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The novel agent amphethinile is shown to inhibit tubulin assembly in vitro. This agent is capable of displacing colchicine but not vinblastine from tubulin and causes a stimulation in GTPase activity in vitro. The affinity constant for the association of this drug with tubulin has been determined (Ka = 1.3 x 10(6) M-1). It is concluded that amphethinile belongs to the class of agents which share a common binding site with colchicine on the tubulin molecule.

The pattern of formation and rejoining of P388F cell DNA single strand breaks revealed by alkaline sedimentation centrifugation studies following treatment with methyl nitro-nitrosoguanidine (MNNG) has been studied. Three periods of single strand break formation have been recognised, an initial period during the first 4 h where breaks are rapidly formed and rejoined, a second (medial) time range of 10–15 h following treatment and a final stage where the formation of breaks appears to be related to cell killing processes. The initial phase is considered to be due to the production and repair of X-ray like DNA lesions whereas those of the medial time range are more comparable to the enzymatic processes associated with the repair of ultraviolet light-induced damage.

The redox potential-pH characteristics of the enzyme l -amino-acid oxidase ( l -amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.2) have been measured in the pH range 6.2 to 8.3 at 4°C. All potentials are reported versus the standard hydrogen electrode. Consistent with the protonation states proposed for the anionic red semiquinone and anionic dihydroquinone forms of the flavin coenzyme, the first electron potential is independent of pH (-0.056 ± 0.006 V), while the second electron transfer potential is pH-dependent, exhibiting a 0.060 V/pH unit slope. At all pH values investigated, the percentage of semiqinone species observed matches closely that calculated from measured potential separations. The semiquinone species is thermodynamically stable, as indicated by formation of semiquinone, similarity of redox potentials in oxidative and reductive directions, and by the slope of Nernst plots.

The cross-linking on the nucleohistone to the associated DNA by a series of methaneusulphonates of the busulphan series (CH3SO2O(CH2) n OSO2CH3) from n=4–9 has been studied by gel electrophoresis, by the use of 35S labelling and by pyrenemaleimide competition. It has been shown that all the dimethanesulphonates react with the Cys 114 of histone H3, and that octamethylene dimethanesulphonate (n=8) cross-links histone H3 with DNA via the sulphydryl group, as well as forming H3-H3 dimers.

Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.

The current status of DNA repair capabilities in mammalian cells is reviewed as well as the possible parameters that may interfere with the repair process. The problems associated with drug-radiation or drug-drug combination treatment are considered with special emphasis on the temporal pattern of repair following the first agent. The effect of MNNG is considered in detail as an example. Mechanical stress alone such as low speed centrifugation is also shown to induce repair synthesis.

Methane monooxygenase has been purified from the Type II methanotroph Methylosinus trichosporium OB3b. As observed for methane monooxygenase isolated from Type I methanotrophs, three protein components are required: a 39.7-kDa NADH reductase containing 1 mol each of FAD and a [2Fe-2S] cluster, a 15.8-kDa protein factor termed component B that contains no metals or cofactors, and a 245-kDa hydroxylase which appears to contain an oxo- or hydroxo-bridged binuclear iron cluster. Through the use of stabilizing reagents, the hydroxylase is obtained in high yield and exhibits a specific activity 8-25-fold greater than reported for previous preparations. The component B and reductase exhibit 1.5- and 4-fold greater specific activity, respectively. Quantitation of the hydroxylase oxo-bridged cluster using EPR and Mossbauer spectroscopies reveals that the highest specific activity preparations (approximately 1700 nmol/min/mg) contain approximately 2 clusters/mol. In contrast, hydroxylase preparations exhibiting a wide range of specific activities below 500 nmol/min/mg contain approximately 1 cluster/mol on average. Efficient turnover coupled to NADH oxidation requires all three protein components. However, both alkanes and alkenes are hydroxylated by the chemically reduced hydroxylase under single turnover conditions in the absence of component B and the reductase. Neither of these components catalyzes hydroxylation individually nor do they significantly affect the yield of hydroxylated product from the chemically reduced hydroxylase. Hydroxylase reduced only to the mixed valent [Fe(II).Fe(III)] state is unreactive toward O2 and yields little hydroxylated product on single turnover. This suggests that the catalytically active species is the fully reduced form. The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase. It thus appears likely that the oxo-bridged iron cluster is capable of catalyzing oxygenase reactions without the intervention of other cofactors. This is a novel function for this type of cluster and implies a new mechanism for the generation of highly reactive oxygen capable of insertion into unactivated carbon-hydrogen bonds.

