Stability of biodegradable radioactive rhenium (Re-186 and Re-188) microspheres after neutron-activation.

Our objective was to determine if microspheres made from the biodegradable polymer poly(lactic acid) that contained rhenium could withstand the conditions of direct neutron activation necessary to produce therapeutic amounts of radioactive rhenium. The radiation damage of the polymer produced by gamma-doses of up to 1.05 MGy from Re-186 and Re-188 was examined by scanning electron microscopy and size exclusion chromatography. At a thermal neutron flux of 1.5 x 10(13)n/cm2/s the microspheres melted after 3 h in the nuclear reactor, but suffered little damage after 1 h of radiation and released less than 5% of the radioactivity during incubation in buffer at 37 degrees C. The radioactive microspheres produced in this manner have a specific activity too low for radioembolization for treatment of liver tumors, but could be injected directly into tumors or applied topically to the wound bed of partially resected tumors.

Dosimetry of a W-188/Re-188 beta line source for endovascular brachytherapy.

PURPOSE: The objective was to determine the dosimetry of a potential endovascular brachytherapy source consisting of a coiled tungsten wire mounted on the distal end of a drive wire and neutron-activated to contain the parent-daughter nuclides tungsten-188 (188W) and rhenium-188 (188Re). METHODS: A coiled tungsten wire 40 mm in length was neutron-activated by double-neutron capture for 78 hours at 1.9 x 10(15) h/cm2/s to contain 925 MBq (25 mCi) of 188W/188Re in equilibrium. The dose-fall off from this source was determined using three independent methods: (a) Thermoluminescence dosimetry with small LiF-100 rods, (b) Gafchromic film dosimetry, and (c) Bang gel dosimetry. In addition, a Monte Carlo simulation was performed to compute the beta-dose. RESULTS: Each of the three measurement methods recorded similar values for the dose fall-off within the distances useful for endovascular brachytherapy. The Monte Carlo calculations closely approximated the measured results in the treatment range between 1 and 3 mm and may thus be useful for evaluating changing geometries in the development of catheters and source setups. A 2 min restenosis treatment delivering 20 Gy at a radius of 2 mm would require a source of 1384.8 MBq/cm (37.4 mCi/cm). CONCLUSIONS: The dose distribution from a 188W/188Re source is similar to that of a 90Y-source. An added advantage of the 188W/188Re source is that it can be used for at least two months and still provides fast treatment times because of the parent isotope's half-life of 69 days. The additional gamma emission from the source is too small to impose a serious radiological hazard. The high atomic number and density of the source material allows direct fluoroscopic imaging without additional markers.

Melanin as a potential cryptococcal defence against microbicidal proteins.

Cryptococcus neoformans is an important fungal pathogen that synthesizes melanin when grown in the presence of phenolic substrates. The ability of C. neoformans to produce melanin is associated with virulence, but the specific role of melanin in the pathogenesis of infection is not clear. In this study the ability of C. neoformans melanin to bind proteins and protect against microbicidal peptides was investigated. Melanin was shown to bind a variety of proteins of fungal and mammalian origin. Melanin-protein interactions were dependent on the pH of the solution and on the amount of protein and melanin present. Melanized cells were less susceptible to killing by three microbicidal peptides: a defensin, a protegrin, and a magainin. Incubation of the microbicidal peptides with melanin particles, followed by removal of the melanin, reduced or abolished fungicidal activity, demonstrating interactions between peptides and melanin. The ability of melanin to bind proteins and to protect against microbicidal peptides suggests a protective function for melanin, whereby it sequesters microbicidal peptides and abrogates their activity.

Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts.

Several laboratories have reported that overexpression of the multidrug resistance (MDR) protein is associated with intracellular alkalinization, and several investigators have reported that cells induced to undergo programmed cell death (apoptosis) acidify quite significantly. Because it is difficult to fully explain the resistance to apoptosis-inducing chemotherapeutic drugs that is exhibited by MDR tumor cells solely via altered drug transport alone [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313], we have investigated whether overexpression of the hu MDR 1 protein alters progression of the apoptotic cascade. LR73 fibroblasts induced to undergo apoptosis either via treatment with the chemotherapeutic drug colchicine or by serum withdrawal exhibit cellular volume changes, intracellular acidification, nuclear condensation, and chromosomal digestion ("ladder formation"), characteristic of apoptosis, in a temporally well-defined pattern. However, multidrug resistant LR73/20E or LR73/27 hu MDR 1 transfectants recently created in our laboratory without selection on chemotherapeutic drug are significantly delayed in the onset of apoptosis as defined by the above criteria, regardless of whether apoptosis is induced by colchicine treatment or by serum withdrawal. Thus, the delay cannot simply be due to the well-known ability of MDR protein overexpression to lower chemotherapeutic drug accumulation in MDR cells. LR73/27V500 "selectants", exhibiting similar levels of MDR protein overexpression but higher multidrug resistance due to selection with the chemotherapeutic drug vincristine, exhibit a slightly longer delay in the progression of apoptosis. Normal apoptotic cascade kinetics are partially restored by pre-treatment of the MDR cells with the MDR protein inhibitor verapamil. Untransfected LR73 cells not expressing MDR protein but elevated in pHi via manipulation of CO2/HCO3- as described [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313] are inhibited in DNA ladder formation, similar to LR73/hu MDR 1 transfectants. These results uncover an additional mechanism whereby MDR protein overexpression may promote the survival of tumor cells and further support the notion that in some systems intracellular acidification may be either causal or permissive for proper progression of the apoptotic cascade.

