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Biomedical subjects

J J Roberts

Publications and source records attributed to J J Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1988

Heterosexual spread of human immunodeficiency virus in Edinburgh.

Heterosexual transmission of human immunodeficiency virus (HIV) was investigated in 123 subjects with no apparent risk factor for infection other than having had heterosexual intercourse with a person who was either infected with HIV or at high risk of being infected with it. Seven subjects were found to be infected with the virus. Risk factors for transmission included being the regular sexual partner of an abuser of intravenous drugs and having a sexual relationship of more than 18 months' duration. Anal intercourse was not a risk factor in the three subjects who admitted to it. There were 41 regular partnerships with abusers of intravenous drugs in which the antibody state and history were fully known for both partners. In these partnerships male to female transmission of the virus occurred in five out of 34 (15%) and female to male in one out of seven. In 30 couples in whom one partner was known to be positive for HIV and an abuser of intravenous drugs four female partners were found to be seropositive at first testing, but there were no new positive results on subsequent serial testing. In six of these 30 couples both partners abused intravenous drugs but the partner who was negative for HIV remained so. Few of the partnerships always practised safe sexual techniques, even after a partner was known to be positive for HIV. Heterosexual transmission of HIV occurred but was incomplete and may be related to the timing of the relationship with the infection.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Inherent sensitivity of cultured human embryonal carcinoma cells to adducts of cis-diamminedichloroplatinum(II) on DNA.

Clinical and experimental data indicate that human embryonal carcinoma cells are unusually sensitive to the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin), but the basis of this sensitivity is unknown. Using colony formation assays we measured survival of cultured human embryonal carcinoma cells following cisplatin treatment and related survival to the amount of platinum bound to DNA, determined by isolation of cellular DNA and flameless atomic absorption spectrometry, over a range of drug doses. Similar measurements were carried out on F9 mouse embryonal carcinoma cells and on a fibroblast cell strain from a patient with the genetic disease Fanconi's anemia, a syndrome associated with hypersensitivity to cytotoxic and clastogenic effects of difunctional DNA-binding agents. These results were compared with similar analyses on a variety of cultured cells from previous studies. The embryonal carcinoma cells and the Fanconi's anemia fibroblast strain were among the most sensitive cells on a dose-response basis. Since the amount of platinum bound to DNA after treatment of these cells was similar to values reported for many other cell types, it follows that mouse and human embryonal carcinoma cells are inherently sensitive to DNA-bound platinum adducts, to a degree approaching the sensitivity of fibroblasts from patients with xeroderma pigmentosum and Fanconi's anemia.

Animals↗
PubMed1987

The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in mammalian cell lines sensitive or resistant to difunctional agents.

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.

Animals↗
PubMed1987

Differential toxicity of cis- and trans-diamminedichloroplatinum(II) toward mammalian cells: lack of influence of any difference in the rates of loss of their DNA-bound adducts.

DNA-bound adducts formed by cis- or trans-diamminedichloroplatinum(II) (cis-DDP or trans-DDP, respectively) have very different effects on DNA synthesis and cell cycle progression in Chinese hamster cells. When the loss of platinum from the DNA of cells treated with pulsed doses of these isomers was corrected for the different effects they produced on DNA replication and cell cycle progression, then adducts formed by either agent were lost slowly from DNA and at comparable rates. The kinetics of accumulation of platinum on the DNA of exponentially growing Chinese hamster cells differed following continuous treatment with a low dose (10 microM) of either cis-DDP or trans-DDP. However, when these DNA binding values were again corrected for the different effects of the two isomers on DNA replication and cell cycle progression, both compounds reacted with DNA to similar extents. The amounts of platinum that accumulated by 30 h on the DNA of stationary-phase Chinese hamster cells treated with a low dose (10 microM) of either cis- or trans-DDP were also similar but resulted in very different effects on cell survival. The amounts of platinum accumulating on the DNA of near confluent African green monkey cells were also similar following continuous treatment with a low dose (10 microM) of either cis-DDP or trans-DDP, after correction for the relatively small amount of DNA synthesis occurring in these cells, and they were similar to the binding of platinum to the DNA of similarly treated Chinese hamster cells. There was no rapid loss of platinum from the DNA of African green monkey cells following treatment with pulsed low or high doses of trans-DDP. It could be concluded that the different cytotoxic effects produced by cis- or trans-DDP resulted from an intrinsic difference in the effects of their respective DNA-bound adducts on DNA replication and were not due to a difference in the rate of repair of such adducts, as previously proposed (R. B. Ciccarelli et al., Biochemistry, 24: 7533-7540, 1985). The accumulation of platinum on the proteins of Chinese hamster or African green monkey kidney cells treated with cis- or trans-DDP was also consistent with the respective toxic effects of the two isomers.