The blood clearances of three platinum compounds, cis-dichlorodiammine platinum II (DDP), cis, trans-dichloro-dihydroxy-bis-(isopropylamine) platinum IV (CHIP), and cis-dichloro-bis-cyclopropylamine platinum II (CP), were determined in nine patients with malignant disease. The complexes were prepared using radioactive platinum (191Pt and 193Pt). A 10-mu Ci dose of each complex, containing the equivalent of 1-2 mg elemental platinum, was injected IV into groups of three patients. Serial blood and urine samples were collected over 72 h. No obvious difference was found between the three complexes for blood clearance, median t1/2a being 16.8 (range 11.2-23.5) min and median t1/2 beta 89 (range 63.7-127) h. The urinary excretion was greatest for CHIP, 60% of injected dose as against 42.6% for CP and 38.8% for DDP. Differences in renal excretion of DDP analogues could indicate potentially less nephrotoxic agents. The use of radioactive Pt will allow in vivo dynamic imaging of the distribution of platinum compounds in areas of interest.

A Yoshida lymphosarcoma line (YMDR8) resistant to methylene dimethane sulfonate (MDMS) showed collateral sensitivity against three halogenated methotrexates: 3'-bromomethotrexate (NSC-98580), 3'-bromo-5'-chloromethotrexate (NSC-98579), and 3',5'-dichloromethotrexate (NSC-29630); however, it was cross-resistant to methotrexate itself. Two other independently derived MDMS-resistant cell lines, YMDR7 and YMDR9, also demonstrated collateral sensitivity against 3'-bromomethotrexate, but with the latter, the origin of the "induced" sensitivity probably was not due to interference with antigenic or oncogenic properties of the cell line. When these agents were used in vivo (in Wistar rats) and in vitro, subpopulation changes within the tumor lines could be observed. The possible importance of this parameter in the development of such sensitivity is discussed.

The redox potentials for both electron transfers for the enzyme lactate oxidase have been measured at pH 7.0 in 0.01 M imidazole buffer at 25 degrees C. Methylviologen is the electrochemically generated reducing agent capable of transferring both electrons to the enzyme in this spectroelectrochemical experiment. The E0' values are as follows: for EFlox + e- = EFl-., E0'1 = -0.067 +/- 0.006 V; for EFl-. + e- + H+ = EFlredH-, E0'2 = -0.231 +/- 0.004 V. All potentials are reported vs. the standard hydrogen electrode (SHE). Both electron transfers are reversible. Consistent with the 164-mV potential separation, 95% of the enzyme anion radical is thermodynamically stabilized at half-reduction in all experiments.

Members of the homologous series of nine antitumour dimethanesulphonic acid esters, with the exception of ethylene dimethanesulphonate (EDMS), were found to cause DNA-DNA interstrand crosslinking in cells derived from the transplantable rodent Yoshida lymphosarcoma. The ability of the series to induce interstrand crosslinks was compared with in vitro cytotoxicity and in vivo antitumour activity and is discussed in relation to their chain length and ability to span critical target distances in DNA.

The intrarenal localisation of platinum following the intravenous administration of platinum-195m-labelled cisplatin, iproplatin and paraplatin was studied using autoradiography. Following injection of cisplatin, platinum was distributed throughout the kidney even up to 14 days after treatment. In the case of iproplatin and paraplatin rapid platinum clearance was noted from the glomeruli, blood vessels and renal medulla within 2 h of administration. Relative cortical and medullary platinum radionuclide concentrations for all three agents were determined by Chalkley grid analysis. This showed greater relative concentrations of platinum in the cortex at increasing times following iproplatin and paraplatin compared with cisplatin. It is suggested that the lower renal toxicity of iproplatin and paraplatin than of cisplatin may be due to reduced platinum retention within the pars recta.

Cells of the methylene dimethanesulphonate-(MDMS)-resistant Yoshida sarcoma cell line contain a low molecular weight “resistance factor” which is present in the culture medium of these cells and may be utilized by MDMS-sensitive Yoshida sarcoma cells either by co-culturing the two cell lines or by culturing the MDMS-sensitive Yoshida cells in a medium containing 20% used medium of MDMS-resistant Yoshida cells or in the presence of dialysed medium from resistant cells. The “resistance factor” does not inactivate the drug itself or its metabolites, and it has no influence on the sensitivity of the cells if added after MDMS treatment. Twenty-four hours seems to be enough time for the transfer of the resistance factor, but its effect on whole populations decreases within 24 hours of ceasing the supply. The relationship between these findings and the known phenomena of metabolic co-operation are discussed.

