Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Recruitment of Ca2+ channels by protein kinase C during rapid formation of putative neuropeptide release sites in isolated Aplysia neurons.

Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.

Developments with targeted enzymes in cancer therapy.

Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.

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)

Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.

Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody.

An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.

Telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.

Telomerase activation is considered to be a critical step in cancer progression due to its role in cellular immortalization. The prevalence of telomerase expression in human cancers makes it an attractive candidate for new mechanism-based targets for cancer therapy. The selective killing of cancer cells can be achieved by gene-directed enzyme pro-drug therapy (GDEPT). In this study we have tested the feasibility of using the transcriptional regulatory sequences from the hTERT and hTR genes to regulate expression of the bacterial nitroreductase enzyme in combination with the pro-drug CB1954 in a suicide gene therapy strategy. hTERT and hTR promoter activity was compared in a panel of 10 cell lines and showed a wide distribution in activity; low activity was observed in normal cells and telomerase-negative immortal ALT cell lines, with up to 300-fold higher activity observed in telomerase positive cancer lines. Placing the nitroreductase gene under the control of the telomerase gene promoters sensitized cancer cells in tissue culture to the pro-drug CB1954 and promoter activity was predictive of sensitization to the pro-drug (2-20-fold sensitization), with cell death restricted to lines exhibiting high levels of promoter activity. The in vivo relevance of these data was tested using two xenograft models (C33a and GLC4 cells). Significant tumour reduction was seen with both telomerase promoters and the promoter-specific patterns of sensitization observed in tissue culture were retained in xenograft models. Thus, telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.

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.

Crystallization and preliminary crystallographic data for an FMN-dependent nitroreductase from Escherichia coli B.

An FMN-dependent nitroreductase enzyme isolated from Escherichia coli B has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with cell parameters a = b = 57.74 A, c = 275.51 A and two molecules per asymmetric unit. Diffraction extends to beyond 1.9 A.

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite.

The bystander effect is an important part of tumor kill using gene-directed enzyme prodrug therapy (GDEPT). Recently, we have described a novel enzyme prodrug system using bacterial nitroreductase and the prodrug CB1954 (NTR/CB1954). We demonstrate here the presence of a cell-permeable cytotoxic activity in the conditioned growth medium of nitroreductase (NTR)-transduced cells treated with CB1954 and show that its appearance corresponds to the appearance of two metabolites of CB1954 previously identified (Friedlos et al., 1992). The degree of bystander effect and the degree of transferred cytotoxicity correlates with the level of NTR enzyme expression. Two other prodrugs for NTR show little bystander killing and do not produce detectable cell permeable metabolites. The elucidation of the mechanism of the bystander effect may allow the more effective use of NTR/CB1954.

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

The chemotherapy of colon cancer.

Despite extensive clinical trials, mortality from colon cancer has remained essentially unchanged since the 1950s. However, the increasing numbers of complete and partial responses seen in clinical trials suggest that colon cancer can be successfully treated by chemotherapy, but only if the antitumour selectivity can be increased by a substantial amount. This will be possible by the introduction of new drugs with more precise mechanisms of action, such as those acting specifically on signalling or cell cycle control pathways shown to be aberrant in colon cancer. Alternatively, the selectivity of present day agents may be increased considerably by the selective activation of prodrugs in tumours (ADEPT) or by targeting them to tumours using polymers. Other new approaches using vaccines or some form of gene therapy will potentiate present chemotherapy, while the introduction of positron emission tomography (PET) scanning will allow the rapid detection of agents with activity that would have been missed by conventional measurements of response.

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954.

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.

Structure-cytotoxicity relationships of nitroimidazoles in an in vitro system.

Correlations of cytotoxicity of nitroimidazoles and their electron affinity depend upon the degree of reduction of the drugs. If the end-point chosen to assess cytotoxicity is one at which incomplete reduction has occurred the slope of the correlation is positive, with respect to E71, but if measurements are taken after complete reduction of the drugs the slope is a negative one. The production of nitrite ion by 5-nitroimidazoles depends on the base composition of DNA suggesting that the active agent responsible for cytotoxicity is the one-electron radical anion, R--NO2-.

The mechanism of nitroimidazole damage to DNA: coulometric evidence.

A high resolution coulometric technique has been developed to measure the electron requirement for reduction of 12 nitroimidazoles, both in the presence and absence of DNA. The cytotoxic species is shown to be a light sensitive intermediate of drug reduction and a common mechanism of cytotoxicity proposed which involves electron transfer from DNA to the one-electron radical anion (R-NO2-).