Animals↗
PubMed1987

Drug resistance and DNA repair.

DNA repair confers resistance to anticancer drugs which kill cells by reacting with DNA. A review of our current information on the topic will be presented here. Our understanding of the molecular biology of repair of 0(6)-alkylguanine adducts in DNA has advanced as a result of the molecular cloning of the E. coli ada gene but the precise role of this lesion in the cytotoxic effects of alkylating agents in mammalian cells is not completely understood. Less progress has been made in understanding the enzymology and molecular biology of DNA cross-link repair even though such lesions are important for the cytotoxic effects of the widely used bifunctional alkylating agents and platinum compounds. It is evident that drug sensitive or resistant phenotypes are as highly complex as are the effects of DNA damage on cell metabolism and various aspects of these effects are discussed. Few clear correlations have been made between quantitative differences in DNA repair capacity and cellular sensitivity but assays which were developed to measure fidelity and intragenomic heterogeneity in DNA repair are beginning to be applied. Such studies may reveal subtle differences between sensitive and resistant cell lines. The molecular cloning of human DNA repair genes by transfection into drug sensitive rodent cells has been attempted. Some success has been achieved in this area but the functions of the cloned genes have yet to be identified.

Animals↗
PubMed1986

CB 1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells.

Walker tumour cells were shown to be uniquely sensitive to CB 1954 when compared with other cells in vitro. CB 1954 forms DNA-DNA interstrand crosslinks in a time-dependent manner in Walker tumour but not Chinese hamster cells. The absence of interstrand crosslinks in hamster cells was not due to a lack of uptake of drug but rather to a failure to convert (probably by bioreduction) CB 1954 to the required reactive difunctional intermediate.

Animals↗
PubMed1986

Epidemic of AIDS related virus (HTLV-III/LAV) infection among intravenous drug abusers.

Stored blood samples from 164 intravenous drug abusers who attended a Scottish general practice were tested for HTLV-III/LAV (human T cell lymphotropic virus type III/lymphadenopathy associated virus) infection. Of those tested, 83 (51%) were seropositive, which is well above the prevalence reported elsewhere in Britain and Europe and approaches that observed in New York City. The timing of taking samples of negative sera and continued drug use suggest that as many as 85% of this population might now be infected. The infection became epidemic in late 1983 and early 1984, thereafter becoming endemic. The practice of sharing needles and syringes correlated with seropositivity, which, combined with the almost exclusive intravenous use of heroin and other behavioural patterns, may explain the high prevalence of HTLV-III/LAV infection in the area. Rapid and aggressive intervention is needed to control the spread of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Walker rat carcinoma cells are exceptionally sensitive to cis-diamminedichloroplatinum(II) (cisplatin) and other difunctional agents but not defective in the removal of platinum-DNA adducts.

Cisplatin binds to cellular macromolecules (DNA, RNA and protein) to the same extent in wild-type Walker rat carcinoma cells and a variant sub-line of these cells resistant to cisplatin and to other difunctional, but not monofunctional cytotoxic agents. Wild-type Walker cells exhibit a unique sensitivity to DNA-bound cisplatin, while the resistant cells have a sensitivity that approximates to that of many normal and other tumour cell lines. Total DNA-bound adducts were lost from both sensitive and resistant Walker cells at similar rates. Equal numbers of DNA interstrand crosslinks and DNA-protein crosslinks were formed in both cell lines, and the rate of loss of both types of crosslinks was similar in the two lines. Therefore the unusual sensitivity of Walker cells to cisplatin is not due to a defect in their ability to remove these platinum-DNA adducts.

Animals↗
PubMed1986

The unique sensitivity of Walker rat tumour cells to difunctional agents is associated with a failure to recover from inhibition of DNA synthesis and increased chromosome damage.

