Biomedical subjects
Publications and source records attributed to M W Taylor.
We reported previously that a housekeeping gene could be converted to a butyrate-inducible gene by replacing its cognate promoter with the Moloney murine sarcoma virus enhancer-promoter element. In this study we report that the activated transcriptional state could be propagated from mother to daughter cells after the withdrawal of the inducer.
Previous in vitro and in vivo studies have shown a synergism between interferon (IFN) and 5-fluorouracil (5-FU) against different tumor cell lines. In the present study we report that the combination of IFN-alpha and 5-FU has a significant effect not only on the inhibition of tumor cell growth but also on the regulation of natural killer cell-mediated cytotoxicity (NK-CMC). The addition of 5-FU to effector cell population neither affects NK cell activity nor activation of NK cells by IFN or by interleukin (IL)-2. However, pretreatment of target cells with 5-FU increased their susceptibility to NK activity and abolished the protective effect induced by IFN against NK-CMC. This dual effect of IFN-alpha and 5-FU was found to be applicable to target cells of different origins including a cervical carcinoma cell line (ME-180), a hairy cell leukemia-like cell line (Eskol), a CML cell line (K-562) and a primary culture of AIDS-related Kaposi's sarcoma cells. Similar results were found with IL-2 treatment of Eskol cells but not other cells. Combination of IL-2 with 5-FU resulted in enhancement of the sensitivity of the cells to NK activity and abolished the protection against NK-CMC. Based on these results we propose that the combination of IFN-alpha and 5-FU not only has a direct growth inhibitory effect on tumor cells but also has a regulatory role on the immunological arm of the NK-CMC. Moreover, since the combination gave the same pattern of response in different tumor cells, both NK-sensitive and NK-resistant, this combination treatment may be a candidate for clinical trials in various types of tumors.
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Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.
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The rate of de novo purine biosynthesis was measured in a series of hypoxanthine guanine phosphoribosyl transferase deficient (HGPRT-) cells from a variety of sources, including human Lesch-Nyhan cells. Under optimum growth conditions, no enhanced purine biosynthesis was detected (in contrast to previous reports). An 'elevated' level of de novo purine biosynthesis could be detected in mutants following starvation for glutamine. However, this was the result of depression of purine biosynthesis in normal cells, with a resulting artifactual overproduction in mutants.
Azaguanine-resistant mutants of Chinese hamster ovary cells were isolated following mutagenesis with ICR-17OG. Of the eight mutant isolates examined, only one, Ag-5 had detectable hypoxanthine(guanine)phosphoribosyltransferase activity. Under normal conditions of growth, de novo purine biosynthesis in the mutants was not significantly different from wild type. However, when the cultures were starved for glutamine over a 2 h period before measuring 5'phosphoribosyl formylglycinamide (a relative measure of de novo purine biosynthesis), elevated levels of 5'-phosphoribosyl formylglycinamide accumulated in some of the mutants, and decreased levels in wild type and Ag-5. The level of purine biosynthesis could be related to the levels of glutamine in the pregrowth medium. The rate of purine biosynthesis correlated with 5-phosphoribosyl pyrophosphate levels, which were enhanced in the mutant (Ag-C) following the starvation period. No alterations were found in levels of 5-phosphoribosyl pyrophosphate synthetase or glutamine synthetase. The extent of feedback inhibition was normal in both mutant and wild type cells. These data suggest that the hypoxanthine (guanine) phosphoribosyltransferase locus is a regulatory gene.
Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.
Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.
The purine salvage pathway enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase EC 2.42.7) has been purified to greater than 85% homogeneity from crude rat liver 100,000 x g supernatant in one step by affinity chromatography. The enzyme binds to an AMP-agarose column and is eluted off the column by 1 mM 5-phosphoribosyl pyrophosphate with a 50 to 80% recovery. Enzyme kinetics indicate that the mechanism of the specific elution is due to competition of the product AMP and substrate 5-phosphoribosyl pyrophosphate for the same site on the enzyme.
When V79 pur 1, a purine-requiring auxotroph of a Chinese hamster cell line, is deprived of adenine, nucleic acid and protein synthesis decline rapidly. However, on continuous starvation RNA and DNA synthesis recommences to reach approximately 30% of the normal level between 12 to 24 h starvation. This is accompanied by a rise in the intracellular nucleotide pool. Utilizing mengovirus, which gives a productive infection in V79 pur 1 cells even under conditions of starvation, we can show that rRNA is preferentially degraded and provides the nucleotides for RNA synthesis. Thus "purineless" death in mammalian cells is accompanied by turnover of stable RNA.
Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.
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Bovine enterovirus-I (BEV-I) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-I lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 80S particles as well as lesser amounts of 160S, 130S, 45S, 14S and 5S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength 130S particles were transformed into 160S particles, and 45S into 80S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 80S particle, although 5S and 14S particles were shown to be precursors of mature virus particles.
A class of purine auxotrophs blocked early in the purine biosynthetic pathway was examined. The inability of these mutants to accumulate formylglycinamide ribotide (FGAR) in the presence of azaserine suggested that one or more of the first three enzymes of the pathway were either missing or defective. By direct enzyme assay, phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14) was found to be absent in extracts of mutant cells. Thus these cells were unable to convert PRPP to phosphoribosylamine (PRA). By reacting ribose 5-phosphate with ammonium ions, PRA was generated nonenzymatically in the incubation mixture, thus enabling us to test for the presence of the two enzymes required to convert PRA to FGAR. It was demonstrated that sufficient amounts of these enzymes, phosphoribosylglycineamide synthetase (E.C. 6.3.1.3) and phosphoribosylglycineamide formyltransferase (E.C. 2.1.2.2), were present in mutant extracts to allow synthesis of FGAR to occur once PRA was so provided.