Thymine ribonucleoside affects the urinary excretion pattern and tissue distribution of 125I-labeled 5-iodo-2′-deoxyuridine (IUdR) and its metabolic derivatives in mice. In the absence of thymine ribonucleoside, 90–95 per cent of the radioactivity administered is excreted in the urine as iodide. A tenfold molar excess of thymine ribonucleoside, administered 30 min before the injection of 125I-IUdR does not alter the pattern of excretion but retards the amount of radioactivity excreted in the first 2 hr. A slight inhibition of the incorporation of IUdR into DNA also occurred at this level. On the other hand, a 100-fold excess of thymine ribonucleoside increased, during the first 2 hr, the urinary excretion of total radioactivity, which was composed mainly of 125I-iodouracil, as well as unchanged 125I-IUdR. The distribution of radioactivity among various tissues after the administration of a 100-fold excess of thymine ribonucleoside indicated a 2–5- to 3-fold greater uptake of radioactivity into the liver, heart, kidney, and muscle. The uptake was altered little or not at all in adrenals or in tissues that show a normal high rate of division, such as small intestine, spleen, and bone marrow. There was no increase in the utilization of IUdR for the biosynthesis of DNA.

In spite of the great advances made during the last thirty years or so in the understanding of organic chemistry, the teaching of the subject, at least at an elementary level, has remained more or less unchanged. It is not uncommon for teachers to adopt exactly the same approach to the subject as was used when they themselves were students and the somewhat dreary repetition of ‘preparations, properties and uses’ is still the most widely used teaching method. Many of the textbooks used have been current for three or more decades and are devoid of modern ideas; even the more recent ones are predominantly old‐fashioned in approach. Good textbooks are, of course, available and they are used to some extent, but unfortunately the old favourites still dominate the field.

Cloned cell lines have been established from Yoshida ascites tumour lines known to be sensitive and resistant to methylene dimethanesulphonate (MDMS) in vivo . The two lines have similar population doubling times and cell cycle times, but the relative lengths of different phases of the cycle differ, the S phase in particular being considerably longer in the resistant line. Cell survival studies by agar cloning and by the back-extrapolation method have shown that the line sensitive to MDMS is cross-sensitive to HN 2 and to UV irradiation. The two cell lines show similar sensitivities to methyl methanesulponate (MMS) and X-rays.

SummaryRepair replication and unscheduled DNA synthesis have been demonstrated in a number of cultured tumour cell-lines of rodent origin. The amount of repair replication observed was less than that observed in HeLa cells when the same labelling procedures were used. u.v.-sensitive mutants of Yoshida cells which showed reduced D0 value and an extrapolation number of 1 showed reduced repair replication. The u.v.-sensitive mutant of L5178Y cells which differed only in its extrapolation number showed the same amount of repair replication as its more resistant mutant. The data show that (1) the amount of repair replication in rodent cells is less than that in HeLa cells and (2) that differences in amount of repair replication between two closely related cell lines are associated with changes in D0 value but not with changes in shoulder size and extrapolation number.

Using alkaline sucrose gradient centrifugation, single-strand breaks can be shown to occur in [ 14 C]thymidine labelled P388F lymphoma cells after treatment with methyl methanesulphonate at levels which allow for a high level of survival of the cells. The pattern of fragmentation of newly synthesized and template labelled DNA differs and these differences are discussed. The “repair” of this damage within 2-h after treatment has been studied and no evidence for rejoining of DNA strands during this time period was obtained. The increase in molecular weight following incubation of treated, newly labelled cells is now considered to be due to normal DNA synthesis on prelabelled fragments. The labelled template DNA shows two new features. A higher molecular weight component which appears to be broken internally by methyl methanesulphonate, but requires further incubation to release the fragments, and a characteristic distribution of labelled fragments which show no evidence of single-strand rejoining within 2-h post-treatment.

A procedure is described, for the alkaline sucrose sedimentation analysis of DNA of high molecular weight from cultured P388F lymphoma cells. Template DNA sedimented rapidly giving a unimodal profile. The sedimentation rate of this material was reduced by treatment of the cells with [3H]thymidine, or with low levels of HN2 and methylenedimethane sulphonate (MDMS). The newly synthesised DNA from P388F cells gave bimodal sedimentation profiles, of which the rapidly sedimenting component cosedimented with template DNA. The medium sedimenting component was unique to the newly synthesised DNA and became disproportionately labelled with [3H]thymidine of high specific activity. On post incubation in fresh medium, the medium sedimenting component was converted into a rapidly sedimenting material. This process was obstructed in cells treated with MDMS in the dose range LD25-LD50.