Basic radiobiological investigations of fast neutrons.

The radiobiological properties of a cyclotron-produced 43-MeV (p----Be) fast-neutron beam relative to gamma rays have been investigated using Chinese hamster V79 cells in culture. As expected, the relative biological effectiveness (RBE) of this neutron beam for cell killing was shown to increase as dose decreased, and the effectiveness per unit dose was slightly less compared to a 25-MeV (d----Be) neutron beam. By tracing single cells that formed microcolonies after irradiation, we found cell proliferation kinetics to be retarded to a greater extent by fast neutrons than by gamma irradiation. Following either neutron or gamma irradiation, a fraction of the irradiated cells failed to divide in the first postirradiation division and another fraction could produce as many as four generations of progeny before proliferation stopped. The properties of these cells presumed to be destined for death suggest that more than one mechanism and/or multistep process underlies the radiation-induced proliferative death. The fast-neutron beam was also found to be more effective quantitatively than gamma rays in producing DNA double-strand breaks (DSBs, measured by nondenaturing filter elution), and G1-phase chromosome fragments (measured by the premature chromosome condensation technique). However, the reverse was observed for DNA single-strand breaks (SSBs, measured by alkaline filter elution or hydroxylapatite uncoiling). Interestingly, both fast neutrons and gamma rays produced a large component of SSBs and DSBs with a fast-rejoining time constant of about 2-5 min, which appears to be independent of dose. The latter results could not resolve the possibility of lengthening the repair-time constant by increasing radiation dose within the range that is reflected by the shoulder of the survival curve, and consequently did not support the idea of repair saturation as a mechanism for the presence of the shoulder. The RBE for the hypoxanthine phosphoribosyl transferase mutation frequency per survivor at the 10% survival level was estimated to be 2.5, a value that is comparable to the RBE (2.1) for cell killing at the same survival level. Although most of the above-mentioned findings are compatible qualitatively with the relatively high-LET (linear energy transfer) nature associated with the fast-neutron beam, the significance of the action attributable to the mixture of LET could not be delineated in these experiments. Further, the biological significance of DSBs and chromosome aberration and the molecular mechanisms responsible for the repair and expression of these damaging processes remain to be elucidated.

A new family of plant antifungal proteins.

Plant seeds contain high concentrations of many antimicrobial proteins. These include chitinases, beta-1,3-glucanases, proteinase inhibitors, and ribosome-inactivating proteins. We recently reported the presence in corn seeds of zeamatin, a protein that has potent activity against a variety of fungi but has none of the above activities. Zeamatin is a 22-kDa protein that acts by causing membrane permeabilization Using a novel bioautography technique, we found similar antifungal proteins in the seeds of 6 of 12 plants examined. A polyclonal antiserum was raised against zeamatin and was used in immunoblots to confirm the presence of zeamatinlike proteins in these seeds. N-terminal amino acid sequencing was carried out on the antifungal proteins from corn, oats, sorghum, and wheat, and these sequences revealed considerable homology with each other. Interestingly, these N-terminal sequences are also similar to those of thaumatin, a pathogenesis-related protein from tobacco, and two salt stress-induced proteins. These results indicate that zeamatin is not unique but is a member of a previously unrecognized family of plant defense proteins that may include some species of pathogenesis-related proteins.

Therapeutic gain factors for fractionated radiation treatment of spontaneous murine tumors using fast neutrons, photons plus O2(1) or 3 ATA, or photons plus misonidazole.

