Low Altitude Solar Magnetic Reconnection, Type III Solar Radio Bursts, and X-ray Emissions.

Type III solar radio bursts are the Sun's most intense and frequent nonthermal radio emissions. They involve two critical problems in astrophysics, plasma physics, and space physics: how collective processes produce nonthermal radiation and how magnetic reconnection occurs and changes magnetic energy into kinetic energy. Here magnetic reconnection events are identified definitively in Solar Dynamics Observatory UV-EUV data, with strong upward and downward pairs of jets, current sheets, and cusp-like geometries on top of time-varying magnetic loops, and strong outflows along pairs of open magnetic field lines. Type III bursts imaged by the Murchison Widefield Array and detected by the Learmonth radiospectrograph and STEREO B spacecraft are demonstrated to be in very good temporal and spatial coincidence with specific reconnection events and with bursts of X-rays detected by the RHESSI spacecraft. The reconnection sites are low, near heights of 5-10 Mm. These images and event timings provide the long-desired direct evidence that semi-relativistic electrons energized in magnetic reconnection regions produce type III radio bursts. Not all the observed reconnection events produce X-ray events or coronal or interplanetary type III bursts; thus different special conditions exist for electrons leaving reconnection regions to produce observable radio, EUV, UV, and X-ray bursts.

Morphological and molecular characterization of spontaneous myogenic differentiation in a human rhabdomyosarcoma cell line.

Skeletal muscle differentiation consists of an ordered withdrawal of committed cells from the cell cycle and their fusion to form multinucleated myotubes. To determine if differentiation of malignant myoblasts parallels that of normal skeletal muscle, a cell line (Rh28) was established from an alveolar rhabdomyosarcoma. Rh28 displays a constant population doubling time of 45-55 h until passage 60, when the doubling time progressively increases until proliferation ceases. Loss of proliferative capacity is associated with morphological evidence of differentiation to multinucleated myotubes, fusion, and the expression of numerous muscle-specific genes. In contrast to normal myogenic differentiation, multinucleated cells continue to synthesize DNA and express abundant c-myc transcripts. These observations suggest synchronous replication and possible arrest in the G2-phase of the cell cycle, since there was no evidence of mitotic activity in differentiated cells. Terminal differentiation of early passage Rh28 cells was induced in the presence of 10% dialyzed fetal calf serum but not by medium containing 2% undialyzed serum, suggesting a role for low molecular weight growth factors in this process. Our data indicate that the Rh28 cell line may be of value in elucidating the relationship between oncogenic transformation and differentiation in rhabdomyosarcoma.

Biochemical mechanisms in colon xenografts: thymidylate synthase as a target for therapy.

Growth of human adenocarcinomas of the colon and rectum in immunoincompetent mice has allowed for a greater understanding of the interaction of 5-fluorouracil, its metabolism, and mechanism(s) of cytotoxicity under conditions of tumor growth in situ. Conversely, this agent has proven to be a useful tool in defining metabolic characteristics in human colon adenocarcinomas. Analysis of tumor sensitivity to 5-fluorouracil (FUra),5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd) suggests that growth inhibition in vivo is related to a DNA-directed event. Resistance, de novo appears to be a consequence of relatively transient inhibition of the target enzyme thymidylate synthase (dTMP-synthase), which may be a consequence of low concentrations of 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) or its polyglutamate forms within tumor cells in situ. In order to study the relationship between inhibition of dTMP-synthase and growth inhibition, mutant cells deficient in their ability to salvage dThd have been selected, and grown as xenografts. Data suggest that transient inhibition of dTMP-synthase and not dThd salvage is responsible for resistance de novo, and that prolonged inhibition of dTMP-synthase would be a lethal event in vivo. This would predict that a cell lacking dTMP-synthase activity would not be tumorigenic. This has been tested directly by selecting clones of GC3 colon adenocarcinoma cells deficient in dTMP-synthase (TS-) activity. Preliminary data indicate that each of 3 TS- clones is tumorigenic in athymic nude mice. The importance of dTMP-synthase as a target for drug development is discussed with respect to these findings.

Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.

Therapeutic selectivity of vinca alkaloids: a role for guanosine 5'-triphosphate?

Tubulin, the protein subunit of microtubules, is considered a target for antimitotic agents such as colchicine, maytansine and the vinca alkaloids vincristine and vinblastine. Of these agents, only vincristine and vinblastine have been found to have clinical utility for treatment of human neoplastic disease. The basis for therapeutic selectivity was examined in a comprehensive model in which human rhabdomyosarcomas were grown as xenografts in mice. This model has allowed a detailed examination of differences between neoplastic and non-neoplastic tissues with respect to binding, retention and metabolism of vinca alkaloids. Of note is that in tumor tissue, vincristine is tenaciously bound whereas vinblastine is not. In non-neoplastic tissue, retention of both agents is poor. The mechanisms responsible for differential retention between vinca alkaloids and between neoplastic and non-neoplastic tissues were examined. Results suggest that guanosine 5-triphosphate may be implicated in the formation and stability of vinca-tubulin complexes in tissue cytosols. Two models consistent with the data are proposed, and the significance to therapeutic efficacy is discussed.

Characterization of cell lines derived from xenografts of childhood rhabdomyosarcoma.