Repair replication has been measured by CsCl density gradient centrifugation in cell lines showing differential sensitivity to mono- and bifunctional alkylating agents. A correlation between cellular sensitivity as measured by the D0 value and amount of repair replication was demonstrated after exposure of Yoshida cells to nitrogen mustard (HN2) and methylene dimethanesulphonate (MDMS). No differences in the amount of repair replication after methyl methanesulphonate (MMS) were observed in two L5178Y cell lines which differed in sensitivity by virtue of the shoulder size only. The Yoshida cell lines showed no difference in sensitivity to MMS and no difference in amount of repair replication. Incorporation of tritiated thymidine 9[3H]TdR) after drug treament was also measured by autoradiography. The qualitative differences observed between the two cell lines were similar to those obtained in density gradient experiments. The temporal pattern of [3H]TdR uptake indicated that the reduced repair replication observed in the sensitive line after HN2 and MDMS is not due to slower synthesis. The kinetics of [3H]TdR incorporation differed for all three mutagens suggesting that different enzymes may be involved in each case.

SummaryThe incorporation of thymidine into the newly-synthesized DNA of P388F lymphoma cells, increases non-linearly with respect to an increasing concentration of 3H-thymidine (TdR) of constant specific activity. As the external TdR concentration rises, in the presence of a constant low level of tritium a progressively greater proportion of the incorporation is hydroxyurea-resistant. These effects are attributed to negative-feedback actions of TdR metabolites on nucleotide biosynthesis.Thymidine incorporation is stimulated at high specific activities of 3H-TdR, largely owing to enhanced hydroxyurea-sensitive DNA synthesis, resulting from DNA damage produced by the incorporated tritium. For concentrations of 3H-TdR up to 8·4 mµM, reduced survival of P388F cells is solely the result of effects of tritium incorporated into newly-synthesized DNA.

SummaryPreparative CsCl density-gradient centrifugation has been used to demonstrate ‘repair synthesis’ in P388F cells after 300 rads x-rays. Repair synthesis was stimulated two-fold when cells were grown in 5-iodo-2-deoxyuridine before irradiation. A time course has been determined by autoradiography and after DNA isolation in cultures treated with hydroxyrea (HU) to inhibit normal DNA synthesis. The rate of HU-insensitive DNA synthesis in irradiated cultures was found to exceed that in control cultures by a factor of two from 0–60 min after irradiation. Autoradiographic studies have shown that the radiation stimulated thymidine incorporation is restricted to S cells.

SummaryThe numbers of single-strand breaks and their rates of rejoining have been compared in two Yoshida cell-lines showing similar sensitivity to X-rays (D0 85 rads, n = 2·5) and two L5178Y cell-lines showing differential sensitivity; the sensitive cell-line having a D0 value of 55 rads, n = 2·5, and the resistant line a D0 value of 160 rads and n = 3·0. After doses of 5 or 10 krads, no differences in the numbers of single-strand breaks produced or in their rates of rejoining were observed when measured by the alkaline sucrose gradient technique. The findings confirm that single-strand breaks in themselves are not lethal events, since similar numbers are produced and virtually all are rejoined in the cell-lines studied. However, a deficiency in some of the processes involved in repair replication could be the cause of their differential sensitivity by allowing damage to be fixed.

A new antitumour agent is described, which has been shown to induce a G2/M block in murine leukaemia cells in vitro. In addition this agent has been shown to be equally toxic toward parental and daunorubicin-resistant P388 cells in vitro. These resistant cells are highly cross-resistant to the established anti-mitotic agents vincristine and vinblastine. Drug accumulation studies in cells have shown that whereas resistance in this cell line is associated with decreased drug accumulation in the case of daunorubicin, vincristine and vinblastine, this effect is much less pronounced for amphethinile. It is proposed that amphethinile is a poor substrate for the drug efflux process associated with the pleiotropic resistance mechanism operating in these cells. The data suggest that cell sensitivity towards amphethinile differs qualitatively from that of the vinca alkaloids and anthracycline. Pharmacokinetic studies in male mice were undertaken. Area under the curve values (AUC), show that levels of approximately 313 micrograms l-1 h-1 were attained at doses equivalent to the LD10. The alpha half life is approximately 8 min after a bolus intravenous injection. The beta half life was approximately 100 min and relatively independent of dose level.