Photosensitive interaction of RSU 1069 with DNA.

RSU 1069 is a 2-nitroimidazole radiosensitizer with an aziridine-containing side chain. In light (360 nm) the absorbance maximum of the nitro group at 325 nm disappears, which is accompanied by expulsion of the nitro group as the nitrite ion. We suggest an intramolecular cyclization of the aziridine side chain and the C2 of the imidazole ring as a possible explanation. This photosensitive effect was used to determine separately the damage to DNA induced by the reduced nitro group and the alkylating property of the aziridine. The aziridine-induced DNA damage is maximized in the dark when the nitro group is either absent (electrolytically reduced prior to the addition of DNA) or non functional (unreduced). In the light, damage is reduced. Typical DNA damage includes helix disruption leading to single strand breaks and the release of thymidine. Alkaline filter elution studies show evidence only for strand breakage and none for cross-linking indicating the drug is capable of mono-functional alkylation only.

Self-immolative prodrugs: candidates for antibody-directed enzyme prodrug therapy in conjunction with a nitroreductase enzyme.

The synthesis and properties of some prodrug candidates for antibody-directed enzyme prodrug therapy (ADEPT) are described. These compounds have been designed to generate the corresponding active drug upon interaction with a bacterial nitroreductase that can be conjugated to antibodies that recognize tumor-selective antigens. The active drugs included in the study are actinomycin D, mitomycin C, doxorubicin, 4-[bis(2-chloroethyl)amino]aniline and 4-[bis(2-chloroethyl)amino]phenol. The prodrugs were all 4-nitrobenzyloxycarbonyl derivatives of these drugs, which upon enzymatic reduction, generated the drug through self-immolation of the 4-(hydroxyamino)benzyloxycarbonyl group. In the case of actinomycin D, the ratio of the dose required between drug and prodrug to give the same cytotoxicity was greater than 100. The prodrug was also much less toxic (20-100x) than actinomycin D to mice in vivo. Therefore this self-immolative prodrug has a potential application in the treatment of cancer using an ADEPT-type approach.

Acceleration of healing of rabbit corneal endothelium by mesodermal growth factor.

Freeze-thaw injuries of rabbit corneas were treated with mesodermal growth factor (MGF) in organ culture and the repair of the endothelium followed by light microscopic examination of endothelial whole mounts. MGF-treated corneas showed an increased number of mitotic figures. In addition, MGF accelerated the rate of cell hypertrophy, formation of spindle-shaped cells, and the rate of their migration into the killed zone so that Descemet's was covered more rapidly than was that of the controls. In MGF-treated corneas, the cells lost their fibroblast-like appearance and became endothelial-like in appearance sooner than did the untreated corneas. A short period of treatment (6 hr) was sufficient to stimulate the repair processes described above. The healing responses obtained in organ culture for control eyes approximately those described in the literature for freeze-thaw injuries to the rabbit endothelium in vivo. The data presented also show that it is possible to accelerate endothelial regeneration in wound healing.

Metabolism of NAD(P)H by blood components. Relevance to bioreductively activated prodrugs in a targeted enzyme therapy system.

NADH was metabolized both by serum components and at the cell surface. The metabolism by serum was either oxidation to NAD+, or hydrolysis of the pyrophosphate to yield nicotinamide mononucleotide (reduced) (NMNH) and AMP. NMNH was further hydrolysed to yield nicotinamide riboside (reduced) (NRH), which was stable. NAD+ was hydrolysed (although at a slower rate than was NADH), but was also reduced to yield NADH. The reduction of NAD+ was catalysed by the enzyme serum L(+)lactate dehydrogenase (EC 1.1.1.27) and was dependent on the concentration of L(+)lactate in the serum. NADPH was hydrolysed in a similar manner to NADH but not oxidized by serum. NADH generated from NAD+ by serum derived from human, foetal calf and horse sources was capable of driving the bioreductive activation of CB 1954 by the enzyme DT diaphorase. Cell surfaces oxidized NADH to NAD+, but did not oxidize NADPH or NRH. These observations suggest that NAD(P)H would be unsuitable as a source of reducing equivalents for the bioreductive activation of prodrugs by a reductase enzyme in Antibody Directed Enzyme Prodrug Therapy (ADEPT). In contrast, NAD+ (which could act as a source of NADH) and NRH could avoid the shortcomings of NAD(P)H, and act as suitable cofactors for an enzyme in an ADEPT system.