The rate and mode of DNA synthesis was examined by thymidine uptake and by flow cytometry in Walker tumour cells highly sensitive to difunctional agents (WS), and in a derived subline of resistant cells (WR) (Rawlings and Roberts, 1986), following their treatment with sulphur mustard. Both cell lines exhibited the same dose-dependent and progressive depression in rate of DNA synthesis for up to 4 h after treatment. Thereafter the depression in rate of synthesis was partially reversed in the WR cells but DNA synthesis continued to decrease in the WS cells resulting in their slower transit through the S phase and a persistent block in the G2/M phase of the cell cycle. Sensitive cells which finally escaped the block in G2 carried more chromosome aberrations than the corresponding resistant cells. Neither cell line was defective in daughter strand-gap repair. In their sensitivity to difunctional but not to monofunctional compounds, their failure to recover from the early depression of DNA synthesis, their apparent lack of a defect in excision repair and their sensitivity to chromosome aberration induction, the Walker cell phenotype closely resembles that of the human Fanconi's anaemia cell.

Animals↗
PubMed1986

Potentiation of sulphur mustard or cisplatin-induced toxicity by caffeine in Chinese hamster cells correlates with formation of DNA double-strand breaks during replication on a damaged template.

Caffeine inhibited the elongation of nascent DNA and induced breaks in the template DNA of sulphur mustard-treated Chinese hamster cells. The sizes of template and nascent DNAs, as indicated by alkaline sucrose gradient sedimentation, were similar suggesting that incision of template DNA occurred opposite gaps formed in nascent DNA by the action of caffeine, forming, effectively, double-strand breaks in DNA. Double-strand break formation was demonstrated, by means of elution of labelled DNA through polycarbonate filters at neutral pH, in both sulphur mustard- and cisplatin-treated cells when they were incubated in the presence of caffeine for 24 h. Double-strand breaks were only formed in that DNA which had been replicated in the presence of caffeine after treatment with sulphur mustard or cisplatin. Non-toxic concentrations of cycloheximide abolished the potentiation by caffeine of sulphur mustard-induced toxicity to Chinese hamster cells and at the same time abolished the formation of the low molecular weight nascent DNA, and as a consequence of its inhibitory effect on DNA synthesis, and the formation of double-strand breaks in DNA. Potentiation of the lethal and clastogenic effects of genotoxic agents by caffeine is therefore due to effects on the rate and mode of DNA synthesis which lead finally to double-strand breaks in DNA.

Animals↗
PubMed1986

Mechanism of cytotoxicity of anticancer platinum drugs: evidence that cis-diamminedichloroplatinum(II) and cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) differ only in the kinetics of their interaction with DNA.

The kinetics of the aquation reactions of cisplatin and carboplatin and their subsequent reactions with DNA, both in vitro and in vivo, have been measured. The results have been extrapolated to indicate the expected cytotoxicity of these compounds in cells obtained from human cancer patients. Rate constants for the aquation at 37 degrees C of cisplatin and carboplatin of 8 X 10(-5) and 7.2 X 10(-7) s-1, respectively, were calculated from the half-life of these compounds in phosphate buffer, pH 7. This difference in their rate of activation was matched by their rates of binding to DNA. By use of a 14C-labeled ligand, carboplatin was shown to bind monofunctionally to DNA, after which there was a time-dependent formation of difunctional interstrand cross-links, formed from some of these initially monofunctional adducts. A similar, although faster, accumulation of cross-links was seen when cisplatin was bound to DNA. The loss of the 14C-CBDCA ligand of carboplatin was calculated to occur with a rate constant of 1.3 X 10(-5) s-1 which was similar to that for the rate of formation of interstrand cross-links and faster than that for the monofunctional reaction with DNA. It was concluded therefore that the CBDCA ligand becomes a more labile leaving group once carboplatin has been monoaquated. In contrast, both chloro-ligands of cisplatin were shown to leave at similar rates. The fact that other difunctional lesions were formed to the same extent, by equal bound doses of cisplatin or carboplatin, was indicated by the unwinding of supercoiled plasmid DNA. The effects of cisplatin and carboplatin on this DNA were the same once bound to the same extent. About a 100-fold larger dose of carboplatin was, as predicted by their rates of aquation, required to produce equivalent binding to plasmid DNA. In vivo, equal binding of the two drugs to DNA of various cell systems resulted in equal cytotoxicity. Again a much larger dose (20- to 40-fold) of carboplatin was required to produce this equal binding. In general a DNA bound platinum level of about 20 nmol/g reduced cell survival by 90%, although certain cell lines were shown to be much more sensitive to DNA bound platinum. Similar binding values, to those above, were obtained in the DNA extracted from cells of human cancer patients treated with cisplatin. It was inferred that the cytotoxic effect of this level of platinum on DNA would be (unless the cells were of a sensitive phenotype) about 90%.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Higher homolog and N-ethyl analog of creatine as synthetic phosphagen precursors in brain, heart, and muscle, repressors of liver amidinotransferase, and substrates for creatine catabolic enzymes.

