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Biomedical subjects

A J Townsend

Publications and source records attributed to A J Townsend.

45 records · Page 3Linked to original sources
PubMed1989

Isolation, characterization, and expression in Escherichia coli of two murine Mu class glutathione S-transferase cDNAs homologous to the rat subunits 3 (Yb1) and 4 (Yb2).

The cytosolic glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of dimeric isoenzymes which catalyze the conjugation of electrophilic substrates with glutathione. We have isolated from a murine cell line (L929) two mu class GST cDNAs, pmGT10 (1.4 kilobases (kb] and pmGT2 (1.1 kb). Analysis of the deduced amino acid sequences revealed 93% homology between pmGT10 and the rat Yb1 (subunit 3) gene and 95% homology between pmGT2 and the rat Yb2 (subunit 4) gene. Furthermore, the 3'-untranslated regions of pmGT10 display a marked degree of homology to the 3' region of the rat Yb1 gene, while this region of pmGT2 displays marked homology to the corresponding region of the rat Yb2 gene. Using probes specific for each gene, northern blot analysis demonstrated that pmGT10 and pmGT2 hybridized to a 1.3-1.4-kb mRNA species present in L929 cells. These two murine cDNAs were subcloned into bacterial expression vectors, and enzymatically active GSTs encoded by pmGT10 (mGTmu1) or pmGT2 (mGTmu2) were purified from transformed Escherichia coli lysates. Western blot analysis of the individual GSTs produced in E. coli indicated that both genes encode GSTs which reacted with antibodies directed against mu class GST, but not with antibodies directed against alpha or pi class GSTs. The isoelectric points of these purified homodimers are 8.7 and 7.1, respectively, which are remarkably similar to those of the GST homodimers of the homologous rat subunits, 3-3 (or Yb1) and 4-4 (or Yb2), which are 8.4 and 6.9, respectively. Furthermore, the two murine subunits can form a heterodimer having a pI = 8.1 after in vitro dissociation with guanidine HCl, followed by dilution and dialysis to allow reassociation. Both homodimers of mGTmu1 and mGTmu2 and an apparent heterodimer are formed in L929 cells, as demonstrated by the isoelectric focusing profile of GST purified from this cell line. Hybridization of gene-specific probes with RNA from normal mouse tissues revealed that mGTmu1 transcripts were highly expressed in murine kidney, heart, lung, liver, and brain. Lower levels of mGTmu2 transcripts were also detected in kidney, heart, and lung. Expression of mGTmu2 RNA was not detected in murine liver or brain, which is in contrast to the regulation of the homologous rat subunit 4 (Yb2), which is expressed in liver and brain.

Amino Acid Sequence↗
PubMed1989

Differential increases in glutathione S-transferase activities in a range of multidrug-resistant human tumor cell lines.

Modified glutathione S-transferase (GST) activity was detected in MCF-7 cell lines selected in vitro for modest levels of resistance (2.6- to 13.7-fold) not only to Adriamycin but also to Novantrone, vincristine (VCR), and etoposide (VP-16). Western blot analysis revealed a qualitative increase in the expression of the pi class GST isozyme and some apparent down regulation of the mu class in these resistant lines relative to the parental line. Overexpression of GST pi protein was also observed after in vitro exposure of MCF-7 cells to fractionated x-irradiation, which resulted in a subline that exhibited 5-fold resistance to VCR and 3-fold resistance to VP-16. Northern blot analysis provided qualitative evidence that these changes in GST pi and mu appeared to be at the transcriptional level in these VCR- and VP-16-resistant MCF-7 sublines. In contrast, similarly selected VCR-resistant, VP-16-resistant, and x-ray-pretreated-resistant sublines, derived from the SuSa cell line established from a human testicular teratoma, did not exhibit significant alterations in GST isozyme profiles compared with their parental cell lines. All parental and resistant SuSa lines that were studied expressed the GST pi isozyme constitutively. Total glutathione content and glutathione peroxidase activities were not modified in parallel with the GST alterations in these human tumor drug resistant sublines. These data suggest that the increased cellular GST activity was associated with selection of resistance to many of the drugs associated with the multidrug resistant phenotype in MCF-7 sublines but that this was not the case for the other human tumor cell line (SuSa) tested.