Therapeutic gain factors (TGFs) have been determined for three spontaneous tumors of the C3H mouse treated by photons + normobaric oxygen (O2(1) ATA), photons + hyperbaric oxygen (O2 3 ATA), photons + misonidazole, or fast neutrons. The tumors were early generation isotransplants of spontaneous tumors: MCaIV, a mammary carcinoma; FSaII, a fibrosarcoma; and SCCVII, a squamous cell carcinoma. The tumors, transplanted to the right leg, were 6 mm at start of treatment. Normal tissue responses studied were acute reaction of normal skin (all treatment modalities) and LD50 following irradiation of the upper abdomen (in test of photons + O2 at 1 or 3 ATA). Thus both the tumor and normal tissues would be classified as "acute responding." All subject tissues were at congruent to 34.5-35 degrees C at irradiation. Treatments were based on d(25)Be or p(43)Be fast neutron beams, 60Co and 137Cs photon beams. Treatments were given in 5 or 15 equal doses in 5 days. For photon treatments, TGFs (air/O2 3 ATA) were substantially and significantly larger than 1 for all three tumor systems treated at small or large doses per fraction when related to skin or abdominal tissue responses. The TGFs (air/O2 1 ATA) were greater than 1 at small doses per fraction for MCaIV and FSaII for skin as the normal tissue; the TGFs for all three tumors and at all doses per fraction would be greater than 1 when related to upper abdominal tissues. TGFs (O2 1 ATA/O2 3 ATA) for photon irradiation greater than 1 were found only for SCCVII and that obtained for both large and small doses per fraction. Misonidazole achieved impressive TGFs (air/air + miso or air/O2 1 ATA + miso); the drug was tested only at 10-12 Gy/fraction and relative to skin. RBEs(FN) for the three tumors were lower at 1.5-2 Gy(FN)/fraction than at 5-6 Gy(FN)/fraction, i.e. the opposite to that reported for normal tissue (RBE increases with decreasing dose per fraction). A TGF (relative to skin reaction) greater than 1 for fast neutron therapy was found only for SCCVII when treated at large doses/fraction; this was true for air or O2 1 ATA conditions.

Survival responses and potentially lethal damage repair of normal 10T1/2 and its transformed TCL 15 cells after irradiation with 43 MeV proton produced neutrons.

Normal 10T1/2 fibroblasts and their transformed counterparts (TCL 15) were used in order to evaluate the RBE, TGF and PLD repair for a therapeutic fast neutron beam (43 MeV proton----Be). For plateau-phase culture, the RBE of 10T1/2 cells were 2.0 and 1.7 at 10 and 1% survival levels respectively and 1.3 from D0. The corresponding RBE values of TCL 15 cells were 2.1, 1.8 and 1.5, and thus the TGF values were 1.05 and 1.1 respectively at 10% and 1% survival levels and 1.8 from D0. For log-phase culture, the survival responses of 10T1/2 and TCL 15 cells to 60Co gamma-rays or neutrons were not significantly different (the RBE at 10% was 2), and thus the TGF value was unity. For neutrons, as a rule no PLD repair has been reported in vitro and in vivo in previous studies. However, in the present study PLD repair occurred in plateau 10T1/2 and TCL 15 cells irradiated with neutrons.

The effects of a static magnetic field on DNA synthesis and survival of mammalian cells irradiated with fast neutrons.

The effects of a static magnetic field (0.75 T) on DNA synthesis and survival were examined with Chinese hamster V79 cells in cultures with and without fast-neutron irradiation. We found that the magnetic field applied alone for up to several hours did not cause a significant effect in either the rate of DNA synthesis or cell viability; the latter was assayed by colony formation. When cells were exposed simultaneously to the magnetic field and fast neutrons, the effects resembled those observed with neutrons alone. This was the case for both inhibition of DNA synthesis and cell killing. Cells irradiated first with neutrons followed immediately by 1 h of magnetic field exposure showed a dose-survival response curve indistinguishable from that of neutrons alone. These data suggest that the biological effect due to the magnetic field is negligible and that the presence of the magnetic field either during or subsequent to fast-neutron irradiation does not affect the neutron-induced radiation damage or its repair.

Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms.

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.

Correlation of microdosimetric measurements with relative biological effectiveness from clinical experience for two neutron therapy beams.

Microdosimetric measurements were made for the neutron therapy beams at the University of Chicago and at the Cleveland Clinic with the same geometry and phantom material using the same tissue-equivalent spherical proportional counter and standard techniques. The energy deposition spectra (dose distributions in lineal energy) are compared for these beams and for their scattered components (direct beam blocked). The model of dual radiation action (DRA) of Kellerer and Rossi is employed to interpret these data in terms of biological effectiveness over this limited range of radiation qualities. The site-diameter parameter of the DRA theory is determined for the Cleveland beam by setting the biological effectiveness (relative to 60Co gamma radiation) equal to the relative biological effectiveness value deduced from radiobiology experiments and clinical experience. The resulting value of this site-diameter parameter is then used to predict the biological effectiveness of the Chicago beam. The prediction agrees with the value deduced from radiobiology and clinical experience. The biological effectiveness of the scattered components of both beams is also estimated using the model.

Isolation and partial characterization of two antifungal proteins from barley.