Tissues of chicks fed 5% N-methyl-3-guanidinopropionate (N-amidino-N-methyl-beta-alanine) for 12 days accumulated the following amounts of free plus phosphorylated derivatives as mumol/g, wet weight: brain, 5.5; heart, 7.3; leg muscle, 21.0; and breast muscle, 24.4. Since total creatine levels remained nearly the same in brain, N-methyl-3-guanidinopropionate-P provided brain with a supplemental reservoir of high energy phosphate. Tissues of rats fed 2% N-ethylguanidinoacetate (N-amidino-N-ethylglycine) accumulated large amounts of N-ethylguanidinoacetate-P, which has thermodynamic properties similar to creatine-P and is the kinetically most reactive synthetic phosphagen yet described. N-Ethylguanidinoacetate derivatives replaced creatine derivatives mole-for-mole, and the fraction of synthetic to total phosphagen after 19 days was 60% in heart, 54% in slow oxidative muscle, 42% in fast glycolytic muscles, and 22% in brain. N-Ethylguanidinoacetate served as a false end product co-repressor of liver arginine:glycine amidinotransferase levels in both chicks and chick embryos; N-methyl-3-guanidinopropionate and N-propylguanidinoacetate were relatively inactive. Creatinine amidohydrolase reversibly cyclized both N-ethylguanidinoacetate and N-propylguanidinoacetate with even lower Km values than for creatine derivatives, but it did not react significantly with N-methyl-3-guanidinopropionate, 3-guanidinopropionate, or 1-carboxy-methyl-2-imino-imidazolidine (cyclocreatine). Creatine amidinohydrolase also hydrolyzed N-acetimidoylsarcosine, but was relatively unreactive toward N-ethylguanidinoacetate, N-methyl-3-guanidinopropionate, 3-guanidinopropionate, and cyclocreatine. Amidinohydrolase can therefore be used to remove interfering creatine in assays of tissues for coexisting N-ethylguanidinoacetate or N-methyl-3-guanidinopropionate. Assays are now available to follow changes during metabolic stresses of any combination or all of the following phosphagens accumulated by the same tissue: creatine-P, N-ethylguanidinoacetate-P, cyclocreatine-P, N-methyl-3-guanidinopropionate-P, and homocyclocreatine-P.

Amidinotransferases↗
PubMed1985

Plasma membrane as target of alkylating agents.

N mustard resistant Walker cells exhibit the same frequency of DNA interstrand cross-links and the same rate of cross-link removal as the sensitive parental line. Employing cytostatically active concentrations of chlorambucil covalently bound to polyethyleneimine, the extent of DNA cross-linking is reduced to levels observed in the presence of nontoxic concentrations of free chlorambucil. It is concluded, therefore, that DNA cross-links alone are not sufficient to explain the inhibition of cell multiplication by alkylating agents and that additional mechanisms have to be considered. Evidence for an interference of alkylating agents with several enzymes of the plasma membrane is presented. An inhibition by N mustard of the furosemide-sensitive Na+/K+/Cl- -cotransport and the Na+/H+-antiport is described in greater detail. Considering the fact that the enzymes which are affected by alkylating agents are controlled by growth factors it was investigated whether a synergism between inhibitors of early growth-factor-controlled reactions and alkylating agents is to be seen. It is demonstrated that mepacrine, an inhibitor of phospholipase C, and the calmodulin binding drugs, chlorpromazine and flunarizine, amplify the action of N mustard.

Adenosine Triphosphatases↗