proteins continues to be an important technology with a variety of applications. In the past year, advances have been made in coupling rational protein design with the power of the display selection process. In addition to the widely used filamentous phage, other bacteriophage surface expression systems have now been developed, some of which may be of particular use for the selection of surface-display cDNA clones.

Antibodies↗

PubMed1995

Chromosomal structure of the human TYRP1 and TYRP2 loci and comparison of the tyrosinase-related protein gene family.

The structures of the human tyrosinase-related protein genes TYRP1 and TYRP2 have been determined and compared with that of the tyrosinase gene (TYR). The TYRP1 protein is encoded in 7 exons spread over 24 kb of genomic DNA. Characterization of a 55-kb contig encompassing the human TYRP2 locus reveals that the protein coding region is divided into 8 exons. All three members of the TYRP gene family share a common C-terminal membrane spanning exon. Examination of the position of other intron junctions suggests that TYRP1 was derived from a TYR duplication and then was itself duplicated to give rise to the TYRP2 gene. The evidence also suggests that at least some of the introns within the TYR, TYRP1, and TYRP2 coding regions were gained after duplication and that intron slippage is unlikely to have occurred.

Amino Acid Sequence↗

PubMed1995

Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein.

The tolerance of bacteriophage lambda morphogenesis for C-terminal additions to the tail tube major protein subunit (the V gene product; gpV) has been investigated. A second modified copy of the lambda V gene, either within a novel phage vector itself or plasmid-borne, was expressed during phage growth. High-level substitution of wild-type gpV by modified gpV bearing a basic C-terminal peptide sequence (RRASV; a target site for cAMP-dependent protein kinase) was possible using multiple repeats of a serine-glycine (SGGG) linker sequence. Highly purified phage bearing copies of gpV-RRASV could be efficiently phosphorylated by the appropriate protein kinase, and the incorporated label was shown to migrate exclusively at the expected size in protein gels. A large tetrameric protein (beta-galactosidase) could be incorporated into active virions in at least one copy, again using a Ser-Gly linker. These studies suggest that with a suitable spacing linker and controlled levels of expression, it is likely that a wide range of protein or peptide substitutents can be fused with gpV at its C terminus and assembled as component subunits of the tail tube.

Amino Acid Sequence↗

PubMed1994

Transcriptional regulation of differentiation, selective toxicity and ATGCAAAT binding of bisbenzimidazole derivatives in human melanoma cells.

To study the relationship between the structure of minor groove ligands and their affinity for specific DNA sequences that regulate gene transcription, three analogues of the A-T-specific DNA minor groove ligands Hoechst 33258 and Hoechst 33342 were synthesized with 5, 8 or 12 carbons in an aliphatic chain attached to the phenolic oxygen of the molecule. There was a striking bimodal relationship between toxicity to HeLa cells and the lipophilicity of the five analogues, toxicity being low for the compounds with a free hydroxyl (Hoechst 33258) or a 12-carbon substituent, yet high for the 5-carbon analogue. Selective killing of human melanoma cells compared with normal fibroblasts was observed for the Hoechst analogue with a 12-carbon chain attached. Hoechst 33258 itself was selectively toxic for the MM96E melanoma cell line compared with other cell lines, induced a highly dendritic morphology, increased tyrosinase activity and tyrosinase mRNA but decreased the level of gp75 (TRP-1) mRNA; message for a third pigment gene, Pmel-17, was unchanged. Tyrosinase activity was decreased in the resistant A2058 melanoma cell line and transcription was affected to a lesser extent than in MM96E. Expression of gp75 protein and two intermediate filament proteins was inhibited by Hoechst 33258 in MM96E cells. There was no major difference in the amount of 125I-Hoechst 33258 taken up by sensitive and resistant cells. Of the five derivatives studied, the parent drug Hoechst 33258 and the 2-carbon analogue (Hoechst 33342) were found to have the most inhibitory effect on affinity of octamer binding proteins for the ATGCAAAT consensus sequence found in the promoter region of certain genes associated with proliferation and differentiation. In contrast to Distamycin A (also an A-T-specific minor groove ligand), Hoechst 33258 displaced proteins already bound to the octamer motif. The G-C ligand chromomycin A3 exhibited a different spectrum of cell toxicity and tyrosinase stimulation compared with Hoechst 33258. Chromomycin A3 but not Hoechst 33258, strongly inhibited the zinc-dependent transcriptional activity of the sheep metallothionein-Ia promoter in reporter gene assays of transfected cells. Since the six metal-responsive elements of the promoter are GC-rich, this provides independent evidence for the sequence-specificity of transcriptional inactivation by one of these drugs in melanoma cells. Overall, the results suggest that Hoechst 33258 acts by inhibiting the transcription of specific genes, cell lines evidently differing in the accessibility to drugs of certain A-T-rich sequences.

Base Sequence↗

PubMed1994

Automated cycle sequencing of PCR templates: relationships between fragment size, concentration and strand renaturation rates on sequencing efficiency.

With the Applied Biosystems 373A automated DNA sequencer, we have systematically investigated the amounts of double-stranded PCR fragments of varying size (200, 564, and 1126 bp) required to give sequence of defined lengths, up to the maximum possible. Sequencing was performed on purified double-stranded PCR products using the dye-terminator chemistry and a thermal cycling procedure. The minimal template concentrations allowing determination of short sequences (< or = 160 bases) were essentially identical for the fragments studied. Maximal possible sequence determination from the 200 bp fragment was achieved over a wide concentration range, despite the fact that within this range a significant fraction of the template renatured by the mid-point of the sequencing reaction time-course. We conclude that the cyclic sequencing process overcomes competitive strand reannealing of double-stranded PCR products. The sequencing concentration-response curves for the 564 bp and 1126 bp fragments were similar to each other, although the minimal template concentrations required to read > 300 bases were slightly increased for the 564 bp fragment. Excess template is undesirable for optimal sequence length determination, but this is unlikely to be solely due to strand reannealing as single-stranded M13 templates in super-optimal concentrations also showed marked reduction in sequencing efficiency.

Automation↗

PubMed1992

Mutant forms of beta-galactosidase with an altered requirement for magnesium ions.

By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.

Amino Acid Sequence↗