We have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, Trichoderma reesei. Using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. Both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. The first protein has a molecular weight of 30 000 and is identical to the 30 kDa ribosome-inactivating protein previously isolated from barley. This protein very effectively inactivates fungal ribosomes and this may explain its antifungal activity and biological role. The second antifungal protein has a molecular weight of 28 000 and is 20-fold more potent than the 30 kDa protein in inhibiting growth of Trichoderma. In addition to Trichoderma, the 28 kDa protein also efficiently inhibits growth of Phycomyces blakesleeanus, Alternaria alternaria and a protoplast-forming mutant of Neurospora crassa. The 28 kDa protein does not inactivate fungal ribosomes and we are currently investigating other possible enzymatic activities of this protein.

Novel 2',5'-oligoadenylates synthesized in interferon-treated, vaccinia virus-infected cells.

2-5A[ppp(A2'p)nA] and related materials accumulate to greater than micromolar concentrations in vaccinia virus-infected HeLa cells and in interferon-treated, vaccinia-infected HeLa, CV1, and L929 cells even when virus replication is not inhibited. A variably complex mixture of authentic 2-5A (n = 2 to 4), nonphosphorylated cores [(A2'p)nA; n = 2 to 5], and additional compounds of unknown structure was observed.

Evidence for similarities between radiation damage expressed by beta-araA and damage involved in the interaction effect observed after exposure of V79 cells to mixed neutrons and gamma radiation.

Plateau-phase V79 cells were exposed sequentially to fast neutrons and gamma rays. A dose-dependent reduction in the shoulder width of the gamma-ray survival curve was observed after preexposure of cells to neutrons. A similar effect was demonstrated on the neutron survival curve when cells were preirradiated with gamma rays. Treatment of cells with 150 microM beta-araA after either gamma or neutron irradiation reduced primarily the shoulder of the survival curve. When beta-araA was given to the cells after exposure to mixed radiation modalities, survival curves similar to those observed after exposure to a single radiation modality and treatment with beta-araA were obtained. The kinetics of loss of the interaction observed after exposure of cells to gamma rays following neutron irradiation was similar to the kinetics of loss of sensitivity to beta-araA (T1/2 = 1 h) measured by delaying drug administration after exposure to gamma rays. The results suggest that the PLD expressed by beta-araA is at least partly involved in the interactive effect observed after combined exposure of plateau-phase V79 cells to neutrons and gamma rays.

Use of a 2-5A analogue probe for detecting RNA ligase and RNA ligase substrates in mammalian cell extracts.

The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.

2-5A accumulates to high levels in interferon-treated, vaccinia virus-infected cells in the absence of any inhibition of virus replication.

We investigated the effects of interferon treatment on virus yield, protein synthesis, and the 2-5A system in vaccinia virus-infected HeLa, L929, and CV1 cells. Under the culture conditions used, vaccinia virus replication was relatively insensitive to the antiviral effects of interferon. In L929 and HeLa cells, interferon at 400 reference units (r.u.) per ml had little effect on viral protein synthesis, the virus-induced inhibition of host protein synthesis, or virus yield: 2,000 to 20,000 r.u./ml were required to inhibit these. Despite this, high levels (up to 5 microM) of 2-5A [ppp(A2'p)nA; n greater than or equal to 2] were found during vaccinia infection of all of these types of cells treated with 400 r.u. of interferon per ml, i.e., at interferon concentrations too low to inhibit significantly virus growth. High levels (up to 5 microM) were also found in non-interferon-treated HeLa cells (which have a high constitutive level of 2-5A synthetase) in which vaccinia virus replicates perfectly well. It can be concluded that high levels of 2-5A per se have no necessary antiviral effect on vaccinia virus in these systems. These results are in marked contrast to those obtained here and previously with encephalomyocarditis virus. For example, in HeLa cells less than 20 nM 2-5A accumulated, but virus replication was inhibited by 50 r.u. of interferon per ml (Silverman et al., Eur. J. Biochem. 124:131-138, 1982). The characteristic cleavage of rRNA by the 2-5A-dependent RNase was delayed relative to 2-5A accumulation in the vaccinia virus-infected cells. This delay was not the result of either defective 2-5A or of a stable virus-induced inhibition of the 2-5A-dependent RNase; 2-5A extracted from the cells had full biological activity when assayed by activation of the 2-5A-dependent RNase in cell extract, and the 2-5A-dependent RNase extracted from the vaccinia virus-infected cells was fully active in vitro. The basis for the delay remains to be determined. High levels of 2-5A were not observed when late (DNA synthesis-dependent) vaccinia transcription was inhibited by either cycloheximide or cytosine arabinoside. The only known activator of the 2-5A synthetase is double-stranded RNA. The presence of 2-5A therefore implies the natural occurrence of double-stranded structures in late viral RNA in intact vaccinia virus-infected cells.

Simian virus 40-infected, interferon-treated cells contain 2',5'-oligoadenylates which do not activate cleavage of RNA.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.