Three human rhabdomyosarcoma cell lines (Rh10, Rh18, and Rh28) have been established from three independently derived xenografts. These lines have been characterized as mesenchymal in origin (reactivity to desmin and vimentin antibodies) and as expressing a human fetal muscle surface antigen recognized by monoclonal antibody 5.1 H11. Measurable levels of creatine phosphokinase have been detected in the cell lines. Rh10 and Rh28 exhibit the same chromosomal translocation and express an atypical lactate dehydrogenase isoenzyme which may be homologous to those previously reported in other tumor types. The karyotype analysis has confirmed that each cell line was derived from its respective tumor and thus provides a unique model for future investigations.

Relationship between binding affinity, retention and sensitivity of human rhabdomyosarcoma xenografts to Vinca alkaloids.

Xenografts of human rhabdomyosarcoma (RMS) have been derived that differ in their degree of sensitivity to Vinca alkaloids. Lines Rh12 and Rh18 demonstrated, respectively, high and moderate sensitivity to vincristine (VCR), but showed little responsiveness to vinblastine (VLB) in vivo. Rh18/VCR-3, a subline of Rh18 selected for resistance to VCR under in situ conditions, was insensitive to further challenge with VCR. Resistance was associated with elimination of the agent in a biphasic manner, whereas sensitivity to VCR corresponded to very prolonged drug retention in sensitive neoplastic tissues. The initial half-times for drug retention in tumors in vivo (t1/2 alpha) correlated with the degree of sensitivity of tumors to Vinca alkaloids, decreasing t1/2 alpha being associated with decreased sensitivity. A single binding species was observed when membrane-free supernatant fractions were incubated at 37 degrees for 15 min with 10.4 nM [3H]VCR and analyzed by gel filtration HPLC. The protein eluted with a retention time of 57 min and corresponded to a molecular weight (Mr) of approximately 113,000 daltons, agreeing very closely with the Mr of dimeric tubulin (approximately equal to 110,000 daltons). Two fractions were collected and eluted on a one-dimensional denaturing gel. Proteins were transferred subsequently to nitrocellulose and probed with an 125I-labeled monoclonal antibody specific for beta-tubulins. Only the fraction containing bound [3H]VCR contained tubulin. Estimates for the dissociation constants (Kd) for the binding affinity of VCR and VLB in crude, membrane-free supernatant fractions from RMS xenografts were obtained by computer curve fitting using a mathematical binding model. Data fitted a two-site binding model, with Kd values for the high-affinity site ranging from 61 to 160 nM, and for the low-affinity site, from 42 to 94 microM. At physiologically achievable drug concentrations, the relationship between binding affinity, drug retention and tumor sensitivity was examined further. A close relationship was apparent between the Kd values for VCR in Rh12, Rh18 and Rh18/VCR-3 tumor supernatant fractions and VLB in Rh12 preparations, and t1/2 alpha values for drug retention. Prolonged drug retention correlated with a low binding constant. As t1/2 alpha decreased, binding affinity also decreased, as demonstrated by an increase in the Kd value. Consequently, the tightness of drug binding in tumors also correlated with the degree of sensitivity of the xenografts to Vinca alkaloids.(ABSTRACT TRUNCATED AT 400 WORDS)

In situ selection of a human rhabdomyosarcoma resistant to vincristine with altered beta-tubulins.

In order to simulate more closely conditions in which resistance to vincristine (VCR) is selected in human solid tumors, a human rhabdomyosarcoma grown as a xenograft in immune-deprived mice has been selected for resistance in situ. Karyotype analysis showed the resistant line, HxRh18/VCR-3, to have a diploid modal number, with no apparent translocations, whereas the predominant population in the parental, sensitive HxRh18 xenograft demonstrated a modal number near-tetraploid with many marker chromosomes. From the rapid rate at which resistance was selected and from karyotypic evidence, data strongly suggest that HxRh18/VCR-3 was a subpopulation within the parent tumor. When grown in the same host, HxRh18/VCR-3 tumors accumulated less drug, and the rate of [3H]VCR loss was 5-fold greater than in HxRh18 tumors. Thus, accumulation and retention of [3H]VCR in HxRh18/VCR-3 resistant tumors was identical to that of [3H]vinblastine (VLB) in HxRh18 xenografts. HxRh18 xenografts are intrinsically resistant to VLB. Analysis by high-performance liquid chromatography of [3H]VCR:protein complexes in HxRh18 cytosols indicated one binding species (Mr 95,000 to 116,000), probably the tubulin heterodimer. Of interest was the observation that beta-tubulin species, identified on Western blots by monoclonal antibody, differed in these tumors. In HxRh18/VCR-3, less acidic beta-tubulins of HxRh18 were decreased or absent, with three additional more acidic isoforms present in the resistant line. As vincristine may bind to the beta-subunit of tubulin, this may have importance to vincristine resistance in vivo.

Cloning efficiency of cultured human tumor cell lines measured with the use of a Coulter particle counter.

A technique is described by which colony formation in agarose may be rapidly and reproducibly determined with the use of a modified Coulter particle counter (CPC). The cloning efficiency of RD human rhabdomyosarcoma cells after exposure to vincristine sulfate or cisplatin has been compared with the CPC method or by conventional visual counting. These techniques give very similar results. In combination with a Coulter Channelyzer, the CPC technique can be used to evaluate drug effects in colony sizes from a median of 20 to greater than 290 cells. The integrity of colonies formed by 3 colon carcinoma cell lines was examined to determine if this method could be applied to other systems.

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.

Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells.

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.