Brian A. Fox is advertising director of the Organization of American Historians and in former days, an occasional substitute teacher. Let's hypothesize for a mo ment. You're coming down with the flu--stomach flu--and you're certain you're not going to make it to school tomorrow. And tomorrow's an important day; exams are quickly approaching, and you must get through the current section by the end of the week. You sigh, realizing that once again another day is going to be wasted. Despon dently you sketch plans for the substitute: reading for periods one through three, the next chapter; and for periods four through six, a videotape (yes, the same one you used last year in this same situation). You plan for a colleague to pick up your instructions, then head for bed feeling worse than ever. Every teacher has faced this situation sometime during his or her career. Most know too well the unpleasant feeling coming in the day after, knowing they must pick up where they left off and somehow compress two days of lessons into one. But with planning and fore sight, those wasted or "lost" substi tute days can be made productive and worthwhile. Here's how.

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml. When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.

We have established four cell lines designated YMDR6 to YMDR9 by in vivo subthreshold treatment of Yoshida lymphosarcoma with methylene dimethanesulphonate (MDMS). These have been transferred to cell culture and grown continuously. Only YMDR8 was cloned.

A pair of cultured rat lymphosarcoma cell lines (Yoshida) with a pronounced differential sensitivity to killing with sulphur mustard (SM), but with the same sensitivity to X-rays, was examined for chromosome damage and DNA repair replication after treatment with these agents. A pair of mouse lymphoma cell lines (L5178Y) with a differential sensitivity to X-rays was similarly investigated. SM-resistant Yoshida cells suffered much less chromosome damage than sensitive cells in spite of equal alkylation of DNA, RNA and protein in sensitive and resistant cells. The pair of Yoshida cell lines sustained the same amount of chromosome damage after X-irradiation. Much less chromosome damage was observed in the radiation-resistant lymphoma cell line than in the sensitive line after X-irradiation. No differences was found between the pairs of cell lines in their capacities for repair replication after SM or X-ray treatment. Thus, the drug and radiation resistance is accompanied by, and perhaps mediated through, a reduced amount of induced chromosome damage but is not quantitatively related to the capacity for DNA repair replication. Apart from small differences in modal chromosome numbers there are no obvious karyotype differences between the sulphur mustard-sensitive and -resistant Yoshida cells or between the radiation-sensitive and -resistant lymphoma cells.

Cells resistant to methotrexate (L1210/R7A) and possessing an increased level of dihydrofolate reductase due to gene amplification can be detected by the technique of flow cytofluorimetry using a new fluorescent derivative of methotrexate (F-MTX) based on a putrescine linker. Comparative studies of dihydrofolate reductase enzyme and cell growth inhibition following treatment with methotrexate and F-MTX suggest that the two agents possess similar modes of action. In an artificially mixed population of cells sensitive and resistant to methotrexate it is possible, using F-MTX, to recognise and separate distinct cell subpopulations showing differential fluorescence using a fluorescence-activated cell sorter (FACS IV). The selective removal of the resistant cells within a mixed population of sensitive and resistant cells has been demonstrated for 5 X 10(-8) M vinblastine by means of flow cytometry. The effectiveness of the vinca alkaloids decreases in the order vinblastine greater than vindesine greater than vincristine, which previously was shown to be the order of effectiveness in producing collateral sensitivity.

SummaryThe nature of the 3H activity released from M. radiodurans after γ-ray doses of 500 krads which cause no decrease in cellular viability has been investigated by gel filtration and alkaline sucrose-gradient centrifugation. Immediately after irradiation, oligonucleotides ∼7·0 × 105 daltons are released which are subsequently rapidly degraded in broth to thymine by exonucleolytic enzymes. Such enzymes are also capable of degrading mammalian DNA. Treatment with iodoacetamide 2 × 10−5 M results in a sensitization expressed as a loss of the shoulder of the dose-response curve. The study of DNA degradation products in M. radiodurans treated with iodoacetamide suggests that sensitization is due to inhibition of an enzyme which excises DNA from a DNA-membrane complex. Iodoacetamide appears not to inhibit the removal of thymine from irradiated DNA. The results suggest that at least two enzymes are involved in the repair of γ-ray damage in M. radiourans and that repair of the DNA-membrane complex is mainly re...

The intracellular accumulation of daunorubicin as determined by flow cytometry correlates well with that as determined by extraction of the drug from cell homogenates. Two P388 mouse leukaemia cell lines showing differential sensitivity to the drug have been used to investigate the transport changes associated with resistance. Resistance to daunorubicin in these cell lines occurs through an alteration in the intracellular accumulation of the drug, resulting from the increased efflux of the anthracycline from the resistant cells. The effect of temperature, drug concentration, pH, and metabolic inhibitors on this process have been investigated. Uptake by a carrier-mediated process of the un-ionised form of the drug (pK = 8.25), coupled with an energy-dependent efflux process, is proposed as the mechanism of cellular accumulation in the case of the resistant cell line.