Antineoplastic Agents↗
PubMed1989

Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins.

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1988

Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer.

The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi). Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene. We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines we found an inverse association between GST pi expression and ER content. We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo. GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples). The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors.

Breast Neoplasms↗
PubMed1988

Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase.

Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.

Antineoplastic Agents↗
PubMed1987

Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerases in vitro: visualization of chain elongation on a defined template.

1-beta-D-arabinofuranosyl-5-aza-cytosine (ara-5-aza-Cyd) is an analog of 1-beta-D-arabinofuranosylcytosine (ara-C), which resembles ara-C in anabolic metabolism, incorporation into DNA, and inhibition of DNA replication. Human T-lymphoblastic cells (Molt-4) incorporate three- to fivefold more ara-5-aza-Cyd than ara-C into DNA during 5-8 hr exposure. Although ara-5-aza-Cyd and its triphosphate metabolite are unstable in aqueous solution, the aza-analog was much more stable in solution when incorporated into native DNA isolated from Molt-4 cells. By using gapped duplex DNA as a substrate for purified human DNA polymerases alpha and beta, inhibition of [3H]-dCTP incorporation by ara-5-aza-CTP and ara-CTP was competitive, with Ki values for alpha of 11 and 1.5 microM, respectively. Ki values for polymerase beta were 39 and 7.6 microM, respectively. A DNA elongation assay was adapted from DNA sequencing technology, using singly primed bacteriophage M13mp19 or M13mp9 (+)-DNA. Elongation of 5'-[32P]-labeled primer by polymerase alpha is slowed considerably by incorporation of one ara-CMP and to a lesser extent after incorporation of one ara-5-aza-CMP. Neither analog significantly affected elongation by polymerase beta after a single incorporation. However, neither polymerase alone could appreciably extend the growing chain if two consecutive ara-5-aza-CMP or ara-CMP analogs were incorporated. Thus, if similar mechanisms are operant in intact cells, the greater incorporation of ara-5-aza-Cyd than ara-C into DNA may be due to a more facile elongation of the nascent DNA strand by polymerase alpha after incorporation of a single analog. The effect in vitro of incorporation of either analog on DNA chain elongation is widely variable, depending on the identity of the polymerase involved and the sequence of the DNA template being copied.

Arabinofuranosylcytosine Triphosphate↗
PubMed1983

Effect of ethanol on alkaline phosphatase activity in HeLa cells.

Incubation of HeLa cells with 10-80 mM ethanol, caused a dose-dependent decrease in alkaline phosphatase specific activity. With 80 mM ethanol, the decrease became apparent after 24-48 hr incubation. The same dose-dependent effect was observed with the nonmetabolizable alcohol t-butanol. The effect of ethanol was not due to toxicity as the cells continued to replicate for weeks in the presence of ethanol and was reversible. There were no differences in the Km values of alkaline phosphatase in crude extracts or solubilized enzyme prepared either from control or 80 mM ethanol-treated cells. Hydrocortisone and choline chloride, which by themselves increase alkaline phosphatase specific activity, altered the effect of ethanol on alkaline phosphatase. In the presence of either 1 microM hydrocortisone or 40 mM choline chloride, neither 10 nor 20 mM ethanol decreased alkaline phosphatase specific activity.

Alkaline Phosphatase↗
PubMed1979

A trypanosomiasis survey of wild animals in the Luangwa Valley, Zambia.

Between 1971 and 1974 546 wild animals of 34 species were examined for trypanosomes; 79 infections (9 mixed) were diagnosed and 29 stocks were cryopreserved. Of 14 stocks of the subgenus Trypanozoon tested by the blood incubation infectivity test three (two from Kobus ellipsiprymnus and one from Phacochoerus aethiopicus) retained their infectivity to rodents. There are indications that the number of animals harbouring trypanosomes is far in excess of those showing detectable parasitaemias even when using a combination of diagnostic methods. The standard microhaematocrit method involving microscopic examination of the buffy layer from a microhaematocrit tube between a slide and coverslip, and the inoculation of experimental animals produced the most satisfactory results.

Animal Population Groups↗