The blood and urinary clearances of cis-diammine 1,1-cyclobutane dicarboxylate platinum(II) (CBDCA, JM8) were determined in four patients with malignancy. A 40 muCi iv injection of 191 Pt/193 Pt (3:1)-labelled CBDCA was followed by serial blood and urine sampling to 72 h. The blood clearance was triphasic, mean values for the fast, intermediate, and slow phases being 10.8 min, 2.5 h, and 125 h, respectively. The fraction of activity excreted in the urine within the first 6 h had a mean value of 66.7%, contrasting with 26.4% for cisplatin. There was only a small fraction of CBDCA excreted by the slow phase (1.5%) as against an average of 20% for CHIP and 27% for cisplatin. The early and rapid renal clearance of CBDCA may account for reduced nephrotoxicity.

A comparative measurement of the transport and localisation of daunorubicin into Yoshida sarcoma cells, was undertaken by a biochemical extraction process and a flow cytometric method. An advantage of this latter procedure would be to identify subpopulation of cells which have enhanced or impaired daunorubicin incorporation as well as the ability to exclude any non-specific incorporation into cell debris, which would otherwise interfere with the overall estimation. It has been possible to use the Biophysics argon ion laser at a wavelength of 488 nm which coincides with the visible absorption bands of daunorubicin and doxorubicin (adriamycin) and the cytofluorimetric estimations of daunorubicin incorporation have now been compared with biochemically determined uptake in Yoshida cells. A high lethal dose of 10 microM was required to achieve the direct measurement by cytofluorimetry procedures on the Biophysics instrument. From cell fractionation and CHCl3/amyl alcohol extraction, it was possible to show that during a 5-h exposure period to daunorubicin (10 microM), the uptake into the nucleus was at first rapid and that into the cytoplasm was much slower. After about 3-h incubation, the level in the cytoplasm decreased, followed by a decrease from the nucleus 1 h later. This could be equated when observed microscopically to the gain in fluorescent cell debris. If all nuclear binding is to DNA, then at the level of (10 microM) concentration in the medium, the number of base pairs to daunorubicin would be 9 : 1, respectively. Cytofluorimetry showed a broad spread of intracellular daunorubicin fluorescence which increases with cell size. Increasing external concentration caused a more rapid incorporation as well as a quicker release from the cells.

Both methylene dimethanesulphonate (MDMS) and methyl methanesulphonate (MMS) cause the template activity of Yoshida cell DNA to decrease in a dose-dependent manner. The MDMS-resistant subline of Yoshida tumour is less sensitive in terms of DNA template activity than the MDMS-sensitive subline towards both agents. Although the difference in sensitivity is not reflected in the survival data after each agent, it does suggest that the DNA from each cell line differs in its capacity to function as an efficient template.

A Yoshida sarcoma cell line (YR/cyclo) showing decreased sensitivity to metabolically activated cyclophosphamide in vitro has been shown to be cross-resistant to phosphoramide mustard, the ultimate alkylating agent formed from cyclophosphamide. Resistance to these alkylating agents has been shown to be associated with increased activity of the glutathione S-transferase group of enzymes, and with elevated levels of glutathione, the cosubstrate of the enzyme. The resistant cell line shows lower levels of cellular damage, as measured by alkaline elution following treatment with phosphoramide mustard, than the parental (YS) line. The mechanism of resistance is ascribed to increased deactivation of potentially damaging metabolites of cyclophosphamide by the glutathione S-transferase enzymes, resulting in decreased cellular damage in the resistant cell line.

Three platinum derivatives, cis-dichlorodiammine platinum (II), (DDP), cis-trans-dichlorodihydroxy-bis-(isopropylamine)platinum (IV) (CHIP) and cis-dichloro-bis-cyclopropylamine platinum (II) (CP), have been prepared with a gamma-emitting platinum label. The distribution of these complexes was studied in male rats.

Instrument design and development has advanced to a greater degree than sample preparation technology over the last decade, and any limitations of liquid scintillation counting appear to be associated more often with the preparation of the counting sample rather than a deficiency in instrumentation. There has however been little apparent progression in the development of new solvents or primary salutes in recent years. Toluene, p-xylene and dioxane-naphthalene are still the most population electron trapping solvents. A useful advance was made by Krumbiegel and Schmidt (1973) who claimed that 97% perdeuterated toluene allows tritium to be assayed almost 90% higher than with normal toluene. A further development in solvent modification was the introduction of organic lead and thallium compounds into scintillant mixtures, first suggested by Ashcroft (1969) as density-increasers to determine y emitting isotopes in liquid scintillation spectrometers. Such solutions can be readily made by shaking saturated lead acetate with a mixture such as Ready Solv VI (Beckman Instruments Ltd) (Helman and Spiehler, 1974) and using the upper layer. Although these scintillants are used by immersing into it a small minivial containing the sample, a more recent development is the direct use of organic thallium or lead loaded scintillant solutions to improve the separation of 1251 and 3H materials mixed directly with them (Lundqvist et.ai., 1976). This combination of isotopes is useful in nucleic acid biochemistry using 1251-iododeoxyuri

The study of tumour resistance to anticancer drugs has been the subject of many publications since the initial discovery of the phenomenon by J. H. Burchenal and colleagues in 1950. Many papers have been published since then reporting development of resistance to most of the well-known anticancer agents in many different animal tumour systems, both in vivo and in vitro. Many different mechanisms of resistance have been described, and it is clear that the tumour cell has a wide diversity of options in overcoming the cell-killing activity of these agents. Definition of the magnitude of the phenomenon in the clinic is, however, much more problematical, and it is with this in mind that the initial chapter, seeks to out line the problem as the clinicians see it. It appears that the phenomenon of true resistance to a drug, as the biochemist would recognise it, is an important cause of the failure which clinicians experience in treating the disease. The extent of the contribution of this phenomenon to the failure of treatment cannot easily be evaluated at the present time, but it is hoped that the development and application of new and more sophisticated techniques for the analysis of cellular sub populations may help to give a more exact estimate and to shed some light on the causes of failure of many of the present therapeutic techniques."

The purification of the hydroxylase component of a 3 component methane monooxygenase from the type II methanotroph Methylosinus trichosporium OB3b is reported. The enzyme (240 kDa) has an (αβγ) 2 subunit structure as observed for hydroxylases isolated from other Type I and Type II methanotrophs, but it exhibits a 5 to 10 fold higher specific activity and is isolated in 2 to 10 fold higher yield. EPR and Mossbauer spectra of the hydroxylase show that it contains a coupled iron center containing an even number of iron atoms. The spectra are similar to those of proteins known to contain oxo-bridged binuclear iron centers. The presence of such a center is unprecedented in a monooxygenase and suggests that a novel mechanism is utilized.

Two P388 cell lines with acquired resistance to daunorubicin have been shown to exhibit cross-resistance to anthrapyrazolone (NSC 357885). The degree of cross-resistance observed in these cell lines (58 and 150 fold) is similar to those observed towards daunorubicin (34 and 142 fold). However the decreased drug accumulation observed for daunorubicin in the resistant cell lines (4-6 fold) is not observed for anthrapyrazolone. Similarly, verapamil can increase daunorubicin accumulation in resistant cells but has no effect on anthrapyrazolone accumulation. It is concluded that in contrast to daunorubicin, decreased anthrapyrazolone accumulation is not the resistance mechanism operative in daunorubicin resistant cell lines towards anthrapyrazolone.

The technique of alkaline elution was employed to study the interactions of methylene dimethane sulphonate (MDMS) and formaldehyde (HCHO) with DNA from Yoshida lymphosarcoma cells treated with these agents. MDMS and HCHO produced a proteinase sensitive filter retention which indicated the presence of DNA-protein cross-links. MDMS also produced some proteinase K-resistant filter retention which was believed to indicate DNA-interstrand cross-linking, whilst only single-strand breaks could be detected following treatment with HCHO. Co-incubation with semicarbazide prevented all DNA-protein cross-links induced by MDMS and HCHO as well as single-strand breaks, most obvious following HCHO treatment. Semicarbazide also reduced HCHO-induced cytotoxicity in the Yoshida lymphosarcoma cell line, while no significant alteration in MDMS-induced cytotoxicity was observed. These results suggest that HCHO-induced DNA-protein cross-links and single-strand breaks do not contribute to MDMS-induced cytotoxicity, and therefore the small but significant level of MDMS-induced DNA-interstrand cross-links is the most likely cytotoxic lesion of this agent.

The effects of the alkylating agents methylene dimethanesulphonate (MDMS), sulphur mustard (SM) and methyl methanesulphonate (MMS) on DNA synthesis have been investigated in Yoshida sarcoma cell lines sensitive (YS) and resistant (YR) to these agents. Measurement of overall DNA synthesis by [3H]thymidine uptake into DNA indicated that both MDMS and SM, but not MMS, induced a greater depression of DNA synthesis in YS cells than in YR cells. Analysis of the sizes of newly synthesised strands of DNA by pulse labelling and alkaline sucrose gradient sedimentation revealed that MDMS inhibited the initation of new replicons and also reduced the size of new DNA strands. The relative extents of inhibition of replicon initation in MDMS-treated YS and YR cells were similar and reduced by caffeine while YR cells were comparatively less sensitive to the effect of MDMS on the sizes of DNA molecules synthesised. Similarly, YR cells apparently synthesised longer DNA strands following SM treatment compared to YS cells suggesting an association between the size of DNA molecules synthesized and the sensitivity of Yoshida cells to bifunctional alkylating agents.

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.

The colloid system has been examined from the point of view of optimisation of the necessary conditions for successful counting. The appearance, viscosity and physical stability of the colloid have been studied throughout all the possible combinations of its components using the triangular co-ordinate system. These parameters are shown to be independent of one another and play no significant role in deciding the best conditions to use. Furthermore, any phase structure designations based on a combination of these parameters is open to question. An instrument Merit Stability Quotient (MISQ) number is suggested which seeks to allow the assignation of a unique number to compare the quality of a suggested combination of detergent, primary solvents and solutes with a particular aqueous solution, so that claims of improved efficiency of different combinations from various authors can be rationally compared independently of local instrument efficiency. The Triton X-100:xylene:water system employing PPO and POPOP is used as an example.

Chromatography on a DEAE-cellulose (DE-52) column of native and denatured DNA from P388F cells has been studied. The main bulk of native DNA is eluted at 0.8 M NaCl and a minor fraction is eluted with 0.5 N NaOH. The proportion of the DNA components obtained depends on the type of isotopic labelling used and the method of storing of the DNA preparation following isolation. Heat-denatured DNA elutes in 2 M NaCl mainly within the pH gradient from 0.1–1.0 M NH4OH. When native DNA is chromatographed on DEAE-cellulose, a small fraction of the DNA elutes at 0.5 N NaOH. This fraction is double-stranded, as determined by hydroxyapatite chromatography. A similar component predominates, however, when “newly synthesised” DNA is fractionated. The profiles obtained with “newly synthesised” and “template” labelled DNA differ in their undenatured and heat-denatured configurations. The presence of formaldehyde during the chromatography of undenatured DNA leads to an increased homogeneity of the profile and during heat denaturation considerable modifications to the profile are observed. Some of the changes can be explained in terms of a decrease in the heterogeneity of the charge distribution on the DNA. The technique appears to combine a high degree of reproducibility with sensitivity to charge clustering along the DNA.

The methane sulfonate esters of dihydric and polyhydric alcohols are biological alkylating agents since their alkyl-oxygen bonds undergo fission and react within the intracellular milieu. The structure of the alkyl group of these esters may vary around the carbon atom adjacent to the oxygen atom where fission is likely to occur. This means that the alkyl group will tend to favor entry into the confines of a sterically favorable nucleophilic site, unlike the ethylene immonium series whose chemically variable grouping will always be separated from the nucleophilic site by a three atom chain and hence exert less of an influence on it. The local structural characteristics of the nucleophile itself would thus assist in conferring a degree of selectivity on the action of the alkane sulfonates.

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [ 14 C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.

MtDNA was extracted by a phenol method from transplanted and primary DAB induced hepatomata in male Wistar rats, normal rat liver, spontaneous human tumours (2 Wilm's tumours, one neuroblastoma and one adrenal carcinoma), as well as 2 specimens of normal human kidney, BNU induced “leukaemias” in mice and CHO fibroblasts in monolayer culture. The proportion of monomers, catenated dimers and oligomers, open dimers and small circles was determined by electron microscopy of the fractions comprising lower and middle DNA bands in a CsCl-EthBr gradient. Tumours were compared where possible with their normal tissue of origin. Open dimers were found in 2 Wilm's tumours and their attached “normal-looking” kidney tissue but not in normal, non-malignant kidney or any other tissue studied. In Wilm's tumours, the occurrence of open dimers is far from being an all-or-none phenomenon. Malignancy produced little change in the relative proportions of catenated dimers and oligomers in the tissues studied. Small circles were found associated with mtDNA from every tissue. Tumour mtDNA was not more heterogeneous in length than monomers from the corresponding normal tissue, neither was the mean length of tumour mtDNA significantly different from its corresponding normal mtDNA. ImagesFig. 4Fig. 3Fig. 5